Hematopathology / FLOW CYTOMETRIC IMMUNOPHENOTYPING IN ANAPLASTIC LARGE CELL LYMPHOMA

Immunophenotypic Analysis of Anaplastic Large Cell Lymphoma by Flow Cytometry Jonathan Juco, MD, Jeannine T. Holden, MD, Karen P. Mann, MD, PhD, Lloyd G. Kelley, MT(ASCP), and Shiyong Li, MD, PhD Key Words: Anaplastic large cell lymphoma; Anaplastic lymphoma kinase; Flow cytometric immunophenotyping DOI: 10.1309/HEFL7KC435KFWEX8

Abstract We studied the antigen expression profiles of 19 anaplastic large cell lymphoma (ALCL) cases by multiparameter flow cytometry. The neoplastic cells expressed CD45, HLA-DR, and CD30 in all cases. At least 1 T cell–associated antigen was expressed in each case (CD2, 12/17 [71%]; CD4, 12/19 [63%]; CD3, 6/19 [32%]; CD7, 6/19 [32%]; CD5, 5/19 [26%]; CD8, 4/19 [21%]). CD25 was expressed in 14 (88%) of 16 cases. CD13 was expressed unexpectedly in 8 (47%) of 17 cases. One CD13+ ALCL also was positive for CD33 and 2 others for CD15. CD19, CD20, CD22, CD14, and CD36 were not expressed. Anaplastic lymphoma kinase protein was detected in about 33% (3/9) of ALCLs examined by flow cytometric immunophenotyping (FCI); expression was validated by immunohistochemical analysis. Of 19 ALCL cases, 12 were diagnosed solely based on FCI findings in conjunction with morphologic evaluation of body fluid (1 case), fine-needle aspirate (3 cases), or excisional biopsy specimen (8 cases). The diagnoses of the remaining 7 cases were suggested strongly by FCI and confirmed by immunohistochemical analysis. FCI is useful to aid in diagnosis of ALCL, particularly along with fine-needle aspiration evaluation. ALCL with aberrant expression of myeloid antigens should not be mistaken for extramedullary myeloid tumor.

Anaplastic large cell lymphoma (ALCL) was first described by Stein et al1 in 1985 as a unique lymphoma characterized by large pleomorphic lymphoid cells, often with horseshoe-shaped or wreath-like nuclei and a sinusoidal growth pattern. It was defined originally by its expression of CD30 (Ki-1) antigen, but a subset of B-cell lymphoproliferative disorders subsequently has been found to express CD30. In the revised European-American lymphoma classification scheme, therefore, ALCL was defined as a disease with a Tcell or null-cell phenotype.2 The vast majority of cases of nullcell type identified by immunohistochemical analysis or flow cytometric immunophenotyping (FCI) display clonal T-cell receptor gene rearrangements by molecular techniques, more definitively defining ALCL as a T-cell lymphoma.3 The recent World Health Organization classification defines ALCL exclusively as a subtype of peripheral T-cell lymphoma.4 Approximately 70% of ALCLs express the anaplastic lymphoma kinase (ALK) protein as a result of chromosomal abnormalities involving the ALK gene. The most frequent genetic abnormality is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5 and the ALK gene on chromosome 2. The ALK gene encodes a tyrosine kinase receptor that normally is expressed in the small intestine, testis, and central nervous system, but not in normal lymphoid cells.5 The genetic sequence is most homologous with the insulin receptor kinase subfamily. The expression of ALK can be seen in both nodal and extranodal ALCLs, with the exception of the primary cutaneous subtype in which ALK expression is rarely seen. The expression of ALK predicts good clinical outcome in ALCL. The overall 5-year survival in ALK-positive ALCL is better than for ALKnegative cases.6,7 Am J Clin Pathol 2003;119:205-212

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Despite the characteristic morphologic features, a definitive diagnosis of ALCL requires ancillary studies such as immunohistochemical analysis. The majority of ALCLs express 1 or more T-cell antigens in addition to the characteristic CD30 staining. Monoclonal antibodies against these antigens have long been available for immunohistochemical and/or flow cytometric analyses. ALK expression frequently is detected by immunohistochemical technique; however, a monoclonal antibody for FCI has become available. The combination of these available markers theoretically should make FCI a useful tool in the diagnosis of ALCL. This would be especially helpful in situations in which paraffin-embedded tissue is not available, as may be the case in fine-needle aspiration (FNA) or body fluid specimens. Scattered case reports have described the immunophenotype of ALCL by FCI,8-12 but no systemic studies have been carried out. We prospectively studied the antigen expression profiles of ALCL by multiparameter flow cytometry and explored the possibility of FCI as an ancillary tool to aid in the diagnosis of ALCL.

