Cytologic and Immunocytochemical Findings of Anaplastic Large Cell Lymphoma

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1 CANCER CYTOPATHOLOGY 33 Cytologic and Immunocytochemical Findings of Anaplastic Large Cell Lymphoma Analysis of Ten Fine-Needle Aspiration Specimens over a 9-Year Period Wai-Kuen Ng, M.B.,B.S. 1 Philip Ip, M.B.,Ch.B. 2 Carolyn Choy, M.B.,B.S. 2 Robert J. Collins, M.B.,B.S. 2 1 Department of Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong. 2 Department of Pathology, Queen Mary Hospital, Hong Kong. Address for reprints: Wai-Kuen Ng, M.B., B.S., Department of Pathology, Pamela Youde Nethersole Eastern Hospital, 3 Lok Man Road, Chai Wan, Hong Kong SAR, China; Fax: ; ngwk@yahoo.com Received May 10, 2002; revision received July 12, 2002; accepted July 18, BACKGROUND. Anaplastic large cell lymphoma (ALCL) has raised much controversy in the field of hematolymphoid pathology. Its nature is becoming better characterized with recent advances in molecular genetics. However, to the authors knowledge, a detailed description of the fine-needle aspiration (FNA) cytology of ALCL is lacking and the application of immunocytochemical study, including immunostaining for anaplastic lymphoma kinase (ALK) protein, to cytology samples has not been studied to date. METHODS. The authors reviewed 10 FNA specimens of ALCL from 8 patients encountered at Pamela Youde Nethersole Eastern Hospital and Queen Mary Hospital in Hong Kong over a 9-year period from early 1993 to the end of The cytologic and immunocytochemical findings (including ALK protein overexpression) of the specimens were correlated with histologic and immunohistochemical findings of surgical biopsy specimens. RESULTS. Six of the eight patients had ALCL of the common variant, whereas the remaining two patients had ALCL of the small cell variant. FNA specimens of ALCL of the common variant yielded many loosely dispersed hallmark cells that contained eccentric kidney-shaped or embryo-like nuclei, several prominent rod-shaped or angulated basophilic nucleoli, and abundant amphophilic cytoplasm. Doughnut cells, tumor cells with multilobated nuclei, and multinucleated giant cells with a wreath-like arrangement of nuclei occasionally were found. A small number of plasmacytoid tumor cells, nondescript small round tumor cells, and reactive polymorphs also was present. In contrast, plasmacytoid cells and nondescript small to medium-sized tumor cells represented the predominant cell population in ALCL of the small cell variant. The plasmacytoid appearance was exaggerated further in air-dried smears. In air-dried smears, small intracytoplasmic vacuoles and scanty azurophilic granules also were noted. On immunocytochemical study performed using the cell block materials, the majority of tumor cells demonstrated membranous and paranuclear dot-like positivity for CD30. The staining for epithelial membrane antigen, leukocyte common antigen, and T-cell markers was variable. Positive staining for ALK protein was demonstrated beautifully in two of the cases. CONCLUSIONS. Despite the wide morphologic spectrum of ALCL, a definitive diagnosis on the basis of FNA cytology is possible on careful interpretation of the cytologic features and a high index of suspicion. The cytologic diagnosis can be confirmed further with proper application of immunostaining to cell block sections. Immunocytochemical study for ALK protein, which provides useful prognostic information, also can be demonstrated satisfactorily using cytology samples. Cancer (Cancer Cytopathol). Cancer (Cancer Cytopathol) 2003;99: American Cancer Society. KEYWORDS: anaplastic large cell lymphoma (ALCL), CD30, ALK, cytology American Cancer Society DOI /cncr.10922

