The transcription factor BCL6 has an important role in

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1 Original Articles Fluorescence Immunophenotyping and Interphase Cytogenetics (FICTION) Detects BCL6 Abnormalities, Including Gene Amplification, in Most Cases of Nodular Lymphocyte-Predominant Hodgkin Lymphoma Alexei G. Bakhirev, MD; Mohammad A. Vasef, MD; Qian-Yun Zhang, MD, PhD; Kaaren K. Reichard, MD; David R. Czuchlewski, MD Context. BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. Objective. To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. Design. We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. Results. BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. Conclusions. To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique. (Arch Pathol Lab Med. 2014;138: ; doi: / arpa oa) The transcription factor BCL6 has an important role in orchestrating normal lymphocyte and immune system function. It is essential for the formation of germinal centers and control of pathways involved in cellular differentiation and proliferation. 1 Rearrangements and mutations of BCL6 at chromosome 3q27 have been implicated in several lymphoid neoplasms. BCL6 translocations occur in up to 30% of diffuse large B-cell lymphomas 2,3 and are among the genetic aberrations present in some cases of double hit B- cell lymphoma. 4 BCL6 rearrangement has also been detected in a subset of follicular lymphomas, especially Accepted for publication June 6, From the Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque. Dr Reichard is now with the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. The authors have no relevant financial interest in the products or companies described in this article. Presented in abstract form (platform presentation) at the European Association for Hematopathology 2010 Lymphoma Symposium and XVth Annual Meeting; September 28, 2010; Uppsala, Sweden. Reprints: Alexei G. Bakhirev, MD, Department of Pathology, University of New Mexico Health Sciences Center, 1001 Woodward Pl NE, Albuquerque, NM ( alexei.bakhirev@tricore.org). some cases of grade 3 follicular lymphoma and those lacking BCL2 rearrangement. 3,5 9 Furthermore, BCL6 mutations occur in diffuse large B-cell lymphomas and other types of germinal center or postgerminal center derived lymphomas; this has been suggested as a mechanism for aberrant BCL6 expression, although the presence of similar changes following somatic hypermutation in benign B cells complicates the interpretation of this finding. 1,10 14 Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is another B-cell lymphoma of germinal center stage differentiation in which BCL6 translocations have been identified. Wlodarska and colleagues 15,16 detected BCL6 translocations in 48% of NLPHL cases and subsequently characterized some of the translocations as t(3;22)(q27;q11), t(3;7)(q27;p12), t(3;9)(q27;p13), complex t(3;7;3;1), and t(3;4)(q27;q32). Renne et al 17 studied 24 cases of NLPHL and identified 5 cases (21%) with the t(3;14)(q27;q32) juxtaposing IGH and BCL6. Because the neoplastic cells are widely dispersed in NLPHL, a technique known as fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) has been used to assist in fluorescence in situ hybridization (FISH) analysis in these cases. 538 Arch Pathol Lab Med Vol 138, April 2014 Fluorescence Immunophenotyping Bakhirev et al

2 Progressive transformation of germinal centers (PTGCs), a presumptive reactive change, may be observed in the background of follicular hyperplasia (FH). Especially when florid or prominent, PTGCs maintain a nebulous relationship with NLPHL. Nodular lymphocyte-predominant Hodgkin lymphoma may occur in up to 15% to 20% of patients with notable PTGCs and may precede, coexist with, or follow the latter diagnosis Morphologically, the differential diagnosis between florid PTGCs and NLPHL can occasionally be challenging. 21 Notably, a single case of PTGCs has been shown to harbor a translocation t(3;22)(q27;q11), potentially involving BCL6 and thus possibly linking PTGCs with NLPHL at the molecular level. 22 Recently, it has been proposed that an intrafollicular variant of NLPHL is an early step in the evolution of the neoplasm and that this stage may bear some relationship to PTGCs. 23 In light of these observations, we analyzed BCL6 by FICTION in a series of well-characterized cases of NLPHL and FH with prominent PTGCs. MATERIALS AND METHODS Case Selection and Review Formalin-fixed, paraffin-embedded tissue blocks corresponding to 24 lymph node biopsy specimens were retrieved from the archives of the Department of Pathology at the University of New Mexico Health Sciences Center, with prior approval by the institutional review board. The samples included 11 cases of NLPHL and 5 cases of FH with prominent PTGCs (ie, enlarged nodules at least twice the size of surrounding germinal centers, containing residual germinal center elements among a proliferation of small lymphocytes with an obscure mantle zone boundary, representing.10% of follicles in a longitudinal section), as well as 8 cases of FH without PTGCs as negative controls. Hematoxylineosin stained slides and routine immunohistochemical studies that were performed at the time of original diagnosis were independently reviewed by 4 hematopathologists for diagnostic confirmation. Nodular lymphocyte-predominant Hodgkin lymphoma was defined according to the 2008 World Health Organization classification. 