Primary Cutaneous Follicle Center Lymphoma Associated With an Extracutaneous Dissemination

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1 AJCP / Case Report Primary Cutaneous Follicle Center Lymphoma Associated With an Extracutaneous Dissemination A Cytogenetic Finding of Potential Prognostic Value Shivakumar Subramaniyam, PhD, Cynthia M. Magro, MD, Swarna Gogineni, MBBS, Wayne Tam, MD, PhD, and Susan Mathew, PhD From the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY. Key Words: Complex translocation; Primary cutaneous follicle center lymphoma; Extracutaneous dissemination; Prognosis Am J Clin Pathol November 2015;144: ABSTRACT Objectives: Cytogenetic studies on cutaneous lymphomas are rare, and very little is known about their prognostic value. We present a rare case of primary cutaneous follicle center lymphoma (PCFCL) with a complex translocation presenting with cutaneous and extracutaneous dissemination in the lymph node. Methods: Morphologic, immunohistochemical, conventional cytogenetic, and fluorescence in situ hybridization (FISH) studies were performed on this patient. Results: A combination of cytogenetic and FISH analysis identified a complex novel four-way t(2;14;9;3) (p11.2;q32;p13;q27) translocation involving rearrangements of BCL6, immunoglobulin light and heavy chain genes, and an unknown gene on 9p. Conclusions: Our report elaborates the morphologic and immunohistochemical features in combination with cytogenetic and molecular cytogenetic analysis of PCFCL, which provide additional insight into the clinical and biologic behavior of this lesion. Primary cutaneous follicle center lymphoma (PCFCL) is the most common form of primary cutaneous B-cell lymphoma, representing approximately 60% of all B-cell lymphomas. Critical to diagnosing any lymphoma as a primary cutaneous one is the absence of any extracutaneous involvement at the time of presentation. 1 PCFCL exhibits a varied morphology that differs between cases. The architecture ranges from nodular to diffuse, while the cytomorphology can (1) be predominated by small cleaved lymphocytes, (2) represent a mixture of small and larger lymphocytes, or (3) be dominated by large cleaved lymphocytes and/or centroblasts. The more common morphologic expression of PCFCL is in the context of a nodular growth pattern recapitulating germinal centers, comprising a mixture of small centrocytes and larger centroblastic forms. The neoplastic cells express clusters of designation (CD), CD20, CD79a, and BCL6. Both BCL2 and CD10 are variable in expression, unlike the ubiquitous expression of both markers in primary nodal B-cell lymphoma. Only 30% of cases will show a phenotypic profile analogous to that noted in nodal follicular lymphoma (FL). Most cases of PCFCL do not express IRF4/ MUM1 and Fox-P As in most indolent lymphoma, BCL2, BCL6, and MYC genes are also involved in cutaneous B-cell lymphoma. 5 Of PCFCLs with a follicular growth pattern, 10% to 40% show BCL2 gene rearrangements detected by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. 6 In addition to BCL2 gene rearrangement, BCL6- IGH translocation (4%) and inactivation of CDKN2B (P15) (10%) and CDKN2A (P16) (30%) tumor suppressor genes by promoter hypermethylation are also reported. 6,7 The presence of BCL2 gene rearrangement is a finding that would be Am J Clin Pathol 2015;144:

2 Subramaniyam et al / Complex Translocation in Cutaneous Lymphoma A B C D Image 1 A, Nodular foci containing numerous large atypical-appearing cells in the 20- to 30-mm range were identified (H&E, 100). B, The large cell dominant nodular foci were positive for BCL2 ( 1,000). C, There was focal staining for BCL6 ( 1,000). D, The CD21 preparation highlighted the remnants of the disrupted follicular dendritic network amid the nodular large cell foci ( 400). more commonly seen in secondary nodal lymphoma involving the skin rather than PCFCL.6 The cytogenetic and molecular cytogenetic data on the PCFCLs are limited.8-11 Hallermann et al10 showed no rearrangement of IGH, BCL6, MYC, BCL2, and MALT1 genes in nine PCFCL cases studied. Array comparative genomic hybridization (acgh) analysis showed amplifications of chromosome 1q23-q25, 2p16.1, 7q21-q22, 10q24.2, 12p13.2-p13.33, 12p11.21-p11.22, 12q13-q14, 14q11.2q12, 18q21.31-q21.33, and 21q11.2-q22.3 regions and deletions of chromosome 6q, 8p23.1, and 