2 34 CANCER (CANCER CYTOPATHOLOGY) February 25, 2003 / Volume 99 / Number 1 Fine-needle aspiration (FNA) biopsy is considered to be a major diagnostic tool in the initial investigation of lymphadenopathy. 1,2 In cases of metastatic malignancies (such as metastatic carcinoma) or granulomatous lymphadenitis due to mycobacterial infection, FNA cytology may provide a definitive diagnosis, sparing the patient incisional or excisional lymph node biopsy. With regard to malignant lymphoma, the cytology diagnosis may be straightforward or problematic. 3 Diffuse large B-cell lymphoma often yields a predominant population of large lymphoid cells with high mitotic activity. Conversely, Burkitt lymphoma produces mainly small to medium-sized lymphoid cells on cytology smears, with frequent mitosis and apoptosis. FNA cytology is diagnostic in patients with Hodgkin lymphoma if classic Reed Sternberg cells are found in an appropriate background that is comprised of lymphocytes, plasma cells, eosinophils, and histiocytes. A cytologic diagnosis of low-grade, B-cell non-hodgkin lymphoma, which includes small lymphocytic lymphoma and follicular lymphoma, occasionally is difficult, even with the help of ancillary investigations. This also is true for some cases of T-cell non-hodgkin lymphoma, such as angioimmunoblastic T-cell lymphoma, because of the heterogeneity of the cell population, resulting in marked cytologic or even histologic overlapping with reactive lymphadenopathy. Immunocytochemistry occasionally is of great help in the diagnosis of non-hodgkin lymphoma. A classic example is lymphoblastic lymphoma, in which diffuse nuclear staining for terminal deoxynucleotidyl transferase (Tdt), coupled with a monotonous population of small lymphoid cells with fine chromatin and high mitotic activity, is virtually diagnostic. With regard to anaplastic large cell lymphoma (ALCL), a diagnosis of high-grade malignancy often is not difficult to make on the basis of cytologic examination. The major diagnostic pitfall lies in its morphologic variations, which may be mistaken for other lymphoid or non-lymphoid malignancies. However, ALCL does carry its own clinical and prognostic characteristics. 4,5 Recognition of this morphologic spectrum, together with proper application of immunocytochemistry, can help clinicians to arrive at a definitive cytologic diagnosis. In the current study, we reviewed the cytologic and immunocytochemical findings of 10 FNA specimens of ALCL occurring in 8 Chinese patients from Pamela Youde Nethersole Eastern Hospital and Queen Mary Hospital in Hong Kong during a 9-year period from early 1993 to the end of 2001, with histologic correlation. Special emphasis was given to the application of anaplastic lymphoma kinase (ALK) immunostaining to cytology samples. MATERIALS AND METHODS Ten FNA specimens of ALCL from 8 patients and the corresponding incisional or excisional biopsies were retrieved from the pathology files of Pamela Youde Nethersole Eastern Hospital and Queen Mary Hospital in Hong Kong during a 9-year period from early 1993 to the end of The cytology preparations and histologic sections were reviewed to assess the morphologic spectrum of ALCL. The demographic data of the patients, sites of FNA biopsies, and initial cytologic diagnoses were tabulated (Table 1). In all eight patients, subsequent incisional or excisional biopsies of s, skin, or soft tissue lesions were performed for histologic confirmation. The FNA biopsies were performed using 22-gauge or occasionally 21-gauge needles, either by pathologists or clinicians. If the material was sufficient, several direct smears were prepared and promptly fixed in 95% ethanol overnight. Additional air-dried smears also were made in case 7. The needles and syringes were rinsed into an equal volume of 50% ethanol and normal saline, which later was centrifuged at a rate of 1500 revolutions per minute for 20 minutes. The sediment obtained, if any, was processed as for paraffin cell blocks. In some cases, cytospin preparations (Shandon, Cheshire, U.K.) also were produced from the supernatant fluid. All the wet-fixed smears and cytospin preparations were stained with either Papanicolaou stain (EA 65) or hematoxylin and eosin (H & E). The air-dried smears were stained with the May- Grünwald-Giemsa stain. Four-micron thick paraffin sections were cut from the cell blocks and stained with H & E. The surgical biopsy specimens were submitted in a fresh state and promptly fixed in 10% neutralbuffered formalin. Representative tissue samples were taken and processed as for routine paraffin blocks. Fourmicron thick sections were stained with H&E.Immunocytochemical/immunohistochemical study using the streptavidin-biotin complex technique for leukocyte common antigen (LCA) (Dakopatts, Glostrup, Denmark); B-cell markers CD20 (Dakopatts) and CD79a (Dakopatts); T-cell markers CD3 (Dakopatts) and CD45RO (Dakopatts); CD30 (Dakopatts); epithelial membrane antigen (EMA) (Dakopatts); and ALK protein (anti-alk1 antibody from Dakopatts) was performed on the paraffin sections from cell block material, if available, and surgical biopsies. Immunostaining for cytokeratin AE1/3 (Dakopatts), S-100 protein (Dakopatts), CD15 (Dakopatts), and CD68 (Dakopatts) also was performed in selected cases. Some of the immunostaining reactions discussed earlier were performed using the Ventana automated immunohistochemistry slide staining device (Ventana, Tucson, AZ). Microwave

