Assessment of t(2;5)(p23;q35) Translocation and Variants in Pediatric ALK+ Anaplastic Large Cell Lymphoma

Transcription

1 Hematopathology / T(2;5)(P23;Q35) AND VARIANTS IN PEDIATRIC ALK+ ANAPLASTIC LARGE CELL LYMPHOMA Assessment of t(2;5)(p23;q35) Translocation and Variants in Pediatric ALK+ Anaplastic Large Cell Lymphoma Xiayuan Liang, MD, 1,2 Sandra J. Meech, MD, 3,4,6 Lorrie F. Odom, MD, 3,4,7 Mitchell A. Bitter, MD, 1,5 John W. Ryder, MD, 1 Stephen P. Hunger, MD, 3,4,8 Mark A. Lovell, MD, 1,2 Lynn Meltesen, 1 Qi Wei, 2 Sara A. Williams, 2 Rebecca N. Hutchinson, 4 and Loris McGavran, PhD 1,2 Key Words: ALK; Anaplastic lymphoma kinase; Anaplastic large cell lymphoma; Lymphoma DOI: /TLE8FN6EYF0NJGP7 Abstract To evaluate t(2;5) and its variants, we studied 21 pediatric cases of anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) by using immunohistochemical staining, fluorescence in situ hybridization, cytogenetics, and reverse transcriptase polymerase chain reaction. Results showed 7 (33%) cases with t(2;5), 6 (29%) with variant gene rearrangements, 7 (33%) with uncharacterized rearrangements, and 1 with ALK protein expression but no ALK rearrangement. Among 6 variant gene rearrangements, 1 had TPM4-ALK/t(2;19)(p23;p13) and 2 had inv(2) with the breakpoint proximate to ATIC-ALK and an unknown partner gene separately. The genetic features of the remaining 3 cases were as follows: ins(8;2) with an unknown partner gene; conversion from ALK at diagnosis to ALK+ at recurrence with unspecified gene rearrangement; complex karyotype without involvement of 2p23, suggesting a cryptic translocation. Concordance between different laboratory results varied from 47% to 81%. These data suggest that ALK variants are not uncommon and underscore the necessity of integrating immunohistochemical, cytogenetic, and molecular genetic approaches to detect, characterize, and confirm t(2;5) and its variant translocations. Anaplastic large cell lymphoma (ALCL) was first described in 1985 by Stein et al 1 as a large-cell non-hodgkin lymphoma characterized by a sheet-like growth pattern of bizarre, CD30+ tumor cells with prominent involvement of the intrasinusoidal and paracortical areas of lymph nodes. It has been recognized as a distinct clinicopathologic entity of T-cell or null cell lineage in the Revised European-American Lymphoma classification 2 and the World Health Organization (WHO) classification. 3 ALCL constitutes approximately 10% to 15% of all non-hodgkin lymphomas 4-9 and 20% to 30% of large cell lymphomas in children. 7,10 A portion of ALCLs was found to be associated with a recurrent cytogenetic abnormality, the t(2;5)(p23;q35) Cloning of the t(2;5) in 1994 by Morris et al 14 led to insights into the pathogenesis of ALCL. The t(2;5) juxtaposes a portion of the NPM (nucleophosmin) gene (located at 5q35) to part of the ALK (anaplastic lymphoma kinase) gene (located at 2p23), resulting in the generation of a novel chimeric protein, NPM-ALK, 14,15 which has tyrosine kinase activity and has been shown to have oncogenic properties in a variety of experimental systems. 14,16-19 Overall, 43% of ALCL cases show t(2;5), including 83% of pediatric and 31% of adult cases. 20 The patients carrying t(2;5) or ALK fusion protein were reported to have good response to chemotherapy and favorable outcome Subsequently, variant rearrangements at the 2p23 ALK locus other than t(2;5) have been identified in ALCL. Six different genes, nonmuscle tropomyosin (TPM3, located at 1q25), 25 TRKfused gene (TFG, 3q21), 26 5'aminoimidazole-4-carboxamide ribonucleotide formyltransferase/imp cyclohydrolase (ATIC, 2q35), clathrin chain polypeptide-like gene (CLTCL, 17q11), 30 tropomyosin 4 (TPM4, 19p13), 31 and moesin 496 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7

