Unknown Case 6. Ann T. Moriarty, MD

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1 Unknown Case 6 Ann T. Moriarty, MD

2 Unknown Case 6 61 year old male with an enlarged cervical lymph node. He has a history of lung carcinoma, renal cell carcinoma and lymphoma.

3 Case 6 Image 1: Fine needle biopsy of cervical lymph node. Romanowsky stained smear, 50x.

4 Case 6 Image 2: Fine needle biopsy of cervical lymph node. Papanicolaou stain 50x.

5 Case 6 What is your morphologic differential diagnosis?

6 Image 3: Compare the cell nuclei to the histiocyte nucleus (Thin Arrow). Most of the nuclei are larger than the histiocyte nuclei. The thick arrow demonstrates cytoplasmic fragments ( lymphoglandular bodies ).

7 Image 4: The arrow points to a histiocyte nucleus. The cells look smaller in ethanol fixed than air-dried preparations. The cells are also more monotonous appearing and there are only rare small lymphocytes present..

8 Morphologic Differential Neoplasms composed of single cells Large Cell Lymphoma Carcinoma Melanoma Small Round Blue Cell Tumor

9 Case 6 What additional information would you like?

10 Image 5: LCA (CD45Ra) immunostain positive in large cells (arrows)

11 Flow Cytometry Histograms of Case 6 A B Image 6: The cells have large side scatter and strong CD45 positivity indicating they are large lymphocytes(a). Histogram B shows the forward scatter versus side scatter characteristics. High forward scatter indicates big cells.

12 A B C D Image 7: Compare the scatter and CD45 characteristics of case 6 in A and B to the CD45 and side scatter characteristics of a small lymphocytic lymphoma in C and D. In C the cells have intense CD45 fluorescence and small side scatter and are not as spread out as those in A. This is accentuated in the forward scatter and side scatter plots, where the small lymphocytes are clustered to the left indicating small size (forward scatter) and little cytoplasmic complexity (side scatter).

13 Flow Cytometry Histograms of Case 6 Image 8: Arrow indicates that the entire population identified in the circled gate is exclusively kappa positive. This is a monoclonal B cell population.

14 Diagnosis: Large cell B cell lymphoma

15 Differential diagnosis Poorly differentiated Carcinoma Small cell undifferentiated carcinoma and other poorly differentiated carcinomas may present as single cells and in patients without a previously known primary. Most non-small cell carcinomas will have more abundant cytoplasm and will be much larger than the cells of large cell lymphoma. While small cell undifferentiated carcinomas may present as single cells, and may even shed cytoplasmic fragments in the background, the fragments are fewer and their nuclear chromatin pattern is coarsely granular compared to the vesicular chromatin and numerous lymphoglandular bodies of large cell lymphoma.

16 Poorly Differentiated Carcinoma Image 9: Small cell undifferentiated carcinoma will frequently fall apart into single cells. However, the nuclei of the cells are usually larger than large cell lymphoma, and the nuclei contain uniformly coarsely granular chromatin without the vesicular pattern and nuclei of large cell lymphomas. Romanowsky stain and Pap stain x 100.

17 Poorly Differentiated Carcinoma A B Image 10: Synaptophysin (A) and Epithelial markers (B) will be positive in most small cell undifferentiated carcinoma, while LCA (Image 6 ) will be negative in carcinomas. Synaptopysin and AE1/AE3 keratin stain x50.

18 Differential diagnosis Melanoma Melanoma can mimic any neoplasm, and should be part of the differential diagnosis of any neoplasm with a single cell pattern. The epithelioid variant of melanoma usually has cells 2 to 3 times the size of large cell lymphoma, has prominent nucleoli and are often binucleate. Helpful immunologic stains include HMB45 as well as melan A.

19 Melanoma Image 11: Romanowsky Stained smear, 50x. Notice how large the cells are and their abundant cytoplasm. The nuclei are several times larger than the histiocyte nuclei. Note the blue green melanin pigment in the histiocytes (arrow.)

20 Differential diagnosis Small round blue cell tumors Small, round, blue cell tumors(srbct) are mostly seen in children. They include, neuroblastoma, Ewing s/pnet tumor, rhabdomyosarcoma and desmoplastic small round cell tumor (DSRCT). Most are associated with characteristic clinical presentations and are most often characterized by distinctive chromosome abnormalities. The clinical presentation of case 6 would be unlikely for a SRBCT. In this 61 year old, Ewing s/pnet tumor and rhabdomyosarcoma may be a consideration. The pattern of expected immunologic reactivity may be seen on the next table. Although lymphoblastic lymphomas may be CD99 positive, they would not demonstrate the flow cytometric pattern in this case.

