According to the World Health Organization (WHO)/

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1 ORIGINAL ARTICLE Primary Cutaneous Follicle Center Lymphomas Expressing BCL2 Protein Frequently Harbor BCL2 Gene Break and May Present 1p36 Deletion A Study of 20 Cases Vanessa Szablewski, MD,* Saskia Ingen-Housz-Oro, MD,w Maryse Baia, MSc,z Marie-Helene Delfau-Larue, MD, PhD,zy8 Christiane Copie-Bergman, MD, PhD,z8z and Nicolas Ortonne, MD, PhDz8z Abstract: The classification of cutaneous follicular lymphoma (CFL) into primary cutaneous follicle center lymphoma (PCFCL) or secondary cutaneous follicular lymphoma (SCFL) is challenging. SCFL is suspected when tumor cells express BCL2 protein, reflecting a BCL2 translocation. However, BCL2 expression is difficult to assess in CFLs because of numerous BCL2 + reactive T cells. To investigate these issues and to further characterize PCFCL, we studied a series of 25 CFLs without any extracutaneous disease at diagnosis, selected on the basis of BCL2 protein expression using 2 BCL2 antibodies (clones 124 and E17) and BOB1/BCL2 double immunostaining. All cases were studied using interphase fluorescence in situ hybridization with BCL2, BCL6, IGH, IGK, IGL breakapart, IGH-BCL2 fusion, and 1p36/1q25 dual-color probes. Nineteen CFLs were BCL2 positive, and 6 were negative. After a medium follow-up of 24 (6 to 96) months, 5 cases were reclassified as SCFL and were excluded from a part of our analyses. Among BCL2 + PCFCLs, 60% (9/15) demonstrated a BCL2 break. BCL2-break positive cases had a tendency to occur in the head and neck and showed the classical phenotype of nodal follicular lymphoma (CD10 +, BCL6 +,BCL2 +,STMN + )compared with BCL2-break negative PCFCLs. Del 1p36 was observed in 1 PCFCL. No significant clinical differences were observed between BCL2 + or BCL2 PCFCL. In conclusion, we show that a subset of PCFCLs harbor similar genetic alterations, as observed in nodal follicular lymphomas, including BCL2 breaks and 1p36 deletion. As BCL2 protein expression is usually associated with the presence of a BCL2 translocation, fluorescence From the *Pathology Department, CHU Montpellier, Gui de Chauliac Hospital, Montpellier; wdermatology Department; yimmunology and Hematology Department; zpathology Department, AP-HP, Groupe Henri Mondor-Albert Chenevier; zinserm, U955 team 9; and 8Medical University, Paris Est Creteil university (UPEC), UMR-S, Cre teil, France. C.C.-B. and N.O. contributed equally. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationship with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Nicolas Ortonne, MD, PhD, Department of Pathology, Hôpital Henri Mondor, 51 Avenue du Mare chal de Lattre de Tassigny, Créteil 94010, France ( nicolas.ortonne@hmn.aphp.fr). in situ hybridization should be performed to confirm this hypothesis. Key Words: primary cutaneous follicle center lymphoma, BCL2, stathmin, FISH, BCL2 rearrangement, del 1p36 (Am J Surg Pathol 2016;40: ) According to the World Health Organization (WHO)/ European Organization for Research and Treatment of Cancer (EORTC) classification, primary cutaneous B-cell lymphomas (PCBCLs) represent a heterogenous group of non-hodgkin lymphomas occurring in the skin without evidence of extracutaneous disease at the time of diagnosis and within the first 6 months after diagnosis. 1 PCBCLs include 3 main histologic subtypes: primary cutaneous follicle center lymphoma (PCFCL), primary cutaneous marginal zone lymphoma (PCMZL), and primary cutaneous diffuse large B-cell lymphomas, leg type (PCLBCL, leg type). PCFCL is a tumor of neoplastic follicle center cells, including centrocytes and variable numbers of centroblasts, with a follicular and/or diffuse growth pattern, which usually involve the head or trunk of middle-aged adults. 2,3 Immunophenotypically, the tumor cells in PCFCL express CD20 and BCL6, whereas CD10 is often positive in cases with a follicular growth pattern and generally negative in cases with a diffuse growth pattern. 