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Flowcytometry Lymphoid Atlas

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Flowctometry Wojciech Gorczyca.4th Edition(2023)

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■■■1Introduction To Flowcytometry

 

FIGURE 1.1 APL: typical flow cytometry pattern. Neoplastic promyelocytes are characterized by hypergranular cytoplasm with Auer rods (a). Flow cytometry features includes high side scatter (b, arrows), lack of CD34 (c), positive CD117 (d), lack of HLA-DR (e), positive CD13 (f), positive CD33 (g), and dimly positiveCD64 (h)

 

FIGURE 1.2 Identification of myeloblasts (acute myelomonocytic leukemia, AMML). Myeloblasts (green dots) show moderate CD45 and low side scatter placing them is "blastic" gate on CD45 versus SSC display (a). They are usually positive for HLA-DR (b), CD34 (b), CD133 (c), and CD117 (c). Monocytes are represented by blue dots

 

FIGURE 1.3 Acute monoblastic leukemia. Monoblasts (green dots, arrow) show bright expression of CD45 and slightly increased side scatter (a). CD34 and CD117 are not expressed (b-c), HLA-DR is positive (d), CD11b is variably expressed with subset of cells being negative and subset showing dim expression (e). There is aberrant, mostly negative CD13 expression (f) and both CD33 and CD64 are brightly expressed (g-h). CD14 is mostly negative (h)

 

FIGURE 1.4 BPDCN shows low side scatter (a), negative to dim CD45 (a), positive CD4 (b), CD56 (c), CD123 (d), and HLA-DR (e), and does not express CD33 (f), CD34 (g), and CD117 (h)

 

FIGURE 1.5 B-lymphoblasts (green dots, arrow) show negative to dim CD45 (a), low side scatter (a), and positive expression of CD34 (b), CD19 (c), and CD10 (d)

 

FIGURE 1.6 T-lymphoblasts (green dots; arrow) are CD45+ (a), have low side scatter (a), express TdT (b), CD7 (c) and are either dual CD4/CD8-negative (d-e) or dual CD4/CD8-positive (not shown)

 

FIGURE 1.7 Identification of clonal B-cells: B-cells (red dots, arrow) show CD20 expression and K restriction (a). There is no expression (b)

 

FIGURE 1.8 Identification of abnormal T-cells: increased forward scatter. PTCL with positive expression of all pan T-cell antigens (a-d). The neoplastic cells have increased forward scatter (compare with residual B-cells (g) and benign T-cells) and show dim CD3 (b). The expression of CD5 is slightly dimmer when compared to benign T-cells (c), and the expression of both CD2 and CD7 is slightly brighter (a and d). Neoplastic T-cells are CD4+ (e-f). Residual B-cells have low forward scatter and positive CD20 (g)

 

FIGURE 1.9 Circulating promyelocytes (APL). (a, b) Blood smear showing atypical promyelocytes. (c) FC showing promyelocytes (green dots) with high side scatter (SSC) and dimmer expression of CD45 when compared to neutrophils

 

FIGURE 1.10 Etiology of hyperleukocytosis: B-ALL (a), AML (b), CML (c), CLL (d), and T-PLL (e)

 

FIGURE 1.11 Hemophagocytic lymphohistiocytosis (HLH)

 

FIGURE 1.12 Burkitt lymphoma involving cerebrospinal fluid (CSF). FC analysis using 1 tube 10-color, 15-antibodies screening panel (CD3, CD4, CD5, CD8, CD10, CD14, CD19, CD20, CD33, CD34, CD45, CD56, CD117, kappa, and lambda). B-cells gated based on CD19 expression (a) show dim kappa expression (b) and co-expression of CD10 and CD20 (c). T-cells gated based on CD3 expression (d) show normal CD4:CD8 ratio (e). Ungated cells (f) show presence of CD5+ T-cells and CD19+ B-cells (f)

 

FIGURE 1.13 t-SNE map (color codes: purple, monocytes; red, T-cells; green, NK-cell; blue, B-cells; and gray, ungated cells). Blood sample stained with CD3, CD10, CD14, CD20, CD27, CD45, CD56, and CD57

 

FIGURE 1.14 (a-b) Gating strategy of flow cytometric samples from lymph node (and other "solid" organs). Non-viable cells, which are labeled by 7-AAD (arrow) are excluded from analysis. Only 7-AAD-negative (viable) cells are further analyzed. Similar results may be achieved with other fluorochromes, e.g., propidium iodide. Both panels (a-b) represent lymph node with B-cell lymphoma: major- ity of cells in sample A are viable (7-AAD-negative) whereas majority of cell in sample B are non-viable (7-AAD-positive). Non-viable cells tend to scatter less light in forward direction and therefore are characterized by low forward scatter, when compared to viable cells. (c-d) Comparison of granulocytes and non-viable cells (c) with eosinophils (d) based on forward scatter versus aqua (viability) staining. Note much brighter aqua staining of non-viable cells

 

FIGURE 1.15 Analysis of percent of kappa and lambda B-cells in this CLL may suggest polytypic process (a, a'), but careful evalu- ation of pattern of antigen expression (CD20 versus kappa and lambda) indicate bi-clonal B-cell lymphoproliferative process: kappa B-cells show variable CD20 expression (b; red arrows) whereas lambda+ B-cells show more cohesive cluster of cells (b'; gray arrow)

 

FIGURE 1.16 Assessing the staining results: the staining intensity for each antibody is compared with the negative control. Myelomonocytic population (green and blue dots) in panel b appears to express CD14, when compared to overtly negative lympho- cytes (red dots). However, the intensity of the expression of CD14 is similar to non-specific staining with isotypic control (panel a), and therefore the result for CD14 have to be interpreted as negative. In this sample, only the cells located within the dotted circle (close to and beyond 103 on x-axis) would be considered positive. These panels show also that the "built-in" negative controls (in this case lymphocytes, which do not express CD14) cannot be used reliably as a negative control, since the population of interest may be char- acterized by a very high non-specific "background" staining (a)

 

FIGURE 1.17 High non-specific (background) staining. Flow cytometry shows three distinct populations: blasts (green dots), granulocytes (gray dots) and lymphocytes (red dots). Based on control staining (left column; a-d) blasts are positive for CD33 (b') and CD2 (c), granulocytes are positive for CD33 (b') and lymphocytes are positive for CD2 (c) and CD3 (d'). All 3 populations do not express HLA-DR (a")

 

FIGURE 1.18 Intensity of antigen expression, see text for details

 

FIGURE 1.19 Co-expression of antigens. (a) Two populations of T-cells are identified by comparing the expression of CD3 and CD7. Normal (benign) T-cells (upper right quadrant) co-express CD3 and CD7, whereas neoplastic cells (lower right quadrant) lack CD3. (b) B-CLL/SLL is characterized by co-expression of CD5 and CD23 (upper right quadrant) in contrast to benign T-cells which have brighter expression of CD5 and do not express CD23 (lower right quadrant). (c) HCL is characterized by co-expression of CD25 and CD103. (d) Immature T-cell population (thymocytes from thymoma) display co-expression of CD4 and CD8 (upper right quadrant). Mature T-cells are either CD4+ (upper left quadrant) or CD8+ (lower right quadrant). (e) Immature T-cell population co-expressing CD2 and TdT (upper right quadrant); benign T-cells express CD2 but are TdT (upper left quadrant). (f) Myeloblasts co-expressing CD34 and CD33 (arrow)

 

FIGURE 1.20 Aberrant antigen expression. (a) DLBCL with aberrant expression of CD56; (b) HCL with aberrant expression of CD2 on a subset of leukemic cells; (c) CLL/SLL with co-expression of CD8 (arrow); (d) PTCL with aberrant expression of CD20 (arrow) on a subset of T-cells; (e) MZL with aberrant expression of CD13 (arrow)

 

FIGURE 1.21 Aberrant lack of antigen expression. (a) Acute monoblastic leukemia with aberrant loss of HLA-DR expression; (b) B-ALL with loss of CD45 expression; (c) DLBCL with loss of CD20; (d) PTCL with aberrant loss of surface CD3 expression

 

FIGURE 1.22 Side scatter (SSC; orthogonal, right angle scatter) on Y axis corresponds to granularity of the cytoplasm (X axis presents CD45 expression). Lymphocytes (a through f; red dots) have low SSC, whereas neutrophils (b-c), cancer cells (e) and atypical promyelocytes (f) have high SSC. Monocytes (b; blue dots), blasts (c; green dots) and HCL cells (d; blue dots) have higher SSC than lymphocytes (compare with red dots)

 

FIGURE 1.23 Forward scatter (FSC). Comparison of FSC (Y axis) between myeloblasts (a-b; green dots) and monoblasts (c-d; blue dots). Myeloblasts are smaller and therefore have lower FSC when compared to monocytic cells (compare with the location of dotted line in upper and lower panels or in relation to small lymphocytes marked with red dots)

 

FIGURE 1.24 Forward scatter (FSC; Y axis): comparison between benign small lymphocytes (red dots, a), large lympho- cytes of diffuse large B-cell lymphoma (blue dots; a) and meta- static cancer cells (orange dots; b)

 

FIGURE 1.25 Classification of Hodgkin lymphomas. (a-f) classic HL: (a) touch imprint with Reed-Sternberg cells (R-S); (b) histo- logic section with typical multinucleated R-S cell; (c) positive CD30; (d) positive CD15; (e) negative CD45; and (f) negative CD20. (g-m) NLPHL: (g) touch imprint showing an atypical cell with large nucleus (LP cells, lymphocyte predominant); (h) low magnification of the lymph node showing nodular pattern; (i) high magnification showing typical LP cells (lymphocyte predominant, syn: "popcorn" or L&H cells); (j) positive CD20, (k) positive EMA; (1) positive CD45 (prominent membranous staining, compare with negative CD45 in R-S cells, e); (m) typical CD3+ T-cell rosettes around LP cells

 

FIGURE 1.26 Classical Hodgkin lymphoma. Reed-Sternberg cells (a) and Hodgkin cells are positive for CD30 (b) and CD15 (c). Flow cytometry analysis (d-j) reveals only benign (polytypic) B-cells (d–f) and benign T-cells with increased CD4:CD8 ratio (f) and normal expression of pan-T-cell antigens (g-j)

 

FIGURE 1.27 Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). The typical nodular patter on low power exami- nation (a). Neoplastic cells (LP cells, also called "popcorn" cells or L&H cells) have large and multilobated or folded nuclei, promi- nent nucleoli and pale vesicular chromatin (b-c). Flow cytometry analysis shows polytypic B-cells (d-e) and T-cells with increased CD4:CD8 ratio (f) and normal pattern of expression of pan-T antigens (g-j)

 

FIGURE 1.28 T-cell-rich large B-cell lymphoma. Atypical lymphoid infiltrate (a-b) with scattered large B-cell expressing CD20 (c) and predominance of small T-lymphocytes expressing CD3 (d)

 

FIGURE 1.29 THRLBL. Tissue section (a) shows rare, atypical large lymphoid cells in the background of small lymphocytes. Flow cytometry (b-c) reveals minute clonal B-cell population (arrow) in the background of predominantly benign (polytypic) B-cells

 

FIGURE 1.30 Diffuse large B-cell lymphoma with necrosis. Histologic section (a) shows necrosis and foci of viable tumor cells around blood vessels. Flow cytometry (b-d) results are negative due to predominance of necrotic cells in the sample (arrow). Rare T- and B-cells are identified (c-d)

 

FIGURE 1.31 Anaplastic large cell lymphoma. Histologic sections (a-b) show numerous large neoplastic cells and areas of necrosis. Tumor cells are positive for CD30 (c). Flow cytometry shows only reactive B (d-e) and T-cells (f-j)

 

FIGURE 1.32 Bone marrow involved by follicular lymphoma (a, histology; b, immunostaining for CD20). Due to paratrabecular location of neoplastic lymphoid aggregates and/or due to reticulin fibrosis which often accompanies lymphoid infiltrate in the bone marrow, flow cytometry often does not reveal clonal B-cell population

 

FIGURE 1.33 Acute megakaryoblastic leukemia (a) with prominent diffuse reticulin fibrosis (b). Fibrosis and predominance of large neoplastic cells (a) contribute to non-diagnostic FC results in many cases

 

FIGURE 1.34 Multiple myeloma. The discrepancy between the number of clonal plasma cells detected by flow cytometry (a; arrow) and actual extent of bone marrow involvement (b) is due to drop-out of neoplastic cells during bone marrow aspiration and sample prepa- ration. Plasma cells have delicate cytoplasm which often get destroyed during tissue processing leading to the under-representation of neoplastic cells on FC displays

 

FIGURE 1.35 Bone marrow aspirate with prominent dyserythropoiesis representing MDS

 

FIGURE 1.36 Classification of hematopoietic tumors. Abbreviations: AITL, nodal TFH cell lymphoma angioimmunoblastic-type; aCML, atypical chronic myeloid leukemia; ALCL, anaplastic large cell lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AMML, acute myelomonocytic leukemia; BCLU, B-cell lymphoma, unclassifiable with features intermediate between BL and DLBCL; BL, Burkitt lymphoma; BPDCN, blastic plasmacytoid dendritic cell neoplasm; CEL, chronic eosinophilic leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; CNL, chronic neutrophilic leukemia; DLBCL, diffuse large B-cell lymphoma; EATL, enteropathy-associated T-cell lymphoma; EB, excess blasts; ET, essential thrombocythemia; HCL, hairy cell leukemia; HL, Hodgkin lymphoma; HSTL, hepatosplenic T-cell lymphoma; LPL, lymphoplasmacytic lymphoma; MBL, monoclonal B-cell lymphocytosis; MCL, mantle cell lymphoma; MLD, multilineage dysplasia; MPAL, mixed phenotype acute leukemia; MDS, myelodysplastic neoplasm; MDS-U, MDS unclassifiable; MF, mycosis fungoides; MPAL, mixed phenotype acute leukemia; MPN, myeloproliferative neoplasm; MZL, marginal zone lymphoma; NHL, non-Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; NOS, not otherwise specified; PBL, plasmablastic lymphoma; PEL, primary effusion lymphoma; PCFCL, primary cutaneous follicle center cell lymphoma; PLL, prolymphocytic leukemia; PMF, primary myelofibrosis; PTCL, peripheral T-cell lymphoma; PV, polycythemia vera; RS, ring sideroblasts; SLD, single lineage dysplasia; SLL, small lymphocytic lymphoma; SPTL, subcutaneous panniculitis-like T-cell lymphoma; SS, Sézary syndrome; TH, T-follicular helper

  

 

■■■【2】Flowcytometry analysis of Blood&Bone marrow

 

 FIGURE 2.1 Bone marrow-normal histology: (a) low magnification; (b) higher magnification; (c) CD34 (blasts+); (d) CD61 (mega- karyocytes+); (e) MPO (myeloid cells+); (f) GPHA (erythroid cells+)

 

FIGURE 2.2 Hematopoiesis (see text for details)

 

FIGURE 2.3 Antigen expression of major cell types during normal hematopoiesis

 

FIGURE 2.4 CD45 expression by granulocytes. The expression of CD45 increases as the cells reach final stages of differentiation into neutrophils (a). The direction of maturation is indicated in dot plot (b) by arrows (benign bone marrow sample from 11-month-old infant). Based on the CD45 versus side scatter (SSC), two populations of granulocytic cells can be identified: one with variable SSC and dimmer CD45 and the other with slightly lower SSC and brighter CD45. The first populations include promyelocytes, myelocytes, metamyelocytes, and bands, and the second population includes mostly mature neutrophils. The distinction between those two popu- lations is more evident in plot (b) (benign bone marrow with relative T-cell lymphocytosis). Blood sample with reactive neutrophilia shows only one population of cells with very bright CD45

 

FIGURE 2.5 The expression of CD10, CD11b, CD16, CD11c, and HLA-DR during normal myeloid maturation (normal bone marrow samples; insets show normal blood samples). Myeloblasts gain CD13 as they mature to promyelocytes, and then lose its intensity as they become myelocytes; and then gain expression along the maturation toward mature neutrophils (a). CD16 expression starts to appear at metamyelocyte stage and is strongest in segmented forms (a-b). CD11b and CD11c expression gradually increases as the cells mature (b-c); neutrophils are brightly positive for both antigens. HLA-DR is positive in blasts and negative in promyelocytes and subsequent stages (c). CD33 is positive in all stages of granulocytic maturation, but the expression decreases as the cells mature (d-e). CD10 is positive in segmented forms (f). Abbreviations: Pro, promyelocytes; Myelo. Myelocytes; Neut, neutrophils

 

FIGURE 2.6 Flow cytometric features of eosinophils. Blood with marked eosinophilia (a; cytospin preparation from flow sample). Eosinophils, similarly to neutrophils, have high side scatter and moderate CD45 (b). Neutrophils are HLA-DR-negative, whereas eosino- phils show dim HLA-DR (c). The expression of CD13 (d) and CD33 (e) is dimmer in eosinophils, when compared to neutrophils. Eosinophils are negative for CD10 (f), show bright expression of CD11b (g) and CD11c (h), and in contrast to neutrophils do not express CD16 (i)

 

FIGURE 2.7 Classification of monocytes based on CD14 and CD16 expression (see text for details)

 

FIGURE 2.8 B-cell development

 

FIGURE 2.9 T-cell development

 

FIGURE 2.10 Gating strategy. Part of the sample from the bone marrow aspirate or blood (tube) is smeared on the microscope glass. slide for morphologic correlation, while the rest is incubated with antibodies, lysed, fixed, and submitted for flow cytometry analysis; see text for details

 

FIGURE 2.11 "Lymphocytic" gate. Benign lymphocytes and the majority of mature lymphoproliferative disorders are characterized by low side scatter (SSC) and bright expression of CD45 (red dots; arrow)

 

FIGURE 2.12 "Monocytic" gate. Benign monocytes and majority of neoplastic monocytic proliferations are characterized by bright CD45 expression and slightly increased side scatter (SSC). They appear above lymphocytic gate on CD45 versus SSC display (arrow). Apart from monocytic cells, similar SSC and CD45 properties are often seen in hairy cell leukemia and occasional lymphoprolifera- tions, mast cell leukemia (unusual and rare form of mast cell disease), and markedly dysplastic granulocytes

 

 FIGURE 2.13 "Hematogones" gate (a). Hematogones are characterized by very low side scatter (SSC) and dimmer expression of CD45 when compared to lymphocytes (red dots); they have variable ("smeared") expression of CD20 (a'; broken arrows), and are posi- tive for CD10 and partially for CD34 (a"). Occasional lymphoproliferative disorders and blasts may have similar to hematogones SSC and CD45 properties (b-e)

 

FIGURE 2.14 "Blast" gate. Myeloid precursors (blasts) have moderate CD45 and low side scatter (SSC). Occasional lymphoprolif- erative disorders, plasma cell tumors, as well as granulocytes with decreased granularity and microgranular APL have similar CD45 versus SSC characteristics

 

FIGURE 2.15 "Plasmacytic" gate. Apart from plasma cell neoplasms, erythroid precursors, occasional AML and B-ALL are charac- terized by negative CD45 and low side scatter. Rare T-PLL may be CD45-negative

 

FIGURE 2.16 "Metastatic" gate. High side scatter and negative CD45 is typical for non-hematopoietic tumors and rare plasma cell neoplasms

 

FIGURE 2.17 "Granulocytic" gate. Granulocytes are characterized by high side scatter (SSC) and moderate expression of CD45. Hematopoietic tumors with similar characteristics include mast cell proliferations, acute promyelocytic leukemia (hypergranular variant), and occasional large cell lymphomas and rare acute myeloid leukemias

 

FIGURE 2.18 Flow cytometry of blood: normal pattern. CD45 versus side scatter (SSC) display (a) shows three major populations: neutrophils (gray dots), monocytes (blue dots), and lymphocytes (red dots). Neutrophils are characterized by high side scatter (SSC, a) and high forward scatter (FSC, b), while lymphocytes show low SSC and FSC. Normal blood sample shows either no blasts or only very few circulating blasts (b). Monocytes and neutrophils are CD11c+ (c), CD11b+ (d), and CD15+ (d). Monocytes show bright expres- sion of CD14 (e) and CD33 (f), while neutrophils show dimmer CD33 when compared to monocytes (f) but are brightly positive for CD10 (g). NK-cells are CD11c (c) and CD16+ (e). Kappa versus lambda displays shows presence of polytypic B-cells (h) with kappa to lambda ratio ranging from 0.5:1 to 3:1 on average. B-cells are positive for CD19 (g, i) and T-cells are positive for CD5 (i); there are no or only very few CD5+ B-cells (the number of CD5+ B-cells is more pronounced in children and young adults). Majority of benign T-cells are positive for both CD3 and CD7 (j). Number of CD7-negative T-cells may increase in certain reactive conditions, such as viral infections. Majority of benign T-cells are positive for CD26, but most normal blood samples show minor fraction of CD26- T-cells (k). Number of benign T-cells with lack of CD26 may be higher when there is increased number of T-LGL cells. Benign B-cells are dimly positive for CD200 (1)

 

FIGURE 2.19 Flow cytometry of BM: normal pattern. CD45 versus side scatter (SSC) display (a) shows six major populations: myeloid cells at different stages of maturation (gray dots), monocytes (dark blue dots), lymphocytes (red dots), hematogones (light blue dots), and blasts (green dots). Myeloid cells (granulocytes) are characterized by high side scatter (SSC, a) and high forward scat- ter (FSC, b), while lymphocytes and hematogones show low SSC and FSC (a-b). Both hematogones and blasts are CD34+, but they differ in FSC (b). B-cells, hematogones and majority of benign plasma cells are CD19+ (c). The expression of CD38 is dim on myeloid cells, moderate to bright on blasts and hematogones, and very bright on plasma cells (c). Hematogones and most mature myeloid cells (neutrophils) are CD10+ (d). Monocytes and most mature myeloid cells (neutrophils, metamyelocytes, and myelocytes) show bright expression of CD11b (d-e) while less mature myeloid precursors are CD11b. CD15 is positive on myeloid cells (granulocytes) and monocytes (e). Myeloid cells (neutrophils, maturing myeloid precursors, and monocytes) are positive for CD13 and CD33 (f); note variable pattern of expression of CD13 at different stages of maturation. Majority of benign T-cells are positive for both CD3 and CD7 (g). Benign B-cells show polytypic expression of surface light-chain immunoglobulins (kappa and lambda, (h), dot plots gated on lym- phocytes only). The proportion of kappa to lambda (K/2 ratio) is within the range of 0.5-3.0:1. HLA-DR is positive on blasts, B-cells, monocytes, and hematogones (i)

  

 

■■■【3】Identification of Clonal B-Cell population

 

 FIGURE 3.1 Benign polyclonal B-cell lymphocytosis in newborn. CD45 versus side scatter (SSC; a) shows predominance of lym- phocytes (red dots). B-cells are polytypic (b) showing kappa+ and lambda populations. B-cells are positive for CD19 and CD20 (c) and show partial expression of CD5 and CD23 (d, arrow). Rare B-cells express also CD10 (e, arrow). B-cells show dim expression of CD81 (f) and moderate expression of CD200 (g). T-cells are positive for CD3 and CD7 (h) and show increased CD4:CD8 ratio (i)

 

FIGURE 3.2 Benign (reactive) lymph node. B-cells show polytypic light chain immunoglobulins expression (a-b). Minor subset of B-cells is CD10+ (c; germinal center cells). Many of small B-cells are CD23+ (d; arrow), whereas activated B-cells are CD71+ (e; arrow)

 

FIGURE 3.3 Florid reactive follicular hyperplasia (lymph node). In reactive lymph node with florid follicular hyperplasia, the major- ity of lymphocytes are small with low forward scatter (FSC, a). Only subset of germinal center cells and activated lymphocytes display increased forward scatter (blue dots). B-cells show polytypic pattern of kappa and lambda expression (b). Benign germinal center cells are CD10+ and show minimally brighter expression of CD19 than the rest of B-cells (c). The dot plots with CD20 versus light chain immunoglobulin (d-e) show few distinct B-cell populations: germinal center B-cells with bright CD20 (arrows) and variable ("smeary") expression of surface light chain immunoglobulins with slight kappa predominance and non-germinal center B-cells with dimmer CD20 and either kappa or lambda positivity. The predominance of kappa+ germinal centers cells is most typical, but some cases with follicular hyperplasia (especially from children and young adults) may show lambda excess (see Figure 3.3). There are only very few CD5+ B-cells (f); the number of B-cells expressing CD5 is more prominent in children. Dot plots g-k show difference in expression of CD20 (g), CD38 (h), CD200 (i), CD71 (j), and CD81 (k) between germinal center cells and other B-cells. In reactive lymph node minor subset of T-cells shows loss of CD26 expression (I)

 

 

 FIGURE 3.4 Follicular hyperplasia with germinal center cells showing lambda excess. Histologic section (H&E, a) shows follicular hyperplasia of tonsil from 4-year-old child. Immunostaining shows CD10+ (b) and BCL2- (c) germinal centers. Flow cytometry (d-f) shows polytypic B-cells with distinct population of bright CD20+ germinal center cells (arrow) displaying variable expression of sur- face light chain immunoglobulins (d-e) with obvious lambda excess (compare d and e) and positive CD10 (f)

 

FIGURE 3.5 Polytypic B-cells in lymph node from HIV+ patient showing poor resolution between surface light chain positive and negative B-cells (a-b). B-cells appear as if they were dual kappa and lambda positive

 

FIGURE 3.6 Clonal B-cells (kappa; a). There is no lambda expression (b)

 

FIGURE 3.7 Partial lymph node involvement by MZL. FC analysis shows minute monoclonal B-cell population (kappa) in the background of predominantly polytypic B-cells. Standard analysis of CD19 versus kappa and lambda (a-b) shows polytypic B-cells with slight kappa excess. A distinct, albeit minute population is noted on CD20 versus CD38 display (c, arrow). It shows brighter CD20 and positive CD38 expression. When analysis is confined to this population (d-d'), it shows kappa+ clone (compare with e-e')

 

FIGURE 3.8 Partial involvement of the lymph node by follicular lymphoma. A histologic section (a) shows a lymph node with reac- tive follicles (*; dotted arrows) and a focus of FL (a; solid arrow). Flow cytometry (b-c) shows two populations of B-cells: those with moderate expression of CD20 (*) and those with bright expression of CD20 (c; arrow). The latter are monoclonal cells (+), representing FL. Residual benign B-cells with moderate CD20 expression are polytypic (compare b and c). The two populations differ not only in respect to the intensity of CD20 expression but also by forward scatter, which corresponds to cell size (d-e): gating on cells with mod- erate CD20 and low forward scatter (d) yields polytypic cells (see histograms on the right). Gating on cells with bright CD20 expression and slightly higher forward scatter (e) yields monoclonal + population (arrow, see histograms on the right)

 

FIGURE 3.9 Diffuse large B-cell lymphoma: identification of clonal B-cells by gating on large cells (cells with high forward scat- ter). (a) Immunohistochemical staining for CD20 on tissue section from flow sample; (b-d) flow cytometry. Despite predominance of large B-cells in the tissue section (a), analysis of lymphocytic gate for kappa and lambda expression (b-c) shows only rare B-cells with suspicious cluster of kappa+ cells (arrow). Gating on large cell population with high forward scatter and brighter CD20 expression (d) shows obvious clonal population