Materials and Methods Case Selection Nineteen cases of ALCL with detailed FCI data were obtained prospectively from July 1999 to March 2002 at Emory Medical Laboratories, Emory University Hospital, Atlanta, GA. Three were FNA-only specimens, 1 was body fluid, and the remaining were excisional biopsy specimens. The clinicopathologic data are summarized in ❚Table 1❚.

❚Table 1❚ Clinicopathologic Data Case No./ Sex/Age (y) 1/M/12 2/M/68 3/M/43 4/F/65 5/F/67 6/F/11 7/F/55 8/F/39 9/M/47 10/M/53 11/M/16 12/F/22 13/M/61 14/M/13 15/F/5 16/F/9 17/F/55 18/M/50 19/F/79

Site/Specimen Type

Cervical lymph node/biopsy Preauricular mass/biopsy Abdominal mass/biopsy Right cervical lymph node/FNA Right-sided neck mass/biopsy Lymph node/biopsy Submandibular mass/FNA Left inguinal mass/FNA Lymph node/biopsy Left axillary lymph node/biopsy Left submandibular lymph node/biopsy Lymph node/biopsy Right axillary lymph node/biopsy Chest wall mass/biopsy Left humeral mass/biopsy Lymph node/biopsy Pleural fluid/thoracentesis Right-sided groin mass/biopsy Right-sided pelvic mass/biopsy

FNA, fine-needle aspirate.

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Yes No No No Yes Yes No No No Yes Yes No Yes Yes No No No No No

Morphologic and Immunohistochemical Examination FNA smears were air dried, fixed in alcohol, and stained with Papanicolaou and Romanowsky methods. Cytocentrifuged slides of the body fluid specimen also were stained with the Romanowsky technique. Excisional biopsy specimens were fixed in buffered formalin solution, paraffinembedded, sectioned, and stained with H&E. Immunohistochemical stains for CD3, CD20, CD30, and CD45 were performed on cases 1, 5, 6, 10, 11, 13, and 14. Immunostain for ALK was performed on cases 2, 3, 5, 6, 10, 13, 14, 15, and 18. Immunostain for CD15 was performed on case 6. The antibodies were purchased from DAKO, Carpinteria, CA, and the standard avidin-biotin complex technique was used for immunohistochemical analysis.13 Flow Cytometric Immunophenotyping FCI was performed on fresh excisional biopsy tissue, body fluid, or FNA specimens collected in RPMI 1640 culture medium. Specimens were processed routinely, and single-cell suspensions were stained with 4 fluorochromeconjugated antibody combinations (fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin). Approximately 10,000 events were acquired on a flow cytometer (FACSCalibur, Becton Dickinson Biosciences, San Jose, CA) and analyzed using the Paint-A-Gate computer software program (Becton Dickinson Biosciences, San Diego, CA). Most of the 19 cases were analyzed by a routine lymphoma panel protocol established in our laboratory and studied for the expression of CD45, Bcell antigens (CD19, CD20, CD22, kappa and lambda light chains), T-cell antigens (CD2, CD3, CD4, CD5, CD7, CD8), myeloid antigens (CD13, CD14, CD33, CD34, CD117), and other markers including CD16, CD25, CD36, CD38, CD56, and HLA-DR (Becton Dickinson Biosciences). Fluorescein isothiocyanate–conjugated CD30 antibody (Becton Dickinson Biosciences) was not part of the routine lymphoma protocol but was added in cases that were highly suggestive of ALCL based on the preliminary FCI results. A permeabilization step was added for phycoerythrin-conjugated ALK antibody (Becton Dickinson Biosciences). Nine of 19 cases had sufficient cells and were studied further for the expression of ALK protein by FCI. ALCL cell lines SUDHL-1 and 2A (provided by Mariusz A. Wasik, MD, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia) were used as positive and negative controls, respectively. Cytogenetic Analysis Cytogenetic studies were performed on only 1 sample (case 6). Fresh tissue was collected in media and subjected to short-term culture without stimulation. Chromosomes were analyzed using the G-banding technique, and the chromosomal © American Society for Clinical Pathology