3 TABLE 1 Demographic Data, Initial Cytologic Diagnoses, and Immunostaining Results of the Reported Cases Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Gender M M M F F M M M Age (yrs) FNA cytology samples Immunocytochemistry FNA Case No. 1a 1b a 5b Site of FNA Right axillary lymph node Another right axillary lymph node Left inguinal Left cervical Right supraclavicular Left paraspinal soft tissue mass Left inguinal lymph node Lower abdominal skin mass Cervical lymph node Right submandibular Initial cytologic diagnosis Anaplastic large cell lymphoma Anaplastic large cell lymphoma Atypical cells Suspicious for lymphoma Suspicious for lymphoma Myeloma Myeloma Suspicious for malignancy Anaplastic large cell lymphoma CD30 EMA ALK1 CD3/CD45RO / / / / / / / LCA Not available Not available Not available Anaplastic large cell lymphoma Surgical biopsy specimens Immunohistochemistry Site of biopsy Right axillary lymph node Left inguinal & pelvic mass Right inguinal & left thigh skin ulcer Right supraclavicular Right inguinal Lower abdominal skin mass Back skin rash CD30 EMA NA ALK1 NA NA CD3/CD45RO / / / / (focal) / / / / LCA (focal) NA Right submandibular M: male; F: female; FNA: fine-needle aspiration; : positive; : negative; EMA: epithelial membrane antigen; LCA: leukocyte common antigen; NA: not available.

4 36 CANCER (CANCER CYTOPATHOLOGY) February 25, 2003 / Volume 99 / Number 1 TABLE 2 FNA Cytology Findings for the 10 Reported Cases Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 FNA Case No. 1a 1b a 5b Cytologic features (smears, cytospin preparations and / or cell blocks) Hallmark cells with kidney-shaped / / or embryo-like nuclei Doughnut cells Multinucleated cells with a wreathlike / arrangement of nuclei Cells with multilobated nuclei / / / Small to medium-sized plasmacytoid cells Nondescript, small to medium-sized cells Prominent rod-shaped/angulated basophilic nucleoli Paranuclear cytoplasmic accentuation Mitotic figures Apoptotic bodies / / / Background polymorphs / / Final histologic diagnosis (based on surgical biopsies) Anaplastic large cell lymphoma, common variant Anaplastic large cell lymphoma, small cell variant FNA: fine-needle aspiration; : positive; / : occasionally present; : negative. heat at 100 C for minutes was employed as the method of antigen retrieval. RESULTS Clinical Findings Six of the eight patients (i.e., all except Patients 5 and 6) initially presented with multiple enlargement and primary systemic ALCL. Their ages ranged from years and there was a marked male predilection. Patients 2, 3, and 7 subsequently were found to have secondary cutaneous involvement by the lymphoma cells. Conversely, Patient 5 had a past history of multiple myeloma that was treated with chemotherapy several years before this presentation. The lymphoma thus most likely represented secondary ALCL. Patient 6 presented with a huge skin ulcer over the lower abdominal wall, with no significant associated lymphadenopathy or systemic disease. This finding corresponded to the category of primary cutaneous, CD30-positive, T-cell lymphoproliferative disorder (precisely, primary cutaneous ALCL). FNA Biopsy Findings (Table 2) The cellularity of wet-fixed smears and cytospin preparations ranged from low to high, depending on the sampling techniques. They contained loosely dispersed, medium-sized to large malignant cells with abundant amphophilic cytoplasm and occasionally illdefined cell borders (Fig. 1). The tumor cells nuclei often were placed eccentrically and varied from round or oval to kidney-shaped or horseshoe-shaped (the so-called hallmark cells) or even multilobated. Some of the kidney-shaped nuclei had a smooth convex surface and slightly irregular concave surface, assuming a jellyfish or embryo-like appearance. The concave border of the nuclei was blurred further by paranuclear cytoplasmic accentuation. Doughnut cells with nuclei containing a large pseudonuclear cytoplasmic inclusion were not uncommon. These inclusions were the result of deep cytoplasmic invaginations into the nuclei. Multinucleated tumor giant cells with nuclei arranged in a wreath-like pattern also were noted. The number, shape, and size of the nucleoli varied. Hallmark cells often contained several prominent rodshaped or angulated basophilic nucleoli. Solitary inclusion-like eosinophilic nucleoli rarely were found. There also was a second tumor cell population comprised of medium-sized cells with eccentrically located oval nuclei and abundant cytoplasm, resembling plasma cells. In general, the tumor cells were