 

FIGURE 3.10 B-cell lymphoproliferative neoplasm composed of two distinct clonal populations, kappa (a) and lambda (b). Kappa positive clone (black arrow) is characterized by bright kappa expression, moderate CD19 (a, b) and moderate CD20 (c, d). Lambda positive clone (green arrow) shows dim CD19 (b) and dim CD20 (d)

 

FIGURE 3.11 Blood with prominent B-cell lymphocytosis. Both kappa+ (a) and lambda+ (b) B-cells are noted mimicking polytypic process. The presence of marked B-cell lymphocytosis and dim CD10 expression (c) on minor subset of kappa B-cells should raise the possibility of a bi-clonal lymphoproliferative process. Molecular testing (PCR) showed clonal rearrangement of IGH

 

 

 FIGURE 3.12 Follicular lymphoma with non-detectable surface immunoglobulins. Clonal B-cells are CD10+ (a) and do not express kappa (b) or lambda (c). Only benign (residual) B-cells show polytypic expression of kappa and lambda (b-c)

 

 

 FIGURE 3.13 DLBCL with non-detectable surface immunoglobulins. When compared to normal (polytypic) B-cells, clonal B-cells (solid arrow) show very high forward scatter (FSC; a-f), slightly dimmer expression of CD19 (a), brighter CD20 (c), and dimmer CD22 (c). They are positive for CD71 (d) and appear both kappa and lambda negative (e-f), although very dim lambda expression cannot be excluded

 

FIGURE 3.14 Burkitt lymphoma in a 4-year-old boy: clonal CD10+ B-cells (a; solid arrow) are negative for surface immunoglobulins (b-c). FISH studies confirmed MYC rearrangement (d)

 

FIGURE 3.15 Follicular lymphoma. Majority of lymphomatous cells are surface immunoglobulin-negative (a−b; blue dots), but a minute subset shows kappa expression (a; arrow). All neoplastic cells express CD10 (c, arrow). Residual benign B-cells are polytypic (a-b; red dots) and do not express CD10 (c)

 

 

FIGURE 3.16 Benign lymph node with follicular hyperplasia. Reactive follicles (H&E section; a) express CD20 (b), and have BCL2- germinal centers (c). Flow cytometry analysis (d-j) shows rather prominent polytypic B-cells composed of two distinct populations: B-cells with moderate CD20 (d-g; green arrows) and B-cells with bright CD20 (d-g; red arrows). B-cells with moderate CD20 rep- resent non-germinal center B-cells, which are CD71- (f; green arrow), CD23+ (g; green arrow), and CD10- (h; green arrow). Germinal center cells are CD10 (h; red arrow), CD71+ (f; red arrow), and CD23 (g; red arrow). The expression of CD19 is similar to slightly brighter when compared to CD10- B-cells (h). Germinal center cells are polytypic but show variable ("smeary") expression of surface light chain immunoglobulins (a-b; i-j; red arrows)

 

 

FIGURE 3.17 Florid follicular hyperplasia from HIV+ patient (H&E section; a); immunohistochemistry (b-c); and flow cytometry (d-e). Germinal center cells are positive for CD10 (b) and are negative for BCL2 (C). Flow cytometry reveals both T-cells (CD20-) and B-cells (CD20+). B-cells show strike predominance of CD10+ germinal center cells (d-g; arrow), which display variable ("smeared") expression of surface light chain immunoglobulins with brighter expression of kappa than lambda (compare d and e). CD10+ B-cells are BCL2(f). This pattern is characteristic for florid follicular hyperplasia and should not be confused with B-cell lymphoma of follicle center cell origin. Clonal B-cells from follicular lymphoma would not display "smeared" pattern of surface immunoglobulin (the clus- ter would be more cohesive) and in the majority of cases would be BCL2+. The expression of CD38 by CD10+ B-cells is often brighter in follicular hyperplasia (g; arrow) when compared to FL

 

FIGURE 3.18 Follicular lymphoma composed of intermediate in size cells. Clonal B-cells display increased forward scatter (a-b; arrow), dim CD19 (a, c), positive CD10 (b-e), and lambda restriction (d-e). Subset of lymphomatous cells is CD71+ (e, arrow)

 

FIGURE 3.19 Follicular lymphoma: clonal B-cell (lambda+; a−b, arrow) are positive for BCL2 (c)

 

 

FIGURE 3.20 MBL with t(11;14) or I-NN-MCL. Flow cytometry (a-e) shows bright CD45 expression and low SSC (a), moderate kappa expression (a, compare with lambda, c), bright CD20 (b-c), co-expression of CD5 (partial) with CD23 (d), and negative CD200 (e; benign B-cells are CD200+). FISH analysis confirmed IGH/CCND1 rearrangement with one green (IGH), one orange (CCND1), and two yellow fusion signals (f)

 

FIGURE 3.21 Aberrant expression of CD13 in MZL. Blood smear (a) shows atypical lymphoid cells. Flow cytometry (b-d) show positive CD19 (b-c), kappa restriction (b, compare with lambda, c), and aberrant albeit partial expression of CD13 (d; arrow; CD13 is positive only on subset of clonal CD20+ B-cells)

 

FIGURE 3.22 Testicular DLBCL with aberrant expression of CD7. Touch smear (a) shows atypical large lymphoid cells. Flow cytom- etry (b-f) shows clonal B-cells (blue dots) with dim CD19 (b), variable CD20 (b), kappa restriction (c), moderate CD200 (d), dim CD71 (e), and aberrant CD7 (f; red dots represent reactive T-cells which are positive for CD3 and CD7)

 

FIGURE 3.23 Plasma cell myeloma: typical phenotype of neoplastic plasma cells (orange dots, arrow) with lack of CD45 (a-b), posi- tive CD56 (a) and CD117 (b, partial), bright expression of CD38 (c), cytoplasmic light chain restriction (c), negative CD27 (d), bright CD138 (e), and positive CD200 (f)

 

 

FIGURE 3.24 Plasma cell myeloma with aberrant expression of CD45 (a; orange dots, arrow), bright CD138 (b), monoclonal cyto- plasmic lambda (c–d), aberrant CD117 (e), aberrant HLA-DR (f), aberrant CD19 (g), and partial CD20 expression (h)

 

FIGURE 3.25 Follicular lymphoma with aberrant phenotype mimicking plasma cells. Patient had history of FL after treatment. Flow cytometry shows clonal B-cells with negative CD45 (a; arrow), positive CD19 (b), negative CD20 (c), negative CD38 (d), positive c.IgM (d), positive (surface) kappa (e), and negative (surface) lambda (f). Lack of CD20 (c) is associated with prior Rituxan treatment. Monocytes (e-f; blue dots) show non-specific staining with kappa and lambda. FISH studies confirmed t(14;18)/BCL2-IGH rearrangement (not shown)

 

FIGURE 3.26 Hematogones (light blue dots; arrow) are negative for light chain surface immunoglobulins (a−b)

 

FIGURE 3.27 B-lymphoblasts (green dots; arrow) show bright CD10 (a), positive CD34 (a-b), and positive CD19 and CD22 (b-d). They are negative for surface light chain immunoglobulins (c-d). Note slightly dimmer expression of CD19 by lymphoblasts (c-d; solid arrow) when compared to residual (polytypic) benign B-cells (c-d; dashed arrows)

 

FIGURE 3.28 Typical FC pattern of hematogones. Hematogones (blue dots; arrow) are characterized by low side scatter (SSC; a), variable CD45 expression, often with distinct two populations (a), low forward scatter (FSC) on majority of cells (b-f) with only minor population of hematogones showing variably increased FSC (arrowheads), moderate CD19 (c), partial CD34 (d), moderate (not bright) CD10 (e), and bright CD38 (f)

  

 

■■■【4】Identification of Clonal T-Cell population

 

 

FIGURE 4.1 T-PLL (peripheral blood). Neoplastic T-cells (red arrow) show slightly increased forward scatter (FSC; a-d) when compared to residual benign T-cells (black arrow). In addition, T-PLL cells show slightly dimmer CD2 (a), dim CD3 (b), and moderate (normal) CD5 (c) and CD7 (d) expression

 

FIGURE 4.2 Identification of abnormal T-cells: aberrant expression of pan T-cell antigens and increased forward scatter. Benign (reac- tive) T-cells (a-d; red dots; *) have low FSC and display normal expression of CD2, CD3, CD5, and CD7. Neoplastic T-cells (a-d; blue dots; arrow) in addition to increased FSC (a-d) show aberrant loss of CD2 (a) and CD5 (c), and brighter expression of CD3 (b) and CD7 (d)

 

FIGURE 4.3 Identification of abnormal T-cells: mild increase in forward scatter and aberrant expression of pan T-cell antigens. T-ALL cells (a-g; green dots) show increased forward scatter (compare to residual small T-cells; red dots), negative expression of all T-cell antigens (CD2, CD3, CD5, and CD7; a-d; green arrow), dual CD4/CD8 positivity (e-f), and positive expression of CD10 (g). Rare residual, benign small T-cells (a-g; red dots) show normal phenotype and low forward scatter

 

FIGURE 4.4 Identification of abnormal T-cells: aberrant expression of T-cell antigens. Tumor cells from ALCL ("null" cell type; a-d; red arrow) are negative for CD2 (a), CD3 (b), CD5 (c), and CD7 (d), have high forward scatter (a-d) and display bright expression of CD30 (e)

 

FIGURE 4.5 Identification of abnormal T-cells: positive expression of all pan T-cell antigens and CD4 restriction. T-PLL shows interstitial BM involvement (a, bone marrow staining for CD3) and clonal rearrangement of TCR gene (b). Flow cytometry immuno- phenotyping (c-g) shows normal expression of all pan-T antigens (d-g) and CD4 restriction (c)

 

FIGURE 4.6 Identification of abnormal T-cells: positive expression of pan-T-cell antigens in T-ALL. The leukemic cells from T-ALL show positive expression of all pan T-cell antigens (a-d) with slightly dimmer CD2 (a; arrow). Positive surface CD3 (b) is rarely seen in T-ALL, as most of the T-leukemias are surface CD3 negative. CD5 shows moderate expression (c) and CD7 is brightly expression (d). Strong expression of both CD34 and CD38 (e) confirms the diagnosis of T-ALL

 

FIGURE 4.7 Identification of atypical T-cell population: positive expression of all pan-T-cell markers in nodal TFH cell lymphoma, angioimmunoblastic-type (AITL). Tumor cells from AITL (a-d; doted circle) show increased forward scatter and minimally brighter expression of CD2 (a) and CD5 (c), as well as minimally dimmer expression of CD3 (b) and CD7 (d), when compared to normal T-cells (arrow)

 

FIGURE 4.8 Aberrant expression of CD7 in patient with mononucleosis. The FC analysis of blood from 21 y/o patient with infectious mononucleosis shows significant population of atypical (activated) T-cells (green dots; arrow) with increased FSC (a-k), reversed CD4:CD8 ratio (a-b) due to predominance of CD8+ T-cells, positive activation markers, HLA-DR (c) and CD38 (d), positive CD2 (e), positive CD3 (f), positive CD5 (g), and aberrant loss of CD7 (h). PCR testing did not reveal clonal T-cell receptor (TCR) gene rearrangement (not shown)

 

FIGURE 4.9 The CD4:CD8 ratio: normal pattern

 

FIGURE 4.10 The CD4:CD8 ratio: predominance of CD4+ T-cells (arrow; red represents residual benign T-cells)

 

 FIGURE 4.11 The CD4:CD8 ratio: CD8 predominance

 

FIGURE 4.12 The CD4:CD8 ratio: dual CD4/CD8+ expression

 

FIGURE 4.13 The CD4:CD8 ratio: dual CD4/CD8-expression

 

FIGURE 4.14 Aberrant expression of CD45: (a) T-PLL with negative CD45; (b) T-PLL with aberrant CD45 (subset of leukemic cells is negative and subset shows moderate CD45); (c) T-ALL with moderate CD45; (d) PTCL with negative CD45; and (e) ALCL with nega- tive CD45 (T-cells are positive for CD2 and negative for CD34 and TdT)

 

FIGURE 4.15 Additional markers in T-cell disorders: T-lymphoblasts (a) display aberrant expression of HLA-DR (b; green dots)

 

FIGURE 4.16 Additional markers in T-cell disorders: CD10 expression in nodal TFH cell lymphoma, angioimmunoblastic-type (AITL; two cases). (a-b) AITL (case #1) showing clusters of clear cells (a) with positive, but slightly dimmer expression of CD7 and positive CD10 (b). (c-m) AITL (case #2) with typical flow cytometry pattern. Tumor cells show partially increased forward scatter, positive CD2 (c), positive but dimmer CD3 (d), positive CD5 (e), negative CD7 (f), positive but dim CD4 (g), negative CD8 (h), partially positive CD56 (i), partially positive CD30 (j), and strongly positive CD10 (k-1). The CD3 versus CD7 display (m) shows loss of CD7 on neoplastic cells and slightly dimmer CD3 expression

 

FIGURE 4.17 Additional markers in T-cell disorders: CD10 expression in T-ALL. Neoplastic cells express CD10 (a; green dots) and CD7 (b; green dots). Surface CD3 is negative (residual benign T-cells are positive for both CD3 and CD7 (red dots)

 

FIGURE 4.18 Additional markers in T-cell disorders: CD30 expression in ALCL. Tumor cells are positive for CD2 (a; red arrow), negative for CD3 (b; black arrow), negative for CD5 (c; black arrow), partially positive CD7 (d; red arrow shows CD7+ cells and black arrow show major population of CD7- cells), positive for CD4 (e; red arrow), negative for CD8 (f; black arrow), positive for CD43 (g; upper quadrants), and mostly positive for CD30 (g; red arrow)

 

FIGURE 4.19 Additional markers in T-cell disorders: Neoplastic cells of adult T-cell leukemia/lymphoma (ATLL; a) are positive for CD25 (b; arrow)

 

FIGURE 4.20 Additional markers in T-cell disorders: CD103 expression in enteropathy-type T-cell lymphoma

 

FIGURE 4.21 Additional markers in T-cell disorders: CD33 expression in T-ALL. Leukemic cells (green dots) are positive for CD3 (a; dim expression), CD5 (b; dim expression), and CD33 (c; heterogeneous expression). Residual (benign) T-cells have moderate CD3 (a) and CD5 (b) and are negative for CD33 (c; red dots). Granulocytes (gray dots) are CD33+ (c)

 

FIGURE 4.22 Additional markers in T-cell disorders: CD117 expression in T-PLL (a–f) and T-ALL (g-h). T prolymphocytes (a) co- express CD8 and CD117 (b; arrow). They do not display aberrant expression of pan-T-cell antigens (c-f). T-lymphoblastic are positive for CD34 (g), CCD3 (g), and CD117 (h)

 

FIGURE 4.23 Additional markers in T-cell disorders: CD16 expression in NK-cells. NK-cells are typically positive for CD2 (a; arrow), negative for surface CD3 (b; solid arrow), and express CD16 (c; solid arrow) and/or CD56 (not shown)

  

 

■■■【5】Identification of Myeloblasts

 

 

 

 

 

  

  

 

 

 

 

 FIGURE 5.1 Algorithm to the diagnosis and subclassification of acute leukemias

 

FIGURE 5.2 Cytomorphologic heterogeneity of blasts: (a) agranular myeloblast, (b) myeloblast with Auer rods, (c) granulated myelo- blast, (d) hypergranular promyelocytes, (e) promyelocyte with numerous Auer rods, (f) agranular promyelocytes, (g) monoblasts, (h) promonocytes, (i) erythroblasts, (j) megakaryoblasts, (k) myeloblasts with hand-mirror features, and (1) BPDCN blasts

 

FIGURE 5.3 Identification of blasts: CD45 versus SSC. Myeloblasts typically show moderate CD45 and low SSC (a; blue dots). Monoblasts (b; green dots) in most cases have bright CD45 and slightly increased SSC when compared to myeloblasts. Neoplastic promyelocytes (c; blue dots) have high SSC and moderate CD45

 

FIGURE 5.4 Case #1 (a–c). Myeloblasts (green dots) are positive for CD34 (a), CD133 (a-b), CD117 (b-c; bright expression), and HLA-DR (c). Case #2 (d–f). Myeloblasts (green dots) are positive for CD34 (d) and CD117 (d–f) and are negative for CD133 (e) and HLA-DR (f)

 

FIGURE 5.5 Blasts are distinguished from mature elements by positive expression of blastic markers (CD34, TdT, and CD117) and lack of expression of markers associated with maturation (CD10, CD11b, CD15, CD16, and CD65). Top panels (a) show myeloblasts negative for CD15 (a, a') and CD65 (a, a") and positive CD117 (a') and CD34 (a"). Lower panels (b) display AML with maturation with blasts (green dots) negative for CD10 (b), CD11b (b'), and CD16 (b"). Note bright expression of those markers by maturing granulocytes (gray dots)

 

FIGURE 5.6 Acute megakaryoblastic leukemia without CD34 and CD117 expression. Megakaryoblasts (green dots) are character- ized by positive CD45 (a), low SSC (a), high FSC (b-h), negative CD13 (b), positive CD33 (c), negative CD34 (d), positive CD41 (e), posi- tive CD61 (f), negative CD117 (g), and negative HLA-DR (h)

 

FIGURE 5.7 Comparison of MPO expression by FC and cytochemistry. Acute myeloid leukemia without maturation (a) shows strong MPO expression by FC (a, isotypic control; a'; MPO) and by cytochemistry (a"). Acute promyelocytic leukemia (b) shows dim expression of MPO by FC (isotypic control b; b', MPO) but is strongly MPO positive by cytochemistry (b")

 

FIGURE 5.8 The expression of CD13 and CD33 in AML. Blasts from AML with or without maturation are most often positive for both CD13 and CD33 (a), the expression of CD13 often being dim- mer than CD33. Subset of leukemias may show negative, dim or partial expression of either CD13 (b) or CD33. Neoplastic promy- elocytes most often show dim CD13 and bright CD33 (c). Neoplastic monocytes have bright CD33 (d-e). Erythroblasts from pure ery- throid leukemia are usually CD13/CD33-negative (f), whereas megakaryoblasts may be CD13 and/or CD33 positive (g)

 

FIGURE 5.9 Identification of myeloblasts: expression of CD4 and CD64 in acute monoblastic leukemia. Monoblasts are posi- tive for CD64 (a; bright expression) and often show variable ("smeary") pattern of CD14, ranging from negative to positive expression (a; arrows). In mature monocytes both CD14 and CD64 are brightly positive (b; arrow)

 

FIGURE 5.10 Identification of myeloblasts: expression of CD11b in acute monoblastic leukemia. Subset of acute monoblastic leuke- mias shows moderate to bright CD11b (a; green dots), but majority of cases have either variable ("smeary") CD11b (b, blue dots) with subset of cells negative and subset with variable positivity

 

FIGURE 5.11 Acute promyelocytic leukemia (APL). Neoplastic promyelocytes (blue dots) are characterized by high side scatter (a-k; y axis), positive CD45 (a) negative CD34 (b), positive CD117 (c), negative HLA-DR (d), negative CD10 (e), negative CD11b (f), positive CD13 (g), negative CD16 (h), positive CD33 (i), negative CD56 (j), and negative to dimly positive CD64 (k). APL often show high non-specific "background" fluorescence and therefore the expression of specific markers need to be correlated with negative or "build-in" controls (e.g., with markers known to be negative such as CD3, CD8, CD19, CD20, etc.)

 

FIGURE 5.12 AML with NPMI mutations. Blasts (a-d; green dots) have low side scatter (a), moderate CD45 (a), negative CD34 (b), positive CD117 (c), and negative HLA-DR (d)

 

FIGURE 5.13 AML without maturation. Blasts (green dots) are characterized by low side scatter (a), dim CD45 expression (a), posi- tive CD34 (b-c), aberrant expression of CD7 (b), positive CD133 (c), positive CD13 and CD33 (d), negative CD71 (e), dim expression of CD81 (e), and positive HLA-DR (f)

 

FIGURE 5.14 AML with maturation. Blasts (green dots) show dim expression of CD45 (a), low side scatter (a-b) and high forward scatter (c). Note the presence of maturing myeloid precursors and neutrophils (a, grey dots, arrow). Blasts display the following phenotype: CD133+ (b), CD13+ (c), CD34+ (d), CD117dim+ (d), HLA-DR+ (e), partially CD11c+ (f), CD11b-(g), CD15-(g), CD14- (h), CD64-(h), and CD56+ (i)

 

FIGURE 5.15 APL-v with typical phenotype and aberrant expression of CD56: CD2+ (a), CD13+ (b), CD33+ (c), CD34+ (d), CD56+ (e), CD64+ (f), CD117+ (g), and HLA-DR- (h)

 

FIGURE 5.16 Four major patterns of APL in flow cytometry analysis. (a) The most common variant, hypergranular APL is char- acterized by high side scatter (SSC). (b) Hypogranular variant (pattern 2) is characterized by low side scatter (similar to majority of non-APL AMLs), positive CD117 and often positive expression of CD34 and CD2. (c) Pattern 3 shows partial involvement of blood or BM by APL with significant mixture of benign neutrophils and/or benign maturing myeloid precursors. (d) Pattern 4 shows presence of both hypergranular and hypogranular clones of APL. Both clones are positive for CD117, but only hypogranular blasts show positive expression of CD2 and CD34

 

FIGURE 5.17 Acute myelomonocytic leukemia. Flow cytometry analysis shows blasts (≥20%; blue dots), monocytes (green dots), and granulocytes (purple dots). Blasts have low side scatter and dim to moderate CD45 (a), positive CD34 (b), CD117, (c), HLA-DR (d), and CD33 (e-f; aberrant variable expression). Monocytes have bright CD45 (a), positive HLA-DR (e), and bright CD33 (e–f)

 

FIGURE 5.18 Acute monoblastic leukemia. Monoblasts (green dots) are characterized by high forward scatter (a-f), aberrant loss of CD13 (a), partially positive CD15 (b), bright expression of CD33 (c), partially positive CD56 (d), bright expression of CD64 (e), positive HLA-DR (f), negative CD34 (g), positive CD117 (g-h), positive CD11c (h), and aberrant loss of CD14 (i)

 

FIGURE 5.19 Acute monoblastic leukemia: monoblasts are CD45+ (a), CD34- (b), CD117-(c), CD56+ (d), CD33+ (e), and CD64+ (f). CD14 is variably expressed (g; arrows) with many blasts being negative

 

FIGURE 5.20 Acute erythroid leukemia (AEL, pure erythroid leukemia). Blasts (blue dots, arrow) are negative for CD34 (a), positive for CD117 (b), and strongly positive for CD71 (c). GPHA is negative (d)

 

FIGURE 5.21 AML (4 cases) with aberrant immunophenotype (LAIP)

 

FIGURE 5.22 LAIP: blasts (green dots) are CD117 positive (a) and show aberrant expression of CD56 (b) and CD7 (c)

 

 FIGURE 5.23 LAIP: two distinct populations of blasts are noted: one population (orange dots) shows dim CD45 (a), variable (mostly negative) TdT (b), bright CD34 (c), bright CD117 (d), negative CD123 (e), dim CD7 (f), bright CD13 (g), and moderate CD33 (h). The second population (green dots) shows moderate CD45 (a), partial TdT (b), dim to moderate CD34 (c), negative CD117 (d), bright CD123 (e), moderate CD7 (f), dim CD13 (g), and dim CD33 (h)

 

FIGURE 5.24 Mixed phenotype acute leukemia (MPAL; AML/T-ALL) - bone marrow. (a) Histologic section with H&E staining shows hypercellular bone marrow showing complete replacement by large blasts. (b) Aspirate smear shows blasts and rare erythroid precursors. (c) Blasts are positive for MPO (cytochemistry). (d-h) Flow cytometry analysis shows the following phenotype of blasts: HLA-DR+ (d), CD117+ (d), CD34+ (e), CD10+ (e), CD7+ (f), cytoplasmic CD3+ (f), CD13+ (g; dim expression), and CD33+ (h)

 

FIGURE 5.25 Differential diagnosis between BPDCN (left col- umn) and acute monoblastic leukemia (right column). BPCDN is positive for CD4 (a), negative or CD11c (b), negative for CD13 (c) [rare cases may be CD13+], positive for CD33 (d) [majority of cases are CD33-], positive for CD56 (e), negative for CD64 (f), and positive for CD123 (g). Acute monoblastic leukemia is positive for CD4 (a), positive for CD11c (b'), positive for CD13 (c'), positive for CD33 (d'), positive for CD56 (e') [subset of cases is negative], positive or CD64 with bright expression (f'), and negative for CD123 (g') [subset of cases may be positive]

 

FIGURE 5.26 Comparison of the expression of CD45 (a-a"), CD10 (b-b"), CD11c (c-c"), CD15 (d-d"), CD16 (e-e"), CD33 (f-f"), and CD117 (g-g") of benign neutrophils (left column; light blue dots), neoplastic promyelocytes from APL (middle column; green dots), and maturing myeloid precursors from benign BM (right column; dark blue dots). Neutrophils are characterized by moderate to birth CD45 (a), and bright expression of CD10 (b), CD11 (c), CD15 (d), and CD16 (e), moderate CD33 (f), and negative CD117 (g). APL is characterized by moderate CD45 (a), negative CD10 (b') and CD11c (c'), negative to partially dim CD15 (d'), negative CD16 (e'), bright CD33 (f) and positive CD117 (g'). Benign maturing myeloid precursors show moderate CD45 (a"), variable CD10 (ranging from negative to positive; b"), variable CD11c (c"), bright CD15 (d"), variable CD16 (e"), moderate CD33 (f"), and negative CD117 (g"). Abbreviation: SSC, side scatter; FSC, forward scatter

 

FIGURE 5.27 Phenotypic differences between hypogranular APL and granulocytes in the BM with partial involvement by APL. Hypogranular APL cells (blue dots) show low SSC (a-g), dim CD45 (a), negative to dim (partial) CD34 (b), positive CD117 (c), negative HLA-DR (d), positive CD2 (e), negative CD11b (f), and bright CD33 (g) whereas benign granulocytes (purple dots) show higher SSC (a), negative CD34 (b), negative CD117 (c), negative HLA-DR (d), negative CD2 (e), brightly positive CD11b (f), and dimmer CD33 (g)

 

FIGURE 5.28 Comparison of acute monoblastic leukemia with high side scatter (left column) with APL (right column). Monoblasts with high side scatter show similar CD45 versus side scatter pattern to APL (a) and similar bright expression of CD33 (b). Acute mono- blastic leukemia differs from APL by brighter expression of CD64 (c), positive CD11c (d), positive HLA-DR (e), and negative CD117 (f)

 

FIGURE 5.29 HLA-DR-negative AML without maturation. Blasts have low side scatter (a) and the following phenotype: CD45+ (a), CD34- (b), CD117+ (c), HLA-DR (d), CD2-(e), CD33+ (f), CD64- (g), and CD11b (h)

 