24 All the reviewers (MV, Q-YZ, KR, DC) agreed with the original diagnosis in all cases. Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms FICTION analysis was performed on 5-lm paraffin sections with modification of a protocol described previously. 25 Briefly, paraffin sections mounted on slides were subjected to antigen retrieval in a pressure cooker using 1mM EDTA solution. Heating time was set at 4 minutes after 1208C was reached. Following the depressurizing and cooling, slides were washed in phosphate-buffered saline and incubated with CD20 1:20 phosphate-buffered saline solution (Dako, clone L26; Dako, Carpinteria, California) for 1 hour. After washing with phosphate-buffered saline 0.5% Tween-20, the slides were subjected to the application of a tyramide signal amplification kit with Alexa-350 according to the manufacturer s protocol (Invitrogen, Molecular Probes, Irvine, California). The slides were then incubated with protease II (0.05 mg/ml in 0.05M Tris-HCl buffer (Sigma-Aldrich, St. Louis, Missouri) for 16 minutes before application of FISH probes to achieve sufficient cell membrane permeability without significant detrimental effect on Alexa-350 membrane signal. FISH analysis used a locus-specific identifiers BCL6 dual-color break-apart rearrangement probe set and, when necessary, a chromosome 3 centromere chromosome enumeration probe (D3Z1) (Vysis; Abbott Molecular, Des Plaines, Illinois). Lymphocyte-predominant cells (LP cells, also known as L&H cells) were identified by relative size (2 times larger than background B lymphocytes), positivity for CD20 or Alexa-350, and a surrounding partial or complete rim of unlabeled T cells. In FH cases, analysis focused on large CD20-positive cells within germinal centers (usually corresponding to centroblasts). In cases with PTGCs, the analysis concentrated on the progressively transformed germinal centers, and the largest CD20-positive cells present in these areas were counted. At least 100 cells were manually analyzed in each case by a single primary reader (AB). In addition, 2 secondary readers and 2 pathologists (DC, KR) experienced in clinical FISH analysis collectively reviewed most cases. BCL6 Immunohistochemistry To assess BCL6 protein expression, immunohistochemistry for BCL6 (Dako) was performed in NLPHL cases using an automated immunohistochemistry protocol (Ventana Medical Systems, Tucson, Arizona) with standard 5-lm paraffin sections. The slides were then digitally scanned using an automated scanner (Aperio Scanscope; Aperio, Vista, California) and quantitatively analyzed for signal intensity in LP cells using the manufacturer s brown (Diaminobenzidine) chromagen pixel intensity algorithm. At least 20 LP cells per case were assessed. Average staining intensity of the LP cells on each slide was then corrected for the background intensity of unlabeled small lymphocytes on the slide, yielding a corrected BCL6 intensity score for each case. RESULTS Characteristics of patients with NLPHL (age, sex, and site of biopsy) are listed in the Table. In all cases, features of NLPHL were present in accord with the 2008 World Health Organization criteria 24 (Figure 1). Successful concurrent immunofluorescent staining with CD20 and hybridization with the BCL6 probe set were achieved in all cases. In 5 of 11 cases of NLPHL (46%), separated signals indicated the presence of BCL6 rearrangement within CD20-positive LP cells (Figure 2). In 4 NLPHL cases, additional fused BCL6 signals were present in LP cells. In 3 of those 4 cases (27% of the total NLPHL cases), the extra signals were tightly clustered; up to 8 extra signals were present in some cells, with an average of 4 to 6 per cell (Figure 3). In these cases, additional analysis with the D3Z1 chromosome 3 centromeric probe revealed no evidence of chromosome 3 aneuploidy, consistent with BCL6 amplification. In the additional case with extra BCL6 signals, the pattern showed fused signals that were widely separated throughout the nucleus, numbering up to 10 per cell (average, 5 per cell). FISH for D3Z1 revealed multiple extra copies of the chromosome 3 centromeric region in this case (average, 6 per cell) on a slide from a separate deeper section, indicating the presence of extra copies of chromosome 3. None of the PTGCs or FH cases showed BCL6 abnormalities. Immunohistochemistry for BCL6 protein in NLPHL cases showed higher levels of expression in cases with BCL6 interphase FICTION abnormalities than in those without (corrected BCL6 expression score, 63 versus 18) (Figure 4); statistical significance was not calculated because of the limited number of cases under review. COMMENT BCL6 translocations have been detected in 21% to 48% of cases of NLPHL in prior studies Evaluation of BCL6 in NLPHL via routine FISH is hindered by the relative scarcity of neoplastic cells. In the prior studies of NLPHL, and in the present study, the FICTION combination has proven valuable. In this assay, a fluorescently labeled monoclonal antibody and FISH probes designed to fluoresce at different wavelengths are simultaneously applied to the tissue section. The cells of interest are identified by immunoflu- Arch Pathol Lab Med Vol 138, April 2014 Fluorescence Immunophenotyping Bakhirev et al 539