2 Hematopathology / ORIGINAL ARTICLE (MSN, Xq11-12), 32 have been cloned as alternative partners to ALK, which generate TPM3-ALK/t(1;2)(q25;p23), TFG- ALK/t(2;3)(p23;q21), ATIC-ALK/inv(2)(p23;q35), CLTCL- ALK/t(2;17)(p23;q11), TPM4-ALK/t(2;19)(p23;p13), and MSN-ALK/t(2;X)(p23;q11-12) chimeric proteins. To date, only a few scattered pediatric cases of ALCL with variant gene translocations have been reported in the literature. 29,31,32 The aim of the present study was to evaluate t(2;5)(p23;q35) and variant gene rearrangements in a larger series of children with ALK+ ALCL by using a comprehensive immunohistochemical staining, cytogenetic, and molecular genetic approach. The usefulness and concordance of different methods for the detection of t(2;5) and variants also were assessed. Materials and Methods Cases A retrospective search of the pathology and hematology-oncology archives of the Children s Hospital, Denver, CO, from January 1979 through January 2003, for ALCL, peripheral T-cell lymphoma, and malignant histiocytosis yielded 22 cases of systemic ALCL. The diagnosis was based on a combination of clinical, morphologic, and immunophenotypic criteria as defined in the Revised Table 1 Clinical Features of ALK+ Lymphoma in Pediatric Patients European-American Lymphoma 2 and WHO 3 classifications. Of 22 cases, 21 (95%) were ALK+ by protein expression and/or gene rearrangement and were included in this study. One case was ALK and was excluded from the study. There were 15 males and 6 females; the median age was 11 years (range, 2-20 years). Patients underwent staging at the initial evaluation through physical examination, chest and abdominal computed tomography scan and gallium scan, and bone marrow evaluation using the Ann Arbor system. 33 The follow-up time ranged from 10 months to 12 years. The clinical information and immunophenotypic profiles are shown in Table 1 and Table 2, respectively. Histopathologic Examination Lymph node and tissue specimens and cell block preparations were fixed in formalin or B-5, paraffin embedded, and stained with H&E. The histologic sections and immunohistochemical staining in all cases were reviewed independently by 2 hematopathologists (X.L. and J.W.R.) who were unaware of the results of the cytogenetic and molecular studies. The morphologic classification was based on the criteria recommended in the WHO classification. 3 Immunohistochemical Studies Immunohistochemical staining was performed on formalin- or B-5 fixed, paraffin-embedded tissue sections using the streptavidin-biotin-peroxidase technique with or without heat-induced antigen retrieval. The diaminobenzidine Case No./Sex/ Other Organs Involved Age (y) Biopsy Site at Diagnosis Stage Treatment and Outcome 1/F/11 Groin LN Skin IV CT; alive, 5 y 2/F/5 Cervical LN Skin IV CT; recurred, 6 mo; BMT, 7 mo; alive, 12 y 3/M/14 Supraorbital ST Bone IV CT, RT; recurred, 8 mo; BMT; died, 1 y 4/M/2 LN Liver, lung, skin IV CT; alive, 6 y 5/F/6 Axillary LN Skin, ST IV CT; alive, 3 y 6/M/13 ST Spleen III CT; alive, 3 y 7/F/14 ST of leg None I E CT; alive, 6 y 8/M/12 Inguinal LN None I CT; disseminated to ST and lung, 1 y; alive, 4 y 9/M/20 Axillary LN None I CT, RT; alive, 6 y 10/F/19 ST LN, skin, uterus IV CT; alive, 5 y 11/M/4 Cervical LN Brain IV CT; alive, 12 y 12/F/16 Cervical LN None I CT, RT; disseminated to salivary gland and appendix, 1.5 y; BMT; alive, 4 y 13/M/5 ST Pleural fluid, LN IV CT; recurred, 6 mo; died, 1.5 y 14/M/7 Axillary LN Skin, SC LN IV CT; recurred, 1 y; BMT, 1.5 y; alive, 6 y 15/M/7 Mediastinal mass Abdominal LN III CT; alive, >3 y 16/M/14 Axillary LN Skin IV CT; alive, 3 y 17/M/2 Bone marrow clot Liver, spleen, PB IV CT; alive, 4 y 18/M/7 Axilla LN Skin IV CT; recurred, 1 y; BMT, 1 y, 5 mo; alive, 2 y 19/M/12 Mesenteric LN Cervical and mediastinal IV CT; alive, 2 y LNs; BM 20/M/6 Cervical LN Abdominal LN III CT; alive, 1 y 21/M/11 LN Skin IV CT; 10 mo ALK, anaplastic lymphoma kinase; BMT, bone marrow transplant; CT, chemotherapy; LN, lymph node; PB, peripheral blood; RT, radiation therapy; SC, supraclavicular; ST, soft tissue. Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7 497