5 Cytology of Anaplastic Large Cell Lymphoma/Ng et al. 37 FIGURE 1. Direct smear from Case 1a demonstrating loosely dispersed large hallmark cells with abundant amphophilic cytoplasm, ill-defined cell borders, and oval to kidney-shaped/embryo-like nuclei. A doughnut cell also is present in left upper corner (Papanicolaou stain, original magnification 800). FIGURE 2. Direct smear from Case 8 demonstrating numerous small to medium-sized tumor cells with eccentrically located round to oval nuclei, indistinct nucleoli, and a moderate amount of cytoplasm, assuming a plasmacytoid appearance (Papanicolaou stain, original magnification 800). mitotically active, with easily discernible mitotic figures, especially in cases with high cellularity. In all the reported cases, various numbers of polymorphs were present in the background. Necrotic debris was not conspicuous, although apoptotic bodies could be identified. In the common ALCL variant, hallmark cells, doughnut cells, cells with multilobated nuclei, and multinucleated tumor giant cells were found easily (Fig. 1). Mononuclear plasmacytoid cells with oval nuclei, although scanty, still were present. In contrast, these small to medium-sized plasmacytoid cells and otherwise nondescript small round cells represented the predominant cell population in the small cell ALCL variant (Fig. 2). These cells contained small inconspicuous nucleoli. Large hallmark cells were found only on diligent search. These plasmacytoid cells were highlighted further in air-dried smears from Case 7 (Figure 3). Tumor cells with elongated cytoplasm, assuming a hand mirror appearance, were not uncommon. Tiny vacuoles and scanty azurophilic granules also were observed in the cytoplasm of some tumor cells. In contrast to true plasma cells in which the cell borders were discrete, small cytoplasmic blebs were identified at the cell periphery. However, lymphoglandular bodies, a feature commonly noted in FNA specimens from lymphoid lesions, were not observed. In general, the cytologic features of secondary systemic ALCL (Cases 5a,5b) and primary cutaneous ALCL (Case 6) did not appear to differ from those of primary systemic ALCL of the common variant. In seven of the FNA specimens in which cell blocks were available, membranous and paranuclear dot-like CD30 positivity was demonstrated in all but two blocks (Fig. 4). Immunocytochemical study for B-cell markers was negative. The staining for LCA, EMA, and T-cell markers was variably positive. In cases in which the tumor cells expressed T-cell markers on the cell block sections, CD45RO was more sensitive than CD3. If performed, the staining for AE1/3, S-100 protein, and CD15 was negative. Cytoplasmic positivity for CD68 was noted in the tumor cells of Case 8. The cell block sections of two cases demonstrated positivity for the ALK1 immunostain (Fig. 5). In Case 7, ALK-positivity took the form of mixed cytoplasmic, nuclear, and occasional nucleolar staining. The nucleolar staining was highlighted beautifully in Case 1a. The ALK protein staining was negative in secondary ALCL (Cases 5a and 5b) and primary cutaneous ALCL (Case 6). Histologic Findings Histologic examination of the surgical biopsy specimens demonstrated that six patients had the common variant of ALCL. The remaining two cases represented the small cell ALCL variant. In the common variant (Fig. 6), the s demonstrated nearly complete replacement by high-grade malignant cells, with a propensity of sinus involvement. In cases of skin biopsies, the dermis and subcutis were found to be heavily infiltrated by similar tumor cells, with focal superficial ulceration. The tumor cells were large in size and contained voluminous and occasionally vacuolated cytoplasm, relatively ill-defined cell borders, and one to several inconspicuous to prominent basophilic, rod-shaped nucleoli. Hallmark cells were