21 Small Round Blue Cell Tumors Tumor Site Age (yrs) Chromosome Desmin MSA CD99 MyoD/ Myogenin Epithelial Rhabdomyosarcoma (Alveolar) extremities Up to 25 9=mean 2 and 13 PAX3(FKHR) PAX7(FKHR) + + Rare weak + - (pos)* Rhabdomyosarcoma (Embryonal) Head/neck 7=mean Loss of 11,changes in 8,20, (pos)* Ewing's/PNET Lower limbs thorax Up to 40 20=mean q12 chrom 22 (EWS) rare scattered Desmoplastic Small Round Cell Tumor (DSRCT) abdomen Up to 48 22=mean t(11;22) (p13;q12) Neuroblastoma ** Adrenal Under 4 No consistent change * Epithelial markers are positive in less than 10% of cases ** Neuroblastoma associated with NSE positivity and serologic increase in catecholamines

22 Rhabdomyosarcoma Image 12: Romanowsky Stained smear, 50x. This is a biopsy from a neck mass in a child. The cells are single and demonstrate rare cytoplasmic fragments (arrow).

23 Rhabdomyosarcoma A B Image 13: (A) is an immunostain for Muscle Specific Actin showing positive cells (arrow) with negative lymphocytes below. (B) shows an LCA stain, reacting with the lymphocytes(arrow) but not the cells of the SRBCT.

24 Large Cell Lymphoma Large cell lymphoma is a morphologic descriptor, not a diagnostic entity. As cytologists, we are usually able to identify that the cells are abnormal, and larger than small lymphocytes. Further classification usually requires ancillary immunological and (sometimes) chromosomal studies. The WHO classification separates lymphocytic disorders into two large subgroups, 1)B cell neoplasms and 2) T cell/natural Killer (NK) cell neoplasms. Case 6 is a B cell neoplasm based upon the immunologic findings.

25 B Cell Lymphoma with Large Cell Size Of the B cell lymphomas, several subtypes have large cells, including: Follicular Lymphoma, Grade 3 Diffuse Large B cell Lymphoma Mediastinal large B cell lymphoma Intravascular Large B cell lymphoma Primary effusion lymphoma

26 B Cell Lymphoma with larger Cell Size Of the B cell lymphomas, several variants have larger cells than small lymphocytes, but are not as large as Large Cell Lymphomas. These are Precursor B lymphoblastic lymphoma Burkitt lymphoma

27 Follicular Lymphoma, Grade 3 Follicular lymphomas are identified by the presence of follicle formation on histologic sections. They are graded based upon the proportion of large cells ( centroblasts ) within neoplastic follicles. The number of large cells with prominent nucleoli are counted within the follicles. Ten follicles are counted. The number of centroblasts is expressed per 40x high power microscope field. Grade 1 follicular lymphoma has 0-5 centroblasts/hpf. Grade 2 follicular lymphoma has 6-15 centroblasts/hpf. Grade 3 has >15 centroblasts/hpf. Presently there is no analogous system for grading proportions of centroblasts in cytologic material.

28 Follicular Lymphoma, Grade 3 Image 14 : Hematoxylin and eosin stained smear, 40x. Grading follicular lymphoma requires counting the number of centroblasts per 40 x field over 10 fields. Centroblast is at arrow. Grade 3 demonstrates > 15 centroblasts/hpf.

29 Follicular Lymphoma, Grade 3 Cytologically, we cannot reliably identify follicular structures, but expression of CD10 on the cells suggests that the neoplasm may have a follicular origin. A B C Image 15: There is weak fluorescent intensity of CD20 characterized as a shift of fluorescence that overlaps the baseline in A (arrow). Lambda chain expression is also of weak or low fluorescent intensity at arrow in B. CD10 in C is usually seen in follicular neoplasms.

30 Mediastinal Large B cell Lymphoma Mediastinal (thymic) large B cell lymphoma is also known as Mediastinal diffuse large cell lymphoma with sclerosis. Most patients are years old and there is a female predominance. Most mediastinal (thymic) B cell lymphomas are limited to the mediastinum upon presentation and may present with superior vena cava syndrome. The cytology and immunophenotyping studies demonstrate large lymphocytes with B cell markers. No CD10 is expressed. The findings of the mediastinal mass with sclerosis and compartmentalized growth pattern distinguishes this type of large cell lymphoma histologically. Cytologically, there are no distinguishing features. With extensive enveloping sclerosis, an inadequate specimen may be obtained with fine needle biopsy.