FIGURE 5.30 Comparison between acute monoblastic leukemia and hypogranular variant of acute promyelocytic leukemia. Monoblasts (left panels; blue dots) are positive for HLA-DR (a), CD11b (b), CD11c (c), CD14 (d, variable expression), and CD64 (e, bright expression), while promyelocytes (right panels; green dots) are negative for HLA-DR (a'), CD11b (b'), CD11c (c'), and CD14 (d') and show dim/partial CD64 (e'). Residual neutrophils (gray dots) express both CD11b (b') and CD11c (c')

 

FIGURE 5.31 MDS: phenotypic features of dysmaturation (flow cytometry). Maturing myeloid precursors and neutrophils (green dots) display prominent features of dysgranulopoiesis in the form of hypogranular cytoplasm and nuclear abnormalities (a). Flow cytometry shows markedly decreased side scatter, placing myeloid cells in "blastic" gate (b; green dots). Blastic markers are negative (c-d) and there is normal expression of CD13 (e), CD33 (f), CD16 (g), and CD10 (h). Lack of CD34 and CD117 and normal expression of CD10 and CD16 help to differentiate dysplastic myeloid cells with low side scatter from myeloblasts

 

FIGURE 5.32 Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL; bone marrow). T-lymphoblasts show hand-mirror appearance (a). They have the following phenotype: CD34+ (b), CD117+ (c), HLA-DR+ (d), CD2+ (e), surface CD3- (f), CD5- (g), CD7+ (h), CD4 (i), CD8 (j), cytoplasmic CD3+ (k), TdT (1) CDla (m), partially CD11b+ (n), CD13+ (0), and CD33 (p). Positive CD117, CD13, CD11b and negative CD5 and CDla are typical for ETP-ALL

 

FIGURE 5.33 Recurrent PCM after therapy with unusual phenotype mimicking myeloblasts (CD45+, CD117+, and HLA-DR†). (a–f) Flow cytometry: plasma cells (magenta dots) show dim expression of CD45 (a), positive CD117 (b) and positive HLA-DR (c), negative CD38 (d), CD15 (e) and CD33 (f). Bright expression of CD138 and cytoplasmic lambda restriction (not shown) in conjunction with negative myeloid markers helped to confirm PCM and exclude myeloblasts. Subsequent BM morphologic analysis showed poorly dif ferentiated PCM with plasmablastic features (g), negative kappa (h), and positive lambda (i), CD138 (j), CD117 (k), and HLA-DR (I)

 

FIGURE 5.34 PCM with blastic morphology. Bone marrow core biopsy (a-b) shows total replacement of normal marrow elements by mononuclear cells with "blastic" morphology (a, original magnification ×200; b, original magnification ×400). Aspirate smear (c-d) shows neoplastic cells with prominent nucleoli, fine chromatin, and scanty basophilic cytoplasm. Occasional mitotic figures are also present. Some of the cells show multinucleation, a typical feature of plasma cell myeloma usually not observed in acute leukemias. Immunohistochemistry (e-g) shows positive CD117, negative CD34, and positive CD138. Flow cytometry (h-n) shows plasma cells (orange dots) with the following phenotype: CD33- (h), CD117+ (i), CD34 (j), HLA-DR- (k), CD45- (1), CD38+ (m-n), and cytoplasmic kappa restriction (m-n). Blastic morphology and positive CD117 expression mimic AML, but lack of CD45, CD34, HLA-DR, and CD33 in conjunction with presence of multinucleated cells and cytoplasmic light chain restriction help to diagnose myeloma rather than acute leukemia

 

FIGURE 5.35 AML without expression of blastic markers (CD34 and CD117). (a) Aspirate smear showing large myeloblast. (b-h) Flow cytometry. Blasts (green dots) are dimly positive for CD45 (b), negative for CD34 (c) and CD117 (d), positive for CD4 (e), negative for CD11b (f) and CD123 (g), and positive for CD56 (h). Myeloid markers (CD13, CD33) were positive and monocytic markers (CD11c, CD14, and CD64) were negative. Lack of CD123 and presence of both CD13 and CD33 does not favor the diagnosis of blastic plasma- cytoid dendritic cell neoplasm

 

FIGURE 5.36 Blast phase of CML with unusual phenotype (lack of blastic markers). Blood smear (a, composite microphotograph) from patient with CML on targeted therapy with imatinib shows leukoerythroblastosis with numerous blasts (arrows). Flow cytometry (b-g) analysis shows blasts (green dots) with low side scatter (b), dim to moderate CD45 (b), high forward scatter (c), positive CD123 (c), lack of CD34, CD117, and CD133 (d-e), co-expression of CD13 and CD33 (f), positive CD56 (g), and negative CD4 (g)

 

 

 

■■■【6】Identificqtion of Lymphoblasts

 

 

 

 

 

 

 

 

 

 

 

 

  

 

FIGURE 6.1 B-cell acute lymphoblastic leukemia (B-ALL): lymphoblasts (green dots) show low side scatter (SSC; a), negative to dim CD45 expression (a), increased, but variable forward scatter (b-g), positive CD34 (b), positive HLA-DR (c), bright expression of CD10 (d), dim expres- sion of CD13 (e), negative CD33 (f), dim (partial) CD20 (g), moderate CD38 (h), and dim CD22 (j). The pattern of CD19 versus CD38 expression (h), CD19 versus CD10 expression (i) and CD22 versus CD34 expression (j), FSC and SSC properties of lymphoblasts, presence of aberrant CD13 or CD33 and lack of very dim CD45 expression often helps in differential diagnosis with hematogones

 

FIGURE 6.2 Identification of B-lymphoblasts B-ALL/LBL. Lymphoblasts (green dots; arrow) are negative for CD10 (a), positive for CD34 (a), and positive for CD19 (b-c). There is no expression of surface light chain immunoglobulins (b-c). Note slightly dimmer expression of CD19 when compared to residual (polytypic) benign B-cells (b-c; dashed arrows)

 

FIGURE 6.3 Identification of B-lymphoblasts. B-lymphoblasts are surface immunoglobulin-negative (a–d) and in majority of cases do not express CD20 (c-d)

 

FIGURE 6.4 Identification of B-lymphoblasts: CD45 expression in B-ALL. Majority of B-ALL cases have dim CD45 (a), but the expression may be negative (b) or moderate (c). Rare cases show bimodal CD45 staining with one population of blasts positive and second population negative (d). The side scatter is typically low in B-ALL

 

FIGURE 6.5 B-ALL - flow cytometry. B-lymphoblasts (blue dots) have the following phenotype: CD45+ (a), CD34+ (b; variable expression), CD117− (c), HLA-DR+ (d), CD20+ (e; variable and partial expression), CD22+ (f), and CD33-(g)

 

FIGURE 6.6 B-ALL with aberrant expression of CD15. B-lymphoblasts (green dots) show positive CD19 (a, b), partially dim CD20 (a), bright CD10 (b), negative surface light chain immunoglobulins (c), moderate CD38 (d), dim CD81 (e), and dim (partial) CD200 (f). Among myeloid antigens, CD13 is negative (g), CD15 is dimly positive (h), and CD33 is negative (i)

 

FIGURE 6.7 B-ALL with mostly negative CD10 (a), aberrant expression of CD13 (b), moderate CD19 (c), aberrant expression of CD33 (d), positive CD34 (e), negative to partially dim CD71 (f), dim CD81 (g), positive CD123 (h), and moderate CD200 (i)

 

 FIGURE 6.8 B-ALL with surface light chain immunoglobulin expression and BCR-ABL1 rearrangement (MYC negative). (a-i) Flow cytometry shows blasts (green dots, arrows) with moderate CD45 and low side scatter (a), positive CD19 (b), variably positive CD20 (b), kappa restriction (c-e), bright CD10 expression (f), negative CD34 (g), positive HLA-DR (g), negative CD71 (h), and positive TdT (i). FISH analysis showed BCR-ABL1 fusion (j: one green (BCR), one orange (ABL1), and two yellow fusion signals) and lack of MYC rearrangement (k: break apart probe with normal pattern, two orange/green fusion signals)

 

FIGURE 6.9 Hematogones (benign B-cell precursors). Two different bone marrow samples (a-c and d-i). Hematogones have very low side scatter and dim to moderate CD45. Based on CD45 and CD34 expression two distinct populations of hematogones can be identified. Less mature hematogones (a; green dots) show dim CD45 and positive CD34 (b). More mature hematogones have moder- ate CD45 expression (a, blue dots) and negative CD34 (b). Hematogones show bright expression of CD10 (b), but expression of CD10 becomes down-regulated as the cells mature (c) Typically for hematogones, the expression of CD20 is variable; earlier forms are CD20, but as the cells mature, they become progressively CD20+ with characteristic heterogeneous ("smeary") pattern of CD20 expression (c). Hematogones are dim positive for cytoplasmic CD22 (d; blue dots) and CD19 (d; blue dots). Surface immunoglobulins are not expressed (e and f; blue dots). Similar to CD34, TdT shows bimodal staining: subset of cells is positive and more mature cells are negative (g). Parts h and I show typical dim expression of cytoplasmic CD22 and positive HLA-DR with smeary expression of CD20

 

FIGURE 6.10 Hematogones (benign B-cell precursors). Hematogones (orange dots) are positive for CD45 and display very low side scatter (a). They are positive for CD10 (b), CD34 (c; partial), CD19 (d), and CD38 (e). One of the characteristic features of hematogones is increased, but variable forward scatter (d; arrows) and variable expression of CD20 (e-f; arrows)

 

 FIGURE 6.11 Hematogones (light blue dots; arrow) with typical pattern of expression of CD10 versus CD20 (a), CD19 versus CD81 (b), and CD19 versus CD200 (c). Note variable expression of CD20 (a), bright CD10 (a), bright CD81 (c), and dim CD200 (d)

 

FIGURE 6.12 Comparison of FC features between hematogones and B-ALL (minimal residual disease). On CD45 versus SCC (a-a') hematogones often show two distinct populations of cells with dim and moderate CD45, whereas B-ALL blasts usually form more cohesive population (a'). Hematogones have distinct forward scatter pattern (FSC, b-d) with major population showing very low FSC and minor population showing slightly increased, variable FSC ("comet"-like). B-ALL blasts are characterized by higher FSC (b'-d'). Hematogones show variable CD34 expression (c) with subset of less mature cells being CD34+ and subset of more mature hematogones being CD34 (c); B-ALL blasts are uniformly CD34+ (c'). Hematogones do not display aberrant expression of CD33 (d) in contrast to B-ALL blasts which may be positive, as illustrated in d'. The pattern of staining with CD20 versus CD10 (e-e') and CD19 versus CD38 (f-f') is also helpful in differential diagnosis: hematogones show dimmer CD10 expression (e), partial CD20 expression (e), and brighter CD38 expression (f). The majority of B-ALL are CD20-

 

FIGURE 6.13 Hematogones versus B-ALL (MRD). Hematogones (blue dots; arrows) usually show two or event three distinct popula- tions, which differ by intensity of CD45 expression (a). B-ALL blasts show homogenous population in regards to CD45 expression (a'). The forward scatter (FSC) of hematogones is variable but mostly low ad minor subset showing higher FSC (b). B-lymphoblasts most often show moderate to high FSC (b'). Only minor subset f hematogones is CD34+ (c), whereas B-lymphoblasts are most often positive for CD34 with rather homogeneous expression (c'). Hematogones show vari- able (heterogeneous) expression of CD20 (d), whereas B-lymphoblasts are usually CD20 (d'). The expression of CD10 is brighter in B-ALL than in hematogones (d-d') and the expression of CD38 is brighter in hematogones (e-e'). In contrast to hematogones, B-lymphoblasts often display aberrant phenotype, including presence of CD33 (f—f′)

 

FIGURE 6.14 BM with mature and immature B-cell populations representing blastic transformation of follicular lymphoma. Blasts are dim CD45+ (a), lack surface light chain immunoglobulin expression (b-c), have bright CD10 (d), positive CD38 (e), negative CD20 (f), positive CD34 (g), and positive TdT (h). Mature B-cell component shows kappa restriction (b-c), moderate CD10 (d), positive CD20 (f), and negative both CD34 and TdT (g-h). Surface CD3 is negative (i)

 

FIGURE 6.15 High grade B-cell lymphoma with TdT expression and rearrangements of MYC and BCL2 representing blastic transfor- mation of follicular lymphoma. (a–c) FISH analysis showing MYC rearrangement (a), BCL2 rearrangement (b) and MYC break-apart dual color with split signals (c; red and green split signals; yellow, normal signal). (d-g) Flow cytometry analysis shows leukemic cells in blastic region (d; arrow) co-expressing CD10 and TdT (e) with clonal lambda expression (f, f'-g, g'), dim CD19 (f-f′), and negative CD20 (g-g')

 

FIGURE 6.16 Identification of T-lymphoblasts. Blasts from T-ALL show moderate CD45 (a), partial CD34 (b), dim TdT (c), negative CDla (d), and positive T-cell markers (CD2, CD3, CD5, and CD7; e-h). In contrast to the blasts in this case, majority of T-ALLs are surface CD3 and have brighter CD7 expression

 

FIGURE 6.17 The pan-T-cell antigens expression in T-ALL/LBL. (a-b; 4 cases) T-lymphoblasts have slightly increased forward scatter when compared to benign T-cells (a-d). Blasts are most often negative for surface CD3 (a, b, c, d') and positive for CD7 (the expression of CD7 is usually brighter than in normal T-cells; a", b", c"). CD2 and CD5 are positive in subset of cases, but are often aberrantly expressed (e.g. dim; a", c, c"). Case d shows loss of all pan-T-cell markers except for CD2

 

FIGURE 6.18 Identification of T-lymphoblasts by flow cytometry. The upper panels (a-c) present T-ALL with dual expression of CD4 and CD8 (a), positive CDla (b), TdT and cytoplasmic CD3 (c). The lower panels (d-f) show T-ALL with dual CD4/CD8-negative phenotype (d) with positive staining for cytoplasmic CD3 (e) and negative TCR (f)

 

FIGURE 6.19 Identification of T-lymphoblasts by flow cytometry: T-lymphoblasts can be identified by moderate CD45 expres- sion and low side scatter (a), positive TdT (b), lack of surface CD3 expression (c), positive CD34 (d), positive CD10 (d-e), and positive CDla (e)

 

FIGURE 6.20 Identification of T-lymphoblasts. T-ALL/LBL blasts are positive for CD45 (a), CD2 (b) and show aberrant expression of HLA-DR (c)

 

 FIGURE 6.21 T-PLL with dual CD4/CD8 expression (a). In contrast to T-ALL, neoplastic T-cells in T-PLL are surface CD3+ (b) and lack CD34 and TdT expression (c)

 

FIGURE 6.22 Comparison between flow cytometric features of thymocytes (a-d) and T-lymphoblasts (e-g). (a-b) Histologic sec- tion of thymoma. Tumor cells are positive for cytokeratin (AE1/AE3). Flow cytometric analysis reveals immature T-cells with dual expression of CD4/CD8 (c) and variable (smeared) expression of surface CD3 (d; black arrows). There is additional population of larger T-cells with negative expression of CD3 (red arrow). (e) T-lymphoblastic lymphoma (mediastinum). Lymphoblasts are dual positive for CD4/CD8 (f). There are distinct populations of benign T-cells expressing CD4 or CD8 (blue arrows). In contrast, thymocytes (c) shows gradual transition from CD4+/CD8 cells to CD4-/CD8+ cells with majority of cells positive for both antigens. T-lymphoblasts (black arrow) show moderate expression of surface CD3 (g) without typical for thymocytes smeared patter (compare with d). Normal (benign) T-cells (blue arrow, g) show brighter CD3 expression and lower forward scatter, when compared to lymphoblasts

 

FIGURE 6.23 Thymocytes (thymoma/thymic hyperplasia) flow cytometric characteristics. Thymocytes show characteristic smeared (variable) expression of surface CD3 on flow cytometric analysis (a; red arrows). Small cells (red dots, more mature cells) have variable expression of CD3, whereas larger cells (green dots; less mature cells) with increased forward scatter are CD3 negative (a). Those larger cells co-express CD10 (b). Both T-cell populations are positive for CD2 (c), CD5 (d), and CD7 (e)

 

FIGURE 6.24 Thymocytes from thymoma are dual CD4/CD8 positive (a), show partial surface CD3 expression (b), and partial CD10 expression (c)

 

FIGURE 6.25 The expression of CD71, CD81, and CD200 by thymocytes. Small, more mature T-cells from thymoma/thymic hyper- plasia (red dots) are CD71-(a), and larger immature T-cells (blue dots) show positive expression of CD71 (a), slightly brighter CD81 (b), and dimmer CD200 (c)

  

 

■■■【7】Immunophenotype Markers

 

FIGURE 7.1 Algorithm for the diagnosis of hematopoietic tumors

 

FIGURE 7.2 CD2 expression - differential diagnosis. CD2 is expressed by mature T-cell lymphoproliferative disorders including T-PLL (a), PTCL (b), and ALCL (c), precursor T-lymphoblastic leukemia/lymphoma (T-ALL, d), subset of blastic plasmacytoid den- dritic cell neoplasm (BPDCN) (e), mast cell disease (f), extranodal NK/T-cell lymphoma, nasal type (ENKTL) (g), acute promyelocytic leukemia (APL), hypogranular variant (h), and monocytic proliferations, e.g., CMML (i)

 

 FIGURE 7.3 The expression of CD4 and CD8

 

FIGURE 7.4 CD7 expression - differential diagnosis. CD7 is positive in significant subset of T-cell lymphoproliferative disorders, including PTCL (a), rare cases of DLBCL (b), rare cases of T-ALL (c), ENKTL (d), subset of APL (e), subset of AML (f), BPDCN (g), and rare cases of CLL (h)

 

FIGURE 7.5 CD10 expression: differential diagnosis includes (a) follicular lymphoma (FL); (b) diffuse large B-cell lymphoma (DLBCL); (c) Burkitt lymphoma (BL); (d) "double-hit" lymphoma (HGBL-R); (e) HGBL, NOS; (f) mantle cell lymphoma (MCL); (g) hairy cell leukemia (HCL); (h) nodal TFH cell lymphoma, angioimmunoblastic-type (AITL); (i) cutaneous CD4 small/intermediate T-cell lymphoma; (j) B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/LBL); and (k) plasma cell myeloma (PCM)

 

FIGURE 7.6 CD14 expression - differential diagnosis. Benign granulocytes (maturing myeloid cells) are negative for CD14 (a). Aberrant (dim) expression of CD14 (up-regulation) may be observed in subset of myeloproliferative disorders (e.g., PV, b; CML, f). Benign monocytes and neoplastic monocytes in majority of cases of CMML (e) and monocytic component of acute myelomonocytic leukemia (c) display bright expression of CD14. In contrast immature monocytic population from acute monoblastic leukemia is either CD14-negative or display variable expression (d, arrows)

 

FIGURE 7.7 CD15 expression - differential diagnosis: (a) HL (classic); (b) CMML; (c) AML; (d) NLPHL (rare cases fixed in B5); (e) ALCL (rare cases); and (f) PTCL, not otherwise specified (rare cases)

 

FIGURE 7.8 B-cell lymphomas with negative CD20 - differential diagnosis: (a) DLBCL (rare cases); (b) primary effusion lymphoma (PEL); (c) DLBCL with ALK-expression; (d) plasmablastic lymphoma (PBL); (e-g) after Rituxan treatment [lymphomatous cells are positive for CD19 (e) and CD22 (g) but display negative staining with CD20 (f; arrow)]

 

FIGURE 7.9 CD25 expression - differential diagnosis: (a) HCL; (b) ATLL; (c) ALCL (bone marrow); and (d) PTCL

 

FIGURE 7.10 CD30 expression - differential diagnosis: (a) activated cells in reactive lymph node; (b) DLBCL; (c) classic HL; (d) pri- mary mediastinal large B-cell lymphoma (PMBL); (e) ALCL; (f) extranodal NK/T-cell lymphoma, nasal type (ENKTL); (g) enteropathy- associated T-cell lymphoma (EATL); (h) Pagetoid reticulosis (variant of MF); (i) primary cutaneous ALCL (C-ALCL); (j) mycosis fungoides (MF) with large cell transformation; (k) plasma cell myeloma (PCM); (1) EBV-associated high grade lymphoma in HIV+ patient

 

FIGURE 7.11 Hematopoietic tumors with negative CD45 expression - differential diagnosis: (a) plasma cell myeloma (PCM); (b) dendritic cell sarcoma; (c) anaplastic large cell lymphoma (ALCL); (d) classic HL; (e) plasmablastic lymphoma (PBL); (f) B-ALL (subset); (g) acute erythroid leukemia (AEL, negative CD45 and strongly positive CD71); (h) T-cell prolymphocytic leukemia (T-PLL) with negative CD45 expression by FC (orange dots. arrow), blood analysis

 

FIGURE 7.12(a) CD56 expression - differential diagnosis (see text for details, section: CD56)

 

FIGURE 7.12(b) CD56 expression - differential diagnosis (see text for details, section: CD56)

 

  FIGURE 7.13 Comparison of CD81 and CD200 expression in CLL (a-b) and MCL (a'-b'). Note negative to partially dim CD81 expression in CLL (a) and strongly positive CD81 expression in MCL (a). The expression of CD200 is strongly positive in CLL (b) and negative in MCL (b')

 

FIGURE 7.14 CD117 expression - differential diagnosis

 

FIGURE 7.15 TdT expression: (a) B-ALL; (b) T-ALL; (c) AML; (d) thymoma; (e) BPDCN; (f) FL (blastic transformation)

  

 

■■■【8】Morphologic  Correlation in Blood

 

 

 FIGURE 8.1 Circulating promyelocytes (APL). (a, b) Blood smear showing atypical promyelocytes; (c) FC showing promyelocytes (green dots) with high side scatter (SSC) and dimmer expression of CD45 when compared to neutrophils

 

FIGURE 8.2 Flow cytometry of blood with marked leukocytosis (WBC=140,000/μL) associated with primary myelofibrosis (PMF). The flow cytometry (FC) pattern of granulocytes in PMF (middle column) is compared with normal blood (left column) and normal BM (right column). Overall, the FC features of blood granulocytes in PMF resemble BM, indicating leftward shift. Subset of granulocytes show decreased expression of CD45 (a; arrows). The expression of CD10 in PMF is down-regulated (b; arrow), but does not show two distinct population seen in normal BM. The expression of CD13 (c) and CD16 (d) in PMF resemble pattern seen in BM and differs from uniformly bright expression of those antigens in normal blood. The staining with CD34 shows rare circulating blasts in PMF (e; arrow). In contrast to normal blood and BM, granulocytes (gray dots) and monocytes (blue dots) display aberrant expression of CD56 on subset of cells (f; arrow)

 

FIGURE 8.3 HCL. Smear from blood shows atypical lymphocytes with many cytoplasmic projections (a). Flow cytometry shows B-cells with bright CD11c and bright CD20 (b), and co-expression of CD25 with CD103 (c)

 

FIGURE 8.4 T-LGL leukemia. Atypical lymphoid cells with cytoplasmic granules (a) show co-expression of CD8 and CD57 (b; arrow)

 

FIGURE 8.5 Flow cytometric features of CML in peripheral blood. The features differentiating CML from reactive neutrophilia include increased blasts expressing CD34, HLA-DR, and CD117 (a-c), increased basophils, expressing CD123 (d), decreased side scat- ter of granulocytes (e-g), down-regulation of CD10 (e; compare with control on e'), up-regulation of CD56 on granulocytes (f; compare with control on f'), and down-regulation of CD16 on granulocytes (g; compare with control on g')

 

FIGURE 8.6 Chronic eosinophilic leukemia (CEL) - flow cytometry (FC) analysis. Eosinophils (a, cytospin preparation) have char- acteristic FC pattern (b-g) with high side scatter (b-g; arrow), positive CD45 (b), CD13 (c), and CD33 (d), negative CD10 (e), positive CD11b (f), and negative CD16 (g)

 

FIGURE 8.7 Lymphocytosis: cytologic-phenotypic correlation. Abbreviations: ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell leukemia/lymphoma; BL, Burkitt lymphoma; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; HCL, hairy cell leukemia; HCL-v, hairy cell leukemia variant; HGBL, high grade B-cell lymphoma; MCL, mantle cell lymphoma; MF/SS, mycosis fungoides/Sézary's syndrome; MZL, marginal zone lymphoma; PBL, plasmablastic lymphoma; PLL, prolymphocytic leukemia; SMZL, splenic marginal zone lymphoma; PTCL, peripheral T-cell lymphoma; SpLL/nucleoli, splenic B-cell lymphoma/leukemia with prominent nucleoli

 

FIGURE 8.8 Thrombocytosis. Comparison between cytologic features of platelets in ET (a) and acute megakaryoblastic leukemia (b). Note strike cytologic atypia of platelets in the latter (arrow). By FC analysis, megakaryoblasts show moderate CD45 (c; green dots; arrow), negative CD34 and CD117 (d), and positive CD61 (e)

 

FIGURE 8.9 Blasts and blast-like cells: cytologic-immunophenotypic correlation

 

FIGURE 8.10 CMML. Neoplastic monocytes are mostly mature (a) and show bright expression of CD14 (b; arrow) and CD64 (c; arrow)

 

FIGURE 8.11 (a-b) Acute monoblastic leukemia. Monocytic cells are immature (a), represented mostly by monoblasts and pro- monocytes. FC analysis (b; green dots) shows bright CD64 expression and variable CD14 expression with majority of monocytic cells negative and minor subset showing variable expression (arrows). (c-f) Blastic plasmacytoid dendritic cell neoplasm (BPDCN). Blasts show slightly eccentric nuclei with irregular outlines, fine chromatin and nucleoli, and pale to vacuolated cytoplasm (c). FC analysis (d-f; magenta dots) shows co-expression of CD4 (d), CD56 (e), and CD123 (f)

 

FIGURE 8.12 Plasma cell leukemia. (a) Blood smear with circulating plasma cells with mature cytologic features. (b) Complex chromosomal changes (including t(11;14)(q13;q32), add(14)(q11.2), add(17)(q25), +der(19)t(1;19)(q23;q12)). (c-h) Flow cytometry shows negative CD45 (c), negative CD117 (d), positive CD56 (e), positive CD38 (f), and positive cytoplasmic kappa (g-h)

  

 

■■■【9】Morphologic Correlation in Bone Marrow

 

 FIGURE 9.1 APL - flow cytometry of hypergranular variant. Neoplastic promyelocytes are characterized by high side scatter and moderate CD45 (a), negative CD34 (b), positive CD117 (c), negative HLA-DR (d; compare with negative staining for CD8, inset), nega- tive CD10 (e), positive CD13 (f), negative CD16 (g), and dim CD64 (h)

 

FIGURE 9.2 AML. Blasts (green dots) are positive for HLA-DR (a), CD117 (a), CD33 (b), and CD34 (b)

 

FIGURE 9.3 Chronic lymphocytic leukemia. Histomorphology (a-c) with three different patterns of involvement: nodular (a); inter- stitial (b); and diffuse lymphoid infiltrate with proliferation centers (c). Flow cytometry (d-f) shows typical immunophenotype of CLL (arrow) with co-expression of CD5 and CD23 (d), negative CD81 (e), and positive CD200 (f)

 