6 38 CANCER (CANCER CYTOPATHOLOGY) February 25, 2003 / Volume 99 / Number 1 FIGURE 4. Immunocytochemical study for CD30 performed on cell block section from Case 8 demonstrating membranous and paranuclear dot-like positivity in many tumor cells (CD30, original magnification 800). FIGURE 3. Direct air-dried smear from Case 7 demonstrating (A) several tumor cells with elongated cytoplasm, assuming a hand mirror appearance. Polymorphs also were noted in the background (May-Grünwald-Giemsa stain). (B) A plasmacytoid tumor cell with eccentric nucleus, tiny intracytoplasmic vacuoles, small number of azurophilic granules, and cytoplasmic blebs at the cell periphery (May-Grünwald-Giemsa, original magnification 800 [A]; 1500 [B], oil immersion). identified readily and the morphology of tumor cell nuclei recapitulated that observed in the FNA specimens. With regard to the small cell variant of ALCL (Fig. 7), the histologic picture was indistinguishable from that of peripheral T-cell lymphoma, not otherwise specified (NOS). The majority of the lymphoma cells were small to medium-sized and contained markedly irregular hyperchromatic nuclei, inconspicuous to small nucleoli, and a scant to moderate amount of amphophilic cytoplasm. However, occasional hallmark cells still were identified on diligent search. Aggregates of larger tumor cells were noted around small vessels. Immunohistochemical study (Table 1) confirmed the diagnosis of ALCL, with membranous and paranuclear dot-like staining for CD30 and occasionally EMA. There was variable staining for LCA and the T-cell markers CD3 and CD45RO. By definition, the tumor cells did not express the B-cell markers CD20 and CD79a. Immunostaining for ALK protein demonstrated a mixed cytoplasmic, nuclear, and nucleolar positivity in some tumor cells in three of the six groups of biopsies in which this ancillary study was performed (Fig. 8). In two of the cases (biopsies of Patients 2 and 3), the tumor cells demonstrated mainly cytoplasmic positivity for ALK protein. There also was perimembranous accentuation of staining in Case 2 (Fig. 9). As expected, the tumor cells of primary cutaneous ALCL (Case 6) did not express ALK protein on immunohistochemical study. DISCUSSION The concept of ALCL has been evolving over time. In the day of the updated Kiel classification, ALCL was subdivided into ALCL of B-cell type and ALCL of T-cell type. 6 Subsequently, it has been discovered that the prognosis of the so-called ALCL of B-cell type is similar to that of diffuse large B-cell lymphoma. 7 The clinical behavior of both types of ALCL also is different. 7 Moreover, instead of demonstrating the classic membranous and paranuclear dot-like staining pattern for CD30, the tumor cells in ALCL of B-cell type often demonstrate a weaker cytoplasmic CD30 positivity. 7 The subgroup of ALCL of B-cell type therefore is removed from the diagnostic category of ALCL in the revised European American classification of lym-

7 Cytology of Anaplastic Large Cell Lymphoma/Ng et al. 39 FIGURE 6. Histologic section of a biopsy from Patient 1 demonstrating large malignant lymphoid cells with kidney-shaped or embryolike vesicular nuclei, prominent nucleoli, and voluminous cytoplasm. Hallmark cells commonly were identified (H & E, original magnification 800). FIGURE 5. (A) Immunocytochemical study for ALK protein performed on a cell block section from Case 7 demonstrating mixed cytoplasmic and nuclear positivity in some tumor cells (ALK1). (B) In a cell block section from Case 1a, the tumor cells demonstrated a peculiar nucleolar staining for ALK protein (ALK1, original magnification 800 [A,B]). phoid neoplasms (REAL) 8-10 and the latest World Health Organization (WHO) classification of hematologic malignancies. 11 In the WHO classification, the differences between primary systemic ALCL and primary cutaneous ALCL, both in terms of clinical presentation and prognostic implication, also are emphasized. In general, ALCL for the most part is of T-cell phenotype and characterized by the classic anaplastic morphology and a peculiar CD30 expression. The phrase anaplastic large cell also has been challenged on discovery of various morphologic variants, especially small cell 12 and lymphohistiocytic variants. 