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abnormalities were listed according to the International System for Human Cytogenetic Nomenclature.

Results The ages of the patients ranged from 5 to 79 years (mean, 41 years; Table 1). Six of the 19 patients were of the pediatric age group (younger than 18 years). The male/female ratio was approximately 1:1 (9 males [(47%]; 10 females [53%]). The majority of the 19 cases manifested with enlarged lymph node(s) or masses adjacent to welldefined lymph node regions. Two cases manifested as extranodal soft tissue masses and 1 as a body cavity effusion. Twelve of 19 cases were diagnosed as ALCL solely by FCI (Table 1) in conjunction with morphologic evaluation (cases 2-4, 7-9, 12, and 15-19). Three of these 12 cases were FNA-only specimens (cases 4, 7, and 8) ❚Image 1❚, 1 was a pleural fluid specimen (case 17), and the other 8 were excisional biopsy specimens (cases 2, 3, 9, 12, 15, 16, 18, and 19). The diagnosis of ALCL in the remaining 7 cases was suggested strongly by the FCI findings and confirmed by immunohistochemical analysis ❚Image 2❚. In case 11, the tumor cells were positive for CD3 by FCI but were negative by immunohistochemical analysis. Except for this discrepancy, the immunophenotypic results of FCI were in complete agreement with those of immunohistochemical analysis. By FCI, the neoplastic cells comprised 4% to 84% of the sample with a mean of about 30%. In almost all cases, the lymphoma cells formed a relatively distinct cluster. On the forward vs right-angle light scatter display, the tumor cells were large with increased right-angle light scatter property,

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resembling monocytes or histiocytes ❚Image 3A❚ . In all cases, the lymphoma cells expressed high-density CD45 ❚Image 3B❚ , again mimicking monocytes. As expected, CD30 was positive in all 19 cases ❚Image 3C❚ and ❚Table 2❚. At least 1 of the T cell–associated antigens was positive in each case ❚Image 3D❚ (Table 2). The most commonly positive T-cell antigen was CD2, which was expressed in 12 (71%) of 17 cases examined. The next most commonly expressed T-cell antigens, in descending order, were CD4 (12/19 [63%]), CD3 (6/19 [32%]), CD7 (6/19 [32%]), CD5 (5/19 [26%]), and CD8 (4/19 [21%]). Except for case 16, the tumor cells expressed an aberrant T-cell phenotype with loss of at least 1 of the T cell–associated antigens (Table 2). More than 60% of cases showed absence of surface CD3 expression. Among those cases, however, 2 cases (6 and 19; Table 2) demonstrated intracellular staining for CD3 by FCI. All but 1 case had loss of CD5, CD7, or both. Of the 19 cases, 9 were negative for CD5 and CD7. The B cell–associated antigens CD19, CD20, and CD22 were not expressed in any of the cases. Myeloid antigen expression was observed unexpectedly in a substantial proportion of ALCL cases (Table 2) ❚Image 4A❚ , ❚Image 4B❚ , and ❚Image 4C❚ . Of the 19 cases, 12 expressed at least 1 of the myeloid antigens (CD13, CD15, or CD33). CD13 was the most frequently expressed myeloid antigen, which occurred in 8 (47%) of 17 cases examined. One CD13+ case coexpressed CD33 (case 3), and 2 others showed CD15 coexpression (cases 11 and 16). The age range for the 12 patients with ALCL aberrantly expressing myeloid antigens was 5 to 79 years (mean, 32 years), which is substantially younger than patients with ALCL not expressing myeloid antigens (mean, 54 years). In fact, half

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❚Image 1❚ Fine-needle aspirate smear of the right cervical lymph node from case 4 stained with the Romanowsky method (A, rapid Romanowsky, ×20; B, rapid Romanowsky, ×50).