FIGURE 9.4 HCL with diffuse BM involvement. Histology (a) and immunohistochemical staining with CD20 (b) show total BM replacement by HCL. Flow cytometry (c-e) shows typical phenotypic profile of HCL (blue dots, arrow), bright expression of CD19 and CD20 (c), co-expression of CD25 and CD103 (d), bright CD200 (e), and negative to dim CD81 (e)

 

FIGURE 9.5 Anaplastic large cell lymphoma (ALCL) totally replacing BM. Histology section of BM core biopsy shows diffuse large cell lymphoid infiltrate (a). Flow cytometry (b-e) shows large cells with bright expression of CD45 (b, magenta dots), bright CD4 (c), negative CD3 (d), and positive CD30 (e). The forward scatter of ALCL cells is high (c-e). Immunohistochemical staining (c'-e') con- firmed flow cytometry findings with positive CD4 (c'), negative CD3 (d'), and positive CD30 (e')

 

FIGURE 9.6 Burkitt leukemia (morphologic, immunophenotypic, and molecular analysis of blood and BM from 45-year-old patient). FC analysis showed 25% clonal B-cells in blood with low side scatter, dim to moderate expression of CD45 (a, green dots), monotypic lambda expression (b), positive CD10 (c), and bright CD20 (d). Aspirate smear (e) shows atypical cells with blastoid appearance. Histologic section from core biopsy (f-g, low and high power) shows diffuse infiltrate by monotonous medium-sized lymphoid cells. Immunohistochemistry analysis (h-I) shows positive CD20 (h) and CD10 (i), negative BCL2 (j), strong expression of MYC (>90%, k), and high Ki-67 index (1). FISH analysis (m) shows one green, one orange, and one yellow/green/orange fusion signal indicative of a chromosomal break in the MYC locus

 

FIGURE 9.7 Mantle cell lymphoma (MCL) with paratrabecular BM involvement. Histology (a) shows atypical lymphoid infiltrate of mostly small lymphocytes with paratrabecular pattern. Lymphomatous cells are positive for BCL1 (cyclin D1; b). Flow cytometry (c-h) clonal B-cells with kappa restriction (c) and typical phenotypic features: moderate expression of both CD19 and CD20 (d), positive CD5 (e), negative CD23 (f), negative CD11c (g), positive CD38 (g), positive CD81 (h), and lack of CD200 (h)

 

FIGURE 9.8 Bone marrow - intrasinusoidal infiltrate. (a) Hepatosplenic T-cell lymphoma (HSTL). Histology (a) shows clusters of large cells, which are CD3+ (a) with intrasinusoidal distribution. Flow cytometry (a"-a") shows atypical T-cells (CD3*) with dim CD7 expression (a") and negative CD5 (a"). (b) DLBCL (b, histology; b', CD20 immunostaining). (c) Splenic marginal zone lymphoma (SMZL). H&E section (c) shows mostly nodular infiltrate, and CD20 immunostaining (c') shows intrasinusoidal lymphoma cells. (d) Mantle cell lymphoma (MCL) with prominent intrasinusoidal pattern (d, histology, d', CD20 staining, and d", BCL1 staining)

 

FIGURE 9.9 Unusual case of metastatic carcinoma to the bone marrow and myeloproliferative neoplasm with eosinophilia and PDGFRB/t(5:12). CBC data revealed prominent leukocytosis. Blood smear (a) showed neutrophilia with eosinophilia. Flow cytometry analysis showed increased eosinophils (b-e; purple dots) with very low forward scatter (b-e), negative CD10 (b), positive CD11b (c) and CD13 (d), and negative CD16 (e). Both FISH and PCR tests were negative for BCR-ABL1 rearrangement. Comprehensive bone marrow analysis showed t(5;12) by metaphase cytogenetics (f; small arrows) and core biopsy showed metastatic carcinoma (g; H&E section; objective ×400), confirmed by immunostaining with cytokeratin (h)

 

FIGURE 9.10 MDS. Dysgranulopoiesis in the form of Pelgeroid changes (a) and dyserythropoiesis with megaloblastoid changes and minute nuclear fragments in the cytoplasm (b). FC analysis shows decreased expression of CD16 (c; arrows), CD33 (d; arrow), and CD11b (e; arrow); compare with benign controls (c'-e')

 

FIGURE 9.11 Metastatic carcinoma to the bone marrow: aspirate smear with cluster of cancer cells (a). Flow cytometry shows cyto- keratin positive (b) and CD45 negative (c) population (orange dots)

 

FIGURE 9.12 Metastatic rhabdomyosarcoma (24-year-old patient with history of rhabdomyosarcoma). Touch smears (a-a') from BM shows clusters of large, highly atypical cells with cytoplasmic vacuoles and hyperchromatic, irregular nuclei. Flow cytometry analysis (b-g) shows cells with low side scatter (SSC, b) and high forward scatter (FSC, c-g). Tumor cells (subset of orange dots, arrow) show the following phenotype: CD45- (b), CD34+ (c), CD117- (d), CD56+ (e), CD38- (f), and CD10+ (h)

  

 

■■■【10】Morphologic correlation in Lymph Node

 

FIGURE 10.1 Flow cytometry pattern of benign (reactive) lymph node. (a-c) Benign lymph node - histology and immunohisto- chemistry. (a) Polarized germinal center with one pole composed of larger lymphocytes with macrophages and the other composed mostly of small lymphocytes (such polarization is typical for reactive follicles and helps to differentiate it from follicular lymphoma). (b) B-cells are visualized by staining with CD20. (c) T-cells are restricted to perifollicular area (CD3 staining). (d-m) Flow cytometry. B-cells are positive for CD19 (d-d') with polytypic pattern of expression of kappa and lambda (d-f). The expression of CD20 is moder- ate (f-f') with minor subset of cells (arrow) showing bright CD20. Rare B-cells express CD10 (g, arrow) and CD5 (h, arrow). Majority of benign B-cells are CD23+ (i, arrow). Both B- and T-cells are CD81+ (dim expression, j). Major subset of B-cells shows dim CD200 expression (k). T-cells are positive for CD3 and CD7 (1) with only few cells showing aberrant loss of CD7 (arrow). Major subset of T-cells is CD4+ and minor subset of T-cells is CD8+ (m)

 

FIGURE 10.2 Flow cytometry pattern of florid follicular hyperplasia. (a) Touch smears from florid follicular hyperplasia often show increased number of large cells (centroblasts) mixed with small lymphocytes and tingible body macrophages. (b-e) Flow cytometry. Germinal center cells from benign follicular hyperplasia (blue dots, arrows) show bright CD20 expression (b), variable expression of surface light chain immunoglobulin (with kappa slightly stronger than lambda; c-c') and increased forward scatter (b-e). Germinal center cells are CD10+ (d) and BCL2-(e)

 

FIGURE 10.3 Flow cytometry pattern of florid follicular hyperplasia (11-year-old patient, lymph node). Based on CD19 expression, B-cells show two distinct populations: non-germinal center B-cells (red dots) with moderate CD19 expression (a-b) and polytypic pattern of kappa and lambda expression (a-b), and germinal center B-cells (blue dots) with bright CD19 and lambda excess (arrow). Germinal center B-cells show positive CD10 and bright CD20 expression (c). CD10+ B-cells are polytypic but show slight excess of lambda+ cells (d-e, arrow). Non-germinal center cells are CD23+ (f)

 

FIGURE 10.4 Small lymphocytic lymphoma. (a) Histology; (b) Cytology. Low power examination (a) reveals diffuse lymphoid infil- trate with typical paler areas (proliferation centers) giving rise to a pseudofollicular pattern. Touch smear (b) shows small round lym- phocytes with regular nuclear outlines and compact, darkly stained chromatin. (c-h) Flow cytometry. SLL/CLL cells are kappa* (c), CD19+ (d), CD20+ (dim expression, d), CD5+ (e), CD23+ (e), CD71- (f), CD81- (g), and CD200+ (h)

 

FIGURE 10.5 Flow cytometry pattern of nodal marginal zone lymphoma (NMZL). (a) Histology section shows diffuse lymphoid infiltrate of small cells. (b-h) Flow cytometry. B-cells show moderate CD20 expression and low forward scatter (b). They express CD20 and CD19 (c) and show moderate, expression of surface light chain immunoglobulin (d; presented case is kappa1). There is no CD5 (e) or CD10 (f) expression. Clonal B-cells in NMZL show dim CD81 expression (g), and negative CD200 (h)

 

FIGURE 10.6 DLBCL, GCB-like. (a) Histology shows diffuse lymphoid infiltrate composed oflarge cells. (b-f) Immunohistochemistry. Lymphomatous cells are positive for CD20 (b), CD10 (c), BCL2 (d), and BCL6 (e). MUM1 is negative (f). (g-i) Flow cytometry shows clonal B-cells with negative kappa (g), dim lambda (h), and strong CD10 expression (i)

 

FIGURE 10.7 DLBCL, ABC-like. (a) Histology shows diffuse lymphoid infiltrate composed of large cells. (b-f) Immunohistochemistry. Lymphomatous cells are positive for CD20 (b), negative for CD10 (c), positive for BCL2 (d). BCL6 (e) and MUM1 (f). (g-l) Flow cytom- etry shows clonal B-cells with dim CD20 (g), moderate CD19 (g), kappa restriction (h), negative CD81 (i), and negative CD200 (j). The forward scatter of B-cells is high (k-1). There is partial dim expression of CD71 (k, arrow), and lack of CD10 (1)

 

FIGURE 10.8 Comparison of FC features of low-grade FL (a, left column) and CD10+ DLBCL with GCB-like phenotype (a', right column). Low-grade FL display low forward scatter (b) and DLBCL shows high forward scatter (b'). Both lympho- mas are positive for CD10 (c-c') and CD38 (d-d') with expres- sion of CD38 being slightly dimmer in FL. In contrast to FL, DLBCL shows CD71 expression (e-e'). The expression of both CD81 and CD200 is often similar in those two lymphomas (f-f' and g-g')

 

FIGURE 10.9 Peripheral T-cell lymphoma (PTCL). (a) Histology shows diffuse large cell lymphoid infiltrate with "clear" appearance. (b-c) Immunohistochemistry shows positive CD5 (b) and lack of CD7 expression (c). (d-g) Flow cytometry. Lymphomatous cells have increased forward scatter, positive CD2 (d), lack of surface CD3 expression (e), positive CD5 (f), and negative CD7 (g)

 

FIGURE 10.10 Lymph node with diffuse and pleomorphic infiltrate. Differential diagnosis includes (a) Kikuchi lymphadenitis; (b) EBV-associated atypical (reactive) hyperplasia; (c) peripheral T-cell lymphoma, unspecified (PTCL); (d) angioimmunoblastic T-cell lymphoma (AITL); (e) T-/histiocyte-rich large B-cell lymphoma (THRLBCL); (f) Hodgkin lymphoma, classic (mixed cellularity); (g) diffuse follicle center cell lymphoma (grade 2); (h) nodal marginal zone lymphoma with increased large cells; (i) EBV+ DLBCL; (j) mediastinal grey zone lymphoma; (k) Langerhans cell histiocytosis; and (1) small lymphocytic lymphoma (SLL/CLL) with large cell transformation (Richter's syndrome)

 

FIGURE 10.11 Flow cytometry of BL. Lymphomatous cells (blue dots, arrow) show increased forward scatter (a-c) and positive expression of CD20 (a), CD22 (b), CD71 (c), and CD19 (d). Kappa versus lambda scattergram (e) shows kappa restriction. The lym- phoma cells are positive for CD10 (f), CD38 (g), and CD81 (h). CD200 is negative (i)

 

FIGURE 10.12 Comparison of flow cytometry pattern in follic- ular hyperplasia and follicular lymphoma (FL). Occasional cases of follicular hyperplasia show prominent population of CD10+ germinal center cells which show excess of kappa expression (a, a'; blue dots, arrow), and less often lambda excess. Follicular hyper- plasia differs from FL by presence of population of B-cells with brighter CD20 expression (b, arrow), expression of CD10 on minor subset of B-cells (c, arrow), positive expression of CD71 on major- ity of germinal center cells (d, arrow), strong expression of CD81 (e, arrow), and lack of expression of CD200 by germinal center cells (f, arrow). Follicular lymphomas usually show homogenous expres- sion of CD20 (b') and CD10 (c'), lack of CD71 (d'), dim expression of CD81 (e'), and negative or dim expression of CD200 (f')

 

FIGURE 10.13 Perifollicular AITL with reactive germinal centers. The histologic section shows lymph node with numerous reac- tive germinal centers (a-c). The reactive B-cell follicles are positive for PAX5 (d). Neoplastic T-cell population is identified by immu- nostaining with PD1 (e-f, low and intermediate magnification), CD3 (g), and CD10 (h). Note dimmer expression of CD3 by neoplastic T-cells (immediately around the follicles) when compared to reactive T-cells in the background (g). The expression of CD10 is stronger among AITL cells than in germinal center cells (h). Scattered EBV-infected cells are noted by EBER (ISH) staining (i)

  

 

■■■【11】 Molecular-Flowcytometry correlation

 

 FIGURE 11.1 Interpretation of FISH results

 

 FIGURE 11.2 FISH analysis for BCR-ABL using three-color probes: aqua for argininosuccinate synthetase 1 (ASSI) gene on chro- mosome 9q34, green for BCR gene on chromosome 22, and red for ABL gene on chromosome 22. Several positive signals (yellow) are seen in case of CML (a); control sample (b) shows two green signals for normal chromosome 22 and two red and blue signals close to each other for normal chromosome 9

 

FIGURE 11.3 AML with NPMI mutations: blasts (green dots) show low side scatter (a), negative CD34 (b), positive CD117 (c), and negative HLA-DR (d), resembling hypogranular variant of APL

 

FIGURE 11.4 AML with RUNX1-RUNXIT1 flow cytometry. Blasts are large with nucleoli and occasional Auer rods (a; smear from flow sample). Flow cytometry (b-g) reveals phenotypic features of AML with maturation (b; blasts are represented by blue dots, and maturing myeloid precursors by purple dots). Blasts are positive for CD45 (b), CD34 (c), CD117 (d), CD33 (e), CD19 (f; partial), and CD56 (g; partial). Granulocytes show aberrant CD56 expression on subset (g)

 

FIGURE 11.5 Acute promyelocytic leukemia (APL) - flow cytometry. Neoplastic promyelocytes (green dots) show high side scatter (a), positive CD117 (b), positive CD13 (c), positive CD33 (d), and positive CD64 (e)

 

FIGURE 11.6 Burkitt lymphoma - flow cytometry. B-cells are CD20 (a-b), kappa positive (a), lambda negative (c), and show partial CD71 expression (d)

 

FIGURE 11.7 Flow cytometric pattern of CML in peripheral blood (CML, left column; control, right column). CML differs from healthy controls (normal blood and reactive neutrophilia) by presence of circulating blasts (a, arrow), basophilia (b, arrow), significant number of CD10-negative (c, arrows) and CD16- negative (D, arrows) granulocytes and up-regulation of CD56 (e, arrow)

 

FIGURE 11.8 T-cell prolymphocytic leukemia. FISH analysis shows the rearrangement of TCL1 gene (a). Flow cytometry shows CD8 restriction (b), dim CD17 on minute subset (c), and positive expression of all pan-T-cell antigens (d-g) with CD7 being slightly dimmer. Cytogenetic (h) shows inversion of chromosome 14 and isochromosome 8q typical for T-PLL, as well as other changes (11q/ ATM deletion and 13q deletion)

 

 FIGURE 11.9 SMZL with del7q. (a-i) Flow cytometry analysis (bone marrow sample) shows clonal lambda B-cells (a, arrow) with bright expression of CD20 (b), negative CD5 (c), positive CD23 (c), negative CD10 (d; hematogones seen as light blue dots are CD10+), negative CD25 (e), partial dim CD103 (e), negative CD71 (f), partially dim CD11c (g), dim to moderate CD81 (h), and moderate CD200 (i). Histologic examination of BM core biopsy shows atypical lymphoid infiltrate with paratrabecular and intrasinusoidal components (j). Intrasinusoidal infiltrate is best visualized by CD20 staining (k). Cytogenetic studies show deletion 7q (1, arrow)

 

FIGURE 11.10 Follicular lymphoma with t(14;18) [IGH-BCL2]: (a) cytogenetics (partial karyotype); (b) FISH. (c-d) Flow cytometry shows clonal B-cells (kappa"; c) with CD10 expression (d)

  

 

■■■【12】Phenotype classification of mature B-Cell neoplasms

  

 FIGURE 12.1 Algorithm for the diagnosis of B-cell lymphoproliferations

 

 FIGURE 12.2 The phenotypic differences between CLL (left column) and MCL (right column). CLL/SLL differs from MCL by dim expression of surface immunoglobulins (a, a'), dim expres- sion of CD20 (b, b'), positive CD23 (c, c'), negative CD81 (d, d'), and positive CD200 (e, e')

 

FIGURE 12.3 Follicular lymphoma. Most lymphomatous cells (red dots) have low forward scatter (a) and only minor subset of cells (blue dots, arrow) show increased forward scatter. The expression of CD19 is dim (a), expression of CD10 is strong (b), and the expres- sion of CD38 is variable (c). Majority of FLs show surface light chain immunoglobulin restriction with moderate or bright expression of either kappa or lambda (d-e). Scattergram f shows co-expression of CD19 with CD10

 

FIGURE 12.4 Mantle cell lymphoma with aberrant expression of CD10. Monoclonal B-cells (a-b) co-express CD10 (c; variable expression) and CD5 (d). FISH studies showed rearrangement of CCND1 and IGH (e; yellow fusion signals)

 

FIGURE 12.5 Follicular lymphoma, unusual case with aberrant CD5 expression. Flow cytometry revealed monoclonal (kappa) B-cells (a-b; arrows) with co-expression of CD5 (c) and CD10 (d). Histology showed nodular infiltrate (e), which by immunohisto- chemistry was positive for CD20 (f), CD5 (g), and CD10 (h). FISH studies confirmed BCL2-IGH rearrangement (i)

 

FIGURE 12.6 Marginal zone lymphoma (blood). Clonal B-cells show moderate expression of kappa (a, arrow), moderate to bright expression of both CD19 and CD20 (b), negative CD5 (c, arrow), positive CD81 (d), dim expression of CD200 (d, arrow), negative CD10 (e), negative CD71 (f, arrow), negative CD11c (g, arrow), and negative CD38 (h, arrow)

 

FIGURE 12.7 FC patterns of HCL-v (left; blue dots) and SMZL (right; magenta dots; inset shows FISH analysis with deletion of 7q: red dot represent 7q arm of chromosome 7 and green dots centromere of chromosome 7). Both HCL-v and SMZL show monotypic expression of surface light chain immunoglobulins (a-a') and non-specific phenotype (CD5, CD10-; b-b'). The expression of CD20 is bright in both neoplasms (c-c'), but FSC is higher in HCL-v (c-c'). CD11c is strongly positive in HCL-v (d) and negative in SMZL (d'). The expression of CD25 (e-e'), CD81 (f-f'), and CD200 (g-g') is similar in both neoplasms. HCL-v differs from SMZL by positive CD103 (h-h'). CD123 is negative in HCL-v and SMZL (i-i')

 

FIGURE 12.8 Comparison of FSC in low grade follicular lymphoma (FL; a) and large B-cell lymphoma (DLBCL; b). Low grade FL show low FSC, which is similar to minimally increased when compared to reactive T-cells (CD19 cells; a). DLBCL is characterized by high FSC (compare to benign CD19-T-cells with low FSC; b). Insets show corresponding smears (Wright-Giemsa, objective x1000)

 

FIGURE 12.9 Pleomorphic variant of MCL. (a-f) Flow cytometry shows clonal B-cells (blue dots) with kappa restriction (a), positive CD19 and CD20 (b), high forward scatter (c), dim CD5 expression (c-d), negative CD23 (d), negative CD200 (e), and dim CD81 (f). (g) Histologic analysis shows diffuse infiltrate of medium to large cells with irregular nuclei. (h-k) Immunohistochemistry shows strong CD20 expression (h), weak CD5 expression (i), positive BCL1 (j), and positive SOX11 (k)

 

FIGURE 12.10 Algorithmic approach to high grade B-cell lymphomas. Abbreviations: ABC, activated B-cell; GCB, germinal center B-cell; HGBL, high grade B-cell lymphoma; HGBL-R, HGBL with rearrangement of MYC and BCL2; BL, Burkitt lymphoma; B-ALL/ LBL, acute B-cell lymphoblastic leukemia/lymphoma; DLBCL, diffuse large B-cell lymphoma; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; SHL, single hit lymphoma; DHT, double hit lymphoma, THL, triple hit lymphoma; PBL, plasma- blastic lymphoma

 

FIGURE 12.11 CLL with plasmacytic differentiation (component) - flow cytometry analysis of the bone marrow aspirate. CLL cells (red dots; red arrows) are positive for CD19 (a, f), CD20 (b; dim expression), CD5 (d-f) and kappa (d). Plasma cells (yellow dots; yel- low arrows) show positive CD19 (a), CD20 (b), CD38 (c; bright expression), and cytoplasmic kappa (g). They are negative for CD56 (i)

  

 

  13Mature B-Cell Neoplasms

  

 

■■■【13-1】CLL/SLL

  

 

FIGURE 13.1 Monoclonal B-cell lymphocytosis (tri-clonal) with dim CD5 expression and partial CD23 expression (a-b); clone #1 (yellow dots): kappa+, CD19+, CD20*; clone #2 (green dots): lambda+, CD19+, bright CD20*; clone #3 (blue dots): kappa, lambda, CD19+, CD20+/- (c-f)

 

FIGURE 13.2 Chronic lymphocytic leukemia - typical immunophenotypic profile (bone marrow). Bone marrow shows prominent lymphocytosis (a; aspirate smear). Flow cytometry shows dim expression of CD20 (b-c), moderate CD19 and CD5 (d), dim CD22 (e), positive CD23 (e), and dim expression of CD38 (f). Bone marrow biopsy from the same patient shows diffuse BM involvement (g) and positive expression of CD20 (h), CD5 (i), and CD23 (j) by immunohistochemistry


FIGURE 13.3 Typical immunophenotypic profile of CLL. Leukemic B-cells (red dots, arrow) show the following phenotype: clonal expression of surface immunoglobulins (kappa in this case, a), positive CD19 and dimly positive CD20 (b), dimly positive CD22 (c), co-expression of CD5 and CD23 (d), negative CD81 (e), and moderate expression of CD200 (f)


FIGURE 13.4 Chronic lymphocytic leukemia - cytology. Bone marrow aspirate with marked lymphocytosis of small lym- phocytes with scanty cytoplasm and small nuclei with dense, clumped nuclear chromatin


FIGURE 13.5 CLL: small lymphocytes (L) and prolymphocytes (P)


 FIGURE 13.6 Small lymphocytic lymphoma - partial involvement of the lymph node (two cases). Case #1 (a-d); (a) Histology (H&E staining). (b-d) Immunohistochemistry. The central, not involved part of the lymph node (*) shows lack of CD20 staining (b), positive CD5 (c, reactive T-cells are slightly darker than neoplastic B-cells), and positive CD3 (d). Neoplastic B-cells at the periphery shows CD20 (b) and CD5 (c) expression and lack of CD3 (d). Case #2; (e) Histology (H&E staining) with follicular hyperplasia. (f-h) Immunohistochemistry. CD20 (f) shows stronger expression by reactive germinal centers. Staining with CD5 (g) and (h) shows interfollicular involvement by SLL. (i-1) Flow cytometry analysis shows lambda+ clone expressing CD5 hidden in the mostly polytypic B-cells

 

FIGURE 13.7 SLL involving tonsil. H&E sections (a–c) show typical pseudonodular pattern with proliferation centers. SLL cells are positive for CD23 (d), CD20 (e), and CD5 (f) by immunohistochemistry. Flow cytometry analysis (g-i) shows typical immunopheno- typic profile with positive CD5 (g-i), CD19 (g), CD20 (h), CD23 (h), and dim lambda expression (i)

 

FIGURE 13.8 Richter syndrome. Two cases of large cell transformation (Richter's syndrome) of SLL/CLL (a-c and d-e). (a) Histologic sec- tion shows diffuse large cell infiltrate. (b) High magnification shows large lymphocytes with several nucleoli. (c) Flow cytometry reveals pre- dominance of lymphoid cells with increased forward scatter (arrow). Normal (small) lymphocytes with bright CD45 have low forward scatter. (d) Lymph node with highly pleomorphic lymphoid infiltrate which still displays co-expression of CD19 and CD5 by flow cytometric analysis (e)

 

FIGURE 13.9 CLL with large cell transformation - flow cytometry. Atypical large cells (a) show higher forward scatter and brighter expression of CD20, when analyzed by flow cytometry (b, blue dots). Residual CLL cells are CD5 and CD23 positive (c), whereas trans- formed cells show negative CD23 (c, blue dots)

  

 FIGURE 13.10 BM with two B-cell neoplasms (B-ALL and DLBCL) in patient with history of CLL. BM biopsy (a) shows prominent lymphoid infiltrate with crush artifacts, composed of mostly CD20 B-cells (b). Aspirate smear (c-d) shows maturing marrow elements with highly atypical large lymphoid cells (c, arrows) and blasts (d, arrows), some of which show "hand-mirror" appearance. (e-k) FC analysis shows blasts (maroon dots) with lack of CD45 (e), positive CD34 (f), and CD10 (g; bright expression); hematogones (light blue dots) with partial CD34 expression (f) and positive CD10 (g); large B-cells with bright CD45 (e), negative CD10 (g), positive CD5 (h), CD19 (1), CD20 (j), and CD23 (k). This may represent either unusual bi-clonal Richter's in the form of B-ALL and large B-cell lymphoma or two separate B-cell neoplasms, Richter's (large B-cell lymphoma) and emerging B-ALL (e.g., therapy-related)

 

FIGURE 13.11 CLL with unusual Richter's transformation in the form of B-ALL. BM aspirate smear (a) shows CLL cells, lympho- blasts and rare normal marrow elements. Histology (b) shows subtotal BM replacement by lymphoid infiltrate composed of small to medium sized blasts and small lymphocytes. Immunohistochemistry analysis (c-g) shows blasts positive for CD34 (c) and CLL cells partially positive for CD20 (e), positive for CD5 (f) and CD23 (g). Flow cytometry analysis (h-o) shows mixed population of maturing myeloid cells (h, gray dots), CLL cells; h, red dots), normal T-cells (h, blue dots) and CD45-B-ALL blasts (h, green dots). Blasts have slightly increased forward scatter and show bright CD10 expression (i), positive CD34 (j), positive CD19 (k), CD38 (k), and CD10 (I). CLL cells have clonal kappa expression (m) positive CD19 (n), partial CD200 (n), positive CD5 (0), and positive CD23 (o)

  

 

■■■【13-2SBLPN/B-PLL

 

 

FIGURE 13.12 SBLPN/B-PLL with unusual phenotype similar to CLL. Leukemic cells (red dots) display increased forward scatter (a), moderate CD20 expression (a), moderate lambda (b), positive (dim) FMC-7 (c), and co-expression of CD5 and CD23 (d). Cytologic analysis of blood or bone marrow smears helps to differentiate SBLPN/B-PLL from atypical CLL

 