3 Liang et al / T(2;5)(P23;Q35) AND VARIANTS IN PEDIATRIC ALK+ ANAPLASTIC LARGE CELL LYMPHOMA reaction was used as the final detection step. The antibodies used (all monoclonal unless specified) were specific for the following: polyclonal anti-cd3 (dilution 1:50; DAKO, Carpinteria, CA), CD15 (dilution 1:50; DAKO), CD20 (dilution 1:100; DAKO), CD30 (dilution 1:40; Immunotech, Miami, FL), CD45 (dilution 1:100; Shandon, Pittsburgh, PA), CD45Ro (dilution 1:100; DAKO), epithelial membrane antigen (EMA; dilution 1:100; DAKO), and ALK-1 (dilution 1:20; DAKO). Some cases were assessed with only a subset of these antibodies. CD45Ro was performed only on cases in which CD3 showed no reactivity. The immunohistochemical staining panel was performed in all recent cases (1999 and after) at the time of diagnosis and was repeated in all remote cases (before 1999). ALK-1 and CD30 expression were defined as at least 5% of tumor cells displaying a membranous, Golgi, or cytoplasmic pattern of staining. 34 Cytogenetic Analysis Giemsa-banded cytogenetic studies were performed from unstimulated cultures of fresh tissue. Samples were prepared by using a direct technique and overnight culture methods as described previously. 35 The karyotypes were written according to the 1995 International System for Human Cytogenetic Nomenclature. 36 Fluorescence in situ hybridization (FISH) was performed using the dual color labeled ALK probe (Vysis, Downers Grove, IL) following the manufacturer s protocol. Fresh tissue samples were used in most of the recent cases (1999 and after). In a few recent cases, owing to the unavailability of fresh tissue, FISH was performed on paraffin Table 2 Immunophenotype in Pediatric Patients With ALK+ Lymphoma sections. For remote cases (before 1999), the diagnostic cytopreparations from conventional cytogenetic cell culture studies were used if they were available; otherwise the diagnostic formalin-fixed paraffin sections were used. Cells with an intact ALK locus have a fused orange-green signal on chromosome 2. If an ALK translocation has occurred, then the probe is split. The proximal Spectrum Green labeled probe remains on the der(2), and the distal Spectrum Orange labeled probe moves to the partner derivative chromosome. Additional FISH studies were performed on case 17 with chromosome 2 and 19 whole chromosome paint probes and with probes for 5p-, 19p-, and 19q-specific subtelomeric sequences under conditions recommended by the manufacturer (Vysis). Both karyotype and FISH results were interpreted by a board-certified cytogeneticist (L. McGavran) who was unaware of the results of the immunohistochemical and molecular analyses, based on the standard cytogenetic criteria of a clone. 37 Molecular Analysis RNA was extracted from fresh or frozen tissue samples in most of the recent cases (1999 and after), from formalinfixed paraffin sections in most of the remote cases (before 1999), and in rare recent cases in which fresh or frozen tissue was not available. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed as described previously 38 using NPM-ALK/t(2;5)(p23;q35), TPM3-ALK/t(1;2)(q25;p23), TPM4-ALK/t(2;19)(p23;p13), and ATIC-ALK/inv(2)(p23;q35) primers. The presence of a distinct, appropriately sized band indicated a positive fusion Case No. CD3 CD45Ro CD15 CD20 CD30 EMA CD ND ND ND ND ND ND ALK, anaplastic lymphoma kinase; EMA, epithelial membrane antigen; ND, not done;, negative; +, positive. 498 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7

4 Hematopathology / ORIGINAL ARTICLE gene transcript. The results were reviewed by a board-certified molecular genetic pathologist (M.A.L.) who was unaware of the results of the immunohistochemical and cytogenetic studies. Results Morphologic and Immunophenotypic Findings The immunophenotypic and morphologic findings are summarized in Table 2 and Table 3, respectively. The morphologic features of the malignant cells were heterogeneous including pleomorphic (common) type (9 cases), small cell variant (5 cases), lymphohistiocytic variant (3 cases), and monomorphic variant (4 cases). All cases were T-cell (CD3+ or CD45Ro+ and CD20 ; 16 cases) or null cell (CD3, CD45Ro, and CD20 ; 5 cases) origin with expression of CD30. EMA was expressed in all cases except 20. CD15 was negative in all cases in which the stain was performed. Identification of t(2;5)(p23;q35) and Variants Table 3 summarizes ALK protein expression and ALK gene rearrangements by 4 different laboratory tests. All cases were ALK+ by either ALK-1 immunohistochemical staining or ALK-FISH probe. Detection of t(2;5)(p23;q35) By using cytogenetics and RT-PCR, t(2;5)(p23;q35) was demonstrated in 7 cases (33%; cases 2, 6, 7, 12, 14, 18, and 19). All cases in this group showed a nuclear and cytoplasmic staining pattern by ALK-1 immunohistochemical staining as described previously. 