13 The tumor cells in ALCL of the small cell variant closely mimic those of peripheral T-cell lymphoma, NOS and are neither large nor anaplastic. The FIGURE 7. Histologic section of a biopsy from Patient 8 demonstrating small to medium-sized malignant lymphoid cells with a markedly irregular nuclear outline and a moderate amount of amphophilic cytoplasm. The morphologic features closely mimicked those of peripheral T-cell lymphoma, not otherwise specified (H & E, original magnification 800). diagnosis relies on recognition of distinctive morphologic clues (such as the presence of occasional hallmark cells, especially around small vessels), as well as immunopositivity for CD30 and occasionally ALK protein. ALK protein overexpression is a timely topic in recent years and has been proven to be helpful in the prognostic subcategorization of ALCL. 14,15 ALK protein is shown to overexpressed in some cases of ALCL (as a result of certain chromosomal translocations), some rare neoplasms (including inflammatory myofi-

8 40 CANCER (CANCER CYTOPATHOLOGY) February 25, 2003 / Volume 99 / Number 1 FIGURE 8. Immunohistochemical study for ALK protein performed on a biopsy from Patient 8 demonstrating mixed cytoplasmic, as well as nuclear and occasionally nucleolar positivity in some tumor cells (ALK1, original magnification 800). broblastic tumor), 16 and occasional normal brain cells. 17 With regard to lymphoma, immunopositivity for ALK protein is almost never demonstrated in non- ALCL cases, with rare exceptions in a peculiar subgroup of large B-cell lymphoma. 18 Among the group of primary systemic ALCL cases, ALK protein-positive tumors often occur in younger age groups, have a male predilection, and tend to carry a more favorable clinical outcome whereas the ALK protein-negative tumors are associated with a poor prognosis. 14,15 Immunostaining for ALK protein also can be correlated with the underlying molecular genetics. 17,19 21 ALCL cells with t(2; 5) tend to demonstrate a mixed cytoplasmic, nuclear, and nucleolar staining pattern because of the production of a nucleophosmin (NPM)- ALK chimeric protein. NPM provides nuclear localization signals for this NPM-ALK fusion gene product, which explains the additional nuclear and nucleolar staining of the ALK protein. In contrast, ALCL cases with translocation lacking the involvement of the NPM gene, such as t(1;2), often demonstrate a pure cytoplasmic staining characteristic. Because of this distinctive ALK protein overexpression in ALCL, some authors have proposed a new diagnostic category of ALKoma. 19,20 To our knowledge to date, the term anaplastic large cell lymphoma still is preferred because it is a widely accepted entity and there also are well documented cases of ALK protein-negative but otherwise classic ALCL. However, a distinction between primary systemic ALCL and primary cutaneous ALCL is warranted because ALK protein is almost never overexpressed in the latter. 17,22 FIGURE 9. Immunohistochemical study for ALK protein performed on a biopsy from Patient 2 demonstrating cytoplasmic positivity with perimembranous accentuation (ALK1, original magnification 800). Despite the recent increased understanding of ALCL, to our knowledge detailed description of FNA cytology findings, including its morphologic variants, is lacking, although occasional case reports and small series have been reported In addition, to our knowledge the application of ALK immunostaining to cytology specimens has never been evaluated to date. The lack of cytology literature regarding this topic is related in part to the previous confusion in the classification of ALCL. FNA cytology of ALCL of the common variant differs from that of the small cell variant. However, considerable overlapping does exist. Cytologically, ALCL of the common variant yields readily identifiable large and malignant-appearing hallmark cells with high mitotic activity. Hallmark cells are characterized by the presence of solitary, eccentrically located nuclei that commonly assume a kidneyshaped or embryo-like appearance, several prominent rod-shaped or angulated basophilic nucleoli, and abundant amphophilic cytoplasm. The cell borders often are ill-defined and on air-dried smears, cytoplasmic blebs are observed at the cell periphery. The airdried smears also help to demonstrate small intracytoplasmic vacuoles and occasional tiny azurophilic granules. The azurophilic granules correspond to sites of cytotoxic molecules characteristically present in ALCL cells The nuclear eccentricity and plasmacytoid appearance also is exaggerated, and lymphoma cells assuming a hand mirror appearance frequently are identified. In addition to hallmark cells, which are considered to be diagnostic of ALCL, other cytologic variants including doughnut cells, tumor cells with multilobated nuclei, and multinucle-