FIGURE 13.13 SBLPN/B-PLL (BM analysis from 76-year-old patient with marked leukocytosis (WBC = 180 k/μL) and mild splenomegaly (no adenopathy and no prior history of lymphoma). (a) Blood smear shows lymphocytosis with cytologic atypia including large nucleoli. (b) BM core biopsy shows diffuse lymphoid infiltrate. (c–d) Immunostaining analysis shows rare cells with expression of BCL1 and negative SOX11 (d-e; inset: FISH is negative for CCND1 rearrangement, orange = CCND1; green = IGH). (f-k) Flow cytometry analysis (BM sample) shows kappa restriction (f), positive CD19 and CD20 (g), negative CD5 and dim CD23 (h), negative CD10 (i), dim CD81 (j), and dim CD200 (k)

  

FIGURE 13.14 SBLPN/B-PLL. (a-c) Blood smear shows medium-sized cells with prominent central nucleoli. (d) BM involvement: lymphoid cells with prominent nucleoli

   

FIGURE 13.15 Differential diagnosis of SBLPN/B-PLL (see text for details). Abbreviations: SBLPN, splenic B-cell lymphoma/ leukemia with prominent nucleoli; PLL, prolymphocytic leukemia, CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; SMZL, splenic MZL; HCL, hairy cell leukemia; SBLPN/HCL-v, splenic B-cell lymphoma/hairy cell leukemia variant; DLBCL, diffuse large B-cell lymphoma; BL, Burkitt lymphoma, HGBL-R, high grade B-cell lymphoma with rearranged MYC and BCL2 genes; LPL, lymphoplasmacytic lymphoma; WM, Waldenström macroglobulin- emia; MF, mycosis fungoides; SS, Sézary's syndrome; T-LGL, T large granular lymphocyte; ATLL, adult T-cell lymphoma/leukemia; GD-TCL, gamma/delta T-cell lymphoma/leukemia; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia

 

FIGURE 13.16 B-chronic lymphocytic leukemia/prolymphocytic leukemia (B-CLL/PLL). Blood smear (a) shows mixed population of small lymphocytes with dense chromatin and larger cells with prominent nucleoli (prolymphocytes). Flow cytometry analysis (b-e) shows two clonal kappat (b-c) B-cell populations: small cells with low forward scatter (d; red dots marked with red dotted arrow) and larger cells with higher forward scatter (d; blue dots marked with solid arrow). Larger cells have brighter expression of surface immu- noglobulin (b), brighter CD20 (b-c) and positive CD5 (d-e) and CD23 (e). Smaller cells show slightly dimmer kappa (b) and CD20 (b-c), negative CD5 (d), and positive CD23 (e)


FIGURE 13.17 Prolymphocytoid transformation of FL to HGBL-R. (a) Blood shows marked lymphocytosis (WBC = 140 k/μL) with large lymphoid cells with prominent nucleoli. (b-g) Flow cytometry analysis shows CD20+ B-cells with high forward scatter (b), clonal kappa restriction (c), positive CD10 (d), negative CD5 (e), positive CD23 (e) bright CD81 (f), and moderate CD200 (g). (h-i) FISH analy- sis shows IGH-BCL2 rearrangement (h, yellow fusion signal indicating BCL2 rearrangement) and MYC rearrangement (i, break apart probe with normal yellow fusion signal and abnormal orange and green signals indicating MYC rearrangement)

  

 

■■■【13-3】NMZL

 

 

FIGURE 13.18 Histologic features of nodal MZL: (a) diffuse pattern without any residual follicles with reactive germinal centers; (b) perifollicular (marginal) zone pattern; (c) prominent monocytoid appearance of lymphomatous cells; (d) NMZL with extensive plasmacytic differentiation. NMZL with diffuse involvement of the lymph node by small, rather monomorphic lymphoid cells (e). (f-j) Immunohistochemistry staining shows positive CD20 (f), negative CD5 (g), negative CD10 (h), positive CD23 (i), and positive for CD43 (j). Flow cytometric analysis shows clonal B-cells (kappa+, k-1) without expression of CD5 (m) or CD10 (n), positive CD11c (0), and positive CD23 (p)

 

FIGURE 13.19 NMZL - flow cytometry analysis. Flow cytometry analysis shows partial lymph node involvement with CD20+ (a–c) monoclonal B-cells (kappat; b, arrow) in the background of polytypic B-cells (kappa and lambda*; b-c). MZL cells are negative for CD71 (d), CD5 (e, arrow), and CD10 (f, arrow)

  

FIGURE 13.20 Nodal marginal zone B-cell lymphoma - flow cytometry. (a) Histology of the lymph node shows residual germinal center surrounded by small lymphocytic infiltrate with plasma cells. Neoplastic B-cells are positive for CD11c (b), kappa immuno- globulins (c-d), and are negative for CD103 (e) and CD25 (f)

 

FIGURE 13.21 Marginal zone lymphoma (blood) with aberrant expression of CD13 - flow cytometry (a-d, all events; e-f, lympho- cytes only). Monoclonal B-cells express CD20 (a) and kappa (b) and display aberrant expression of CD13 (on subset of monoclonal B-cells with brighter CD20; d, f). CD5 and CD10 are not expressed (e)


FIGURE 13.22 NMZL with lack of surface immunoglobulin expression - flow cytometry. Lymphomatous cells (arrow) are kappa and lambda negative (a-b; residual benign B-cells are either kappa or lambda), do not express CD5 (c) or CD10 (d), show dim expres- sion of CD81 and dim expression of CD200

 

FIGURE 13.23 Gastric marginal zone B-cell lymphoma (MALT-lymphoma) with prominent lymphoepithelial lesions (a–c). The lymphoepithelial lesions can be easily identified by immunohistochemical staining with cytokeratin (d)

  

FIGURE 13.24 Extranodal MZL (a, colon; b, thyroid, and c, salivary gland). Flow cytometry (right panels) analysis show kappa+ clonal B-cell population with moderate CD19 and CD20 expression

  

 

■■■【13-4】SMZL

  

 

FIGURE 13.25 SMZL with characteristic biphasic pattern (histology at low intermediate and high magnification)

 

FIGURE 13.26 Bone marrow involvement by SMZL– nodular and intrasinusoidal pattern (a, H&E staining; b-c, CD20 staining)

  

 

■■■【13-5】SBLPN/HCL-v

  

 

FIGURE 13.27 SBLPN/HCL-v. Leukemic cells (magenta dots, arrow) are characterized by bright CD45 and increased side scatter (a), bright CD20 (b-d), bright surface light chain immunoglobulin expression (b-c), moderate CD19 (d), negative CD10 (e), negative CD5 (f), moderate CD22 (g), moderate CD11c (h), negative CD38 (i), negative CD25 (j), positive CD103 (k), and negative to partially dim CD71 (1)

 

 

■■■【13-6】HCL

 

 

FIGURE 13.28 Hairy cell leukemia - cytology (blood smear of several cases of HCL)

 

FIGURE 13.29 HCL - histology (BM): interstitial infiltrate (low, intermediate and high magnification)

 

FIGURE 13.30 Hairy cell leukemia – lymph node. (a) Low magnification shows prominent perifollicular/paracortical infiltrate. (b-c) Higher magnification shows atypical lymphoid cells with moderate amount of pale cytoplasm and irregular nuclei. Hairy cell leukemia cells are positive for CD20 (d), negative for CD5 (e) and positive for bcl-1 (f), DBA44 (g), and CD25 (h)

 

FIGURE 13.31 Hairy cell leukemia - flow cytometry (FC). delete this part FC analysis shows increased orthogonal side scatter (a; SSC, arrow), which places the cells in the "monocytic region", above normal lymphocytes (red dots). HCL cells show moderate to bright expression of CD20 (b-e), surface immunoglobulin (lambda, c), bright expression of CD11c (d), and co-expression of CD25 and CD103 (e-f)

  

 FIGURE 13.32 HCL flow cytometry analysis (blood). HCL cells (arrow) are strongly positive for CD19 (a), CD20 (b), CD11c (c), CD25 (d), CD103 (e), and CD123 (f)

 

 FIGURE 13.33 HCL with aberrant expression of CD5. FC analysis shows show bright CD20+ HCL cells (a, arrow) with co-expression of CD25 and CD103 (b) and aberrant expression of CD5 (c; gated on lymphocytes only)

 

FIGURE 13.34 HCL with aberrant expression of CD10. FC analysis of BM shows leukemic cells with bright CD45 and increased side scatter (a, blue dots), kappa restriction (b-c), bright CD10 expression (d)

  

FIGURE 13.35 Hairy cell leukemia (HCL) with no detectable surface immunoglobulins (a) and HCL with aberrant (partial) CD2 expression and aberrant CD10 expression (b)

  

FIGURE 13.36 Comparison of flow cytometry characteristics of HCL (left column, red dots), HCL-v (middle column, blue dots), and SMZL (right column, red dots). All three disorders show moderate expression of surface light chain immunoglobulins (a-a") and moder- ate to bright expression of CD19 and CD20 (b-b"). HCL is characterized by bright expression of CD11c (c), while the expression of CD11c in SBLPN/HCL-v is moderate (c') and negative to partially dim in SMZL (c"). HCL and SBLPN/HCL-v are positive for CD103 (d-d') and SMZL shows negative to minimally dimly positive CD103 (d"). CD25 expression is positive only in HCL (d). SBLPN/HCL-v are always CD25-(d'), while SMZL are either negative (d") or positive (not shown). CD200 is strongly expressed in HCL (e) and is negative in SBLPN/ HCL-v (e'). The expression of CD200 in MZLs are usually negative or dimly positive, but SMZL often shows moderate CD200 (e")

  

 

■■■【13-7】SDRPL

  

   

 

■■■【13-8】LPL

  

  

 FIGURE 13.37 Lymphoplasmacytic lymphoma (bone marrow aspirate): typical mixture of small lymphocytes, plasmacytoid lym- phocytes and plasma cells (a). Many plasma cells show Dutcher bodies (intranuclear inclusions; b)

 

FIGURE 13.38 LPL paratrabecular BM involvement (a, histology; b, immunohistochemistry). CD20 immunostaining shows prominent paratrabecular accumulation of lymphoid cells (b)

  

FIGURE 13.39 LPL (MYD88) with blood involvement. (a) Blood smear with small lymphocytes and occasional plasma cells. (b-d) Flow cytometry analysis shows clonal B-cells with CD20 and kappa expression (b, arrow) and smaller population of clonal plasma cells with bright CD38 (c-d), cytoplasmic kappa expression (c, arrow) and c.IgM expression (d, arrow)

 

FIGURE 13.40 Lymphoplasmacytic lymphoma. (a) Aspirate smear with lymphocytes, plasmacytoid lymphocytes and plasma cells (inset: flame cell). (b) Core biopsy with atypical paratrabecular lymphoid aggregate. (c-g) FC analysis shows clonal B-cells and clonal plasma cells. B-cells (arrow) display strong expression of surface immunoglobulins (kappa, c-d) and dim to moderate expression of cytoplasmic IgM (f). Plasma cells (*) are relatively rare. They have bright expression of cytoplasmic IgM (f, compare with cytoplasmic IgG on display e) and bright cytoplasmic kappa (g)

 

FIGURE 13.41 LPL with partial CD5 expression - immunohistochemistry. The BM core biopsy shows atypical interstitial and paratrabecular lymphoid infiltrate (a). Lymphoid cells are positive for CD20 (b) and PAX5 (c) and display aberrant expression of CD5 (d-e; two different areas, compare with CD3 staining on f). Plasma cells are positive for IgM (g) and kappa (h-i)

 

FIGURE 13.42 LPL (IgG) with aberrant expression of CD10 (FISH analysis was negative for BCL2, BCL6, and MYC rearrange- ments; molecular studies showed MYD88 mutation). (a) Aspirate smear with prominent lymphocytosis. (b) BM core biopsy with dif- fuse lymphoid infiltrate of mostly small lymphocytes (immunostaining with CD138 showed only rare monotypic plasma cells). (c–d) FC analysis shows clonal (kappa+) B-cells (c) with CD10 expression (d)

 

FIGURE 13.43 IgA+ LPL. (a) Aspirate smear with lymphocytosis with occasional plasma cells. (b). Core biopsy shows atypical para- trabecular lymphoid infiltrate. (c-f) Immunohistochemistry. B-cells are CD20+ (c) and plasma cells are CD138 (d), kappa (e, inset lambda staining), and IgA+ (f)

   

 

■■■【13-9】FL

  

 

FIGURE 13.44 Follicular lymphoma (FL) - lymph node. (a) FL with prominent nodular pattern. (b) FL with vague nodularity. (c) FL with nodules of different sizes. (d) FL with irregular nodules

  

FIGURE 13.45 Morphologic variants of FL. (a) Floral variant. (b) FL with pale (clear) cytoplasm (monocytoid B-cell appearance). (c) Reversed pattern. (d) FL with prominent sclerosis, obliterating nodular architecture. (e-e') Signet ring cell variant (low and high magnification). (f-f') Diffuse variant of FL. (g-g') FL with Reed-Sternberg-like cells. (h-h") FL with prominent plasmacytic differen- tiation. (i-i') FL with increased vascularity

  

FIGURE 13.46 Leukemic peripheral blood involvement by follicular lymphoma (a, blood smear). Flow cytometry (b-c) shows clonal expression of kappa light chain immunoglobulin

 

FIGURE 13.47 Follicular lymphoma - patterns of bone marrow involvement. (a) Characteristic paratrabecular pattern of involve- ment with lymphomatous cells expressing CD20, CD10, and BCL2 (a', low magnification; a" CD20 immunostaining; a"" CD10 immu- nostaining; a""" BCL2 immunostaining). (b) Nodular bone marrow involvement

 

FIGURE 13.48 Follicular lymphoma (FL) - immunohistochemistry. Typical FL (a) is positive for CD20 (b), CD10 (c), and BCL2 (d)

 

FIGURE 13.49 (a) FLBL with unusual lack of CD10 (a) and BCL2 (a"). BCL6 is positive (a"). (b) Classic FL with positive CD20 (b'), lack of BCL2 (b") and strong CD10 (b")

 

FIGURE 13.50 Follicular lymphoma - flow cytometry analysis. Neoplastic B-cells (black arrow) show increased forward scatter (a-b), dim expression of CD19 (a; compare with benign B-cells), positive CD10 (b; d-f) and positive CD71 (c). CD10+ population is lambda positive (compare e-f). Increased forward scatter and positive CD71 is usually associated with FLBL (CFLs show low forward scatter and negative CD71)

 

FIGURE 13.51 Follicular lymphoma - flow cytometry. (a-d) Analysis of lymph node with FL shows polytypic B-cells (red dots; red arrows), clonal B-cells (blue dots; blue arrow) and reactive T-cells (CD19-). The lymphomatous cells show dim CD19 (a-b) and bright kappa (a). Clonal B-cells show expression of CD10 (c-d). In another example (e-g) follicular lymphoma cells (red arrow) are surface light chain immunoglobulin negative (e-f) and show positive CD10 expression (g). Rare polytypic B-cells show kappa and lambda expression (e-f, black arrow). Numerous T-cells (blue arrow) are also noted (kappa, lambda and CD19 negative; e-g)

 

 

 FIGURE 13.52 FL with aberrant expression of CD5. Flow cytometry (a-c) shows monoclonal (kappa) B-cell population co-expressing CD5 (b) and CD10 (c)

  

FIGURE 13.53 FL with lack of surface light chain immunoglobulin expression. (a) Histology with atypical nodular proliferation. (b-d) Flow cytometry shows co-expression of CD20 with CD10 (b, arrow), and lack of both kappa (c) and lambda (d) expression. Rare polytypic

(*)B-cells are noted

 

 FIGURE 13.54 Follicular lymphoma concurrent flowcytometry analysis of the bone marrow (BM; left column) and lymph node (LN; right column) showing discrepant phenotype of clonal cells between those two sites. Both populations are CD19+ (a-b), CD20+ (a-b), and CD10+ (c-h), but clonal B-cells is the BM are kappa (e) and show moderate intensity of CD10 expression (c), whereas clonal B-cells in the lymph node are mostly lambda+ (h) with only minor population being kappa+ (f), and show bright CD10 expression (d)

 

FIGURE 13.55 Follicular lymphoma (FL) – grading. (a) Low grade FL (grade 1) is composed predominantly of small cells with irregular nuclei. (b) Intermediate grade FL (grade 2) has mixture of small (centrocytes) and larger (centroblasts) lymphocytes. (c-d) High grade FL (grade 3) has predominantly large lymphocytes. Depending on the presence of few scattered small lymphocytes grade 3 FL can be subdivided into 3a (small cells present) and 3b (small cells absent). Grade 1, 2 and 3a are now classified as CFL and grade 3b FL is now classified as FLBL

 

FIGURE 13.56 Flow cytometry of low-grade follicular lymphoma (histologically grade 1-2 of 3). Lymphomatous cells display low forward scatter (a, arrow), positive CD20 (a), kappa restriction (b), positive CD10 (c), dim CD81 (d) and CD200 (e), and negative CD71 (f)

 

FIGURE 13.57 Flow cytometry of FLBL. Lymphomatous cells display high forward scatter (a, blue dots, arrow), positive CD20 (a), kappa restriction (b), positive CD10 (c), bright CD81 (d), negative CD200 (e), and positive CD71 (f)

 

FIGURE 13.58 FL with transformation to HGBL-R (double-hit lymphoma). Patient with history of FL. Flow cytometry of blood (a-f) shows neoplastic cells (orange dots, arrow) with moderate CD45 expression (a), high forward scatter (b), positive HLA-DR (b), negative CD34 (c), bright CD20 (d-e), lack of surface light chain immunoglobulins expression (d-e), dimly positive CD19 (f), and positive CD10 (f). FISH studies show MYC rearrangement (g; break-apart probe showing one orange, one green and one fusion) and BCL2-IGH rearrangement (h; one green, one orange and two yellow fusion signals)

 

FIGURE 13.59 Blastic transformation of FL. (a–c) FISH analysis showing MYC rearrangement (a), BCL2 rearrangement (b) and break-apart dual color with split signals (c; red and green split signals; yellow, normal signal). (d-g) Flow cytometry analysis shows leukemic cells in blastic region (d; arrow, green dots) co-expressing CD10 and TdT (e) with clonal lambda expression (f-g), dim CD19 (f), and negative CD20 (g)

 

FIGURE 13.60 Benign lymph node. Flow cytometry analysis shows polytypic B-cells (a-c, arrows) which are mostly CD5- (d). Rare B-cells express CD10 (e) and CD38 (f). Majority of show dim expression of CD23 (g), CD81 (h), and CD200 (i)

 

FIGURE 13.61 Flow cytometry (FC) of florid follicular hyperplasia (a, H&E; b–e, immunohistochemistry; f-i, flow cytometry). Reactive lymph node with florid follicular hyperplasia (a; H&E section) shows numerous large CD20+ follicles (b) with scattered CD3+ T-cells (c) and irregular CD10+ germinal centers (d) which are BCL2-(e). FC shows two distinct B-cell populations, one with moderate CD20 and second (predominant) population with bright CD20 expression (f-g; red arrowheads). Those bright CD20+ B-cells represent CD10+ germinal centers cells. They show dim CD10 expression (h-i) and typically a "smeary" expression of surface light chain immunoglobulins with kappa usually brighter than lambda (f−i). This pattern should not be confused with a clonal (kappa1) B-cell population in the background of polytypic B-cells

 

FIGURE 13.62 Reactive lymph node with florid follicular hyperplasia showing distinct kappa+ B-cells mimicking clonal population. Flow cytometry shows polytypic, kappa and lambda+ B-cells (a-c) with distinct two populations, one with moderate CD20 expres- sion of polytypic kappa and lambda expression (b-c, red dots/red arrows) and another with brighter CD20 and predominantly kappa expression (blue dots/blue arrows). The bright CD20* germinal center cells (blue dots/blue arrows) show moderate expression of CD19 (d), dim CD71 (e), dim CD10 (f), bright CD38 (g), bright CD81 (h), and negative CD200 (i)

 

FIGURE 13.63 MCL with prominent paratrabecular BM involvement (a, histology; b, CD20 immunostaining; c, CD5 immunostaining)

 

FIGURE 13.64 Tonsil with ISFN. (a) Histology shows mostly reactive germinal centers. One neoplastic follicle (arrow) shows bright expression of CD10 (b), positive Ki-67 without overt polarization (c), and bright expression of BCL2 (d)

 

FIGURE 13.65 ISFN. (a-e) Flow cytometry shows predominant population of benign (polytypic) B-cells (a-b), T-cells (a) and minor population of CD10+ B-cells (c). Analysis of CD10 versus kappa and lambda (d-e) shows clonal CD10+ B-cells with dim lambda expres- sion. (f) Histology of the lymph node shows scattered lymphoid aggregates. (g-i) Immunohistochemistry (low and high magnification) shows strong expression of CD20 (g-g'), CD10 (h-h'), and BCL2 (i-i') by neoplastic B-cells

 

FIGURE 13.66 Pediatric-type follicular lymphoma (PTFL). Inguinal lymph node from 7-year-old patient shows atypical nodular infiltrate (a-b) composed highly atypical large cells (c). Nodular pattern is visualized by CD10 staining (d; low magnification) and CD20 (e; intermediate magnification). Lymphomatous cells are positive for CD10 (f), negative for BCL2 (g) and positive for p53 (h). Molecular studies (PCR) confirmed clonal rearrangement of IGH gene (1)

 

FIGURE 13.67 Primary cutaneous follicle center lymphoma(PCFCL). Flow cytometry of scalp lesion from 38-year-old patient showing clonal kappa B-cell population (a-b, arrow) with CD10 expression (c)

 

FIGURE 13.68 Composite lymphoma (PCFCL and small lymphocytic lymphoma) involving scalp from 71-year-old patient. Larger SLL clone shows dim expression of CD20 (a), dim CD5 (b), negative CD10 (c), and moderate kappa expression (d). Minor PCFCCL clone shows moderate CD20 (a), negative CD5 (b), positive CD10 (c), and moderate expression of lambda (e). Benign (reactive) T-cells show brighter expression of CD5 when compared to SLL cells (b and f)

  

 

■■■【13-10】MCL

  

 

FIGURE 13.69 Mantle cell lymphoma (MCL) - histology and cytology. Histologic examination (a) shows diffuse lymphoid infiltrate composed of lymphocytes with irregular nuclear contours. Note the presence of histiocytes, often present in MCL [inset: BC1 immu- nostaining with positive nuclei]. Cytologic analysis (b) shows irregular nuclear outlines (cleaved nuclei)

 

FIGURE 13.70 Extranodal MCL

 

FIGURE 13.71 Mantle cell lymphoma - BM (paratrabecular pattern). (a) Histology of BM core biopsy shows a dense paratrabecular lymphoid infiltrate. (b) Higher magnification displays nuclear contour irregularities. (c) Neoplastic lymphocytes are positive for BCL1

 

FIGURE 13.72 In situ mantle cell neoplasm (two cases). (a-d) Case #1. (a) Histologic section shows "reactive" looking lymphoid infiltrate composed of mostly small cells. (b-d) Immunohistochemistry shows CD3- (b), CD20+ (c), and BCL1+ (d) subtle infiltrate around reactive germinal center. (e-h) Case #2. (e-f) Histologic section (low and higher magnification) shows lymph node with reac- tive follicles. Staining with BCL1 (cyclin D1; g-h) helps to differentiate between reactive follicular hyperplasia and early MCL

 

FIGURE 13.73 In situ mantle cell neoplasm (lymph node). (a) Minute fragment of lymph node (H&E section, X20; with reactive germinal centers (b, H&E section x100) and focal infiltrate by BCL1+ mantle cells (c); FC analysis (d-e) shows a minute lambda+ clonal population with bright CD20 expression in the background of mostly polytypic B-cells

 

FIGURE 13.74 Aggressive variants of mantle cell lymphoma. (a) Pleomorphic variant of mantle cell lymphoma with irregular nuclei and few inconspicuous nucleoli. (b) Blastoid variant with monomorphic features and prominent nucleoli

 

FIGURE 13.75 Mantle cell lymphoma (MCL) with classic mantle zone pattern. Neoplastic B-cells surround benign (residual) ger- minal center (a; H&E section). Lymphomatous cells express CD20 (b), CD5 (c), BCL1 (d-e, low and high magnification), CD43 (f), and BCL2 (g), and are negative for CD23 (h) and CD10 (i)

 

FIGURE 13.76 Mantle cell lymphoma - flow cytometry (blood). Lymphomatous cells display moderate expression of CD20 (a, d) and kappa (b), positive CD5 (a-b), negative CD23 (c), and positive CD38 (e). FISH studies performed on blood sample confirms the diagnosis of MCL by positive rearrangement of CCND1 (f; yellow fusion signal)

 

FIGURE 13.77 MCL - typical flow cytometry pattern. MCL is characterized by moderate expression of surface immunoglobulins (a) and CD20 (b), positive CD5 (b-c), negative CD23 (c), negative CD10 (d), positive CD38 (e), positive CD81 (f), negative CD71 (g), and negative CD200 (h)

 

FIGURE 13.78 CD5-negative mantle cell lymphoma (a, histology). Neoplastic cells are positive for CD20 (b) and BCL1 (d); they lack CD5 (c). (e-g) Flow cytometry analysis (e-g) shows positive CD19, CD20 and lambda, and negative CD5. CD5-negative MCL comprise approximately 11% of all MCL cases

 

FIGURE 13.79 CD10+ mantle cell lymphoma (MCL). (a) histology; (b-c) immunohistochemistry; (d-e) flow cytometry. MCL cells express BCL1 (b) and co-express CD5 and CD10 (c-e)

 

FIGURE 13.80 Mantle cell lymphoma with aberrant CD23 expression (two cases). (a-e) Spleen. Histology shows atypical lymphoid infiltrate (a). (b-e) Immunohistochemistry. Lymphomatous cells express CD20 (b), BCL1 (c), and CD23 (d) and are negative for CD43 (e). (f-k) Blood. Flow cytometry analysis shows kappa restriction (f), co-expression of CD19 with CD5 and CD23 (g-i), and partial expression of CD11c (j). (k) FISH analysis shows IGH/CCNDI rearrangement: one green (IGH), one orange (CCNDI) and one yellow fusion signal

 

FIGURE 13.81 Blastoid variant of MCL - flow cytometry and FISH studies. Lymphomatous cells have increased forward scatter (a, arrow). They are positive for CD20 (a), partially CD71 (b, arrow), CD5 (c-d), partially CD10 (d; arrow), lambda (e-f) and are negative for CD23 (g). FISH studies (h) showed rearrangement of CCNDI (red) and IGH (green) giving yellow fusion signals


FIGURE 13.82 Large cell (pleomorphic) variant of mantle cell lymphoma involving lung. The lymphomatous cells (blue dots; blue arrow) show high forward scatter (a), bright CD20 expression (a), moderate CD19 (b–f), moderate lambda (c), positive CD5 (d), negative CD23 (e), and positive CD38 (f)

 

FIGURE 13.83 Flow cytometry features of CD23+ MCL and CLL. MCL differs from CLL by stronger expression of surface immunoglobulins (a), lack of CD11c (b), bright expression of CD20 (c), positive CD81 (e), and lack of CD200 (f). Only minor subset of MCL shows CD23 expression imitating CLL (d)