26,27,30 Detection of ALK Variant Gene Rearrangements Variant gene rearrangements were found in 6 cases (29%; cases 1, 3, 8, 11, 17, and 21). Four (cases 8, 11, 17, and 21) showed a cytoplasmic staining only pattern by ALK- 1 immunohistochemical staining as described previously. 26,27,30 ALK-1 immunohistochemical staining was negative in case 3. Case 1 was ALK-1+ according to the diagnostic pathology report; however, the staining pattern could not be assessed during the present study because the slides and blocks were unavailable. All 6 cases with variant gene rearrangements are described separately. Case 1. Cytogenetic study showed ins(8;2) (q22;p21p23) Image 1. A split ALK gene rearrangement signal was detected by FISH (not shown). The partner gene on chromosome 8 is unknown. Case 3. ALK protein was not detected, but ALK gene rearrangement was found by FISH Image 2A. The cytogenetics showed inv(2)(p13;q33) Image 2B and Table 4. The breakpoint appears distinct from that of ATIC- ALK/inv(2)(p23;q35), and RT-PCR could not confirm Table 3 ALK Protein Expression and Gene Rearrangement by Immunohistochemical Stain, and Cytogenetic and Molecular Genetic Analyses Case No. Morphologic Variant ALK-1 Immunostain Karyotype ALK-FISH t(2;5) RT-PCR 1 Small cell + (NA) Variant * + ND 2 Monomorphic + (N, C) t(2;5)(p23;q35) ND ND 3 Common Variant * + ND 4 Small cell + (N, C) ND ND 5 Lymphohistiocytic + (C) 46,XX + ND 6 Common + (N, C) 46,XY Monomorphic + (N, C) ND + 8 Common (initial diagnosis) ND Common (recurrence) + (C) 46,XY + 9 Common + (N, C) ND + ND 10 Common + (N, C) 46,XY + ND 11 Common + (C) Variant * + ND 12 Small cell + (N, C) t(2;5)(p23;q35) + ND 13 Common + (N, C) ND + ND 14 Small cell + (N, C) t(2;5)(p23;q35) + ND 15 Monomorphic + (C) 46,XY + ND 16 Common + (N, C) ND + ND 17 Monomorphic + (C) Variant * + 18 Small cell + (N, C) ND Common + (N, C) 46,XY Lymphohistiocytic + (C) 46,XX 21 Lymphohistiocytic + (C) Variant * + ALK, anaplastic lymphoma kinase; C, cytoplasmic staining; FISH, fluorescence in situ hybridization; N, nuclear staining; NA, not available; ND, not done; RT-PCR, reverse transcriptase polymerase chain reaction;, negative; +, positive. * See Table 4 for detailed karyotype. Specimen failure. Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7 499

5 Liang et al / T(2;5)(P23;Q35) AND VARIANTS IN PEDIATRIC ALK+ ANAPLASTIC LARGE CELL LYMPHOMA X Image 1 (Case 1) Giemsa-banded cytogenetic study illustrates ins(8;2)(q22;p21p23). A Normal ALK der(2) inv(2) 18 ATIC-ALK translocation because of an inadequate RNA sample. Owing to the proximity of these breakpoints, involvement of ATIC is still possible. Case 8. Neither ALK protein nor ALK gene rearrangement was detected by immunohistochemical staining Image 3A or FISH Image 3B at diagnosis. RT-PCR was also negative for t(2;5) at diagnosis. The lymphoma recurred 1 year later. Both cytoplasmic ALK protein expression and ALK gene rearrangement were observed by immunohistochemical staining Image 3C and FISH Image 3D at recurrence. RT-PCR still failed to demonstrate t(2;5). These data indicated the presence of ALK variant translocation at recurrence. To further identify the specific variant gene rearrangement, additional 3 ALK variant primers, TPM3- ALK/t(1;2)(q25;p23), TPM4-ALK/t(1;19)(p23;p13), and ATIC-ALK/inv(2)(p23;q35) were tested by RT-PCR. None of the results were positive (not shown). Case 11. ALK protein expression and gene rearrangement were demonstrated by immunohistochemical staining X Y Image 2 (Case 3) ALK split-apart fluorescence in situ hybridization probe (A) and Giemsa-banded cytogenetic study (B) illustrate split orange and green signals and 3 copies of chromosome 2 with 1 normal, 1 inv(2)(p13;q33), and 