9 Cytology of Anaplastic Large Cell Lymphoma/Ng et al. 41 FIGURE 10. Algorithm for the correct cytologic diagnosis of primary systemic anaplastic large cell lymphoma. LCA: leukocyte common antigen; : positive; EMA: epithelial membrane antigen; - negative. ated tumor giant cells with a wreath-like arrangement of nuclei are variably found. In contrast to the apparent cohesiveness noted in histologic sections, ALCL cells are dispersed widely. Unlike other types of lymphoma, ALCL does not produce lymphoglandular bodies, which represent cytoplasmic fragments of friable lymphoid cells, on FNA. Conversely, reactive polymorphs are noted in the background. An extreme example is the neutrophil-rich variant of ALCL, in which numerous polymorphs are observed admixed with scattered lymphoma cells, mimicking a suppurative inflammatory process In ALCL of the common variant, a small number of small to medium-sized plasmacytoid cells or nondescript round cells (occasionally resembling centrocytes or centroblasts) also are identified. They lack the classic clock face chromatin pattern and paranuclear hof of true plasma cells. These plasmacytoid cells and nondescript small round cells become the predominant tumor cell population in ALCL of the small cell variant. However, occasional mediumsized hallmark cells with kidney-shaped nuclei still are found on diligent search. The FNA cytology of ALCL variants represents a morphologic spectrum, with various compositions of a different cell population. The morphologic diagnosis of ALCL can be confirmed reliably by immunocytochemical study performed on the cell block sections. However, the sensitivity is less than that for histology specimens, which most likely is related to the partial disruption of some membrane-associated antigens during the centrifugation process in cell block preparation. The characteristic membranous and paranuclear dot-like positivity for CD30 is maintained for the most part. The diagnosis of ALCL is supported further by positivity for EMA, LCA, and occasionally T-cell markers, especially CD45RO. In contrast, CD15 (a marker sometimes expressed by Reed Sternberg cells in patients with Hodgkin lymphoma), S-100 protein (a marker commonly expressed by melanoma cells), and the epithelial marker cytokeratin are negative. Cell block sections also are useful for ALK protein immunostaining. ALK protein overexpression helps to confirm the diagnosis of ALCL further. In addition, cases of ALCL with ALK positivity tend to have a more favorable clinical course. 14,15 Therefore, careful morphologic interpretation of FNA cytology, as well as proper application of ancillary investigations, can provide a definitive diagnosis of ALCL and provide clues regarding useful prognostic information, sparing patients more invasive surgical biopsy procedures. With regard to cytologic differential diagnosis, ALCL of the common variant may mimic Hodgkin lymphoma (especially with regard to lymphocyte depletion subtype), sarcomatoid carcinoma, malignant melanoma, other high-grade non-hodgkin lymphoma (such as T-cell-rich B-cell lymphoma and CD30-positive diffuse large B-cell lymphoma), and even true sarcoma. The morphologic overlaps with Hodgkin lymphoma are well known and the controversy is highlighted further by a previous report of the existence of a poorly defined entity called Hodgkin-like ALCL, 8 10 which recently was shown to carry immunophenotypic and clinical characteristics similar to those for nodular sclerosing (syncytial variant) or lymphocyte depletion subtypes of Hodgkin lymphoma. 37 Classic Reed Sternberg cells often are identified in patients with Hodgkin lymphoma. Unlike hallmark cells of ALCL, classic Reed Sternberg cells are binucleated and contain solitary inclusion-like eosinophilic macronucleoli surrounded by clear chromatin. The nuclear membrane often is thickened. This binucle-