2,3 PCFCL is considered a distinct entity from nodal follicular lymphoma (NFL) in the WHO classification. Nevertheless, the classification of follicular lymphoma (FL) with initial skin involvement (cutaneous follicular lymphoma [CFL]) into PCFCL or secondary cutaneous follicular lymphoma (SCFL) remains challenging, but a complete clinical staging at diagnosis, including bone marrow biopsy, is actually controversial. Initially, most studies emphasized that, contrasting with NFLs, PCFCLs do not express BCL2 protein and lack t(14;18), 4 6 considered to be the hallmark of NFL. Thus, the distinction between PCFCL and SCFL in skin biopsies is usually based on BCL2 protein expression by Am J Surg Pathol Volume 40, Number 1, January

2 Szablewski et al Am J Surg Pathol Volume 40, Number 1, January 2016 neoplastic B cells, which usually correlates with the BCL2 translocation. 7,8 However, evaluation of BCL2 protein expression in CFL neoplastic cells is challenging because of numerous reactive T cells that normally express the BCL2 protein. Moreover, recent studies on BCL2 status in PCFCL have given conflicting results, some reporting the presence of BCL2 protein expression and/or BCL2 gene break in a significant number of cases. 2,9 11 Besides t(14;18), other genetic alterations have been reported in NFL, including BCL6/3q27 rearrangement 12,13 and 1p36 deletion (del 1p36), 14,15 the incidence of which in PCFCL has not been well investigated. The aim of the study was to better characterize a series of CFLs expressing BCL2 protein compared with BCL2-negative cases using both immunohistochemistry, including double BOB1/BCL2 immunostaining and the recently described germinal center marker STMN1 (stathmin), and interphase fluorescence in situ hybridization (FISH) for BCL2, BCL6, IGH, IGK, IGL breaks, and 1p36 deletion. MATERIALS AND METHODS Patient Selection Nineteen patients with CFL expressing BCL2 protein, diagnosed in the Department of Pathology, Henri Mondor Hospital, Cre teil, France, between 2009 and 2013, were prospectively included. Six cases of CFL without BCL2 protein expression were also included and served as the control group. In each case, the following clinical data were collected: age, sex, clinical presentation, site(s) of involvement, number of lesions, size of lesion, initial therapy, relapse, follow-up, and clinical status at last follow-up. The staging procedure at the time of diagnosis in all cases included physical examination, computed tomography and/or gallium scan, peripheral blood cell count, and serum lactate dehydrogenase. Eleven patients had bone marrow biopsy. Cases were classified as PCFCL when there was no evidence of systemic disease at diagnosis, within the first 6 months, and at the end of follow-up; otherwise, cases were classified as SCFL. Pathologic Review The histopathologic diagnosis of CFL was made on hematoxylin-eosin-safran stained tissue sections. All cases were reviewed by 3 pathologists (C.C.-B., N.O., and V.S.). The histopathologic diagnosis of cutaneous FL was based on the WHO Classification of Hematopoietic and Lymphoid Neoplasm (4th ed, 2008) and the EORTC classification. Cases were classified into 3 grades, based on the numbers of large cells (centroblasts), according to the criteria of Mann and Berard. Grades 1 and 2 were included within a single group, denominated grade 1/2 (low-grade lymphoma). Immunohistochemistry Three-mm-thick tissue sections cut from formalinfixed and paraffin-embedded cutaneous biopsies were subjected to antigen retrieval and were immunostained using a BOND-III Autostainer (Leica Microsystems, Newcastle-upon-Tyne, UK). The following antibodies were used after appropriate antigen retrieval: CD20 (clone L26; Dako Cytomation, Glostrup, Denmark), CD5 (clone 4C7; Leica Microsystems), CD10 (clone 56C6; Leica Microsystems), BCL6 (clone LN22; Leica Microsystems), MUM1 (clone MUM1p; Dako Cytomation), CD23 (clone 1B12; Leica Microsystems), CD21 (clone 2G9; Leica Microsystems), and Ki-67/MIB1 (clone MIB-1; Dako