 

FIGURE 13.84 Cytologic features of mantle cell lymphoma (a) and follicular lymphoma (b) in blood smear

   

 

■■■【13-11】DLBCL

  

 

FIGURE 13.85 DLBCL: centroblastic (a), immunoblastic (b), anaplastic (c), and T-cell-rich (d; inset: CD20 immunostaining showing scattered large B-cells)

  

FIGURE 13.86 Immunophenotypic algorithms for subclassification of DLBCL

  

FIGURE 13.87 DLBCL with centroblastic morphology. (a) Histology; (b-f) Immunohistochemistry. Lymphomatous cells express CD20 (b), CD79a (b), BCL6 (d), CD10 (e), and are negative for MUM1 (f)

  

FIGURE 13.88 Diffuse large B-cell lymphoma (DLBCL) - immunoblastic variant. (a) Lymph node with diffuse large cell lymphoid infiltrate. Neoplastic cells are positive for CD20 (b) and PAX5 (c). High magnification shows large monomorphic cells with prominent central nucleoli (d)

  

FIGURE 13.89 THRLBL. (a-b) Low and high magnifications show scattered large cells in the background of small lymphocytes and histiocytes. Lymphomatous cells are positive for CD20 (c). T-cells express CD3 (d) and histiocytes express CD68 (e)

  

FIGURE 13.90 Large B-cell lymphoma. Tissue section (a) shows atypical large lymphoid cells in the background of small lympho- cytes. Flow cytometry (b-c) reveals only rare neoplastic cells (arrow); benign(polytypic)B-cells predominat(*)e

  

FIGURE 13.91 DLBCL - flow cytometry (lymph node). Neoplastic B-cells show increased forward scatter (a–f, black arrow; com- pare with residual reactive T-cells, red arrow). Lymphomatous cells express CD20 (a; bright expression), lambda (c), CD10 (d), CD38 (e), and CD71 (f)

 

FIGURE 13.92 DLBCL with GCB-like phenotype and negative BCL2. (a) Histomorphology. (b) Touch smear cytology showing large atypical lymphoid cells. (c-d) FC analysis showing clonal lambda+ B-cells (c; blue dots) expressing CD10 (d). BCL2 is negative (d). FISH studies in this cases showed only BCL6 rearrangement (both MYC and BCL2 were negative)

 

FIGURE 13.93 DLBCL (ABC-like). Clonal B-cells (kappa+, a, blue arrow) are negative for CD10 (b) and CD5 (c), show negative to partially dim CD71 (d), dim CD81 (e) and negative CD200 (f). Rare benign (polytypic) B-cells are also noted (red dots, red arrows)

 

FIGURE 13.94 DLBCL - bone marrow. Lymphomatous cells display bright CD45 (a), moderate CD19 (b), bright CD20 (c), moderate CD22 (d), and moderate CD38 (e). The forward scatter is increased (d-e), being higher than in granulocytes/maturing myeloid precur- sors (purple dots)

 

FIGURE 13.95 DLBCL of the testis with aberrant expression of CD5 and CD7. Tumor cells show centroblastic cytomorphology (a, touch imprint). By flow cytometry analysis, they are characterized by high forward scatter (b, blue arrow), bright CD45 expression (b), kappa restriction (c), positive CD19 (d), variable expression of CD20 (d), positive CD71 (e), positive CD5 (f; residual benign T-cells show brighter CD5 expression), positive CD81 (g), and positive CD200 (h). Comparison of CD3 versus CD7 expression shows that clonal B-cells have slightly dimmer expression of CD7 when compared to benign T-cells (i)

 

FIGURE 13.96 Comparison of FC analysis of BL (left column) and DLBCL (GCB-like; right column). When compared to BL, DLBCL differs by higher forward scatter (a-d; a'-d'). Both BL and DLBCL with germinal center B-cell phenotype express CD10 (a, a'), CD20 (b-b') and CD38 (c-c'). The expression of CD71 is brighter in DLBCL than in BL (d-d')

  

FIGURE 13.97 Lymph node with de novo CD5+ DLBCL. (a-f) Flow cytometry shows major population of large B-cells (blue dots) with high forward scatter and positive CD20 (a) and residual small lymphocytes with low forward scatter (red dots). Large B-cells show lambda restriction (b), while small B-cells are polytypic (c). Lymphomatous cells are positive for CD5 (d; partial expression) and CD81 (e), and negative for CD200 (f). FISH for CCND1/IGH shows two green signals (IGH) and two red signals (CCNDI) indicating lack of CCNDI rearrangement (g). Lack of history of CLL or small clonal CD5+ B-cells excludes Richter's transformation

 

 FIGURE 13.98 Intravascular large B-cell lymphoma (IVLBCL; bladder) (a) Histology shows large atypical lymphoid cells within large vascular space. (b-f) Immunohistochemistry. Tumor cells are positive for CD45 (b), CD20 (c), PAX5 (d), and CD5 (e). CD34 staining shows vessel wall (f)

 

FIGURE 13.99 Primary DLBCL of CNS. Lymphomatous cells (blue dots, arrow) show high forward scatter and bright CD20 expres- sion (a), lambda restriction (b), and are positive for CD10 (c), CD56 (d), CD38 (e), CD71(f), CD81 (g), and CD200 (h)

  

 

■■■【13-12】BL

  

 

FIGURE 13.100 Algorithm for differential diagnosis of Burkitt lymphoma versus DLBCL versus HGBL with MYC and BCL2 (ad/or BCL6) rearrangements. HGBL with MYC and BCL2 (double hit lymphoma) is diagnosed with rearrangement of MYC and BCL2 or MYC and BCL6, and triple hit lymphoma is diagnosed with rearrangement of all three genes (MYC, BCL2, and BCL6). Presence of BCL2 and BCL6 rearrangement without MYC does not qualify as double hit lymphoma

  

FIGURE 13.101 Burkitt lymphoma: Blood smear (a) and touch smear from the lymph node (b) shows highly atypical lymphoid cells with increased nuclear/cytoplasmic ratio, vacuolated, basophilic cytoplasm, slightly granular chromatin and several small nucleoli

 

FIGURE 13.102 Burkitt lymphoma. (a-c) Lymph node: low magnification shows diffuse lymphoid infiltrate with scattered histio- cytes creating "starry-sky" pattern (a); high magnification shows medium sized monomorphic lymphomatous cells with increased nuclear-cytoplasmic ratio (b). Tingible body macrophages are present. (c-g) Immunohistochemistry reveals the typical phenotype: positive expression of CD10 (c), CD20 (d), and CD43 (f) and negative staining with BCL2 (h). Proliferation index as determined by staining with Ki-67 (MIB-1) approaches 100% (i)

 

FIGURE 13.103 BL: diffuse BM involvement. Aspirate smear (a) and touch smear (b) shows typical cytomorphologic features of BL. Histologic section (c) shows diffuse replacement of BM by atypical lymphoid infiltrate. Tumor cells are positive for CD20 (d) and BCL6 (e), and do not express BCL2 (f)

 

FIGURE 13.104 Burkitt leukemia (morphologic, immunophenotypic and molecular analysis of blood and BM from 45-year-old patient). FC analysis showed 25% clonal B-cells in blood with low side scatter, dim to moderate expression of CD45 (a, green dots), monotypic lambda expression (b), positive CD10 (c), and bright CD20 (d). Aspirate smear (e) shows atypical cells with blastoid appearance. Histologic section from core biopsy (f-g, low and high power) shows diffuse infiltrate by monotonous medium-sized lymphoid cells. Immunohistochemistry analysis (h-l) shows positive CD20 (h) and CD10 (1), negative BCL2 (j), strong expression of MYC (>90%, k), and high Ki-67 index (1). FISH analysis (m) shows one green, one orange, and one yellow/green/orange fusion signal indicative of a chromosomal break in the MYC locus

 

FIGURE 13.105 Extranodal BL. (a) BL - endometrium. (a) Dense monomorphic lymphoid infiltrate with somewhat "blastoid" appearance composed of medium-sized cells with high nuclear-cytoplasmic ratio. Tumor cells are positive for CD20 (a'), CD10 (a"), negative for BCL2 (a"), and positive for CD43 (a). (b) BL stomach, expressing CD20 (b′), BCL6 (b"), CD10 (b), and Ki-67 (b). (c) BL- small intestine (low and high magnification). (d) BL- mandible. (e) BL- kidney. Tumor cells are negative for keratin (e'), express CD20 (e"), and PAX5 (e"), are negative for BCL2 (e) and show strong Ki-67 staining (e)

 

FIGURE 13.106 Burkitt lymphoma - flow cytometry (lymph node). Neoplastic B-cells show increased forward scatter (a-f, arrow), positive CD20 (a), CD10 (b), CD38 (c), CD71 (d; partial), and do not express surface light chain immunoglobulins (e-f)

 

FIGURE 13.107 BL. Lymphomatous cells (blue dots, arrow) show increased forward scatter (a), bright CD20 expression (a), lambda restriction (b), and positive expression of CD10 (c), CD71 (d), and CD81 (e). CD200 is negative (f)

 

FIGURE 13.108 BL with unusual CD5 expression (43-year-old man with mediastinal mass and circulating "blasts"). Blood smear shows highly atypical lymphoid cells (a). FC analysis shows B-cells with high forward scatter and positive expression of CD20 (b), CD10 (c), kappa (d; compare with lambda, e), and CD5 (f)

 

FIGURE 13.109 Burkitt lymphoma. (a) FISH analysis for c-MYC (break-apart probe); (b) FISH analysis shows MYC-IGH fusion. (c) Conventional cytogenetics shows complex changes including t(8;14)

  

 

■■■【13-13】HGBL-R

  

 

FIGURE 13.110 Composite high-grade B-cell lymphoma: Burkitt lymphoma (BL) and high-grade B-cell lymphoma, NOS. (a–c) Histology with monomorphic infiltrate with starry sky appearance. The BL cells are slightly smaller than non-BL (compare a and b). (c) Low poser shows several core biopsy, some of which show Burkitt lymphoma component (BL) and some non-Burkitt lymphoma component (non-BL). (d-k) Immunohistochemistry shows different phenotype of two components of this composite lymphoma, BL is positive for CD20 (d), EBC (e), MYC (g), CD10 (1, strong expression), BCL6 (j) and Ki-67 (k), while BCL2 (f) and MUM1 (h) are negative. Non-BL component is positive for CD20 (d), EBV (e), BCL2 (f), MYC (g, partial expression), MUM1 (h), CD10 (I, dim expression), and Ki-67 (k). The expression of BCL6 is negative (g)

 

FIGURE 13.111 HGBL-R (double hit lymphoma: MYC and BCL2 rearrangement): lymphoid cells are positive for CD20 (b), CD10 (c), and BCL2 (d; dim expression). The proliferation fraction is high (<90% ; e). CD43 is not expressed (f). Flow cytometry shows B-cells with increased forward scatter (g-h), positive CD19 (g), CD10 and CD71 (i). FISH studies revealed rearrangement MYC-IGH (j) and IGH-BCL2(k)

 

FIGURE 13.112 HGBL-R (double hit) with extensive BM involvement. Core biopsy (a) shows complete BM replacement by necrotic lymphoma. Clot section (b) shows aggregates of highly atypical, viable lymphoid cells. Aspirate smear (c; oil magnification) shows group of neoplastic cells with "blastoid" appearance. Immunohistochemistry stainings (clot) show positive CD20 (d), CD10 (e), BCL6 (f), BCL2 (g), and p53 (i). Majority of cells express Ki-67 (h). FISH analysis showed rearrangement of MYC (j; break-apart probe) and BCL2 (IGH-BCL2 probe)

 

FIGURE 13.113 Triple hit lymphoma(MYC and BCL2 and BCL6 rearrangement) (71-year-old patient with adenopathy and lymphocytosis). Flow cytometry analysis (a–c) shows monoclonal B-cell population (arrows) with expression of CD20 (a-b), kappa (a), CD10 (c), and BCL2 (d). FISH studies showed rearrangement of MYC (e; MYC, red; IGH, green; centromere 8, blue), BCL2 (f; IGH, green; BCL2, red), and BCL6 (g; break-apart probe)

 

FIGURE 13.114 HGBL, NOS (BM involvement). Aspirate smear (a) show highly atypical lymphoid cells with blastoid appearance. Core biopsy of the BM (b-d) shows prominent involvement by atypical CD20+ (c) B-cell infiltrate with paratrabecular pattern pro- gressing to diffuse pattern. FC analysis (e-g) shows clonal kappa+ (e-f) B-cells without CD5 or CD10 expression (g)

   

 

■■■【13-14】PBL

  

 

FIGURE 13.115 Plasmablastic lymphoma, oral mucosa. (a-b) histology (a, low power; b, high power); (c-f) immunohistochemistry: lymphomatous cells are positive for EBER (c) and CD138 (d), and negative for CD45 (e) and CD20 (f)

 

FIGURE 13.116 PBL of the buccal mucosa. Histologic section (a-c; different magnifications) shows diffuse highly atypical infiltrate composed of large cells with irregular nuclei, scanty cytoplasm and several prominent nucleoli. Tumor cells express EBV/EBER (by ISH; d), CD138 (e), MUM1 (f), and are negative for CD56 (g), PAX5 (h), and CD20 (i)

 

FIGURE 13.117 PBL with plasmacytic differentiation (a, histologic section). Tumor cells express EBV/EBER (by ISH; b), MUM1 (c), CD19 (d), CD56 (e), CD10 (f), and lambda (g; dual kappa and lambda staining with kappa brown and lambda red); they are negative for CD43 (h), CD117 (1), CD20 (j), PAX5 (k), and BCL1 (cyclin D1; I)

 

FIGURE 13.118 PBL involving BM. (a-b) Aspirate smear shows highly atypical mononuclear cells with blastoid features (plasma- blasts) with macronucleoli, fine chromatin and abundant basophilic cytoplasm with occasional vacuoles. Some cells resemble more mature plasma cells. (c-d) Histology shows diffuse BM involvement (low and high power). (e-i) Immunohistochemistry. PBL cells show the following phenotype: MUM1+ (e), CD20 (f), CD79a (g), Ki-67+ (h), and EBV+ (i)

 

FIGURE 13.119 PBL (presenting as nasal polyp). (a–c) Histology (low, intermediate and high power). (d-l) Immunohistochemistry. Tumor cells are positive for MUM1 (d), CD138 (e), CD45 (f), BOB1 (g), OCT2 (h), EBV (i), CD117 (j), and weakly positive for CD56 (k). CD20 is negative (1)

  

 

■■■【13-15】PEL

  

 

FIGURE 13.120 PEL - immunohistochemistry. (a) Cell block preparation shows large atypical lymphoid cells. (b–d) Immunohistochemistry. Tumor cells are negative for CD20 (b) and PAX5 (c), and are positive for CD30 (d), MUM1 (e), Ki-67 (f), CD43 (g), HHV-8 (h), and EBER(i)

 

FIGURE 13.121 Primary effusion lymphoma (PEL). Lymphomatous cells are large with irregular nuclei and prominent nucleoli (a). Flow cytometry and immunohistochemical staining (cell block) showed tumor cells positive for HHV-8 (b), EBER (c), EMA (d), BOBI (e), and CD3 (f), negative for CD20 (g), positive for CD38 (h), CD45 (i-k), CD43 (i-j), CD30 (m-n) and negative for kappa (o-p), lambda (r-q) and PAX5 (s)

 

FIGURE 13.122 Primary effusion lymphoma - cytology (cytospin preparation) and immunohistochemistry. The tumor cells are large and pleomorphic with increased nuclear-cytoplasmic ratio, basophilic cytoplasm and irregular nuclei (a) and express CD43 (b) CD30 (c), HHV8 (d) and lack CD45 (e). Immunocytochemical staining was performed on smears

 

 

■■■【14】Plasma cell Neoplasms

 

 

 FIGURE 14.1 PCM: radiograph of the skull demonstrating lytic bone lesions (a), radiograph of the vertebrae demonstrating com- pressive fracture (b), and radiograph of the humerus with pathologic fracture (c)

 

FIGURE 14.2 Plasma cell myeloma - cytologic features. (a) Mature plasma cells with abundant cytoplasm and eccentric nuclei with clumped chromatin; (b) Plasma cells with mild anisocytosis and inconspicuous nucleoli; (c) Plasma cells with intranuclear inclusions; (d) plasma cells with crystalloid cytoplasm mimicking Gaucher cells (e, e') Atypical plasma cells with prominent nucleoli and mitotic figures; (f) Multinucleated cells; (g) Mott cell with numerous cytoplasmic vacuoles; (g) “flame” cell

 

FIGURE 14.3 Plasma cell myeloma - histologic features. (a) Diffuse plasma cell infiltrate completely replacing bone marrow ele- ments. (b) Russell bodies. (c) Dutcher bodies. (d-e) Anaplastic (immature) variants of plasma cell myeloma (two cases). Note promi- nent nuclear pleomorphism, hyperchromasia, macronucleoli, and multinucleation. (f) Plasma cell myeloma with prominent nucleoli and mitotic figures

 

FIGURE 14.4 Plasma cell myeloma – immunohistochemistry. Plasma cells (a) express CD138 (b), lambda (c), PAX-5 (d), and BCL1 (cyclin D1; e)

 

FIGURE 14.5 Benign plasma cells flow cytometry (blood). Benign plasma cells (orange dots, arrow), are positive for CD45 (a), show polytypic expression of cytoplasmic light chain immunoglobulins (b-c), and are most often dimly CD19+ (d), CD27+ (e), CD56 (f), CD81+ (g), CD17 (h), and CD200-(i)

 

FIGURE 14.6 PCM - flow cytometry. Plasma cells (orange dots) show clonal expression of cytoplasmic lambda (a-b), bright expres- sion of CD38 (a−b) and typical aberrant phenotype associated with plasma cell myeloma with negative CD45 (c-d), positive CD117 (d; partial expression), positive CD56 (e-f), negative CD27 (f), and negative CD81 (i)

 

FIGURE 14.7 PCM - flow cytometry. Plasma cells (orange dots) are negative to dimly positive for CD45 (a), show cytoplasmic kappa restriction (b-c) and aberrant expression of CD33 (d), CD56 (e), and CD117 (f). The expression of CD81 (g), CD19 (h), CD20 (i), and CD27 (j) is negative (as seen in most plasma cell neoplasms). Many plasma cell myelomas display aberrant (positive) expression of CD200, but presented case is CD200 ̄ (k)

 

   FIGURE 14.8 PCM flow cytometry. Plasma cells (orange dots) show cytoplasmic kappa restriction (a-b), positive cytoplasmic IgG (c), bright expression of CD38 (d) and CD138 (f), negative to partially positive CD27 (f), negative CD56 (g), positive CD81 (h), and positive CD200 (i)

 

 FIGURE 14.9 Plasma cell myeloma with unusual high side scatter (a-c; orange dots), bright CD38 (a), bright cytoplasmic lambda (b), and bright CD56 (c)

 

 FIGURE 14.10 PCM - flow cytometry. Plasma cells (blue dots) display aberrant (positive) expression of CD45 (a), normal (bright) expression of CD38 (b) and CD138 (c), aberrant expression of CD20 (d), monotypic kappa restriction (e-f), negative CD19 (g), aberrant positivity for CD27 (h) and CD56 (i), lack of CD81 (j), and positive expression of CD117 (k) and CD200 (1)

 

FIGURE 14.11 PCM with unusual CD27 expression. Plasma cells (orange dots) are monotypic (lambda+; a) and show positive CD27 expression (b)

 

FIGURE 14.12 Plasma cell myeloma with immature (plasmablastic) cytomorphology. Blastic morphology (a, BM aspirate smear; b, BM core biopsy) in conjunction with positive CD45 (c), CD4 (d, dim), CD33 (e), and CD56 (f) mimic acute myeloid leukemia or blas- tic plasmacytoid dendritic cell neoplasm (BPDCN). Bright expression of CD38 (g), monotypic expression of cytoplasmic light chain. immunoglobulins (h-i), and lack of CD34 (j), CD117 (k) and HLA-DR (k) helps to exclude both AML and BPDCN and establish the diagnosis of PCM

 

FIGURE 14.13 Plasma cell myeloma with aberrant lack of CD38 expression (a-c; arrow), lambda restriction (b) and positive CD56 (c). Activated T-cells and NK-cells are CD38+ (a-b; red dots)

 

FIGURE 14.14 PCM - FC analysis. This "lymphocytic" variant of PCM (a) shows bright CD45 expression (b), bright CD38 (c) and cytoplasmic kappa expression (c), negative CD19 (d), positive CD20 (e), negative CD22 (f) and positive CD23 (g), CD56 (h), and CD117 (i)

 

FIGURE 14.15 Plasma cell myeloma - flow cytometry. Plasma cells (histology section of the bone marrow, a) are negative for CD45 (b), positive for CD38/CD138 (c) and CD56 (d) and show clonal expression of lambda and IgD (e-f)

 

FIGURE 14.16 Biclonal plasma cell myeloma. Both kappa (orange dots) and lambda (purple dots) populations are CD38+ (a). Monoclonal kappa* plasma (b) have low side scatter and negative to dim CD56 (d), whereas monoclonal lambda plasma cells (c) have high scatter and bright CD56 expression (d)

 

FIGURE 14.17 Biclonal plasma cell myeloma. Flow cytometry shows two population of neoplastic IgG+ plasma cells: larger popula- tion express cytoplasmic kappa (a; black arrow) and smaller population express cytoplasmic lambda (b; red arrow). Both population show bright expression of CD38, but careful evaluation reveals that kappa population has slightly dimmer CD38 when compared to lambda+ cells (compare a and b). The difference in the intensity of CD38 expression is more evident on panel c showing expression of cytoplasmic IgG versus CD38: kappa+ cells (black arrow) and lambda+ cells (red arrow) express IgG (there was no expression of other heavy chain immunoglobulins). Kappa+ cells are positive for CD20 (d). Bone marrow core biopsy (e-h) confirmed flow cytometry find- ings: two plasma cell populations are evident. Kappa cells are occur individually and in small clusters (black arrows), whereas lambda+ cells form large aggregate (red arrow). Lambda population has strong expression of CD56 (g) and is CD20- (h)

 

FIGURE 14.18 PCM with unusual co-expression of kappa and lambda. (a–c) Immunohistochemistry. (d-e) FC analysis. Highly atypical plasma cells (including multinucleated giant plasma cells) are strongly positive for CD138 (a) and positive for kappa (b, d) and lambda (c, e). This unusual co-expression of kappa and lambda was confirmed by electrophoresis analysis of serum and urine

 

FIGURE 14.19 PCM differential diagnosis. (a) Plasmablastic lymphoma (PBL). (b) Metastatic breast carcinoma (bone marrow aspirate smear, histology, and immunostaining for cytokeratin). (c) Acute erythroid leukemia (AEL). (d) Mantle cell lymphoma (MCL). (e) Metastatic adenocarcinoma (prostate). (f) Osteoblasts. (g) MZL with extensive plasmacytic differentiation. (h) Metastatic small cell carcinoma. (i) Solitary plasmacytoma. (j) Lymphoplasmacytic lymphoma (LPL). (k) Castleman's disease (plasma cell type). (1) AML. (m) Large B-cell lymphoma with ALK expression. (n) Primary effusion lymphoma (PEL)

 

FIGURE 14.20 B-ALL with negative CD45 and partial expression of CD56. B-lymphoblasts (blue dots) show negative CD45 (a) and dim CD56 (a), mimicking PCM. Lack of cytoplasmic light chain immunoglobulin expression (b-c), and co-expression of CD19 (d), CD10 (d), cytoplasmic CD22 and CD79a (e), and TdT (f) confirms the diagnosis of B-ALL

 

FIGURE  14.21 IgM+ PCM. Neoplastic plasma cells (orange dots represent FC analysis with surface antibodies and brown dots represent FC with cytoplasmic antibodies) show lack of CD45 (a), monotypic expression of cytoplasmic lambda (b-c), positive cyto- plasmic IgM (d), negative expression of surface light chain immunoglobulins (e), CD19 (f), CD20 (g), HLA-DR (h), CD56 (i), CD117 (j), bright CD38 (k), and aberrant positivity for CD33 (1). Lack of CD45, CD19, CD20, HLA-DR, and surface light chain immunoglobulins excludes B-cell lymphoma

 

FIGURE 14.22 Plasmacytoma of the thyroid. Histologic section shows prominent atypical plasma cell infiltrate (a-b), which by immunohistochemistry are positive for CD45 (c), CD20 (d), MUM1 (e), and CD138 (f), and do not express PAX5 (g). Flow cytometry analysis (h-n) showed plasma cells (arrow) with bright CD38 expression (h), positive CD20 (i) negative CD22 (j) and HLA-DR (j), positive CD38 (k-n), positive CD19 (k), cytoplasmic kappa restriction (1-m), positive IgG (n), surface markers (h-k), and cytoplasmic markers (1-n)

 

FIGURE 14.23 Plasma cell leukemia. (a−b) Blood smear with circulating plasma cells with prominent atypia (immature features) mimicking acute (monoblastic leukemia). (c-i) Flow cytometry revealed the following phenotype of plasma cells (orange dots): CD45- (c), CD34- (d), CD20-(e), CD117- (f), CD38+ (g), CD138+ (h), and CD56- (i). Plasma cells were monoclonal (lambda+; not shown)

 

FIGURE 14.24 Plasma cell leukemia. (a) Blood smear with circulating plasma cells. (b-h) Flow cytometry shows plasma cells with positive CD45 (b) and variable although mostly low side scatter (b; arrows), kappa restriction (c-d), and negative expression of CD19 (d), CD20 (e), CD56 (f), and CD117 (h)

 

FIGURE 14.25 Prominent (reactive) plasmacytosis in blood from a 25-year-old patient with AITL. Plasma cells comprise >50% cells in peripheral blood and display cytologic atypia (a). They are CD45+ (b), CD19+ (c), CD20- (d), CD38+ (e; bright expression), and CD56- (f). Staining with cytoplasmic kappa and lambda confirmed benign (polytypic) nature of plasma cells (g-h)

 

 

■■■【15】Phenotype classification of mature T-cell neoplasms

 

FIGURE 15.1 Algorithmic approach to the diagnosis of T-cell lymphomas. Abbreviations: ATLL, adult T-cell lymphoma/leukemia; AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ANKL, aggressive NK-cell leukemia; EATL, enteropathy-associated T-cell lymphoma; HSTL, hepatosplenic T-cell lymphoma; LGL, large granular lymphocyte; LyP, lymphoma- toid papulosis; MEITL, monomorphic epitheliotropic intestinal T-cell lymphoma; MF, mycosis fungoides; PTCL, peripheral T-cell lymphoma; SS, Sézary's syndrome; TFH, SPTCL, subcutaneous panniculitis-like T-cell lymphoma; T follicular helper; T-PLL, T-cell prolymphocytic leukemia

 

 FIGURE 15.2 Algorithmic approach to lymph node with atypical T-cell infiltrate. Abbreviations: AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell lymphoma/leukemia; cHL, classic Hodgkin lymphoma; FTCL, follicular T-cell lymphoma; LP cells, large cells from NLPHL; MF, mycosis fungoides; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; PLL, prolymphocytic leukemia; PTCL, peripheral T-cell lymphoma; SS, Sézary's syndrome; T-ALL, T-cell acute lymphoblastic leukemia; T-LBL, T-cell acute lymphoblastic lymphoma, TFH, T-follicular helper