10 42 CANCER (CANCER CYTOPATHOLOGY) February 25, 2003 / Volume 99 / Number 1 ated morphology may on occasion give rise to a mirror image owl eye appearance. In Hodgkin lymphoma, the background contains plasma cells, lymphocytes, eosinophils, and histiocytes. Immunocytochemical study also is helpful in controversial cases. Reed Sternberg cells are CD30- and occasionally CD15-positive. In contrast, EMA immunostaining often is negative. Reed Sternberg cells occasionally may demonstrate variable and weak expression of B-cell markers. To our knowledge, ALK protein positivity has not been reported to date. In approximately 50% of cases of Hodgkin lymphoma, the Reed Sternberg cells demonstrate positive nuclear signals for Epstein Barr virus (EBV)-encoded RNA on in situ hybridization study. 41 In contrast, ALCL most likely is not EBV-related, although occasional EBVpositive and ALK-negative cases have been reported. 42 Conversely, sarcomatoid carcinoma may produce markedly pleomorphic or even multinucleated tumor cells on FNA, not unlike those observed in patients with ALCL of the common type. Moreover, the tumor cells in sarcomatoid carcinoma may be dispersed loosely with minimal cohesiveness. The immunoprofile of ALCL further can mimic that of carcinoma because both tumors often are EMA-positive and ALCL cells may fail to express lymphoid markers including LCA and CD3. Correct cytologic diagnosis can be achieved only with a high index of suspicion and the proper application of immunostaining for CD30, ALK protein, and cytokeratin. It also is worthwhile to note that metastatic embryonal carcinoma cells can express CD In contrast to ALCL, these tumor cells are cytokeratin-positive and EMA-negative. With regard to amelanotic malignant melanoma, the tumor cells also are loosely cohesive and contain prominent nucleoli. Brown-colored melanin pigments may not be conspicuous. In contrast to ALCL cells, melanoma cells often demonstrate nuclear positivity for S-100 protein and they are negative for CD30 and lymphoid markers. Another cytologic differential diagnosis of ALCL is diffuse large B-cell lymphoma. The lymphoma cells in diffuse large B-cell lymphoma may closely resemble those of ALCL. The classic examples are T-cell-rich B-cell lymphoma and CD30-positive diffuse large B-cell lymphoma, in which the lymphoma cells may contain multilobated nuclei and prominent macronucleoli. However, they demonstrate a different immunophenotype compared with ALCL, which for the most part is a T-cell non-hodgkin lymphoma. Conversely, the nondescript medium-sized tumor cells noted in the small cell variant of ALCL also may be mistaken for tumor cells of diffuse large B-cell lymphoma (centroblastic lymphoma, according to the updated Kiel classification). In fact, the possibility of ALCL must be considered if the FNA specimen is found to demonstrate a significant amount of centroblast-like cells, which have been reported to express T-cell markers on immunocytochemical study. The plasmacytoid ALCL cells also may mimic tumor cells of true plasma cell neoplasm, neuroendocrine tumor, or even poorly differentiated adenocarcinoma. Finally, cytologic distinction between ALCL and true histiocytic lymphoma may be difficult, 44 especially if some ALCL cells also express the histiocytic marker CD68 (as in Case 8). Immunocytochemical study using a more comprehensive panel of antibodies is essential to arrive at a correct diagnosis. A definitive diagnosis of ALCL is possible on careful interpretation of cytologic features and the proper application of immunocytochemical study to cytology samples (Fig. 10). Immunocytochemical study performed on cell block sections can achieve a relatively satisfactory result, although the sensitivity is less when compared with that performed on surgical biopsy specimens. Immunostaining for ALK protein, a useful investigative tool that helps to provide important diagnostic and prognostic information, also can be applied to cell block materials. The different staining patterns, which correlate with underlying molecular genetics of lymphoma cells, can be demonstrated. Some authors also have demonstrated that ALCL cells with overexpression of the ALK protein often are positive for EMA. 19 Therefore, immunostaining for EMA may provide some prognostic clues in cases in which the anti-alk protein antibody is not available in the laboratories. 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