Cytomation). BCL2 protein expression was studied with 2 anti-bcl2 antibodies, clone 124 (Dako Cytomation) and clone E17 (Epitomics, Burlingame, CA). Moreover, to assess BCL2 protein expression by neoplastic B cells and to discriminate from reactive BCL2 + T cells, double staining with BOB1 (clone TG14, mouse monoclonal antibody; Leica Microsystems) and BCL2 (clone 124) was performed. BCL2 was detected using immunoperoxidase, and BOB1 staining relied on alkaline phosphatase. For MUM1 and Ki-67, we evaluated the percentage of positive B cells. The usefulness of the germinal center marker STMN1 (stathmin) in distinguishing FLs from marginal zone lymphoma has been recently reported. 16 Thus, we studied the expression of STMN1 in our CFL series and in 5 PCMZLs and 4 PCLBCLs, leg type, and in 1 case with skin involvement by B-cell chronic lymphocytic leukemia, including foci of large cell transformation in the skin (Richter syndrome), which were retrospectively retrieved from archival material. STMN1 expression was quantified using a Z score system as follows: Z score = P+I, with P being the proportion of positive B cells, evaluated semiquantitatively into 4 categories (1, <5%; 2, 5% to 50%; 3, >50%; and 4, 100%), and I being the staining intensity, evaluated semiquantitatively into 3 categories (1, low; 2, intermediate; and 3, strong). Interphase FISH Interphase FISH was performed on 3-mm-thick tissue sections using split-signal FISH DNA probes for BCL2/18q21, BCL6/3q27, IGH/14q32, IGK/2p11, IGL/ 22q11 (probes Y5407, Y5408, Y5406, Y5416, Y5412; Dako A/S), dual-color, dual Fusion Translocation Probe Vysis LSI IGH/BCL2 (probe 08L60; Abbott Molecular diagnostic, Rungis, France), and dual-color probe Zytolight SPEC 1p36/1q25 (probe Z-2075; Zytovision, Clinisciences, France) according to the manufacturer s instructions. At least 100 intact nuclei per case were evaluated independently by 2 scorers (M.B. and V.S) on a Zeiss AxioImager Z1 (Zeiss, Go ttingen, Germany) fluorescence microscope. Images were captured with a 40 objective using a Hamamatsu digital camera attached to the fluorescence microscope and Visilog 6.9 software (FEI, Les Ulis, France). For BCL2 and BCL6 split-signal FISH DNA probes, scoring of the hybridization signals was performed as previously described. 17 For IGH, IGK, and IGL, cases were considered positive when >20% of cells displayed split signals. For del 1p36 assessment, the ratio between orange (1p36) signal and green signal (1q25) was 128

3 Am J Surg Pathol Volume 40, Number 1, January 2016 BCL2+ Primary Cutaneous Follicle Center Lymphomas TABLE 1. Clinical Features of the 25 Patients With CFL CFL PCFCL (n = 20) All Patients (n = 25) All Patients BCL2 + (n = 15) BCL2 (n = 5) SCFL (n = 5) Mean age (range) (y) 65 (40-80) 63.5 (40-80) 57 (40-77) 69 (65-80) 70 (54-73) Male sex (n [%]) 15 (60) 11 (55) 8 (53.3) 3 (60) 4 (80) Localization (n [%]) Head and neck 16 (64) 12 (60) 11 (73.3) 1 (20) 4 (80) Other 9 (6) 8 (40) 4 (26.7) 4 (80) 1 (20) P = Type of lesion (n [%]) Nodules 21 (84) 17 (85) 12 (80) 5 (100) 4 (80) Plaques 2 (8) 1 (5) 1 (6.7) 0 (0) 1 (20) Plaques and nodules 2 (8) 2 (10) 2 (13.3) 0 (0) 0 (0) Initial management (n [%]) Observation 1 (4) 1 (5) 1 (6.7) 0 (0) 0 (0) Excision 8 (32) 7 (35) 5 (33.3) 2 (40) 1 (20) Excision+radiotherapy 8 (32) 7 (35) 5 (33.3) 2 (40) 1 (20) Excision+rituximab 3 (12) 3 (15) 2 (13.3) 1 (20) 0 Excision+chemotherapy 3 (12) 1 (5) 1 (6.7) 0 (0) 2 (40) Rituximab alone 1 (4) 1 (5) 1 (6.7) 0 (0) 0 (0) Chemotherapy alone 1 (4) 0 (0) 0 (0) 0 (0) 1 (20) Outcome (n [%]) RC at last follow-up 21 (84) 19 (95) 14 (93.3) 5 (100) 2 (40) AWD at last follow-up 4 (16) 1 (5) 1 (6.7) 0 (0) 3 (60) Relapse or progression 7 (28) 5 (25) 3 (20) 2 (40) 2 (40) P = Median follow-up (range) (mo) 24 (6-96) AWD indicates alive with disease. evaluated in 100 tumor nuclei. The cutoff level for del 1p36 was defined as the mean ratio plus 3 SDs in 5 formalin-fixed and paraffin-embedded non-neoplastic tissue (2 reactive lymph nodes and 3 