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❚Image 2❚ Histologic sections of the lymph node and right-sided neck mass from cases 6 and 5 (A, H&E, ×20; B, CD30, ×20; C, anaplastic lymphoma kinase [ALK], ×20; D, H&E, ×20; E, CD30, ×20; F, ALK, ×20).

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of the 12 patients were 16 years old or younger at the time of diagnosis. The activation marker, HLA-DR, was expressed in all 19 cases, while the interleukin-2 receptor, CD25, was expressed in 14 (88%) of 16 cases studied (Table 2) ❚Image 4D❚ . No mature monocytic antigens, such as CD14 and CD36, were expressed. Owing to limitations in the amount of material available, ALK was tested by FCI in only 9 cases. After a permeabilization procedure, 3 of the 9 cases were positive for ALK by FCI (Table 2). These same 9 cases subsequently were subjected to ALK immunohistochemical staining. The same cases that were positive for ALK by FCI also were positive by immunohistochemical analysis ❚Image 5A❚ (Image 2C). No additional ALK-positive cases were detected, consistent with a perfect correlation between FCI and immunohistochemical techniques for ALK expression ❚Image 5B❚ (Image 2F). As expected, the SUDHL-1 cells were positive for ALK and the 2A cells were negative by FCI ❚Image 5C❚ and ❚Image 5D❚. Normal lymphocytes and monocytes did not express ALK protein by FCI. Cytogenetic analysis performed in 1 case (case 6) demonstrated the t(2;5) involving the ALK gene on 2p23 and an unknown partner gene on 5q23. This case also was positive for ALK protein by both FCI and immunohistochemical methods.

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❚Image 3❚ Representative scatter plots showing the anaplastic large cell lymphoma cells (yellow dots) with high forward and right-angle light scatter properties and high-density CD45 expression simulating monocytes (green, A and B). The tumor cells express CD30 (C) and one of the T cell–associated antigens CD4 (D). Red dots, background normal T cells; gray dots, cellular debris or nonviable cells. APC, allophycocyanin; FITC, fluorescein isothiocyanate; IC, intracellular; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

Discussion ALCL has a morphologic appearance that in some cases can be quite distinct, but in others may be difficult to differentiate from other large cell lymphomas, Hodgkin lymphoma, and even metastatic carcinoma.1,2,4,8,14 Immunohistochemical ❚Table 2❚ Flow Cytometric Immunophenotyping Results Case No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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+ – + – – + ND ND – – + + – + + + – – –

– – + – – – – – – – ND – – – – – – – –

– + – – – – ND ND – – + ND + – – + – + +

+ – + + + + + + ND ND ND + – + + + + + +

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ALK

+ + + + + + + + + + + + + + + + + + +

ND – + ND – + ND ND ND – ND ND – – + ND ND – ND

ALK, anaplastic lymphoma kinase; ND, not done; +, positive; –, negative; ±, surface negative, intracellular positive.

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❚Image 4❚ Representative scatter plots showing the anaplastic large cell lymphoma cells (yellow dots) with aberrant expression of myeloid antigens CD13 (A), CD33 (B), and CD15 (C). The tumor cells also express CD25 and HLADR (D). Green dots, monocytes; red dots, background T cells and a few polytypic B cells; gray dots, cellular debris or nonviable cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

❚Image 5❚ Representative scatter plots showing the expression of anaplastic lymphoma kinase (ALK). A, Case 6. B, Case 5. C, Negative control anaplastic large cell lymphoma (ALCL) cell line 2A. D, Positive control ALCL cell line SUDHL-1. Yellow dots, neoplastic cells; red dots, background normal T and polytypic B cells; gray dots, cellular debris or nonviable cells. APC, allophycocyanin; FITC, fluorescein isothiocyanate; IC, intracellular; PE, phycoerythrin.