1 der(2)inv(2)(p13;q33)t(2;3)(p13;p14). Arrows indicate breakpoints. ALK, anaplastic lymphoma kinase. Table 4 Karyotypes of Patients With Variant ALK Translocations B Case No. Karyotype 1 46,XX,ins(8;2)(q22;p21p23) 3 49,XY,+1,i(1)(q10),inv(2)(p13;q33),+der(2)inv(2)(p13;q33)t(2;3)(p13;p14),+5,der(18)t(7;18)(q22;q23) 11 47,XY,der(1)t(1;5)(q23;q13),der(2)t(2;4)(p21;q25),der(4)t(4;12)(q25;q15),der(5)t(1;5)(q42;q13),t(?6;9)(q25.3;q34.1), +2xder(12)t(2;12)(p21;q15)inv ins(12;1)(q15;q42q23) 17 46,XY,t(2;19)(p23;p13.1),der(5)t(5;19)(p15.3;q13.1) 21 50,XY,add(2)inv(2)(?:2p23 q3?5::2p23 pter)x2,add(10)(p12),del(11)(q14),+12,+17,+21 ALK, anaplastic lymphoma kinase. 500 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7

6 Hematopathology / ORIGINAL ARTICLE A B C (not shown) and FISH Image 4A. Cytogenetics revealed a very complex karyotype without abnormality at the 2p23 locus Image 4B (Table 4), suggesting a cryptic translocation. Case 17. ALK protein was detected by immunohistochemical staining (not shown). Both cytogenetics Image 5A and FISH Image 5B showed t(2;19)(p23;p13.1). DNA sequencing identified the partner gene, TPM4, on chromosome Case 21. ALK protein and gene rearrangement were identified by immunohistochemical staining (not shown) and FISH Image 6A. Cytogenetics showed a hyperdiploid karyotype with inv(2)(?:2p23 q3?5::2p23 pter) Image 6B (Table 4). The partner gene is unknown. RT-PCR did Rearranged Extra 2p sequence Normal cell Image 3 (Case 8) A and B, Specimen at diagnosis. A, Negative staining in tumor cells (anaplastic lymphoma kinase [ALK]-1, 400). B, ALK split-apart fluorescence in situ hybridization (FISH) probe illustrates a normal fused orange-green signal in the paraffin section. C and D, Specimen at recurrence. C, Positive cytoplasmic staining in tumor cells (ALK-1, 400). D, ALK splitapart FISH probe shows split orange and green signals. D not detect t(2;5). To further specify the variant gene rearrangement, the same 3 ALK variant primers as used in case 8 were tested by RT-PCR. None of the results were positive (not shown). Detection of an Uncharacterized ALK Translocation One third of the cases (4, 5, 9, 10, 13, 15, and 16) had ALK protein expression, ALK gene rearrangement, or both, but neither t(2;5) nor variants were identified owing to lack of available specimens or unsuccessful retrospective assays. ALK protein expression by immunohistochemical staining displayed a combined nuclear and cytoplasmic pattern in 5 cases (4, 9, 10, 13, and 16), suggesting NPM-ALK fusion, Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7 501

7 Liang et al / T(2;5)(P23;Q35) AND VARIANTS IN PEDIATRIC ALK+ ANAPLASTIC LARGE CELL LYMPHOMA A B and a cytoplasmic-only pattern in 2 cases (5 and 15), suggesting fusion to a variant partner gene. Case 20. An unusual pattern was found in this case with cytoplasmic ALK protein expression shown by immunohistochemical staining but no ALK gene rearrangement shown by cytogenetics or FISH. RT-PCR was also negative for t(2;5)(p23;q35), t(1;2)(q25;p23), t(1;19) (p23;p13), and inv(2)(p23;q35). Concordance Between Methods for ALK Detection ALK-1 Immunohistochemical Staining vs FISH In our study, 21 cases (representing 20 patients) were analyzed by both ALK-1 immunohistochemical staining and ALK-FISH. We found that 17 cases (representing 16 patients) showed consistent results by both methods. Four cases (representing 4 patients) were discrepant. ALK protein was detected, but ALK gene rearrangement was not seen by FISH in cases 4, 7, and 20; the opposite discrepancy was present in case 3. The concordance rate between immunohistochemical staining and FISH was 81% (17/21). ALK-1 Immunohistochemical Staining vs Cytogenetics Fifteen cases (representing 15 patients) were studied by both ALK-1 immunohistochemical staining and cytogenetics. Consistent results between ALK protein expression and the presence of t(2;5) or variant gene rearrangements were obtained in 7 cases (1, 2, 11, 12, 14, 17, and 21) by both methods. The concordance was 47% (7/15). Discrepant results between these methods were found in 8 cases (3, 5, 6, 8, 10, 15, 