14q32 regions.11 These findings suggest that the molecular pathogenesis and disease progression in PCFCLs are unclear. We present a case of a PCFCL with a novel four-way t(2;14;9;3) 806 Am J Clin Pathol 2015;144: (p11.2;q32;p13;q27) translocation involving BCL6, IGH, and IGK genes and an unknown candidate gene on 9p. To our knowledge, complex translocations of such type have not been described in PCFCLs. Materials and Methods Cytogenetic Analysis Lymph node cells were cultured unstimulated overnight. Harvesting, slide preparation, and GTG banding were performed as per standard methods. A minimum of 20 metaphase cells were evaluated, and karyotypes were written

3 AJCP / Case Report according to the guidelines of the International System for Human Cytogenetic Nomenclature (2013). 12 FISH FISH analysis was performed on the methanol/acetic acid (3:1 ratio) fixed cell pellets and formalin-fixed, paraffin-embedded (FFPE) tissue sections of the skin punch/ biopsy sample using the following probes: LSI IGK (Cytocell, Cambridge, England); MYC, BCL6, and IGH dualcolor break-apart probes (Abbott Molecular, Des Plaines, IL); CDKN2A (P16)/CEP 9 and IGH-BCL2 dual-color dual-fusion probes (Abbott Molecular); and PAX5/CEP 9 dual-color probes (Empire Genomics, Buffalo, NY) as per standard protocols. Two hundred interphase nuclei and five metaphase cells were analyzed for each probe. The identities of the chromosomes involved were established by the reverse 4,6-diamidino-2-phenylindole banding pattern obtained by the imaging analysis. Case Report A 62-year-old woman sought treatment for a left posterior auricular lesion in Initially, she thought it was a pimple, as the lesion did not extrude pus and did not resolve by itself. In 2008, she noticed growth of the lesion and onset of pruritus and tenderness locally and consulted a dermatologist for further evaluation. Her initial CBC was within normal limits. Histopathology and Immunohistochemistry A punch biopsy of the skin lesion was obtained from the left posterior ear in September The skin biopsy showed diffuse and nodular lymphohistiocytic and eosinophilic infiltrate involving the entire sample thickness. Nodular foci containing numerous large atypical-appearing cells in the 20- to 30-mm range were identified Image 1A. Immunohistochemical stains showed large cell dominant nodular foci positive for CD20 and CD79a with extensive staining for BCL2 Image 1B and CD43 and focal staining for BCL6 Image 1C. There was no immunoreactivity for CD10. Significant staining was not observed for MUM-1. CD23 was extensively positive, while the CD21 preparation highlighted the remnants of the disrupted follicular dendritic network amid the nodular large cell foci Image 1D. PCR studies for immunoglobulin heavy chain and T-cell receptor g chain gene rearrangements showed monoclonality for both genes. The morphologic and immunophenotypic findings on the skin biopsy specimen were diagnostic of PCFCL with a background of reactive T-cell rich lymphoid hyperplasia. Computed tomography (CT) scans and positron emission topography (PET) of the whole body performed after 7 months Image 2 Giemsa trypsin G-banding karyotype showing the abnormalities 47,XX,+X,t(2;14;9;3)(p11.2;q32;p13;q27),der(15) t(1;15)(q21;p11.2). identified no systemic disease. She was treated with local radiation therapy, and the skin lesions completely resolved. A repeat PET/CT scan after 8 months revealed a mildly enlarged hyperactive (standardized uptake value of 10) supraclavicular lymph node on the opposite side. Fine-needle aspiration cytology of the supraclavicular lymph node showed an atypical lymphoid population with atypical lymphocytes and macrophages. After 2 weeks, an excisional biopsy of the cervical lymph node was performed. The lymph node biopsy specimen demonstrated prominent follicles that were positive for BCL- 6, CD21, and immunoglobulin D but negative for CD10 and BCL2. Flow cytometric/immunophenotypic analysis on CD45 bright gating showed positivity for CD19, CD20, CD23, HLA- DR, and CD52. The B cells expressed polytypic surface immunoglobulin. PCR showed monoclonality for the IGH gene. PCR on the skin biopsy sample also showed monoclonality for the IGH gene and the same band as observed in the cervical lymph node. The morphologic and immunophenotypic findings on the cervical lymph node biopsy specimen