tonsils) and in 5 classical FLs lacking 1p36 deletion demonstrated by conventional cytogenetics and was determined to be Statistical Analyses The Fisher exact test was used to calculate the P-value with GraphPad Prism 6.0, GraphPad. Results were considered significant at P < RESULTS Clinical Features and Follow-up Clinical features and follow-up are summarized in Table 1. Twenty-five patients were included. After a median follow-up of 24 months (range, 6 to 96 mo), 20 patients were retrospectively classified as having PCFCL. The 5 remaining patients were classified as SCFL because of systemic disease revealed during follow-up and were excluded from a part of our analyses. The mean age of patients with PCFCL was 63.5 years (range, 40 to 80 y) with a male predominance (sex ratio: M:F 1.22:1). PCFCL with BCL2 protein expression (BCL2 + PCFL, n = 15) had a tendency to involve the head and neck more frequently compared with PCFCL without BCL2 protein expression (BCL2 PCFCL, n = 5) (73.3% vs. 20%, P = ). No significant difference was observed in either group with respect to initial management, and most patients underwent local therapy. At last follow-up, all patients including BCL2 and BCL2 + PCFCL except 1 were in complete remission (RC) (93.3% vs. 100%, ). Pathologic and Phenotypic Findings The main pathologic and phenotypic findings are summarized in Table 2. Representative pictures of 2 BCL2 + PCFCL, with or without BCL2 rearrangement, are shown in Figure 1. BCL2 protein expression was clearly established in 19 cases using exhaustive immunohistochemistry (anti- BCL2 clones 124 and E17, and BOB1/BCL2 double immunostaining). The 6 remaining cases were confirmed as negative, and no BCL2 pseudonegative CFL was actually diagnosed using the E17 clone. In most samples 129

4 Szablewski et al Am J Surg Pathol Volume 40, Number 1, January 2016 TABLE 2. Pathologic, Phenotypic, and Cytogenetic Features of the 25 Patients With CFL Groups Morphology (Positive/Total [%]) Architecture Histologic Grade Phenotype (Positive/Total [%]) Some Follicular Area Purely Diffuse P Grade 1/2 Grade 3 P CD20 CD5 CD10 P STMN1 Total CFL (N = 25) 13/25 (52) 12/25 (48) 21/25 (84) 4/25 (16) 25/25 (100) 1/24 (4.2) 17/25 (68) 20/20 (100) PCFCL (N = 20) 10/25 (50) 10/20 (50) 18/20 (90) 2/20 (10) 20/20 (100) 0/19 (0) 13/20 (65) 15/15 (100) PCFCL BCL2 + 8/15 (53.3) 7/15 (46.7) /15 (93.3) 1/15 (6.7) /15 (100) 0/14 (0) 11/15 (73.3) /10 (100) (N = 15) PCFCL BCL2 2/5 (40) 3/5 (60) 4/5 (80) 1/5 (20) 5/5 (100) 0/5 (0) 2/5 (40) 5/5 (100) (N = 5) SFCL (N = 5) 3/5 (60) 2/5 (40) 3/5 (60) 2/5 (40) 5/5 (100) 1/5 (20) 4/5 (80) 5/5 (100) Phenotype (Positive/Total [%]) Cytogenetics (Positive/Total [%]) Groups BCL6 BCL2 CD23 MUM1 BCL2 Break P IGH/BCL2 Translocation BCL6 Break IGH Break IGK Break IGL Break 1p36 Deletion Total CFL (N = 25) 23/24 (95.8) 19/25 (76) 13/25 (52) 7/20 (35) 12/25 (48) 11/24 (45.8) 2/25 (8) 0/12 (0) 0/12 (0) 0/12 (0) 1/23 (4.3) PCFCL (N = 20) 19/19 (100) 15/20 (75) 11/20 (55) 6/17 (35.3) 9/20 (45) 8/19 (42.1) 0/20 (0) 0/10 (0) 0/10 (0) 0/10 (0) 1/18 (5.6) PCFCL BCL2 + (N = 15) 15/15 (100) 15/15 (100) 9/15 (60) 4/13 (30.8) 9/15 (60) /14 (57.1) 0/15 (0) 0/5 (0) 0/5 (0) 0/5 (0) 1/13 (7.7) PCFCL BCL2 4/4 (100) 0/5 (0) 2/5 (40) 2/4 (50) 0/5 (0) 0/5 (0) 0/5 (0) 0/5 (0) 0/5 (0) 0/5 (0) (N = 5) SFCL (N = 5) 4/5 (80) 4/5 (80) 2/5 (40) 1/3 (33.3) 3/5 (60) 3/5 (60) 2/5 (40) 0/2 (0) 0/2 (0) 0/2 (0) 0/5 (0) double staining helped in the analysis of BCL2 positivity within neoplastic B cells. In some BCL2 + PCFCL cases, we observed a heterogenous BCL2 expression, with a higher intensity in the neoplastic B cells, as compared with the surrounding reactive lymphocytes. No significant difference was observed between BCL2 + PCFL and BCL2 PCFL concerning the growth pattern and histologic grade, with, respectively, 53.3% and 40% of cases having a nodular architecture () and 93.3% and 80% being grade 1/2 (P =0.4474). Interestingly, BCL2 + PCFCL had a tendency to express CD10 more frequently, in addition to BCL6 (classical FL phenotype), compared with BCL2 PCFL (73.3% vs. 40%, P = 0.29). MUM1 was also frequently expressed (19/22, 86%), although in most samples it was expressed in <25% of cells. In 1 case, the neoplastic B cells diffusely expressed MUM1 (70%). This case was an SCFL with a diffuse growth pattern on skin biopsy and with a diagnosis of diffuse large B-cell lymphoma (DLBCL) on a lung biopsy. The mean proliferative index evaluated with Ki-67 was 25%, ranging from 5% to 80% of neoplastic B cells. In most cases, the Ki-67 index was <25% (15/21, 71%). Among the 6 remaining cases, 2 were grade 3A, including the 1 with the highest Ki-67 index. All evaluable cases of CFL (BCL2+PCFCL, BCL2 PCFCL, and SCFL) expressed STMN1. No differences were observed between BCL2+ and BCL2 CFL. The 5 PCMZL cases showed STMN1 expression restricted to the residual germinal centers, as shown in Figure 2. Unexpectedly, all 4 cases of PCLBCL, leg type, strongly expressed STMN1. The mean Z scores were 4 in PCFCL (range, 2 to 6) versus 2.4 (range, 2 to 4) in marginal zone lymphomas and 5.3 (range, 3 to 6) in PCLBCL, leg type. FISH Studies BCL2 gene rearrangement was observed in 12 CFL cases (12/25, 48%), including 9 PCFCL and 3 SCFL cases, with additional copies of BCL2 gene (4 to 5 copies) in 2 cases (1 PCFCL, 1 SCFL) (Table 2 and Fig. 3). Eleven of these 12 cases were evaluable with a dual-color fusion probe for t(14;18)(igh/bcl2) and were positive. Cases showing no BCL2 rearrangement were studied with IGH, IGL, and IGK break-apart probes to search for other cytogenetic alterations that may involve the immunoglobulin genes. We did not observe any immunoglobulin gene breaks in these remaining cases. Nine BCL2+PCFCLs demonstrated a BCL2 break (9/15, 60%), whereas all BCL2 PCFCLs showed normal FISH signal pattern (Fig. 4). Two SCFLs displayed concurrent BCL2 and BCL6 gene rearrangements. A unique case harbored a deletion involving the 1p36 chromosomal region (Fig. 4). It was a BCL2 + PCFCL without any additional chromosomal abnormality of BCL2, BCL6, IGH, IGK, and IGL genes detectable with the present approach. Comparison of BCL2-break positive and BCL2-break negative PCFCL PCFCL cases with BCL2 breaks were compared with PCFCLs without BCL2 breaks (Table 3). No significant difference was observed with regard to the clinical outcome; most cases in both groups were in RC at last follow-up (88.9% vs. 100%, P = 0.45), and few patients 130

5 Am J Surg Pathol Volume 40, Number 1, January 2016 BCL2+ Primary Cutaneous Follicle Center Lymphomas FIGURE 1. PCFCLs showing a nodular growth pattern and CD10 and BCL2 expression. A, C, E,G, BCL2 + PCFCL without a BCL2 break. The nodular growth pattern is highlighted by the STMN1 staining shown in the inset (A). Neoplastic cells are small to medium-sized centrocytes grouped in nodules with no tingible bodies or macrophages, and few mitoses (C). BOB1 (red)/bcl2 (brown) double immunostaining showing strong BCL2 expression by neoplastic centrocytes (arrows) within the nodules, whereas BCL2-positive T cells are present in the vicinity. In contrast, the neoplastic cells in a BCL2 PCFCL (arrows) are shown in the inset (E). A heterogenous but significant expression of CD10 is observed in the neoplastic follicles (G). B, D, F, H, BCL2 + PCFCL with BCL2 break. This case shows a mixed, diffuse, and nodular growth pattern (B). The neoplastic cells are a mixture of small to large centrocytes and centroblasts (D). The BCL2 staining with the clone 124 shows a diffuse expression, encompassing both neoplastic B cells (arrows) and reactive T cells, the former showing a stronger expression (F). A significant CD10 expression is seen (H)

