CCND1-IGH Fusion-Amplification and MYC Copy Number Gain in a Case of Pleomorphic Variant Mantle Cell Lymphoma

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1 AJCP /CASE REPORT CCND1-IGH Fusion-Amplification and MYC Copy Number Gain in a Case of Pleomorphic Variant Mantle Cell Lymphoma Yuan Miao, MD, 1,2 Pei Lin, MD, 1 Wei Wang, MD, 1 L. Jeffrey Medeiros, MD, 1 and Xinyan Lu, MD 1 From the 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston; and 2 Department of Pathology, The First Affiliated Hospital and the College of Basic Medical Science of China Medical University, Shenyang. Key Words: Mantle cell lymphoma (MCL); CCND1-IGH gene fusion-amplification; MYC copy number gain; CD5 negative; Pleomorphic variant Am J Clin Pathol December 2016;146: DOI: /AJCP/AQW194 ABSTRACT Objectives: Mantle cell lymphoma (MCL) may present de novo or undergo progression to a clinically aggressive variant, known as a blastoid or pleomorphic variant. We report an unusual case of classic MCL in a 78-year-old man with a typical immunophenotype, including CD5 positivity, who subsequently relapsed with CD5-negative pleomorphic variant MCL. Methods: Biopsy specimens were evaluated using Wright- Giemsa stained or H&E-stained sections, flow cytometry, immunohistochemistry, conventional cytogenetic, nextgeneration sequencing, and fluorescence in situ hybridization. Results: The patient continued to be refractory to intensive chemotherapy and radiation therapy. Initial conventional cytogenetic analysis showed a complex karyotype with amplification of the CCND1-IGH fusion gene on the der(14): 44, Y, t(x;2)(p22.3;q21), del(2)(p21), del(6)(p23), add(7)(p22), 9, del(9)(p22), add(11)(q13), 13, add(14)(p11.2), der(14)t(11;14)(q13;q32)hsr(14)(q32), add(18)(q23), add(21)(p11.1), 22,þmar[12]. A repeat biopsy revealed MCL, pleomorphic variant, with loss of CD5 expression and extra copies of the MYC. Mantle cell lymphoma (MCL) represents 3% to 10% of all cases of non-hodgkin lymphoma. Virtually all cases carry t(11;14)(q13;q32)/ccnd1-igh and overexpress cyclin D1. 1,2 Morphologically, the classic type is characterized by small cells with hyperchromatic nuclei. Blastoid and pleomorphic variants of MCL are aggressive variants and usually accompanied by complex genetic changes and sometimes an atypical immunophenotype. 3 The presence of MYC abnormalities, including MYC copy number gain and MYC rearrangement, have been correlated with these morphologic variants and predict poorer survival in patients with MCL. 4 The occurrence of an amplified CCND1-IGH fusion is very rare in lymphomas. This event has been described in one case of MCL and one case of plasma cell leukemia; both patients had an aggressive clinical course. 5,6 To our knowledge, there are no reports of patients with MCL who had CCND1-IGH fusion gene amplification associated with blastoid or pleomorphic variant MCL. In this study, we report a unique MCL case with an amplified CCND1-IGH fusion gene. At the time of relapse, the MCL had morphologic features of a pleomorphic variant, CD5 expression was lost, and MYC copy number gain was identified. Conclusions: CCND1-IGH fusion-amplification with MYC copy number gain is extremely rare and may play a role in disease progression in a subset of MCL cases. Case Report A 78-year-old man developed symptoms of increased fatigue, dyspnea, and drenching night sweats in September American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com 747 Am J Clin Pathol 2016;146: DOI: /ajcp/aqw194