were diagnostic of follicular lymphoma grade 3b. A bone marrow biopsy was performed 1 month after the lymph node biopsy specimen was studied. The aspiration yielded dry tap, and the marrow was clotted and inadequate for staging purposes. Cytogenetics and FISH Chromosomal analysis performed on the cervical lymph node biopsy sample exhibited the following karyotype: 47,XX,+X,t(2;14;9;3)(p11.2;q32;p13;q27),der(15)t(1;15) (q21;p11.2)[8]/46,xx[12] Image 2. FISH analysis did not detect rearrangements of MYC, PAX5, and CDKN2A (P16) genes in this sample. Interphase FISH analysis showed rearrangements of BCL6, IGH, and IGK genes. Metaphase FISH showed 3 IGK on der(14q), 5 IGH on der(9p), CDKN2A (P16) and PAX5 on der(3q), and 5 BCL6 on Am J Clin Pathol 2015;144:

4 Subramaniyam et al / Complex Translocation in Cutaneous Lymphoma A B 5 BCL6 5 IGK 5 IGH 3 IGH 3 IGK C 3 BCL6 PAX5 and CDK2NA der(2)t(2;3) der(14)t(2;14) der(9)t(9;14) der(3)t(3;9) Image 3 A, Partial karyotype showing abnormal chromosomes identified by Giemsa trypsin G-banding (upper panel). B and C, Fluorescence in situ hybridization and reverse 4,6-diamidino-2-phenylindole images of abnormal chromosomes (middle and lower panels). Figure 1 Ideogram (400-band level) of the chromosomes involved in the t(2;14;9;3) (p11.2;q32;p13;q27) translocation with the fluorescence in situ hybridization signals. Arrows indicate the translocated regions from one chromosome to the other. der(2p) chromosomes, establishing the four-way t(2;14;9;3) (p11.2;q32;p13;q27) translocation in this patient Image 3 and Figure 1. FISH assay ruled out IGH-BCL2 gene rearrangements in this sample (data not shown). FISH analysis on the FFPE skin tissue sections showed rearrangement of IGH and BCL6 genes, suggesting that the clone observed in the lymph node is of PCFCL in origin (data not shown). No rearrangement of IGH-BCL2 was observed in the skin biopsy specimen. The IGK FISH assay failed due to suboptimal hybridization quality. The patient completed three cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) with pegfilgrastim support followed by local irradiation. Follow-up CT/PET scans 1 year after initial treatment identified no significant lymphadenopathy or other masses. The patient is currently in remission 6 years after initial diagnosis and is being monitored for disease status. Discussion The diagnosis and classification of cutaneous B-cell lymphomas can be challenging, including the absolute distinction between primary cutaneous B-cell lymphoma and nodal B-cell lymphomas secondarily involving the skin. Furthermore, while most primary cutaneous B-cell lymphomas follow an indolent clinical course, remaining as isolated cutaneous lesions without any tendency toward extracutaneous dissemination, a subset of cutaneous B-cell lymphomas can manifest extracutaneous dissemination. Most of these lymphomas represent diffuse large cell B-cell lymphomas (DLBCLs) of leg type. 13 The more indolent lymphomas, specifically marginal zone lymphoma and follicle center lymphoma, rarely exhibit extracutaneous dissemination except those cases demonstrating blastic transformation. 14 An attempt has been made to predict the clinical course of primary cutaneous B-cell lymphomas based on cytogenetic and molecular cytogenetic studies. Perhaps the most important finding cytogenetically is the presence of a 9p21 deletion in cases of leg-type DLBCL, a finding that predicts a more aggressive clinical course. In fact, those cases of leg-type lymphoma without the 9p21 deletion are similar prognostically to marginal zone lymphoma and PCFCL. In our case, the dissemination to lymph nodes was a very unexpected finding for a PCFCL. Conversely, the morphology was not at all compatible with an aggressive variant of primary cutaneous B-cell lymphoma of leg type, a lymphoma that could be associated with nodal dissemination. Despite the large cell dominant morphology, features that were not supportive of the leg-type lymphoma included the nodularity of the infiltrate, the lack of greater positivity for BCL-6, and MUM-1 negativity. The absence of BCL2 translocation by FISH and lack of staining for CD10 would be more in keeping with a PCFCL as opposed to a nodal FL secondarily involving the skin. 15 We identified a novel four-way translocation, t(2;14;9;3) (p11.2;q32;p13;q27), involving IGK, IGH, BCL6, and an unknown gene on 9p in this patient. It is quite possible that 808 Am J Clin Pathol 2015;144:

5 AJCP / Case Report this unusual cytogenetic profile conferred a tendency toward a more aggressive disease. The presence of identical IGH and BCL6 gene rearrangements in the skin and lymph node biopsy specimens points toward a common B-cell clone. Even though BCL6 rearrangements are often reported in systemic lymphomas, such rearrangements are not described in PCFCL. However, the phenotypic and cytogenetic profile was typical for a PCFCL and not consistent with a systemic nodal FL based on the lack of staining for CD10 and absence of a BCL2 rearrangement. 3 Cytogenetically, there are two translocations, t(2;3) and t(9;14), occurring concurrently, which might have contributed to the pathogenesis of PCFCL in this patient. The molecular consequence of this four-way translocation results in deregulated expression of the BCL6 gene and an unknown gene on the 9p. Even though translocations t(2;3) and t(9;14) are described in FL, this case is unique because of a four-way translocation as well as the involvement of IGK and IGH genes. The BCL6 gene is rearranged with IGK, and IGH is rearranged with an unknown partner gene on 9p. The involvement of PAX5 and CDKN2A (P16) was ruled out by FISH (data not shown). The involvement of CDKN2B (P15) was not tested. The short arm of chromosome 9 is mostly deleted and rarely rearranged in lymphoid malignancies. 7 Chromosome 9p harbors PAX5, CDKN2B (P15), and CDKN2A (P16) genes, which are abnormal in most B-cell lymphomas. Translocations involving the 9p13 locus and IGH gene are described in 28 cases of B-cell lymphoma. 16 Of these, 21 cases showed PAX5-IGH gene rearrangement. The t(9;14) translocation deregulates the PAX5 gene and contributes to the pathogenesis of these lymphomas. Of the remaining seven cases, the partner gene on 9p13 is unraveled. In our case, PAX5 was not involved in the rearrangement but instead was translocated to the der(3) chromosome, which might have resulted in a cryptic loss of 9p, which could not be established in this study. Molecular analysis using chromosomal acgh will be helpful in elucidating a cryptic deletion on 9p. A 9p21 deletion is highly predictive of an aggressive clinical course, with a tendency toward extracutaneous dissemination in the setting of primary cutaneous B-cell lymphoma of leg type. Rearrangements of multiple genes in systemic lymphomas indicate poor prognosis, mainly when the MYC gene is involved. However, PCFCLs tend to have a good prognosis irrespective of their genetic status. 17 Despite the presence of a complex rearrangement involving several genes, our patient is currently in remission 6 years after initial diagnosis, although clearly this clinical course of nodal involvement is highly unusual in PCFCL, a neoplasm that in most cases remains confined to the skin. In the realm of primary cutaneous B-cell lymphoma, any lymphoma associated with extracutaneous dissemination as noted here would be considered a primary cutaneous hematopoietic neoplasm associated with an unusual and aggressive clinical course even though in this patient, multiagent chemotherapy induced clinical remission. However, this case emphasizes the importance of cytogenetic studies on primary cutaneous B-cell lymphomas as a means of predicting biological behavior. Just as a 9p21 deletion is prognostically significant in the setting of primary cutaneous B-cell lymphoma of leg type, one might speculate that multiple cytogenetic hits could also predict a more aggressive clinical course in the setting of PCFCL similar to the aggressive clinical course observed in the double-hit and triple-hit lymphomas that arise in lower grade nodal FL. 18 This finding must be studied in a larger cohort of patients with PCFCL, perhaps focusing on those PCFCLs that exhibit a more common indolent cutaneous confined course vs those manifesting extracutaneous dissemination. Corresponding author: Shivakumar Subramaniyam, PhD, Dept of Pathology and Laboratory Medicine, Winthrop University Hospital, 222 Station Place North, Suite 609, Mineola, NY 11501; ssubramaniyam@winthrop.org. References 1. Willemze R, Swerdlow SH, Harris NL, et al. Primary cutaneous follicle centre lymphoma. In: Swerdlow SH, Campo E, Harris NL, et al, eds. 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