19, and 20). FISH vs Cytogenetics Fourteen cases (representing 14 patients) were analyzed by ALK-FISH and cytogenetics. Eight cases (1, 3, 11, 12, 14, 17, 20, and 21) had consistent results with ALK split signals by FISH and the presence of t(2;5) or variant partner genes by cytogenetics. The concordance between these methods was 57% (8/14). Inconsistent results were found in 6 cases (5, 6, 8, 10, 15, and 19). Discussion X Y Image 4 (Case 11) A, ALK split-apart fluorescence in situ hybridization probe shows split orange and green signals (arrow) in paraffin section. B, Giemsa-banded cytogenetic study shows a complex karyotype without involvement at the 2p23 locus. Arrows indicate breakpoints. ALK, anaplastic lymphoma kinase. The t(2;5)(p23;q35) is present in a substantial portion of ALCLs 12,13,39,40 and causes the NPM gene located at 5q35 to fuse with a gene at 2p23 encoding the receptor tyrosine kinase, ALK. As a consequence, the ALK gene comes under the control of the NPM promoter, which induces a constitutive transcription of the NPM-ALK hybrid gene, resulting in the production of an 80-kd chimeric protein, NPM-ALK or p80. 14,16 The C-terminal NPM domain carries nuclear localization signals involved in shuttling of ribosomal proteins into the nucleus, leading to positive staining for ALK in both the cytoplasm and the nucleus. 41 Since the cloning of the genes involved in the t(2;5)(p23;q35), a number of variant ALK translocation partners have been identified in ALCL. To date, 6 variant translocations, TPM3-ALK/t(1;2)(q25;p23), TFG-ALK/t(2;3)(p23;q21), ATIC-ALK/inv(2)(p23;q35), CLTCL-ALK/t(2;17)(p23;q11), TPM4-ALK/t(2;19) (p23;p13), and MSN-ALK/t(2;X)(p23;q11-12) have been cloned ,42-44 These variant gene rearrangements also cause dysregulated ALK expression, but ALK protein 502 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7

8 Hematopathology / ORIGINAL ARTICLE A B der(5) X Y staining is confined to the cytoplasm except in the t(2;x)(p23;q11-12), which shows cell membrane restricted ALK expression. In our study, t(2;5)(p23;q35) was detected in 7 of 21 ALK+ cases by cytogenetics or RT-PCR. All 7 cases showed a combined nuclear and cytoplasmic staining pattern by ALK-1 immunohistochemical staining. An additional 5 cases (4, 9, 10, 13, and 16) with uncharacterized ALK gene rearrangements also showed nuclear and cytoplasmic staining, suggesting the presence of an NPM-ALK translocation. Taken together, 12 cases had evidence to suggest the der(19) Image 5 (Case 17) A, Giemsa-banded cytogenetic study illustrates t(2;19)(p23;p13.1),der(5)t(5;19)(p15.3;p13.1). Arrows indicate breakpoints. B, Fluorescence in situ hybridization with dual-color whole chromosome paint probes shows No. 19 sequences (orange) translocated to der(2) and der(5) and No. 2 sequences (green) on der(19). A { Inversion (2) Normal (2) Inversion (2) 2 inv der(2) inv(2) 1 2 inv(2) X Y Image 6 (Case 21) ALK split-apart fluorescence in situ hybridization probe (A) and Giemsa-banded cytogenetic study (B) illustrate 3 copies of chromosome 2 with 1 normal and 2 inv(2)(?:2p23 q3?5::2p23 pter). ALK, anaplastic lymphoma kinase. B presence of an NPM-ALK translocation. Owing to the unavailability of fresh tissue samples, unsuccessful cell culture, or unsuccessful extraction of RNA from paraffin blocks, we could not confirm t(2;5)(p23;q35) by cytogenetics and/or RT-PCR in the latter 5 cases. ALK variants were reported from 10% to 25% in some large studies. 26,41,45,46 Six of 21 cases (1, 3, 8, 11, 17, and 21) in our study displayed variant ALK gene rearrangements. Our data and the literature indicate that variants of the NPM-ALK fusion gene are not uncommon. In accordance with previous observations, cytoplasmic staining of ALK-1 was seen in 4 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7 503