31 Mediastinal(thymic) Large B cell Lymphoma A B Image 16: Mediastinal large B cell lymphoma is composed of large cells with compartmentalizing fibrosis [A]. The flow cytometry histogram in [B] shows CD20 positive B-cells and no evidence of follicular origin indicated by lack of CD10 expression in C. C

32 Primary Effusion Lymphoma Body cavity based lymphomas are usually seen in immunosuppressed individuals and are composed of large cells, often with an immunoblastic or plasma cell appearance. The patients present with effusions and no other adenopathy. Image 17: There is a monotonous population of large cells in the fluid of this HIV positive male. The cells are large (compare them to the polymorphonuclear leukocytes at the arrow.) The cells have eccentric cytoplasm and an immunoblastic appearance. Pap stain,100x

33 Primary Effusion Lymphoma Primary effusion lymphomas are often CD45 positive and demonstrate wide side scatter, but frequently do not express pan B markers or surface light chains. Often, plasma cell markers (CD38) are seen on the surface. This lymphoma is associated with Herpes virus 8/Kaposi sarcoma herpes virus. Immunocytochemistry will demonstrate viral reactivity in the nuclei of the lymphoma cells. A B Image 18: Note the wide side scatter in [A] at arrow. The population is negative for any light chain in [B].

34 B Cell Lymphoma with larger Cell Size Precursor B lymphoblastic lymphoma is a malignant lymphoma which is identical in cell type to acute lymphoblastic leukemia. This lymphoma is usually a disease of children, although there is a second peak at the age of 70. This is an aggressive lymphoma with immature phenotype, no surface immunoglobulin and dim CD45 expression. They can express the Ewing's/PNET marker CD99. The cells are usually intermediate in size between small lymphocytic lymphomas and large cell lymphomas. Most lymphoblastic lymphomas are precursor T cell lymphoblastic lymphomas; 20% of those lymphomas with lymphoblastic features are precursor B lymphoblastic lymphoma. T and B cell lymphoblastic lymphoma are indistinguishable morphologically.

35 Lymphoblastic Lymphoma Image 19: The lymphocytes of lymphoblastic lymphoma are intermediate in size between large cell lymphoma and lymphomas of smaller size. (Compare the cell size to neutrophils or red cells). The cells have delicate blast like chromatin and inconspicuous nucleoli, similar to lymphoblastic leukemia. Romanowsky stain and Pap stain, x100.

36 Lymphoblastic Lymphoma A B C D E F Image 20: The cells of lymphoblastic lymphoma have dim CD45 intensity(b).the other blast characteristics are positivity for CD34 and HLADR(D). The dim CD20 (F)compared to CD19(E) is characteristic of B lymphoblasts as is the presence of CD10(C).

37 B Cell Lymphoma with larger Cell Size Burkitt lymphoma is another lymphoma with cells smaller than large cells, that might be considered in the morphologic differential diagnosis of large cell lymphoma. This lymphoma occurs endemically in Africa and is associated with the jaw and face. The sporadic cases usually seen in the United States are associated with the gastrointestinal tract and abdomen. The sporadic Burkitt lymphoma occurs in children and young adults. Immunodeficiency states are also associated with Burkitt lymphoma. Burkitt lymphoma is an aggressive neoplasm with a rapid growth rate. The cells are medium size, have classic midnight blue cytoplasm with vacuoles and coarse chromatin with multiple chromocenters. It is a B cell lymphoma with brilliant light chain fluorescence.

38 Burkitt Lymphoma Image 21: The lymphocytes of Burkitt lymphomas are intermediate in size between large cell lymphoma and lymphomas of smaller size. The cells have coarse chromatin and deep blue cytoplasm with vacuoles. The multiple chromocenters are more easily seen on Pap stain. Romanowsky stain and Pap stain, x100. (Histiocyte nucleus at arrow.)

39 Large Cell Lymphoma Large cell lymphomas also occur in the T cell/nk cell lymphomas. The T cell/nk disorders usually have characteristic clinical syndromes and are more difficult to define immunologically. Frequently T cell gene rearrangement studies are performed to confirm the diagnosis of a T cell lymphoma if aberrant T cell phenotyping studies are not seen. T cell Lymphomas that may fall into the large cell category include: Peripheral T cell lymphoma,nos Adult T cell leukemia/lymphoma syndrome Extranodal NK/T cell lymphoma, nasal type Anaplastic large cell lymphoma

40 Peripheral T cell lymphoma, NOS This category is a wastebasket of T cell lymphoma. Histologically, there is effacement of the lymph node with a mixture of cells. Although large cells may predominate, there are usually a spectrum of cell sizes. Eosinophils, histiocytes and plasma cells may also be present which can be confused as Hodgkin s Lymphoma. This is the most common type of T cell lymphoma seen in the Western Hemisphere. It occurs in adults and is associated with advanced disease and poor response to therapy. Immunologically the cells display mature T cell antigens. CD3 is positive. There may be loss of normal antigen expression ( aberrant phenotype.)