2 Miao et al / CCND1-IGH FUSION-AMPLIFICATION AND MYC COPY NUMBER GAIN IN MCL Physical examination revealed a 4-cm left groin mass and a 5-cm upper right arm mass. Laboratory studies showed that the WBC count was /L (reference range, /L) with many lymphoma cells identified and a serum lactic dehydrogenase level of 1,100 IU/L (reference range, IU/L). Subsequent bone marrow aspiration and biopsy confirmed the diagnosis of MCL (classic, CD5þ). The patient underwent six cycles of bendamustine and rituximab and two cycles of rituximab and hyperfractionated cyclophosphamide but did not have any response. In July 2015, the patient was treated with three cycles of rituximab, gemcitabine, cisplatin, and dexamethasone but again failed to respond, and his disease continued to progress. In October 2015, after progression, a repeat biopsy sample taken from the lymph node of the left pelvis and a lateral retroperitoneal mass showed CD5- negative pleomorphic MCL. The patient received radiation therapy to the right upper arm, left groin, and left abdominal mass. However, a subsequent positron emission tomography scan displayed overall progression. The patient then received salvage chemotherapy with dexamethasone, rituximab, lenalidomide, and bortezomib. The chemotherapy was eventually discontinued due to development of severe and persistent neuropathy. The patient was discharged to an inpatient hospice in December Materials and Methods Morphology and Immunohistochemistry Wright-Giemsa stained bone marrow aspirate smears and H&E-stained histologic sections of bone marrow, left pelvic lymph node, and lateral retroperitoneal mass were reviewed. Immunohistochemical stains were performed using formalin-fixed, paraffin-embedded tissue sections. The antibodies used were specific for PAX5, CD5, CD10, CD79a, and Ki-67 (DAKO, Carpinteria, CA); SOX11 and c-myc (Ventana Medical Systems, Tucson, AZ); and cyclin D1 (NeoMarkers, Fremont, CA). Flow Cytometry Immunophenotypic Analysis The diagnostic bone marrow (BM), peripheral blood (PB), and lateral retroperitoneal biopsy specimens at relapse were assessed by flow cytometry immunophenotyping. The antibody panel included reagents specific for CD5, CD11c, CD19, CD20, CD22, CD23, CD25, CD30, CD38, CD43, CD44, CD79b, CD81, CD103, CD200, FMC-7, and surface immunoglobulin j and k light chains. All antibodies were obtained from Becton-Dickinson Biosciences (San Jose, CA). Conventional Cytogenetic and Fluorescence In Situ Hybridization Analyses Conventional cytogenetic analysis was performed at the time of the initial diagnosis, according to the standard laboratory procedures. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature Fluorescence in situ hybridization (FISH) analysis using a dual-color/dual-fusion probe set for CCND1-IGH rearrangements; a dual-color, break-apart rearrangement probe for BCL2 and MYC; and a panel of five locus-specific probes (ATM, TP53, D12Z3, D13S319, andlamp1 loci) for chronic lymphocytic leukemia (CLL) were performed on cultured initial diagnostic bone marrow cells. All probes were purchased from Abbott Molecular (Des Plaines, IL). Molecular Analysis Genomic DNA was extracted from the initial diagnostic bone marrow aspirate. A panel of 53 genes targeting TP53, ATM, KIT, NPM1, KRAS, NRAS, IDH1, APC, TET2, ASXL1, and other genes was assessed using a next-generation sequencing based assay as described previously. 8 Results Morphology The diagnostic bone marrow aspirate smears showed many monotonous small lymphoma cells, and the biopsy section showed complete replacement by lymphoma cells with mature chromatin and scant cytoplasm. After progression, left pelvic lymph node biopsy and lateral retroperitoneal mass biopsy specimens showed diffuse sheets of large lymphoid cells with irregular nuclear contours, occasionally prominent nucleoli, and moderate cytoplasm Image 1. Immunophenotypic Analysis Initially, flow cytometric immunophenotyping of the BM aspirate specimen showed that the lymphocytes were positive for CD5, CD19, CD20, CD22, FMC7, and immunoglobulin k light chain and were negative for CD11c, CD23, CD25, CD43, CD103, CD200, and immunoglobulin j light chain. Flow cytometric immunophenotyping in the PB showed 83% lymphoma cells positive for CD5, CD19, CD20, CD22, CD43 (dim), CD79b, CD81, and immunoglobulin k light chain and negative for CD3 and immunoglobulin j light chain. After progression, flow cytometric analysis of the lateral retroperitoneal biopsy specimen showed a population of aberrant B cells positive for CD11c (partial), CD19 (dim), CD20 (dim), CD22 (partial), CD Am J Clin Pathol 2016;146: American Society for Clinical Pathology 748 DOI: /ajcp/aqw194

3 AJCP / CASE REPORT B C D E F A Image 1 Morphology and immunocytochemistry. H&E staining in bone marrow: low-power (A) and high-power (B) H&E staining in a lateral retroperitoneal biopsy specimen (C) and immunohistochemical stains of cyclin D1 (D), MYC (E), and Ki-67 (F) in a lateral retroperitoneal biopsy specimen (A, 100; B-F, 400). American Society for Clinical Pathology 749 Am J Clin Pathol 2016;146: DOI: /ajcp/aqw194

4 Miao et al / CCND1-IGH FUSION-AMPLIFICATION AND MYC COPY NUMBER GAIN IN MCL A B C E D Image 2 Fluorescence in situ hybridization (FISH) and cytogenetic analysis. Conventional karyotype results (A). FISH with the CCND1-IGH dual-color, dual-fusion, locusspecific probes in the bone marrow aspirate showing the presence of the t(11;14)(q13;q32) and amplification of CCND1-IGH fusion gene (B). Chronic lymphocytic leukemia (CLL) panel FISH results showed a normal signal patterns for ATM (green) and TP53 (red; C). CLL panel showed a heterozygous D13S319 deletion (red) and no evidence of trisomy 12 (green) or deletion of the LAMP1 gene (aqua; D). MYC FISH using the dual-color break-apart probe showing four copies of the MYC gene (arrows) with no evidence of rearrangement (E). (1,000) 750 Am J Clin Pathol 2016;146: American Society for Clinical Pathology 750 DOI: /ajcp/aqw194