 

FIGURE 15.3 Preferential involvement by cutaneous lympho- mas based on body area. Abbreviations: MF, mycosis fungoides; FMF, folliculotropic MF; CD8+ C-ATLC, primary cutaneous acral CD8+ T-cell lymphoma; CD4+ C-PTCLPD, primary cutaneous CD4+ small/medium pleomorphic T-cell lymphoproliferative disor- der; PCFCL, primary cutaneous follicle center lymphoma; PCMZL, primary cutaneous marginal zone lymphoma; SPTCL, subcuta- neous panniculitis-like T-cell lymphoma; SS, Sézary's syndrome; C-DLBCL-LT, cutaneous diffuse large B-cell lymphoma, leg type

 

FIGURE 15.4 ATLL with partial expression of CD30 and leukemic blood involvement (63-year-old female with WBC of 34k). Neoplastic T-cells show the following phenotype: CD2+ (a), surface CD3- (b), CD4+ (c), CD5+ (d), CD7- (e), CD25+ (f), CD26+ (g), and CD30+ (partial; g)

  

 

■■■【16】Mature T-Cell neoplasms

   

 

■■■【16-1】T-PLL

 

 

FIGURE 16.1 T-prolymphocytic leukemia (T-PLL) – peripheral blood films. Small to medium-sized lymphocytes with scanty baso- philic cytoplasm and irregular nuclei with nucleoli predominate

 

FIGURE 16.2 T-prolymphocytic leukemia (T-PLL) - bone marrow. Core biopsy shows prominent lymphocytosis of predominantly small lymphocytes (a) with positive expression of all four T-cell antigens (b-e). The expression of CD2 and CD7 (a, e) is slightly dimmer than the expression of CD3 and CD5 (c, d)

 

FIGURE 16.3 T-prolymphocytic leukemia (T-PLL) – lymph node. (a) Low magnification shows diffuse lymphoid infiltrate spar- ing of the follicle. (b) High magnification shows predominance of small, mature-appearing lymphocytes. Neoplastic cells show CD4 restriction (c and d)

 

FIGURE 16.4 T-PLL - flow cytometry. (a) Peripheral blood film with marked lymphocytosis. (b-k) Flow cytometry analysis of blood shows predominance of lymphocytes (red dots). Note the paucity of granulocytes and monocytes. Lymphocytes display CD4 subset restriction (c) and bright expression of CD2 (d), lack of surface CD3 (e), bright CD5 (f), and positive CD7 (g). Cytoplasmic CD3 is positive (h). Neoplastic lymphocytes do not co-express CD25 (i), TCR alpha/beta, TCR gamma/delta (j) or CD57 (k)

 

 

FIGURE 16.5 T-PLL - flow cytometry. Lymphomatous cells (black arrow) show increased forward scatter (a-e; compare to residual normal T-cells), positive CD2 (a), positive (dim) CD3 (b), positive CD5 (c), negative CD7 (d), and positive CD4 (e)

 

FIGURE 16.6 T-PLL with positive expression of all pan T-cell antigens. (a) Blood film shows atypical lymphoid cells with nucleoli. (b-d) BM shows prominent lymphoid infiltrate (b) expressing CD3 (c), and TCL1 (d). (e-i) Flow cytometry shows lymphoid cells (red dots, arrow) with normal expression of all pan T-cell antigens (e–h) and dual CD4/CD8 positivity (i)

 

FIGURE 16.7 T-PLL with aberrant (partial) expression of CD45 (a) and normal expression of T-cell antigens (CD2, CD3, CD5, and CD7; b-e) [green dots represent T-PLL; red dots represent benign B- and T-cells; gray dots represent granulocytes; arrows indicates CD45-negative cells]

 

FIGURE 16.8 CD45-negative T-PLL flow cytometry. T-PLL cells are negative for CD45 (a), surface CD3 (c), CD8 (f), and TdT (h); they express CD2 (b), CD5 (d), CD7 (e), CD8 (f), and cytoplasmic CD3 (g)

 

FIGURE 16.9 Lymph node involved by T-PLL with aberrant expression of CD117. (a) Histology: diffuse small lymphocytic infiltrate. (b-c) Immunohistochemistry. (d-i) Flow cytometry. Lymphomatous cells show aberrant expression of CD117 (b, immunohistochem- istry; d, flow cytometry) and are CD4-/CD8+ (c, immunohistochemistry; e, flow cytometry). Leukemic cells show normal expression of all pan T-cell antigens (CD2, CD3, CD5, CD7; f-i)

 

FIGURE 16.10 T-PLL flow cytometry. Leukemic cells (red dots) display the following phenotype: CD2- (a), CD3+ (b), CD5+ (c), CD7+ (d), CD25 (e), CD26+ (f), CD81* (g), CD52+ (h), CD200 (i), dual CD4/CD8+ (j), and TCR alpha/beta* (k)

 

FIGURE 16.11 T-PLL with aberrant loss of CD3 and positive HLA-DR - flow cytometry analysis. 74-year-old man with WBC of 64 × 10/L. Leukemic T-cells (orange dots) show the following phenotype: CD2+ (a), CD3- (b, rare benign T-cells are positive red dots), CD4+ (c), CD5+ (d), CD7- (e, only rare leukemic cells are positive), and HLA-DR+ (f)

 

 FIGURE 16.12 T-PLL. FISH analysis confirmed the rearrangement of TCLI gene (a). Flow cytometry shows CD8 restriction (b), dim CD117 on minute subset (c), and positive expression of all T-cell antigens (d-g) with CD7 being slightly dimmer

 

FIGURE 16.13 T-PLL with an abnormal male karyotype positive for translocation t(X;14)(q28;q11.2). Cytogenetic analysis shows an abnormal hypodiploid clone (3 out of 20 cells) characterized by a reciprocal translocation between chromosomes Xq28 and 14q11.2, loss of one copy of chromosomes 8, 10, 13, 16, 17, 20, and 22, addition of material of unknown origin to 14q24, and the presence of 4-6 marker chromosomes. Seventeen normal cells were found during the course of analysis. Translocation t(X;14) is associated with TCR alpha/delta (14q11.2) and MTCP1 (Xq28) gene rearrangement, which leads to overexpression of MTCP1

 

FIGURE 16.14 T-PLL differential diagnosis (blood): (a) chronic lymphocytic leukemia (B-CLL); (b) Splenic B-cell lymphoma/ leukemia with prominent nucleoli (SBLPN); (c) marginal zone lymphoma (MZL); (d) adult T-cell lymphoma/leukemia (ATLL); (e) Sézary's syndrome; (f) peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS); (g) leukemic phase of follicular lym- phoma (FL); (h) mantle cell lymphoma (MCL); (i) leukemic phase of DLBCL; (j) T-LGL leukemia; (k) B-ALL; (1) T-ALL; and (m) acute myeloid leukemia (AML)

 

 

■■■【16-2】ATLL

 

 

FIGURE 16.15 Adult T-cell leukemia/lymphoma (ATLL). Typical medium to large neoplastic cells with prominent nuclear pleo- morphism ("flower cells")

 

FIGURE 16.16 ATLL lymph node. Diffuse infiltrate (a) of small to medium-sized lymphocytes with nuclear irregularities (b). Neoplastic T-cells are CD2 (c), CD3- (d), and CD25+ (e)

 

FIGURE 16.17 ATLL-lymph node with paracortical/perifollicular pattern of involvement by ATLL cells (a-b; H&E section at low and high magnification). Neoplastic cells are positive for CD2 (c), CD3 (d), and CD5 (e), do not express CD7 (f), and express CD4 (g) and CD25 (h)

 

FIGURE 16.18 ATLL - flow cytometry of the bone marrow (a-b, without granulocytes; c-f, with granulocytes). ATLL cells show CD4 subset restriction (a) and CD25 expression (b). CD2 (c), CD3 (d), and CD5 (e) are positive, and CD7 is negative (f)

 

FIGURE 16.19 ATLL - flow cytometry of the bone marrow. Leukemic cells (black arrow) are CD3+ and CD25+ (a). They show high forward scatter (FSC; b-f; black arrows) similar to FSC of granulocytes (black dots). Normal (benign) T-cells (red arrowhead; b-f) show low FSC. The expression of CD2 is moderate (b), comparable to normal T-cells, the expression of CD3 is dim (c), the expression of CD5 is bright (d), and CD7 is negative (e). CD4 is positive (f)

 

 

■■■【16-3】T-LGLL

 

 

FIGURE 16.20 T-LGL leukemia. Peripheral blood films (a-b) show several LGLs with prominent cytoplasmic granules. Compare with monocytes and normal small lymphocytes (a)

 

 FIGURE 16.21 T-LGL leukemia – bone marrow. (a) T-LGL shows interstitial bone marrow involvement. Leukemic cells express CD3 (b), CD8 (c), CD57 (d), and granzyme B (e)

 

FIGURE 16.22 T-LGLL (blood). Leukemic T-cells (magenta dots) display the following phenotype: CD2+ (a), CD3+ (b), CD4 (c), CD5+ (d, dim expression), CD7+ (f, dim expression), CD8+ (g), and CD57+ (h-i). Note dimmer expression of both CD5 and CD7 when compared to normal T-cells (j) [dot plots a-f, open gate, dot plots g-i, lymphocyte gate]

 

FIGURE 16.23 T-LGLL - FC analysis. Neoplastic cells have the following phenotype: CD2+ (a, red dots), CD3+ (b), CD4 ̄ (c), CD5+ (d, dim expression), CD7- (e), CD8+ (f), CD56 (g), and CD57+ (h)

 

FIGURE 16.24 T-LGLL-flow cytometry. Leukemic cells (red arrow) are positive for CD2 (a), CD3 (b), and CD5 (c; dim expression), negative for CD7 (d), positive for CD8 (e), positive for CD11c (f), negative for CD56 (g), and positive for CD57 (h)

 

FIGURE 16.25 T-LGLL - FC analysis. Leukemic cells are positive for CD2 (a) and CD3 (b), show negative to partially dim CD5 (c), dim CD7 expression (d), and dim CD16 expression (e)

 

FIGURE 16.26 T-LGLL with CD4 expression. FC analysis shows CD4+ T-LGL cells (a), co-expression CD3 with CD56 (b) and CD57 (c)

 

FIGURE 16.27 Early T-LGLL with gamma-delta phenotype (peripheral blood). Gamma-delta+ T-cells (green dots) are character- ized by bright CD3 expression (a-b), negative to dim CD5 (a), variable CD7 (b), positive gamma-delta (c), and positive CD56 (d), and CD57 (c, e). PCR testing confirmed the clonality of T-cells (f)

 

 

■■■【16-4】CLPD-NK

 

 

FIGURE 16.28 CLPD-NK-flow cytometry. Atypical lymphocytes (a) are positive for CD56 (b), negative for CD4 and CD8 (c-d). Characteristically, NK-LGL cells are positive for CD2 and CD7 and lack the expression of CD3 and CD5 (e-h)

 

FIGURE 16.29 CLPD-NK - FC analysis (blood). Leukemic NK-cells show dimmer expression of CD45 (a, green dots)) when com- pared to normal lymphocytes (red dots). Leukemic NK-cells show the following phenotype: CD2+ (b), CD3-(c), CD4-(d), CD5-(e), CD7 dimly* (f), CD8 dimly* (g), CD11b dimly+ (h), CD11c (i), CD16+ (j), CD38 (k), CD56 (I), and CD57 variably+ (m)

 

FIGURE 16.30 Chronic NK cell lymphocytosis - bone marrow. NK-cells (arrow) show triple-positive phenotype (CD16+, CD56+, CD57+; a-c), and positive expression of CD2 (d), CD7 (e), CD11b (f), and CD11c (g)

 

FIGURE 16.31 Immature NK-cells with CD117 expression. FC analysis of blood in patient with CMML after therapy. Monocytes (blue dots) have bright expression of CD45 (a) and immature NK-cells (a, green dots) show dimmer CD45 expression. NK-cells are positive for CD2 (b), CD7 (c), CD56 (d, bright expression), and CD117 (e, dim expression). Surface CD3 is negative (f)

 

 

■■■【16-5】PTCL

 

 

 FIGURE 16.32 PTCL, NOS. Diffuse lymph node involvement by medium-sized to large cells with irregular nuclei and nucleoli (a). Tumor cells are positive for all pan-T-cell markers (CD2, CD3, CD5, and CD7, b-e) with CD7 being dimly expressed. They are CD4+ (f) and CD8- (g)

 

FIGURE 16.33 PTCL. (a-b) Interfollicular pattern of involvement (T-zone variant). (c) High magnification shows pleomorphic lymphomatous cells with irregular nuclear outlines. CD20 staining shows preserved B-cell areas (d). Neoplastic cells are positive for CD3 (e) and CD4 (f) Only rare CD8+ small T-cells are present (g)

 

FIGURE 16.34 PTCL - comparison of immunohistochemistry and flow cytometry. (a) Tissue section shows diffuse lymphoid infiltrate. (b) Touch smear shows medium-sized lymphoid cells with inconspicuous nucleoli. Immunohistochemistry (c-g) and flow cytometry (h-1) shows the following phenotype of neoplastic cells: CD2+ (dim; a, h), CD3-(d, i), CD5* (e, j), CD7+ (f, k), and CD56+ (g, 1)

 

FIGURE 16.35 Leukemic blood involvement by PTCL flow cytometry analysis. Tumor cell display aberrant lack of CD45 (a; orange dots), CD4 restriction (b; arrow), positive, although slightly dimmer CD3 (c; orange dots) and loss of CD7 (c; orange dots). Normal T-cells (red dots) are positive for both CD3 and CD7 (c)

 

FIGURE 16.36 PTCL FC analysis of lymph node. Lymphoma cells show (blue dots) shows positive expression of all pan-T-cell antigens (a-d). Only high forward scatter and CD4 restriction (e) allows for identification of abnormal T-cell population

 

FIGURE 16.37 PTCL - flow cytometry. Lymphomatous cells (arrow) show positive expression of all T-cell markers. The expression of CD2 (a) is dim, CD3 is moderate (b), CD5 is bright (c), and CD7 is moderate (d). Forward scatter is increased when compared to benign T-cells (*)

 

FIGURE 16.38 PTCL with clear cell cytoplasm (a) and prominent phenotypic atypia. Immunohistochemistry (b-g) and flow cytometry (b'-g', blue dots) show positive CD2 and CD5 only and lack of CD3, CD7, CD4, and CD8

 

FIGURE 16.39 PTCL (lymph node from 34-year man) with prominent phenotypic atypia. Neoplastic cells (blue dots, arrow) shows increased forward scatter), positive CD2 (a), dim CD3 (b), and negative CD5 and CD7 (c-d). Benign T-cells (red dots) show normal expression of T-cell markers


FIGURE 16.40 PTCL - flow cytometry (lymph node). Lymphomatous cells (arrow) show high forward scatter (a-e), positive CD2 (a), CD3 (b), and CD5 (c), negative CD7 (d), and bright expression of CD38 (e)


 

 FIGURE 16.41 PTCL - flow cytometry. Blood with predominance of granulocytes (gray dots), benign T-cells (red dots) and minor population of atypical T-cells. Despite minimal involvement by PTCL, lymphomatous cells (blue dots) are easily identifiable by FC based on increased FSC (a-e), brighter CD2 expression when compared to normal T-cells (a), negative CD5 (c) and CD7 (d), and CD8 restriction (e)

 

FIGURE 16.42 PTCL with CD8 expression (lymph node). (a) Histology shows highly atypical pleomorphic large cell infiltrate with numerous mitoses. Immunohistochemistry (b-d) and flow cytometry (e-g) shows positive CD3 (b, e), negative CD4 (c, f), and positive CD8 (d, g)

 

FIGURE 16.43 PTCL differential diagnosis (lymph node). (a) Extramedullary myeloid tumor (EMT). Lack of pan-T antigens and positivity for CD34 and pan-myeloid antigens (MPO, muramidase, CD68) excludes PTCL. (b) Anaplastic large cell lymphoma (ALCL). (c) Dermatopathic lymphadenitis. It is characterized by the presence pigmented cells and clusters of atypical $100+ interdigitating reticu- lum cells. (d) DLBCL with unusual interfollicular distribution. (e) Hodgkin lymphoma (HL, classical). Neoplastic cells in HL are negative for CD45, pan-T cell markers and CD4/CD8. (f) Blastic plasmacytoid dendritic cell neoplasm (BPDCN). (g) Kikuchi disease. Prominent histiocytic infiltrate with necrosis and karyorrhectic nuclear debris without neutrophils. (h) Nodal TFH cell lymphoma, angioimmuno- blastic-type (AITL). (i) Mycosis fungoides (MF) involving the lymph node. (j) Adult T-cell lymphoma/leukemia (ATLL). (k) T-cell prolym- phocytic leukemia (T-PLL)

 

 

■■■【16-6】AITL

 

 

FIGURE 16.44 AITL. (a) Low power shows effacement of the architecture by a polymorphous lympho-vascular infiltrate. (b) Higher magnification shows the mixture of small and medium-sized lymphocytes, plasma cells, eosinophils, and blood vessels. (c) Another area shows clusters of atypical lymphocytes with clear cytoplasm. (d) CD4+ T-cells predominate. (e) Only rare CD8+ T-cell are present. (f) T-cells display aberrant expression of CD10. (g) B-cells are sparse and tend to accumulate at the periphery of the lymph node. (h) Scattered B-immunoblasts are expressing EBER. (i) Staining for CD23 highlights the expansion of follicular dendritic cells

 

 FIGURE 16.45 AITL. (a) Low magnification of the lymph node shows preserved architecture with secondary follicles surrounded by rim of clear cells. (b) Immunostaining with CD21 shows rather cohesive follicular dendritic meshwork, which does not expand into interfollicular area. (c) Intermediate magnification shows reactive germinal center surrounded by neoplastic cells with clear cyto- plasm. (d-k) Immunostaining of the area depicted in d, shows residual germinal center positive for CD20 (e), CD10 (f), and BCL6 (g) surrounded by neoplastic T-cells (AITL), which express CD10 (f), BCL6 (g), CD2 (h), CD3 (i), CD5 (j), and CD4 (k; inset: CD8 staining for comparison). (1-r) High magnification focusing on the border of germinal center shows two distinct populations: benign B-cells (l; germinal center, GC) and lymphomatous cells with clear cytoplasm (1; AITL). Timor cells are negative for PAX5 (m) and positive for CD10 (n; note brighter expression compared to B-cells), CD2 (0), CD3 (p), and CD5 (q). CD7 is not expressed (r)

 

FIGURE 16.46 Perifollicular AITL with reactive germinal centers. The histologic section shows lymph node with numerous reac- tive germinal centers (a-c). The reactive B-cell follicles are positive for PAX5 (d). Neoplastic T-cell population is identified by immu- nostaining with PD1 (e-f, low and intermediate magnification), CD3 (g) and CD10 (h). Note dimmer expression of CD3 by neoplastic T-cells (immediately around the follicles) when compared to reactive T-cells in the background (g). The expression of CD10 is stronger among AITL cells than in germinal center cells (h). Scattered EBV-infected cells are noted by EBER (ISH) staining (i)

 

FIGURE 16.47 AITL - paratrabecular BM involvement. (a–e) Histology. Prominent paratrabecular involvement of BM by polymor- phic lymphoid infiltrate of small to medium to large cells mixed with eosinophils. (f-I) Immunohistochemistry. Lymphomatous cells are positive for CD3 (f), PD1 (g), CD5 (h), and CD4 (I)

 

FIGURE 16.48 Blood with marked polytypic plasmacytosis in young patient with history of AITL. (a) Blood smear shows highly atypical plasma cells with occasional immature features. (b-d) Flow cytometry shows plasma cells with low side scatter and positive CD45 (b) and bright CD38 (c-d). Analysis of cytoplasmic light chains shows polytypic pattern with both kappa (c) and lambda (d) expression

 

FIGURE 16.49 AITL - immunohistochemistry. Histology show typical features of AITL (a). Lymphomatous cells express all pan T-cell markers (CD2, CD3, CD5, and CD7; b-e), are CD4+/CD8- (f-g) and show aberrant expression of CD10 (h). Scattered B-cells (CD20+; i), CD30+ cells (j), and EBV-infected cells (k) are noted

 

FIGURE 16.50 AITL with strong CD10 expression by both flow cytometry and IHC. (a) FC analysis shows significant T-cell popula- tion showing CD5 and CD10 co-expression (arrows). (b) Histology. (c-e) Immunohistochemistry. Only rare CD20+ B-cells are present, mostly in the subcapsular area (c). T-cells show aberrant expression of CD10 (d-e)

 

FIGURE 16.51 AITL (perifollicular pattern) - flow cytometry. (a-d) FC analysis shows predominance of small T-cells with nor- mal expression of T-cell antigens. Only minor subset of T-cells with high forward scatter (arrow) show positive CD2 expression (a) and lack of surface CD3 (b), CD5 (c), and CD7 (d). (e) Histology shows atypical clusters of clear cells around lymphoid follicles. (f-j). Immunohistochemistry. Neoplastic T-cells are positive for CD2 (f), CD3 (g), and negative for CD5 (h) and CD7 (I)

 

FIGURE 16.52 AITL-FC analysis (two cases). (a-e). Case #1. AITL cells have increased forward scatter. They are positive for CD2 (a, arrow), negative for CD3 (b), positive for CD5 (c, arrow), negative for CD7 (d), and positive for CD10 (e, arrow; benign germinal center cells are CD5 and CD10*, *). (f−k) Case #2. AITL cells (blue dots) have increased forward scatter. They are positive for CD2 (f, arrow), negative for CD3 (g), positive for CD4 (h, arrow), positive for CD5 (i, arrow), and negative for CD7 (j)

 

FIGURE 16.53 AITL. Neoplastic T-cells (black arrow) show partially increased forward scatter and subtle changes in the expres- sion of pan-T-cell antigens, including minimally dimmer expression of CD3 (b) and CD7 (d). Other changes typical for AITL are easily identifiable, such as CD4 restriction (e) and aberrant expression of CD10 (g). Molecular testing confirmed T-cell clonality (PCR; h)

 

FIGURE 16.54 AITL flow cytometry. Neoplastic T-cells show increased forward scatter (a-g; arrow), positive CD2 (a) and CD5 (c), negative CD3 (b) and CD7 (d), dimly positive CD4 (e), negative CD8 (f), positive CD71 (g), positive CD10 (h), and positive CD56 (i). Molecular testing (PCR) revealed clonal rearrangement of both IGH (j) and TCR (k)

 

 

■■■【16-7】ALCL

 

 

FIGURE 16.55 Anaplastic large cell lymphoma (ALCL) - common variant. (a) Pleomorphic large cell lymphoid infiltrate. Many nuclei are irregular with a horseshoe shape, so-called hallmark cells (arrow). (b) Low power shows focal intrasinusoidal distribution of tumor cells. (c) Intrasinusoidal clusters of tumor cells show strong nuclear and cytoplasmic expression of ALK

 

FIGURE 16.56 Extranodal ALK ALCL. (a) Skin. (b) Soft tissue. (c) Bone. (d) Lung. (e) Pleural effusion with concurrent involvement of the lymph node. (f) Testis

 

FIGURE 16.57 ALCL-two patterns of ALK expression: (a) nuclear, nucleolar, and cytoplasmic staining; (b) cytoplasmic staining

 

FIGURE 16.58 ALCL immunohistochemistry. Neoplastic cells are positive for CD30 (a-b, strong membranous and Golgi-area staining), EMA (c), and UCHL-1 (d)

 

FIGURE 16.59 ALCL flow cytometry. ALCL (cervical lymph node from 12-year-old boy) shows negative expression of T-cell antigens (a-c), except for partial CD7 expression (d). CD30 is strongly positive (e). FISH analysis showed ALK rearrangement (f; break- apart probe)

 

FIGURE 16.60 ALCL-flow cytometry. Lymphomatous cells are positive for CD2 (a), CD4 (e), CD30 (f), and CD71 (f). CD3 (b), CD5 (c), and CD7 (d) are negative

 

 FIGURE 16.61 ALCL-leukemic phase (blood involvement). Flow cytometry of blood shows population of large cells (increased forward scatter; arrows) expressing CD2 (a), CD7 (b), CD8 (c), and CD30 (d), Benign small T-cells (") are positive for CD2, CD7, CD8, and negative for CD30

 

FIGURE 16.62 ALCL with extensive BM involvement. Histology of the core biopsy (a) shows diffuse infiltrate of large atypical lymphoid cells. (b-d) Immunohistochemistry analysis shows lack of CD3 expression (b) and positive CD4 (c), and CD30 (d). (e-j) Flow cytometry shows lymphomatous cells (magenta dots) positive for CD2 (e), negative for CD3 (f), and positive for CD4 (g), CD25 (h), CD30 (i), and partially CD71 (j)

 

FIGURE 16.63 Pleural effusion - ALCL. (a) Cytospin preparation shows numerous malignant cells with large nuclei, dense cyto- plasm with occasional vacuoles. (b) High magnification shows detailed cytologic features. (c) Immunohistochemical staining with ALK (effusion). (d) Flow cytometry shows T-cells co-expressing CD30 (arrow). (e) Histologic section of the lymph node from the same patients shows diffuse large cell infiltrate. (f) Tumor cells show nuclear, nucleolar, and cytoplasmic staining with ALK

 


FIGURE 16.64 ALCL - differential diagnosis. (a) Langerhans cell histiocytosis. Tumor cells have abundant cytoplasm, characteris- tic nuclear features (grooves) and are positive for CD1a and $100. (b) DLBCL, immunoblastic variant. It may resemble monomorphic subtype of ALCL, but is expresses B-cell markers, including CD20 and PAX5. (c) Large B-cell lymphoma with cytoplasmic ALK expres- sion. This unusual variant of DLBCL is negative for CD20 and expresses CD138, IgA and ALK (cytoplasmic). (d) Malignant melanoma. (e) Anaplastic large cell carcinoma. (f) Angiosarcoma. Tumor cells express vascular markers and are negative for ALK. (g) Histiocytic sarcoma. The neoplastic cells lack reactivity with pan-T antigens, B-cell markers and show variable staining with CD68 (and other his- tiocytic markers) and CD15. With the use of immunostaining and FISH/molecular tests, majority of those tumors are now diagnosed as ALCL. (h) Follicular dendritic cell sarcoma. Tumor cells are positive for CD21. (i) Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). CD30 expression is variable, but ALK-1 is negative. (Continued)

 

FIGURE 16.64 (Continued) (j-k) Classic HL. (1) Mediastinal grey zone lymphoma. (m) Anaplastic variant of plasma cell myeloma (PCM). (n) DLBCL with anaplastic features. (o) Primary cutaneous ALCL (C-ALCL)

 

FIGURE 16.65 Breast-implant associated ALCL. (a–c) Section of the capsule surrounding breast implant showing large cell infil- trate (low, intermediate, and high magnification). (d–j) Immunohistochemistry. Anaplastic lymphoma cells show null cell phenotype (CD2-, CD3-, CD5-, and CD7-; d-g). They are positive for CD4 (h), CD30 (i), and CD43 (j)