9 Liang et al / T(2;5)(P23;Q35) AND VARIANTS IN PEDIATRIC ALK+ ANAPLASTIC LARGE CELL LYMPHOMA cases (8, 11, 17, and 21). The ALK staining pattern was not recorded in the pathology report in case 1, and the slides and blocks were unavailable for reevaluation in the present study. In case 3, ALK protein was negative by immunohistochemical staining, probably owing to protein degradation in prolonged preservation of the specimen. Case 3 exhibited inv(2)(p13;q33) in cytogenetic analysis. The breakpoint is proximate to that of ATIC-ALK/inv(2)(p23;q35). Although RT-PCR could not confirm ATIC-ALK translocation because of inadequate RNA, involvement of ATIC is still possible. Case 21 also exhibited inv(2), but the breakpoint was uncertain. RT-PCR did not detect ATIC-ALK/inv(2)(p23;q35), suggesting that a gene other than ATIC was involved in this case and would require further molecular characterization. Case 17 showed TPM4-ALK/t(2;19)(p23;p13.1), which has been described previously. 31 Three cases (1, 8, and 11) showed genetic features that, to our knowledge, have not been reported in the literature. Case 1 exhibited ins(8;2)(q22;p23) by cytogenetics. The specific partner gene located at 8q22 is unknown at the present time. In case 8, the tumor cells converted from ALK to ALK+ during disease progression. At diagnosis, neither ALK protein nor ALK gene rearrangement was detected by immunohistochemical staining or FISH. RT-PCR was also negative for t(2;5). The diagnosis of ALCL was based on presence of intrasinusoidal infiltration and sheets of large bizarre null lymphoid cells with expression of CD30 and EMA. Interestingly, when the lymphoma recurred 1 year later, both ALK protein and ALK gene rearrangement were detected by immunohistochemical staining and FISH, although the tumor cells retained the same morphologic features and immunophenotype as the primary tumor. ALK protein expression in this case was characterized by cytoplasmic distribution. Cytogenetics was unsuccessful owing to specimen failure. Again, RT-PCR was unable to detect t(2;5) at recurrence. These data suggest that the tumor cells at recurrence carried an ALK variant gene rearrangement. However, none of the additional RT-PCR assay results using TPM3-ALK/t(1;2)(q25;p23), TPM4-ALK/t(1;19)(p23;p13), and ATIC-ALK/inv(2)(p23;q35) primers were positive. MSN- ALK/t(2;X)(p23;q11-12) was ruled out because this translocation has a unique cell membrane staining pattern of ALK rather than the cytoplasmic staining observed in this case. We did not test for 2 other identified ALK variant translocations in this case, TFG-ALK/t(2;3)(p23;q21) and CLTCL- ALK/t(2;17)(p23;q11). Several factors might be relevant to the inconsistent ALK status between diagnosis and recurrence in this case. Technical problems might have caused false-negative results at diagnosis. This possibility was unlikely, since all the tests were repeated and the same results were obtained by different methods with appropriate controls. Alternatively, clonal evolution might contribute to the development of a novel ALK translocation at tumor recurrence. Because the tumors at diagnosis and recurrence have identical morphologic and immunophenotypic features except for ALK, it is unlikely that recurrence represents a second malignancy. Case 11 carried a very complex karyotype without evidence of cytogenetic involvement of 2p23. However, ALK-1 cytoplasmic staining and ALK-FISH split signal suggested the presence of a cryptic ALK variant gene rearrangement. Unfortunately, further cytogenetic and molecular studies were unable to be performed in this case because the material was preserved inadequately. An additional 2 cases (5 and 15) with uncharacterized ALK gene rearrangements also showed cytoplasm-only ALK- 1 staining by immunohistochemical staining. They also might represent ALK fusion protein(s) other than NPM-ALK. Case 20 was an unusual case characterized by the presence of cytoplasmic expression of ALK-1 protein and the absence of ALK gene rearrangement by cytogenetics, FISH, and RT-PCR. Because it did not carry the ALK translocation, it is not included in the ALK variant category. A subset of diffuse large B-cell lymphoma that expresses the full-length form of the ALK receptor kinase molecule with the absence of t(2;5) has been reported. 