6 Szablewski et al Am J Surg Pathol Volume 40, Number 1, January 2016 FIGURE 2. STMN1 expression in PCBCLs. A, Grade 3A PCFCL showing a diffuse expression of STMN1 (Z score = 5). B, Grade 1/2 PCFCL with a weaker and heterogenous pattern of expression (Z score = 2). C, In this PCMZL, STMN1 expression is restricted to residual dissociated germinal centers. D, Strong STMN1 expression in a primary cutaneous DLBCL, leg type (Z score = 6). Immunohistochemistry with 3,3 -diaminobenzidine tetrahydrochloride chromogen. in both groups had relapse or progression (22.2% vs. 27.3%, P = ). Median follow-up was 48 months (6 to 54 mo) in the BCL2-break positive group versus 28 months (15 to 96 mo) in the BCL2-break negative group. BCL2-break positive PCFCLs had a tendency to involve the head and neck compared with BCL2-break negative 132

7 Am J Surg Pathol Volume 40, Number 1, January 2016 BCL2+ Primary Cutaneous Follicle Center Lymphomas FIGURE 3. FISH results for BCL2 and 1p36. A, PCFCL showing BCL2 rearrangement using a break-apart probe, with isolated green and red signals (arrows) in most cells. B, PCFCL with BCL2 rearrangement and amplification (arrow). C and D, In this PCFCL, a del 1p36 is seen with unique red signals (1p36) (arrows), whereas 2 green signals (1q25 control) are observed in tumor cell nuclei. PCFCLs (77.8% vs. 45.5%, P = ) and to significantly express the classical FL phenotype (CD10 +, BCL6 +, BCL2 + ) (100% vs. 18.2%, P = ). DISCUSSION In the present study, we reported the immunophenotypic and genetic features of PCFCLs with BCL2 protein expression (BCL2 + PCFCL) and compared them with PCFCLs without BCL2 protein expression (BCL2 PCFCL). We found that a majority of BCL2 + PCFCLs harbored the t(14;18)(igh-bcl2) translocation (9/15, 60%). We also reported for the first time that del 1p36 may be observed in PCFCL and is not limited to NFL. The characterization of BCL2 protein expression in CFL is often challenging because the neoplastic B cells are admixed with numerous reactive T cells that normally express this protein. This may explain why the real proportion of PCFCLs with BCL2 protein expression still remains controversial in the literature, some reporting that a large proportion of cases are seen to express BCL2 protein on immunohistochemistry and others showing that most, if not all, PCFCLs are BCL2 negative. 5,6,8,11,21 Double staining of BCL2 with BOB1 allowed to overcome this issue and to clearly distinguish BOB1 + /BCL2 + neoplastic B cells, BOB1 + /BCL2 nontumoral B cells, and BOB1 /BCL2 + bystander T cells. Another challenge regarding BCL2 protein expression using immunohistochemistry are the gene somatic mutations that may alter the antigen recognition by monoclonal antibodies. Lymphomas harboring such abnormalities were termed BCL2 pseudonegative NFL in the literature. 16,22 The use of 2 different anti-bcl2 clones is thus recommended. To note, we did not observe any pseudonegative case of CFL in this series. After a median follow-up period of 24 months, we could retrospectively classify CFLs into PCFCLs and SCFLs with good confidence and then focus our analyses on PCFCL. Using this approach, we observed that some PCFCLs do express 133

8 Szablewski et al Am J Surg Pathol Volume 40, Number 1, January 2016 TABLE 3. Comparison of PCFL BCL2-break Positive and Negative Groups BCL2-FISH Positive (N = 9 [%]) BCL2-FISH Negative (N = 11 [%]) P Histologic grade 1/2 8 (88.9) 10 (91) 1 3A 1 (1.1) 1 (9) Growth pattern Follicular 6 (66.7) 4 (36.4) Diffuse 3 (33.3) 7 (63.6) Immunopositive CD10 9 (100) 4 (36.4) BCL6 9 (100) 10/10 (100) BCL2 9 (100) 6 (54.5) Classical NFL 9 (100) 2 (18.2) phenotype (CD10 +, BCL6 +, BCL2 + ) Status at last follow-up CR 8 (88.9) 11 (100) 0.45 PR 1 (1.1) 0 (0) Relapse or progression Yes 2 (22.2) 3 (27.3) No 7 (77.8) 8 (72.7) Location of lesions Head and neck 7 (77.8) 5 (45.5) Other 2 (22.2) 6 (54.5) Median follow-up (range) (mo) 28 (15-96) 48 (6-54) CR indicates complete remission; PR, partial remission. BCL2 protein. Among 19 CFL cases with confirmed BCL2 protein expression, 15 (15/19, 79%) were finally classified as PCFCL, whereas 4 (4/19, 21%) were SCFLs. Thus, BCL2 staining is not sufficient in routine practice for the diagnosis of SCFL. Comparison of BCL2 + PCFCL with BCL2 PCFCL showed that the former had a higher tendency to involve the