41 Peripheral T cell Lymphoma, NOS Image 22 : The cytologic spectrum of Peripheral T cell lymphoma is wide. Usually there is a mixture of small and large cells, epithelioid histiocytes may be abundant and there are few normal appearing lymphocytes. Morphologically, most of these are confused with a reactive lymph node because of the spectrum of cell size. Romanowsky and Pap stain x 100.

42 Peripheral T cell Lymphoma, NOS Image 23 : CD3 is strongly positive in the malignant cells. Notice the size and shape variation. CD3 antibody stain x 100 (Cell block material).

43 Anaplastic Large cell Lymphoma A cutaneous and a systemic form of Anaplastic Large Cell Lymphoma (ALCL) exist. Although B cell lymphomas may have anaplastic morphology, the characterization of a lymphoma as ALCL is reserved for those lymphomas of T cell or Null cell type. These lymphomas are characterized by large, hyperlobated cells that are Ki-1 (CD30) positive and and express anaplastic large cell lymphoma kinase (ALK). The systemic variety of ALCL occurs in young patients (the first three decades of life) and is more aggressive than the cutaneous variety. The cutaneous form is recurrent, occurs later in life and is more often ALK negative. Variants of anaplastic large cell lymphoma have been described (including a small cell variant.) These variants are characterized by the same immunologic features. Confusingly, anaplastic large cell lymphomas are usually epithelial membrane antigen positive, and may be LCA negative. A panel of immunologic markers, including ALK and CD30 should be performed on cases with hyperlobated cells.

44 Anaplastic Large Cell Lymphoma A B Image 24: A demonstrates Ki1(CD30) positivity in ALCL in the typical cytoplasmic and golgi distribution. B is a Papanicolaou stained fine needle aspirate demonstrating the large size and nuclear lobulations. CD30 antibody and Pap stain x 100.

45 Hodgkin's Lymphoma Although Hodgkin s lymphoma is not high on the differential diagnosis of Large Cell Lymphoma, it may be a consideration when entertaining the diagnosis of ALCL. Reed Sternberg (RS) cells are usually relatively rare in aspirates of Hodgkin's lymphoma. There is a background of reactive follicles and eosinophils, histiocytes and plasma cells may accompany the follicular hyperplasia. Flow cytometry is not helpful in the diagnosis of Hodgkin s Lymphoma. Immunochemical stains are most important. The classic RS cells should be LCA negative, LeuM1(CD15) positive and CD30 positive. Image 25 : This cytospin demonstrates the RS cells with a background of eosinophils and lymphocytes. The immunostain demonstrates the characteristic LeuM1 positivity in a Golgi distribution (Arrow). Cytospin Pap stain and CD30 stain x 50.

46 Summary In summary, unknown Case 6 is a case of Diffuse large B cell lymphoma. The cells are larger than histiocyte nuclei, with vesicular chromatin and nucleoli. Numerous lymphoglandular bodies(cytoplasmic fragments) were present in the background. An LCA stain(cd45ra) was positive, confirming the lymphocytic origin. Although, the diagnosis of Large cell lymphoma is descriptive, the flow cytometric analysis demonstrated a B cell origin without evidence of follicular derivation (CD10 negative.) With the full complement of morphology, immunocytochemistry and flow cytometry, a diagnosis of Large B Cell lymphoma, most likely Diffuse B cell lymphoma could be entertained. There are numerous lymphomas of both T/NK cell and B cell which may appear large morphologically

47 Summary Diffuse Large B cell Lymphoma comprise 30-40% of all lymphomas in the United States. The median age group is 60 years, but the age range is wide. Usually patients present with isolated node or extranodal involvement. The histology demonstrates diffuse replacement of the node without follicle formation. There are several morphologic variants described. These include: centroblastic, immunoblastic, T cell rich B cell, and anaplastic lymphoma. Although these types are recognizable as a Large B cell lymphoma, interobserver variability is broad and the prognostic differences between these types is not established.

48 References Harris NJ, Jaffe ES, Stein H et al. Revised European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group. Blood. 1994;84: Harris NJ, Jafffe ES, Diebold J et al. Lymphoma Classification-from controversy to consensus: The REAL and WHO classification of lymphoid neoplasms. Ann Oncol 2000;11(Suppl 1): Isaacson PG. The current status of lymphoma classification. Br J Haematol 2000;109: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds.) Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer (IARC) Press; Lyon, Kempson Rl, Fletcher CD, Evans HL, Hendrickson MR, Sibley RK. Tumors of the Soft Tissues. Atlas of Tumor Pathology Series, no 30, third series. AFIP: Washington DC, Young NA, Al-Saleem T. Diagnosis of lymphoma by fine-needle aspiration cytology using the Revised European-American classification of lymphoid neoplasms. Cancer(Cytopathol) 1999;87:

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