techniques are the mainstay in the immunophenotypic characterization of ALCL. The characteristic CD30 (Ki-1) marker can help differentiate ALCL from most diffuse large B-cell lymphomas and peripheral T-cell lymphomas. ALK expression is an important prognostic indicator, warranting immunophenotypic, cytogenetic, or molecular testing for the ALK antigen in all cases of ALCL.6,7 Immunohistochemical techniques permit testing of formalin-fixed, paraffinembedded tissue samples and, hence, permit the cells in question to be examined in a “morphologic setting.” Immunostains, however, require sufficient tissue, and in most cases the acquisition of this tissue requires an open incisional biopsy. Depending on the location of the tumor, an open biopsy may be difficult or impossible to perform and, in most cases, necessitates the use of general anesthesia. FCI has become an essential component in the accurate diagnosis of hematologic malignant neoplasms, including malignant lymphoma. As with immunohistochemical analysis, the expression of certain markers can be indicative of a specific

disease and useful as a prognostic indicator. Flow cytometric techniques, in contrast with immunohistochemical techniques, permit immunophenotyping in a shorter time and, in some cases, without an open tissue biopsy. In the present study, we demonstrated that FCI is a useful ancillary tool in the diagnosis of ALCL. Staining for CD45, CD30, and at least 1 T-cell marker was the most common immunophenotype detected by FCI. CD30 is not normally part of the lymphoma panel protocol in our laboratory. Rather, anti-CD30 antibody was added after the morphologic and initial FCI results suggested a diagnosis of ALCL. At least 1 T-cell marker was positive in each case. The most common T-cell antigen detected by FCI was CD2, which was positive in approximately 71% of cases (12/17). CD3 was detected in only about a third of the cases examined (6/19). The fact that fewer than half of the 7 cases suggestive of ALCL by FCI were CD3+ by immunohistochemical analysis conceivably could have contributed to some initial phenotypic confusion if FCI results had not

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been available. Although CD45 results were available for the 7 cases that had immunohistochemical confirmation, the lack of staining of the neoplastic cells for either CD20 or CD3 by immunohistochemical analysis could have caused misinterpretation before the CD30 results were available. The availability of many T-cell markers increases the likelihood that ALCL will be diagnosed as a T-cell as opposed to a null-cell process. The results of the T-cell markers from our cases closely follow those of previous reports3,4,15 The usefulness of FCI in the diagnosis of ALCL is best exemplified by its use in conjunction with FNA samples. By the very nature of the sample (single-cell suspension), an FNA biopsy specimen is ideal for FCI analysis. Immunohistochemical analysis of cell blocks from FNA specimens often is difficult to interpret and displays confounding staining patterns. Despite potential problems with the acquisition of large cells for FCI owing to the inherent fragility of the cells, we demonstrate that the use of flow cytometry with FNA-only specimens may eliminate the requirement for excisional biopsy. The apparent reliability of CD30, CD45, T-cell, and ALK markers permits FCI to be an important tool. However, this usefulness is not exclusive of FNA samples but also can be used for open biopsy specimens. If the diagnosis of ALCL is suspected after morphologic examination of an excisional lymph node biopsy specimen, markers not usually part of the “in-house” flow cytometric lymphoma panel, such as CD30 and ALK-1, can be added quickly, bypassing the usual lengthy turnaround time for immunohistochemical stains. We are not suggesting the replacement of immunohistochemical analysis by FCI. Immunohistochemical analysis is and likely will remain the method of choice to characterize ALCL. However, when faced with FNA-only samples or time issues, FCI can be an effective aid in the diagnosis of ALCL. The aberrant expression of myeloid markers has been described previously in ALCL, mostly in case report form.8-12,16 In a case report, Meech et al16 described an 18month-old boy with atypical manifestation of ALCL. The neoplasm in this child expressed both myeloid (CD13 and CD33) and natural killer cell antigens (CD56). A case report by Popnikolov et al11 describes a 12-year-old boy with CD13+ ALCL that initially was called extramedullary myeloid tumor because of the expression of CD13 and HLADR and the absence of surface T-cell markers by FCI. In our series, the myeloid antigen CD13 was expressed unexpectedly in 8 (47%) of 17 cases. The presence of myeloid markers, especially with the loss of surface T-cell antigens, may result in the misdiagnosis of ALCL as an extramedullary myeloid tumor. Misdiagnosis is more likely to occur if a limited FCI panel is performed. The prognostic significance of the expression of CD13 and other myeloid markers is unknown. Coexpression of CD30 and CD15 is