head and neck. No significant difference was observed according to the histologic growth pattern and histologic grade, but interestingly BCL2 + PCFCL had a 19 patients with BCL2+ CFL (cases) Positive N=12/25 9 PCFL, 3 SCFL 25 patients Histological and FISH evaluation FISH analysis t(14;18) 6 patients with BCL2- CFL (controls) Clinical evaluation Negative N=13/25 11 PCFL, 2 SCFL 20 patients with PCFL: BCL2+ n=15 BCL2- n=5 5 patients with SCFL: BCL2+ n=4 BCL2- n=1 FIGURE 4. Flow chart showing the distribution of CFL with BCL2 rearrangement in the PCFCL and SCFL groups. tendency to express more frequently both germinal center markers CD10 and BCL6 compared with BCL2 PCFCL. Moreover, they usually harbored t(14;18)(igh- BCL2), considered to be the cytogenetic hallmark of NFL. 3 By contrast, none of the BCL2 PCFCLs disclosed any BCL2, IGK, IGL, or IGH rearrangement. These results suggest that FISH studies should be indicated only when a significant expression of BCL2 protein is shown in neoplastic B cells. In summary, neoplastic cells of PCFCL can express BCL2 protein and, when positive, usually harbor t(14;18)(igh-bcl2). Thus, the diagnosis of SCFL cannot rely with high confidence either on BCL2 immunostaining or on BCL2 gene alterations. We also compared BCL2-break positive and BCL2- break negative PCFCLs. Whereas for most authors, the finding of a t(14;18) in a CFL is a rare event and strongly suggests a secondary NFL, 4 8,11,21,23,24 others found that t(14;18) may be present in a subset of PCFCL. 2,10,18 20 Interestingly, Streubel et al 10 studied a series of 27 PCFCLs by means of FISH for t(14;18) and found this cytogenetic abnormality in 11 cases (41%). More recently, in a series of 53 patients with CFL unselected for BCL2 expression, Pham-Ledard et al 25 reported that the proportion of BCL2 + PCFCL carrying the t(14;18) translocation was 8.5%. They also observed that these variants expressed CD10. In our series, we clearly identified PCFCL associated with t(14;18). Whether they should be considered as a particular variant of PCFCL remains an interesting question. In our series, they had a tendency to more frequently occur on the head and neck, to more frequently demonstrate a follicular growth pattern, and to significantly express the typical NFL phenotype (CD10 +, BCL6 +, BCL2 + ). Besides t(14;18), found in about 85% of NFLs, other gene abnormalities have been described, such as BCL6 gene rearrangement and del 1p36. 12,26 28 BCL2 gene amplifications have rarely been reported in NFL. 29,30 Interestingly, we observed additional copies of the BCL2 gene (4 to 5 copies) associated with BCL2 gene break in 2 cases, 1 PCFCL and 1 SCFL. Other genetic alterations included BCL6 gene rearrangement, observed in 2 SCFLs, both in association with a t(14;18). In their series of PCFCLs, Streubel et al 10 found a translocation affecting BCL6 in 7% of cases (2/27). However, BCL6 translocation was the sole abnormality and so far occurred in cases of PCFCL. We did not identify isolated IGH, IGK, origl gene rearrangement but found, for the first time to our knowledge, a del 1p36 in 1 PCFCL that occurred as the sole genetic event. Katzenberger et al 26 previously identified a distinct subgroup of NFLs with a predominantly diffuse growth pattern, involving the inguinal region, lacking the classical t(14;18) and harboring del 1p36. They speculated that the del 1p36 may constitute a primary cytogenetic aberration in this particular subtype of NFL. Nevertheless, 1p36 alteration is also one of the most frequent genetic abnormalities in NFL occurring in 30% to 67% of cases in addition to the classical t(14;18). 15,31,32 Altogether these results show that PCFCLs share cytogenetic abnormalities with NFLs, in