 

FIGURE 16.66 ANKL. BM with atypical lymphoid infiltrate of large cells (a) and areas of necrosis (b). Tumor cells are positive for CD3 (c), CD56 (d), CD30 (e, focal expression), and EBV (f, EBER-ISH)

 

 

■■■【16-8】ENKTL

 

 

FIGURE 16.67 Extranodal NK/T-cell lymphoma, nasal type - nasopharynx. (a-b) Diffuse lymphoid infiltrate within nasal mucosa (inset: pleomorphic lymphoid cells). Lymphomatous cells are positive for EBER (c), CD56 (d), and T-cell antigens CD2 and CD3 (e-f), and do not express CD5 and CD7 (g-h)

 

FIGURE 16.68 ENKTL composed of medium-sized cells (nasal cavity). Flow cytometry analysis (a-g) shows predominance of reac- tive small T-cells (red dots) and minor population of atypical cells (green dots) with slightly increased forward scatter, positive CD56 (a), negative CD4 (b), negative CD8 (c), positive CD2 (d), positive CD3 (e), negative CD5 (f), and negative CD7 (g). Histologic section shows diffuse infiltrate of medium sized cells (h-i), expressing EBV/EBER (j), CD56 (k), CD2 (I), and granzyme B (m)

 

FIGURE 16.69 ENKTL, right nasal mass from 28-year-old man (FC analysis). (a) Touch smear shows highly atypical large neoplas- tic cells with irregular nuclei, basophilic cytoplasmic and "dirty" background. (b-h) Flow cytometry shows strong CD56 expression (b), positive CD2 (c), negative CD3 (d), CD4 (e) and CD5 (f), positive CD7 (g), and negative CD8

 

FIGURE 16.70 Differential diagnosis of ENKTL. (a) Plasmacytoma. Atypical diffuse plasma cell infiltrate within nasal cavity may suggest NK-cell lymphoma. Expression of light chain immunoglobulins and negative expression of EBER helps in diagnosis of plasma cell neoplasm. (b) Plasmablastic lymphoma. Positive expression of EBER and CD56 is common for both plasmablastic lymphoma and NK-cell lymphoma. Lack of angiocentrism and lack of expression of pan-T antigens (e.g., CD2) and cytotoxic granule associated proteins (granzyme B, TIA1) distinguish these two neoplasms. (c) Poorly differentiated nasopharyngeal carcinoma. Pleomorphic infil- trate with expression of EBV in nasopharyngeal carcinoma may be confused with nasal NK-cell lymphoma. Expression of cytokeratin establishes the correct diagnosis. (d) DLBCL: EBV-associated DLBCL (e.g., in immunocompromised or elderly patients) may be mis- taken for nasal NK-cell lymphoma. (e) AML with CD56 expression. (f) Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) with CD56 expression. (g) Acute monoblastic leukemia. (h) Blastic plasmacytoid dendritic cell neoplasm (BPDCN)

 

 

■■■【16-9】EATL

 

 

 FIGURE 16.71 EATL - small intestine. Histology shows pleomorphic lymphoid infiltrate with scattered large cells resembling Reed-Sternberg cells (a-b). Tumor cells are positive for CD7 (c), CD45 (b), and CD30 (e)

  

FIGURE 16.72 EATL - small intestine and liver. (a) Ulcerated intestinal mucosa with dense, atypical lymphoid infiltrate. (b) Tissue section from liver shows prominent involvement by lymphoma.Flowcytometry (c-d) shows positive CD103 and CD56 expression

  

FIGURE 16.73 EATL-flow cytometry. Tumor cells show the following phenotype: CD2+ (a), surface CD3- (b), CD4-(c), CD5-(d), CD7+ (e), CD8+ (f), CD10- (g), CD25- (h), CD26+ (i), CD30- (j), CD56+ (k), and CD71-(1)

 

FIGURE 16.74 EATL- differential diagnosis. (a) DLBCL. (b) HL (large intestine). (c) ALCL. (d) PTCL

 

 

■■■【16-10】MEITL

 

 

FIGURE 16.75 MEITL - small intestine. Histologic section shows diffuse lymphoid infiltrate of rather monomorphic appearing cells (a-b). They are positive for CD2 (c), CD3 (d), CD7 (f), CD8 (g), and CD56 (h) and show aberrant loss of CD5 (e)

 

FIGURE 16.76 MEITL with ulceration (a). Tumor cells are mostly medium in size and show slight variation in nuclear shape. By immunohistochemistry, this case displays the following phenotype: betaF1* (c), CD2 (d), CD3+ (e), CD4 (f), CD5 (g), CD7* (h), CD8+ (i), TIA1+ (j), perforin (k), granzyme B- (1), and CD56+ (m)

 

FIGURE 16.77 MEITL (a-c) Histology shows monomorphic lymphoid infiltrate of medium-sized cells with epitheliotropism. (d-k) Immunohistochemistry. Tumor cells are positive for CD2 (d, weak staining), CD3 (e; strong staining), CD7 (g, CD8 (1), CD56 (j), and TIA1(k). They are negative for CD5 (f) and CD4 (h)

 

FIGURE 16.78 MEITL with involvement of large intestine (a-m) and small intestine (n-p). Neoplastic cells display prominent tropism to epithelium (a-b; o). Cytokeratin staining (c) shows prominent intraepithelial lymphocytosis. The tumor cells are small to medium in size with pale cytoplasm (a-b; o). They are positive for CD2 (d and p, immunohistochemistry; j, flow cytometry), CD3 (dim; e and k), CD7 (bright; g and m), TIA1 (h) and CD56 (i); they are negative for CD5 (f and I)

 

FIGURE 16.79 ENKTL - small intestine. Large polypoid mass (a) shows highly atypical and diffuse lymphoid infiltrate (b-c). Tumor cells are positive for CD2 (d), CD3 (e; weak expression) and EBV (f; EBER-ISH); CD5 and CD7 were negative (not shown)

 

 

■■■【16-11】MF

 

 

FIGURE 16.80 MF (early stage) showing prominent epidermotropism (a-c) with characteristic accumulation of lymphomatous cells within epidermis (c). Tumor cells are positive CD2 (d) and CD4 (e) and negative CD8 (f)

 

FIGURE 16.81 MF - Pautrier microabscesses (a-b) Histology sections show prominent lymphoid aggregated within epidermis (a, low magnification; b, high magnification of different area). (c-f) Immunostaining with cytokeratin (c-d) and CD3 (e-f) helps to visual- ize lymphoid aggregates in the epidermis

 

FIGURE 16.82 MF - lymph node involvement. (a) Low magnification shows prominent paracortical/interfollicular infiltrate. (b) High magnification shows medium-sized to large lymphoid cells with nuclear pleomorphism and nucleoli. Neoplastic cells show lack of CD2 (c), positive CD3 (d) and CD5 (e), and lack of CD7 expression (f)

 

FIGURE 16.83 Large cell transformation of MF. Dense lymphoid infiltrate involving dermis (a), composed of pleomorphic cells with numerous large anaplastic lymphocytes (b-c). Neoplastic cells are positive for CD2 (d), CD5 (e), and CD30 (f)

 

FIGURE 16.84 Pagetoid reticulosis variant of MF. (a-b) Histology shows prominent intraepidermal lymphoid infiltrate (a, low mag- nification; b, high magnification). Tumor cells show the following phenotype: CD30+ (c), CD2+ (d), CD3+ (e), CD5+ (f, CD7- (g), CD4+ (h), and CD8-(i)

 

FIGURE 16.85 Mycosis fungoides (MF) involving the lymph node. Lymphomatous cells (arrow) display increased forward scatter (FSC; a-e), positive CD2 (a), positive (dim) CD3 (b), positive CD5 (c), negative CD7 (d), and positive CD4 (e). Residual (benign) T-cells (*) show normal expression of T-cell antigens and low FSC

 

 

■■■【16-12】SS

 

 

FIGURE 16.86 Sézary's syndrome. Blood smear with atypical lymphocytes (a-b). Flow cytometry (c-d) shows lymphocyte with predominance of CD4+ cells

 

FIGURE 16.87 Sézary's syndrome (blood) - flow cytometry. Neoplastic T-cells (blue dots) are positive for CD2 (a), CD3 (b), CD5 (c), CD7 (d), and CD4 (e) and are negative for CD8 (f). The expression of CD2 is dim (a; compare with benign T-cells - red dots) and expression of CD5 is brighter than in normal T-cells (c)

 

FIGURE 16.88 SS (history of MF, blood with 15x10/L. Sézary's cells). Flow cytometry analysis shows the following phenotype of Sézary's cells (arrows): CD2+ (a), CD3+ (b, bright expression), CD5+ (c, bright expression), CD4+ (d), and CD7-(e-f)

 

 

■■■【16-13】HSTL

 

 

FIGURE 16.89 Spleen - HSTL. (a-f) Case #1: (a) low magnification shows clusters of atypical lymphoid cells; (b) high magnification shows intrasinusoidal distribution of neoplastic cells; (c-f) immunohistochemistry shows positive CD3 (c), negative CD8 (d), negative CD5 (e), and positive TIA-1 (f). (g-j) Case #2: (g) atypical lymphoid infiltrate; (h-j) immunohistochemistry shows positive CD8 (h), negative CD4 (i), and positive CD3 (j). (k-r) Case #3. HSTL in 17 y/o male patient. Low magnification (k) shows expanded red pulp with diffuse atypical lymphoid infiltrate. Tumor cells are larger when compared to residual germinal center cells (1, arrow points to benign lymphocytes). High magnification (m) shows large atypical cells with irregular nuclei, nucleoli and pale cytoplasm. Mitotic figures are easily identifiable. Lymphomatous cells are positive for CD2 (n) and CD3 (o), negative for CD5 (p), and positive for CD7 (r)

 

FIGURE 16.90 Liver - HSTL. Atypical, mostly intrasinusoidal infiltrate of medium-sized cells

 

FIGURE 16.91 Bone marrow with typical intrasinusoidal involvement by HSTL. (a-b) Histology (BM core biopsy). (c-d) Immunohistochemistry. Tumor cells express CD3 (c) and are negative for CD5 (d)

 

FIGURE 16.92 HSTL-flow cytometry analysis. Lymphomatous T-cells show the following phenotype: CD2- (a), CD3+ (b), CD5-(c), CD7+ (d), CD4-(e), CD8+ (f), CD56+ (g), CD71+ (h, partial), TCRys+ (i-j), and CD103- (k)

  

 

■■■【17】B-Cell Acute Lymphoblastic Leukemia

 

 

 FIGURE 17.1 B-lymphoblastic leukemia - cytology. BM aspirate smears from four B-ALL cases (a-d) show lymphoblasts with increased nuclear-cytoplasmic ratio, scanty cytoplasm, nucleoli, and occasional cytoplasmic vacuoles (Wright-Giemsa, x1000)

 

FIGURE 17.2 B-ALL - bone marrow (histology and immunohistochemistry). (a) Bone marrow core biopsy shows complete replace- ment of marrow elements by lymphoblasts. (b) Higher magnification shows rather monomorphic blasts with scanty cytoplasm, evenly distributed chromatin and nucleoli. (c-j) Immunohistochemistry: blasts are negative for CD20 (c), and positive for CD79a (d), PAX-5 (e), CD43 (f), CD10 (g), Fli-1 (h), and BCL2 (i). MPO is not expressed (j)

 

FIGURE 17.3 Precursor B-cell lymphoblastic lymphoma - lymph node. (a) Touch smear shows blasts with round regular nuclei and few inconspicuous nucleoli. (b-c) Histology section of the lymph node shows sheets of lymphoblasts (low and high magnification). Residual small lymphocytes are also noted (left side). (d–i) Immunohistochemistry. Blasts are negative for CD20 (d) and are positive for CD22 (e), CD10 (f), TdT (g), CD34 (h), and Fli-1 (i)

 

FIGURE 17.4 B-ALL - flow cytometry (bone marrow from 5-year-old patient). Blasts (green dots) have the following phenotype: CD45-/dim* (a), CD10+ (bright; b), CD13+ (dim/partial; c), CD19+ (d), CD22+ (e), CD33- (f), CD34+ (g), and CD38+ (h). Note typical for B-ALL bright expression of CD10 (i) and negative CD20 (j). Majority of B-ALL are positive for CD81 (k). The expression of CD200 var- ies, ranging from positive (1) to negative

 

FIGURE 17.5 Identification of B-lymphoblasts by flow cytometry. B-lymphoblasts (magenta dots) are negative for CD45 (a), positive for CD34 (a-b), negative for CD117 (b), negative for surface light chain immunoglobulins (c), positive for CD19 (d), and negative for CD20 (d)

 

FIGURE 17.6 B-ALL - flow cytometry. Blasts (green dots) show low side scatter (a), negative CD45 expression (a) and increased forward scatter (b-h). They are positive for CD34 (b), CD19 (c), HLA-DR (d), CD10 (e), and CD33 (f), whereas CD71 (g) and CD123 (h) are negative. Note very bright CD10 expression (e; much brighter when compared to neutrophils represented by gray dots)

 

FIGURE 17.7 B-ALL - flow cytometry. Blasts (green dots) show bright expression of CD34 (a), positive CD19 (b), negative CD20 (c), negative surface light chain immunoglobulins (d), negative CD71 (e), positive CD123 (f), positive TdT (g), and positive CD13 (h)

 

FIGURE 17.8 B-ALL with aberrant expression of pan-myeloid antigens - flow cytometry. B-lymphoblasts (blue dots) are positive for CD45 (a), CD34 (b), CD10 (c; bright expression), CD19 (d; bright expression), CD22 (e), and partially for both CD13 (f; arrow) and CD33 (g; arrow). Negative cytoplasmic MPO excludes mixed phenotype acute leukemia (MPAL). FISH (g; inset) shows several copies of BCR-ABL fusion; (ASSI-aqua, ABL1-orange, BCR-green)

 

FIGURE 17.9 B-ALL with aberrant expression of CD15. B-lymphoblasts (green dots) show positive CD19 (a, b), partially dim CD20 (a), bright CD10 (b), negative surface light chain immunoglobulins (c), moderate CD38 (d), dim CD81 (e), and dim (partial) CD200 (f). Among myeloid antigens, CD13 is negative (g), CD15 is dimly positive (h), and CD33 is negative (i)

 

 FIGURE 17.10 B-ALL with mostly negative CD10 (a), aberrant expression of CD13 (b), moderate CD19 (c), aberrant expression of CD33 (d), positive CD34 (e), negative to partially dim CD71 (f), dim CD81 (g), positive CD123 (h), and moderate CD200 (i)

 

FIGURE 17.11 B-ALL flow cytometry. B-lymphoblasts (blue dots) are positive for CD45 (a), CD34 (b; variable expression), HLA-DR (d), CD20 (e; variable and partial expression), and CD22 (f). CD117 (c) and CD33 (g) are not expressed

 

FIGURE 17.12 Early (emerging) B-ALL - blood (WBC = 18k/μL). Blasts (green dots) show moderate CD45 expression (a), mostly negative CD34 (b; only minor population of blasts is positive), bright CD38 (c), moderate CD19 (c), positive CD10 (d), and variable expression of CD20 ranging from negative to moderate (d)

 

FIGURE 17.13 Flow cytometry features of hematogones. On the CD45 versus side scatter (SCC; a) hematogones show two distinct populations, one with dim CD45 and one with moderate CD45 expression (arrows). Subset of hematogones is positive for CD34 (b). Forward scatter (FSC) is characteristically variable (b-d; arrows) ranging from low to high, with majority of hematogones display low FSC. Hematogones are positive for CD10 (c, f-g) and CD38 (d-e), with CD10 being dimmer than in B-ALL and CD38 being brighter than in B-ALL. The expression of CD20 (f) is variable, ranging from negative (majority of hematogones) to dimly positive (minor subset)

 

FIGURE 17.14 Comparison of the expression of CD10 and CD38 versus CD19 in hematogones (a, b) and B-ALL (a, b). Hematogones (a-b; blue dots) and B-ALL (a'-b'; green dots) are CD19+, CD10+, and CD38+, but deferrer in the intensity of staining: CD10 is much brighter in B-ALL and CD38 is dimmer in B-ALL, when compared to hematogones

 

FIGURE 17.15 Comparison of FC pattern of hematogones (blue dots, left column) and B-ALL (green dots, right column)

 

FIGURE 17.16 B-ALL-measurable (minimal) residual disease. B-lymphoblasts (orange dots, orange arrows) are negative for CD45 (a) and positive for CD34 (b), CD19 (c), and CD33 (d). Rare hematogones are also noted; they show bright expression of CD38 (e; blue dots, blue arrow)

  

FIGURE 17.17 B-ALL - measurable (minimal) residual disease. B-lymphoblasts (green dots) show dim to moderate CD45 expres- sion (a), positive CD34 (b), partial CD33 expression (b), bright CD10 expression (c), positive CD19 (c-d), and negative to partially dim CD38 expression (d) [flow data represent higher number of events collected when compared to standard flow cytometry analysis]

 

FIGURE 17.18 B-ALL differential diagnosis. (a) Follicular lymphoma (leukemic phase in the peripheral blood). (b) DLBCL involving blood. (c) T-LGL leukemia (gamma/delta). (d) DLBCL involving BM. (e) Burkitt lymphoma (surface immunoglobulin nega- tive). (f) High grade lymphoma with MYC and BCL2 rearrangement (HGBL-R; double hit lymphoma, BM aspirate and clot section). (g) Plasmablastic lymphoma (chest wall). (Continued)

 

FIGURE 17.18 (Continued) B-ALL- differential diagnosis. (h) Plasma cell myeloma (PCM). (i) T-ALL. (j) AML. (k) Acute erythroid leukemia. (I) B-PLL. (m) T-PLL. (n) Metastatic small cell carcinoma. (o) Metastatic neuroblastoma

 

 FIGURE 17.19 Follicular lymphoma without expression of surface light chain immunoglobulins. Lymphomatous cells (arrow) are positive for CD20 (a-b), CD19 (c-d), CD10 (c), and CD38 (d). Surface light chain immunoglobulins are negative (a-b; residual benign B-cells marked with (*) show normal expression of kappa and lambda). Note slightly brighter expression of CD20 by lymphomatous cells when compared to polytypic B-cells. In contrast to typical B-ALL, follicular lymphomas show dimmer expression of CD10 and CD38 and strong (homogeneous) expression of CD20

 

FIGURE 17.20 High grade B-cell lymphoma (CD10+) with rearrangement of BCL2 and MYC and complex karyotype. B-cells (green dots) show bright CD45 (a), lack of surface light chain immunoglobulins (b-c), negative CD34 (c), bright CD38 (d), and positive CD10 (e). FISH studies show BCL2 (g) and MYC (h) rearrangement. Metaphase cytogenetics shows complex karyotype (i): 50,XX,+X,+X,add(3) (q12),der(4)t(1;4)(q12;q31.1), t(6;17)(p10;q10),+7, +8, t(8;22)(q24.1;q11.2), add(9)(p22), add(9)(q22), t(14;18)(q32;q21.3)[5]/50,sl,-add(3) (q12), +der(3)ins(3;?)(q12;q12),-der(4)t(1;4)(q12;q31.1),+4[3]/46,XX[12]

 

FIGURE 17.21 High-grade TdT blastic B-cell lymphoma/leukemia with MYC and BCL2 rearrangement. Neoplastic B-cells (green dots) are positive for CD19 (a) and CD10 (b), negative for CD34 (c), dimly positive for TdT (d) and show moderate expression of CD45 and slightly increased side scatter placing them in "blastic" region" on CD45 versus side scatter display (e). CD20 is partially posi- tive (dim) without expression of surface light chain immunoglobulins (f-g). Metaphase cytogenetics (h) shows an abnormal female karyotype positive for translocation t(2;8)(p11;q24) and translocation t(14;18)(q32;q21). The translocation t(14;18) identified in this specimen is associated with BCL2-IGH gene rearrangement and is a characteristic finding in follicular lymphoma and diffuse large B-cell lymphoma of follicular center cell origin. Translocation between 8q24.2 (where the MYC gene is located) and 2p11.2 (where the Immunoglobulin Kappa gene is located) has been reported mostly in Burkitt lymphoma, diffuse large B-cell lymphoma, and also in other B-cell lymphomas

  

 

■■■【18】 T-Cell Acute Lymphoblastic Leukemia

 

  

 

 

 FIGURE 18.1 T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) - cytology (a-b, two cases). Bone marrow aspirate with numer- ous blasts with delicate chromatin, round eccentric nuclei with nucleoli, and occasional "hand-mirror" appearance

 

FIGURE 18.2 Precursor T-cell acute lymphoblastic leukemia (T-ALL) – bone marrow. (a) Histology shows replacement of the bone marrow by blasts. (b-g) Immunohistochemistry shows expression of CD3 (b), CD4 (c), CD8 (d), CD10 (e), CD43 (f), and TdT (g)

 

FIGURE 18.3 T-LBL - lymph node. (a) Histologic section shows monotonous lymphoid infiltrate with blastic appearance. (b-k) Immunohistochemistry. T-lymphoblasts have the following phenotype: CD2+ (b), CD3 (c), CD5+ (d), CD7+ (e), CD4- (f), CD8- (g), CD10+ (h), CD43+ (i), CD1a+ (j), and BCL6+ (k)

 

FIGURE 18.4 T-LBL with T-zone pattern (paracortical, interfollicular lymph node involvement; a-b). Blasts are positive for TdT (c) and CD7 (e)

 

FIGURE 18.5 Identification of T-lymphoblasts by flow cytometry: T-lymphoblasts can be identified by moderate CD45 expression (a), positive TdT (b), lack of surface CD3 expression (c), positive CD34 (d), positive CD10 (d-e), positive CD1a (e), and by either dual CD4/CD8 negativity (f) or dual CD4/CD8 positivity (g)

 

FIGURE 18.6 T-ALL with positive expression of all pan-T-cell antigens. Blasts (green dots) show dim expression of TdT (a), positive CD3 (b), positive surface CD3 (c), positive CD5 (d), and dimly positive CD7 (e)

 

 FIGURE 18.7 T-lymphoblastic lymphoma (lymph node) - flow cytometry (two cases). Case #1 (a–e): blasts show increased forward scatter and the following phenotype: CD2+, CD3-, CD5+, CD7+, and CD34+. Case #2: (f-j): blasts show loss of all T-cell antigens (f-h) except for CD7 (i). CD34 is positive (j)

 

FIGURE 18.8 Partial blood involvement by T-ALL. Blasts (green dots, arrow) show moderate expression of CD45 (a), low side scat- ter (a), positive CD34 (b), negative CD4 and CD8 (c), low forward scatter (d-g), partial expression of CD2 (d), negative surface CD3 (e), negative CD5 (f) and bright expression of CD7 (g). Normal (benign) T-cells (red dots) show bright CD45 (a), either CD4 or CD8 expres- sion (c), and moderate expression of T-cell markers (d-g)

 

FIGURE 18.9 T-ALL/LBL - lymph node. Flow cytometry analysis shows T-lymphoblasts (green dots) with partial expression of CD4 and CD8, negative CD3 and lack of CD7 on majority of blasts. The forward scatter is increased and variable. The lymphoblasts have the following phenotype: CD45+ (a; moderate expression), CD4/CD8+ (b; partial positive expression), CD34+ (c), CD38+ (d; bright expression), CD2+ (e), surface CD3- (f), CD5+ (g), CD7- (h; minor subset dimly+), and CD10 (i; dim expression)

 

FIGURE 18.10 Precursor T-cell lymphoblastic lymphoma of the mediastinum from 4-year-old boy. Blasts (magenta dots) show dim to moderate CD45 (a), positive TdT (b) and dual CD4/CD8 negativity (c)

 

FIGURE 18.11 Precursor T-cell lymphoblastic lymphoma of the mediastinum - flow cytometry. Blasts show "cortical" T-cell phe- notype with positive expression of all T-cell markers (including surface CD3): dual CD4/CD8+ (a), CD1a+ (b), CD10+ (c), CD34 ̄ (d), CD2+ (e), CD3+ (f), CD5+ (g), and CD7+ (h)

 

FIGURE 18.12 T-ALL with CD4 expression. Blasts (blue dots) are positive for CD45 (a), TdT (b), CD4 (c), CD7 (d), CD5 (e), and CD10 (e). They are negative for surface CD3 (d), CD34 (f), and HLA-DR (f)

 

FIGURE 18.13 T-ALL/LBL - bone marrow. Flow cytometry analysis shows predominance of T-lymphoblasts (blue dots) with the following phenotype: CD45+ (a), CD34+ (b; partial expression), CD10+ (c; partial expression), CD38+ (d; strong expression), CD2+ (e; partial expression), surface CD3- (f), CD5+ (g), CD7+ (h; bright expression), CD4- (i), CD8- (j), CD33+ (k), and cytoplasmic CD3+ (1)

 

 FIGURE 18.14 T-ALL/LBL with positive HLA-DR

 

FIGURE 18.15 Precursor T-lymphoblastic lymphoma with TCRyō phenotype. Lymphoblasts are positive for CD2 (a), TdT (a), and TCRyô (b)

 

FIGURE 18.16 Comparison of flow cytometry patterns of thy- mocytes (immature T-cells from thymoma; left column) with T-lymphoblasts (middle and right columns). Thymocytes are dual CD4/CD8 positive (a) and show positive expression of all pan-T- cell markers (b-e). Based on forward scatter properties (b-f), two distinct populations are easily identifiable: more mature cells with low forward scatter and less mature cells with high forward scatter (arrow). More mature cells with low forward scatter show variable (mostly positive) expression of surface CD3 while less mature cells with high forward scatter are mostly surface CD3- (c, arrow) and show dim expression of CD10 (f, arrow). T-lymphoblasts are either dual CD4/CD8+ (a') or dual CD4/CD8- (a") and are homogeneous on forward scatter analysis (b'/b" to f'/f"). Most T-ALL cases are surface CD3-, but minor subset may be surface CD3+ (c'). The expression of pan T-cell markers is often aberrant (either dim or negative) and sub- set of cases may be CD10+ (f"). A case of T-lymphoblastic lymphoma (a"-f") from lymph node shows many residual (benign) T-cells with normal expression of CD4, CD8 and pan-T-cell antigens

 

FIGURE 18.17 ETP-ALL. (a-h) Flow cytometry shows blasts (blue dots) with positive CD34 (a), negative CD1a (b), positive CD5 (c; <75% ), positive CD7 (d), positive cytoplasmic CD3 (e), partially positive CD13 (f), positive CD33 (g), and negative MPO (h). (i) Karyotype is normal by metaphase cytogenetics. (j) PCR testing confirmed T-cell clonality

 

FIGURE 18.18 ETP-ALL (flow cytometry from BM). (a) Aspirate smear shows lymphoblasts with hand-mirror appearance. Blasts display typical phenotype for ETP-ALL: CD11b+, CD13+, HLA-DR+, CDla, CD34+, CD117+, CD2+, surface CD3, CD5, CD7+, and cytoplasmic CD3+

 

FIGURE 18.19 T-ALL/LBL with expression of CD56 and CD117. Blasts (green dots) are positive for CD7 (a), CD10 (b), CD34 (c), CD56 (d), and partially CD117 (e)