relatively rare but may suggest classic Hodgkin lymphoma in the differential diagnosis. The expression of high-density CD45, at least 1 T-cell marker, and in some cases ALK, however, essentially rules out the diagnostic possibility of classic Hodgkin lymphoma. High-density CD25 was observed in the majority of our cases (Image 4D). This interleukin-2 receptor has been reported previously in ALCL.16-20 In a study by Rubie et al,20 all 10 pediatric cases tested for CD25 were positive. Our study closely follows these results, with 88% (14/16) expressing CD25. Prognostically, CD25 does not seem to be significant. The frequency with which CD25 is positive in ALCL suggests that CD25 may be a useful marker in the immunophenotypic diagnosis of ALCL and a potential therapeutic target. The t(2;5)(p23;q35) cytogenetic abnormality was first described in ALCL in 1989.21,22 The actual chromosomal breakpoints were cloned, showing that the translocation fuses the NPM gene on chromosome 5q35 to the ALK gene on chromosome 2p23. The result is the 80-kd NPM-ALK fusion protein, also referred to as p80.5 Antibodies that specifically detect the p80 or NPM-ALK fusion protein, referred to as p80 or ALK-1, were developed and shown to be specific for cases with t(2;5).23 The expression of p80 or ALK is associated with a young pediatric age group and a good prognosis.7,14,24 ALK staining has been shown not to occur in normal or reactive lymphoid tissue.24,25 Variant translocations other than t(2;5) have been described, but all involve the ALK gene on chromosome 2. These include translocations with chromosomes 1, 3, 17, and 19 and inversion of chromosome 2.4,7,15,26 ALK immunostains are positive in both the cytoplasm and nucleus in t(2;5) but usually are limited to the cytoplasm in variant translocations. Monoclonal antibodies against ALK protein for use in FCI have become available. Owing to the cytoplasmic localization of the NPM-ALK fusion protein, a permeabilization step is required for the detection of ALK. Of the 9 cases with material available for ALK testing by FCI, 3 were positive. There was complete correlation with ALK detection by immunohistochemical analysis, which suggests that ALK detection by flow cytometry is at least as sensitive as by immunohistochemical analysis. Expression of ALK by FCI also is confirmatory, but not obligatory, in the diagnosis of ALCL. Normal lymphocytes and monocytes or macrophages did not express ALK by FCI, confirming the usefulness of ALK in flow cytometry and suggesting it as a specific marker for ALCL. FCI can be an important adjunct in the diagnosis of ALCL. CD45, CD30, and at least 1 T-cell marker are the most commonly expressed antigens as detected by FCI in our study. The T-cell immunophenotype often is aberrant, as shown by the abnormal T-cell marker expression in the majority of cases in our series. Aberrant expression of myeloid markers is not uncommon in ALCL and may result Am J Clin Pathol 2003;119:205-212

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in misdiagnosis of ALCL as an extramedullary myeloid tumor if a limited panel is performed. Expression of the important prognostic indicator ALK also can be analyzed reliably by multiparameter flow cytometry. From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA. Address reprint requests to Dr Li: Dept of Pathology and Laboratory Medicine, Emory University School of Medicine, Emory University Hospital, Room F143D, 1364 Clifton Rd NE, Atlanta, GA 30322.