5 AJCP /CASE REPORT (partial), CD44, CD79b (dim), and monotypic immunoglobulin k light chain and negative for CD5, CD10, CD23, CD30, CD43, and CD200. Immunohistochemical analysis of the left pelvic lymph node biopsy sections showed the lymphoma cells were positive for CD79a and cyclin D1 and negative for CD5 and CD10. In the lateral retroperitoneal mass biopsy slides, the lymphoma cells were positive for PAX5, cyclin D1, and MYC and negative for SOX11. The proliferation (Ki-67) index was about 80% (Image 1). Conventional Cytogenetic, FISH, and Molecular Analyses Conventional cytogenetic analysis on the diagnostic BM aspirate specimen showed a complex karyotype containing t(11;14)(q13;q32) with a homogeneously staining region (HSR) involving an CCND1-IGH amplification: 44, Y, t(x;2)(p22.3;q21), del(2)(p21), del(6)(p23), add(7)(p22), 9, del(9)(p22), add(11)(q13), 13, add(14)(p11.2), der(14) t(11;14)(q13;q32)hsr(14)(q32), add(18)(q23), add(21) (p11.1), 22,þmar[12]. Amplified nuclear fusion signals were observed in 90% of the interphases Image 2A. FISH on the initial BM aspirate specimen confirmed the CCND1-IGH fusion-amplification tobeanhsronchromo- some 14q32 Image 2B. A CLL panel showed no evidence of TP53 or ATM deletions Image 2C but a heterozygous D13S319 deletion in 76% of cells, which was consistent with the monosomy 13 Image 2D. The results of other loci for the CLL FISH panel were all negative. The 53-gene mutation panel showed no evidence of any mutations for the genes targeted, including TP53 and ATM. After progression, additional FISH analysis on the lateral retroperitoneal biopsy specimen showed no evidence of MYC or BCL2 rearrangement but with extra copies of MYC (arrows; Image 2E ). No materials from the lymph node biopsy specimen were available for chromosome analysis or for other FISH and molecular testing. Discussion WereportauniquecaseofMCLwiththeamplificationof the CCND1-IGH fusion and MYC copy number gain. The patient had aggressive disease and a refractory clinical course. After treatment, the tumor lost CD5 expression and histologically progressed from classic MCL to a pleomorphic variant. Previous studies have shown that aggressive variants of MCL arise in a patient with a previously documented classic MCL. However, the immunophenotype of the transformed disease usually is similar to its predecessor. 9 Although CD5 expression of lymphoid cells is sometimes negative in aggressive variants, CD5 loss has not been associated specifically with a pleomorphic variant. 10 Our patient represents a rare event in MCL with CD5 expression converting from positive to negative during morphologic transformation. Pleomorphic and blastoid variants of MCL are nearly always accompanied by great genetic complexity. 3 MYC abnormalities, including MYC copy number gain and MYC rearrangement, have been associated with aggressive variants of MCL and likely contribute to its adverse clinical outcome. 4 Interestingly, in the present case, using conventional karyotyping and FISH, the amplified CCND1-IGH rearrangement formed an HSR at 14q32. Gene fusion-amplification in general is a very rare event in hematologic malignancies. To our knowledge, only two cases of CCND1-IGH fusionamplification have been reported, one in plasma cell leukemia and one in leukemic MCL. 5,6 Including our case, both CCND1-IGH fusion-amplification MCL cases showed additional complex chromosomal changes. However, the previously reported MCL case also had TP53 gene mutation and amplification of the ATM gene; the composite changes make it difficult to ascertain whether CCND1-IGH fusionamplification was the cause for the rapid clinical progression. 5 In our patient, although there were very complex chromosomal aberrations, the patient had no other mutations or amplifications except for extra MYC copies or heterozygous D13S319 deletion, and it is likely that the CCND1-IGH fusion-amplification along with a complex karyotype may lead to an aggressive clinical course. In summary, we present an unusual case of MCL associated with CCND1-IGH fusion-amplification, CD5 antigen loss, transformation to a pleomorphic variant, and MYC copy number gain. This patient had a very aggressive clinical course. To our knowledge, this is the first case of MCL associated with CCND1-IGH fusion-amplification that subsequently transformed to a pleomorphic variant. Although a single unusual case report cannot lead to a conclusion that CCND1-IGH gene fusion-amplification contributes to the histologic or immunophenotypic evolution in MCL, an integrated approach to MCL diagnosis, by incorporating clinical features, morphology, immunophenotype, and genetic/genomic findings, is needed to accurately diagnose and risk-stratify these unusual cases. Corresponding author: Xinyan Lu, MD, Dept of Hematopathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; xlu4@mdanderson.org. References 1. Vose JM. Mantle cell lymphoma: 2015 update on diagnosis, risk-stratification, and clinical management. Am J Hematol. 2015;90: American Society for Clinical Pathology Am J Clin Pathol 2016;146: DOI: /ajcp/aqw194

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