47 To our knowledge, expression of the full-length form of this molecule in ALCL has not been described. Determining whether this case might represent expression of the full-length form of ALK molecule will require further studies to detect the extracellular portion of the ALK molecule by immunohistochemical staining and molecular weight of the ALK protein by Western blot analysis. In our study, 4 different methods immunohistochemical staining, conventional cytogenetics, FISH, and RT- PCR were used to detect ALK protein expression and translocations. The concordance was 47% between immunohistochemical staining and cytogenetics, 57% between cytogenetics and FISH, and 81% between immunohistochemical staining and FISH. The pattern of concordance in our study parallels that reported in the literature, with the highest concordance between immunohistochemical staining and FISH and the lowest concordance between immunohistochemical staining and cytogenetics. 20 Several factors might be responsible for discrepancies between methods. Many of our cases were examined retrospectively, and prolonged tissue storage often was suboptimal for comprehensive analysis. Sampling artifact might result in the absence of the tumor cells in the specimen submitted for certain tests. Cell culture difficulties might lead to specimen failure for cytogenetic studies. Since RNA is labile during prolonged tissue storage, it was recovered successfully from only a few recent cases. Therefore, the concordance between RT-PCR and other methods was not evaluated in the present study. Based 504 Am J Clin Pathol 2004;121: DOI: /TLE8FN6EYF0NJGP7

10 Hematopathology / ORIGINAL ARTICLE on our experience, optimally, multiple laboratory tests should be done to identify, characterize, and confirm ALK expression, t(2;5), and variant translocations. In accordance with previous observations, our data showed a varied morphologic appearance of tumor cells. No morphologic correlation was seen between the ALK-1 immunohistochemical staining pattern, t(2;5), or other translocation variants. ALK expression is known to be associated with a favorable prognosis in ALCL. 23,24 The majority of our patients (17/21) were alive at 2- to 12-year follow-up periods. Two recent cases were in complete remission at 10 and 12 months of follow-up. Only 2 cases (3 and 13) died of disease. Falini et al 42 also found that the prognosis of ALCLs expressing non NPM-ALK fusion proteins resembled that of typical NPM-ALK+ cases and proposed that the expression of any ALK fusion protein in a T cell (or possibly NK cell) sets the same process of oncogenic transformation in motion regardless of the molecular mechanism. They also pointed out that these tumors together constitute a homogeneous group of good-prognosis neoplasms that should be distinguished from ALK ALCL. In our study, all patients (7/7) with confirmed NMP-ALK/t(2;5)(p23;q35) were alive at 2 to 12 years of follow-up. Among 6 cases of ALK variant, 4 patients were alive at 3 to 12 years of follow-up. One was in complete remission at 10 months of follow-up. Another (case 3) died 1 year after diagnosis. However, the number of patients in our study is too small to make any conclusions regarding prognosis. A larger study with long-term followup is required to confirm the observation by Falini et al. 42 Our study confirms that most pediatric ALCL cases are ALK+. At the mutational level, ALK variants are not uncommon. Our study also underscores the usefulness of integrating immunohistochemical, cytogenetic, and molecular genetic approaches for the detection, characterization, and confirmation of t(2;5) and its variants. From the Departments of 1 Pathology and 3 Pediatrics, University of Colorado School of Medicine, Denver; 2 Department of Pathology and 4 Section of Hematology/Oncology, the Children s Hospital, Denver; 5 UniPath, Denver; 6 Division of Pediatric Hematology/Oncology, Rhode Island Hospital/Brown Medical School, Providence; 7 Childhood Hematology/Oncology Associates, Denver; and 8 University of Florida College of Medicine, Gainesville. Address reprint requests to Dr Liang: Dept of Pathology, the Children s Hospital, 1056 E 19th Ave, B120, Denver, CO References 1. 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