References 1. Stein H, Mason DY, Gerdes J, et al. The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood. 1985;66:848-858. 2. Harris NL, Jaffe ES, Stein H, et al. A revised EuropeanAmerican classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;84:1361-1392. 3. Foss HD, Anagnostopoulos I, Araujo I, et al. Anaplastic largecell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules. Blood. 1996;88:4005-4011. 4. Delsol G, Ralfkiaer E, Stein H, et al. Anaplastic large cell lymphoma. In: Jaffe ES, Harris NL, Stein H, et al, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001:230-235. World Health Organization Classification of Tumours. 5. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in nonHodgkin’s lymphoma. Science. 1995;263:1281-1284. 6. Gascoyne RD, Aoun P, Wu D, et al. Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma. Blood. 1999;93:3913-3921. 7. Falini B, Pileri S, Zinzani PL, et al. ALK+ lymphoma: clinicopathological findings and outcome. Blood. 1999;93:26972706. 8. Weiss LM, Picker LJ, Copenhaver CM, et al. Large-cell hematolymphoid neoplasms of uncertain lineage. Hum Pathol. 1988;19:967-973. 9. Carbone A, Gloghini A, Volpe R, et al. KP1 (CD68)-positive large cell lymphomas: a histopathologic and immunophenotypic characterization of 12 cases. Hum Pathol. 1993;24:886-896. 10. Simonitsch I, Panzer-Gruemayer ER, Ghali DW, et al. NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas. Br J Haematol. 1996;92:866-871. 11. Popnikolov NK, Payne DA, Hudnall D, et al. CD13-positive anaplastic large cell lymphoma of T-cell origin: a diagnostic and histogenetic problem: a case report and review of the literature. Arch Pathol Lab Med. 2000;124:1804-1808.

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12. Dunphy CH, Gardner LJ, Manes JL, et al. CD30+ anaplastic large cell lymphoma with aberrant expression of CD13: case report and review of the literature. J Clin Lab Anal. 2000;14:299-304. 13. Hsu SM, Raine L, Fanger H. Use of the avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577-580. 14. Falini B, Bigerna B, Fizzotti M, et al. ALK expression defines a distinct group of T/null lymphomas (“ALK lymphomas”) with a wide morphological spectrum. Am J Pathol. 1998;153:875886. 15. Stein H, Foss HD, Durkop H, et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000;96:3681-3695. 16. Meech SJ, McGavran L, Odom LF, et al. Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4–anaplastic lymphoma kinase gene fusion. Blood. 2001;98:1209-1216. 17. Sato N, Sato K, Yagi E, et al. Primary cutaneous Ki-1+ anaplastic large cell lymphoma: a morphologic, immunohistochemical and genetic study of an indolent case. J Dermatol. 1995;22:441-449. 18. Aoki M, Niimi Y, Takezaki S, et al. CD30+ lymphoproliferative disorder: primary cutaneous anaplastic large cell lymphoma followed by lymphomatoid papulosis. Br J Dermatol. 2001;145:123-126. 19. Delsol G, Al Saati T, Gatter KC, et al. Coexpression of epithelial membrane antigen (EMA), Ki-1, and interleukin-2 receptor by anaplastic large cell lymphomas: diagnostic value in so-called malignant histiocytosis. Am J Pathol. 1988;130:59-70. 20. Rubie H, Gladieff L, Robert A, et al. Childhood anaplastic large cell lymphoma Ki-1/CD30: clinicopathologic features of 19 cases. Med Pediatr Oncol. 1994;22:155-161. 21. Rimokh R, Magaud JP, Berger F, et al. A translocation involving a specific breakpoint (q35) on chromosome 5 is characteristic of anaplastic large cell lymphoma (“Ki-1 lymphoma”). Br J Haematol. 1989;71:31-36. 22. Kaneko Y, Frizzera G, Edamura S, et al. A novel translocation, t(2;5)(p23;q35), in childhood phagocytic large T-cell lymphoma mimicking malignant histiocytosis. Blood. 1989;73:806-813. 23. Shiota M, Fujimoto J, Takenaga M, et al. Diagnosis of t(2;5)(p23;q35)-associated Ki-1 lymphoma with immunohistochemistry. Blood. 1994;84:3648-3652. 24. Shiota M, Nakamura S, Ichinohasama R, et al. Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity. Blood. 1995;86:1954-1960. 25. Pulford K, Lamant L, Morris SW, et al. Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1. Blood. 1997;89:1394-1404. 26. Trinei M, Lanfrancone L, Campo E, et al. A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATICALK) in a case of ALK-positive anaplastic large cell lymphoma. Cancer Res. 2000;60:793-798.

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