9 Am J Surg Pathol Volume 40, Number 1, January 2016 BCL2+ Primary Cutaneous Follicle Center Lymphomas cluding BCL2 rearrangement and amplification, and 1p36 deletion, which therefore cannot be used for the characterization of SCFL in skin biopsies. In this series, we also investigated the expression of the germinal center marker STMN1 in CFL. The differential diagnosis between PCFCL and other cutaneous B- cell malignancies may represent, in some instances, a diagnostic challenge, as PCFCLs typically lack CD10 and BCL2 protein expression that are phenotypic markers of NFL. STMN1 expression has been studied in nodal lymphomas and now represents a novel useful diagnostic marker in distinguishing NFL from marginal zone lymphoma. 16 Nevertheless, it has never been investigated in CFL. We demonstrated that all PCFCLs from this series expressed STMN1, with no differences between BCL2 + and BCL2 PCFCL. The 5 PCMZL cases, used as the control group, were negative, except in the residual germinal centers. Therefore, STMN1 also appears to be a useful marker for discriminating between PCFCL and PCMZL. Another diagnostic pitfall is the differentiation of PCFCL predominantly composed of large centrocytes/ centroblasts from PCLBCL, leg type. The latter may actually express BCL6, and, as shown in this series, PCFCL like PCLBCL, leg type may comprise a proportion of MUM1 + cells, may have a high proliferative index, and can express BCL2. Surprisingly STMN1 was strongly expressed in the 4 PCLBCL, leg type cases studied. Interestingly, it has been recently shown that PCLBCL, leg type is associated with somatically hypermutated immunoglobulin variable genes, indicating that neoplastic B cells have experienced germinal center reaction. 33 Whether these findings will explain why neoplastic B cells in this lymphoma can express some germinal center markers such as BCL6 and STMN1 is an interesting issue. On pathologic grounds, even if further investigations are needed, it appears that STMN1 is not a discriminant marker to differentiate between PCFCL and PCLBCL, leg type. The search for the MYD88 L265P mutation remains thus the best tool in this context, shown to be present in 70% of PCLBCL, leg type and absent in PCFCL. One of the most challenging issues in CFL is the final classification into PCFCL or SCFL. This differentiation has a strong prognostic impact, as PCFCLs most often show an excellent prognosis and an indolent behavior, whereas NFLs may transform into aggressive DLBCLs with a median survival rate of 40% to 85% at 10 years, depending on the International Prognosis Index at diagnosis. 34 In the classification proposed by the EORTC-Cutaneous Lymphoma Project Group, PCFCL is described as having a diffuse pattern of growth in the great majority of cases, with no expression of CD10 and BCL2 protein in most cases and without t(14;18). 1,35 Nevertheless, in this series half of the PCFCLs had a follicular growth pattern, and 45% showed a BCL2 translocation. Although our cases were included on the basis of BCL2 protein expression, PCFCL may share more morphologic, phenotypic, and genetic features with NFL than initially believed, and clinical staging and follow-up appears to be the main criterion for the classification of CFL into PCFCL or SCFL. These results further raise the question about the clinical staging at diagnosis in PCFCL, including bone marrow biopsy that is actually not systematically performed. Whether a bone marrow biopsy and/or a closer follow-up should be performed in patients with BCL2 + PCFCL appears questionable, as recently discussed by others. 25 To note, most patients with PCFCL were in RC at last follow-up, including those with BCL2 rearrangement with a median follow-up of 48 months (6 to 54 mo) in this subgroup. Therefore, the molecular abnormality is not sufficient to drive malignancy. In agreement with this hypothesis, it was actually found in minor B-cell populations of normal healthy individuals. In conclusion, we showed that t(14;18) may be observed in PCFCL and is not the hallmark of secondary CFL. The presence of t(14;18) in PCFCL strongly correlates with head and neck involvement and with the classical FL immunophenotype CD10 +, BCL6 +, BCL2 +, and STMN1 +. 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