3 Patient No. orescence microscopy, and the pattern of FISH probes in these cells is assessed using filters for the appropriate wavelength (Figure 2). This permits targeted assessment of neoplastic cells even when these cells are few in number or exist in a reactive cellular background. Our study demonstrated BCL6 translocations in 45% of NLPHL cases, consistent with prior reports. 15,16 However, to the best of our knowledge, the BCL6 gene amplification that was identified in 27% of our NLPHL cases is a novel finding that has not been previously reported. The explanation for the absence of this finding in the 47 cases previously described in the literature is not clear. Notably, data from one of the prior studies include evidence of some cells with extra BCL6 signals that were not further characterized, leaving open the possibility that amplification was present. 15 Because our protocol for FICTION was somewhat different from the protocols previously used, it is important to note that additional BCL6 signals consistent with amplification were not seen in any small, reactive lymphocytes in the NLPHL cases or in any case of FH. Thus, we are confident that our modified FICTION protocol did not result in falsepositive results for gene amplification. The detection of multiple copies of a given gene in interphase FISH analysis does not always indicate pathologic gene amplification; in particular, extra copies of the chromosome on which the gene resides may be present, and the detected extra gene copy may be simply a passenger on the extra chromosome. We detected a case in which extra copies of BCL6 correlated with extra copies of chromosome 3 (as shown with D3Z1), indicating that aneuploidy for chromosome 3 may have accounted for the BCL6 signal pattern. In contrast, 3 cases showed large, tight clusters of multiple BCL6 probe signals, with up to 8 signals per cluster, in the absence of increased copies of the D3Z1 region (Figure 3). This pattern is consistent with gene amplification. We hypothesized that BCL6 rearrangement or amplification could lead to changes in protein expression that could be detected by immunohistochemistry. In fact, BCL6 expression, as determined by digital image analysis, was higher in NLPHL cases with BCL6 FISH abnormalities than in those without BCL6 abnormalities. However, because so few of our cases fell into the latter category, the significance of this observation is uncertain. In addition, the difference in Characteristics of Patients With NLPHL and FISH Results Age, y Sex Site of Biopsy FISH Result BCL6 Probe Pattern CEP3 Probe Pattern 1 30 Male Left axillary mass BCL6 rearranged Separated NT 2 32 Female Left inguinal lymph node BCL6 rearranged Separated NT 3 29 Male Right inguinal lymph node BCL6 rearranged Separated NT 4 62 Female Right submandibular BCL6 rearranged Separated NT lymph node 5 55 Female Right iliac lymph node Polyploidy for chromosome 3 10 Signals, no clustering (mode ¼ 5) Increased (mode ¼ 6) 6 59 Female Right axillary lymph node BCL6 amplified 8 Clustered signals Normal (mode ¼ 4) 7 36 Male Left submandibular lymph Normal Normal NT node 8 66 Male Left parotid lymph node BCL6 amplified 8 Clustered signals Normal (mode ¼ 6) 9 61 Male Right axillary lymph node BCL6 amplified 8 Clustered signals Normal (mode ¼ 6) Female Left cervical lymph node BCL6 rearranged Separated NT Female Left axillary lymph node Normal Normal NT Abbreviations: CEP3, centromeric probe for chromosome 3; FISH, fluorescence in situ hybridization; NLPHL, nodular lymphocyte-predominant Hodgkin lymphoma; NT, not tested. BCL6 staining intensity was difficult to detect on visual microscopic inspection (Figure 4). BCL6 encodes a zinc finger transcriptional repressor protein essential for B-cell maturation in the germinal center. 1 As a transcriptional repressor, BCL6 decreases expression of its targets, which may include as many as 500 genes. 26 BCL6 protein is expressed in germinal center B cells and is thought to facilitate an environment of physiologic genomic instability associated with the germinal center environment. 27 Various B-cell lymphomas, including follicular lymphoma and diffuse large B-cell lymphoma, have been shown to harbor abnormalities of BCL6 that result in constitutive expression and consequent inhibition of apoptosis and differentiation. 1,27 BCL6 translocation is a frequent mechanism of this dysregulation. More than 20 translocation partners have been described, including both immunoglobulin and nonimmunoglobulin genes, 28 making targeted assessment of the translocation partner challenging. In this clinical setting, a break-apart probe strategy to identify BCL6 rearrangements by interphase FISH is commonly used. Progressive transformation of germinal centers is a reactive process, but in some cases, especially if florid, it may exhibit morphologic and clinical overlap with NLPHL and pose a diagnostic challenge. 29 No specific marker to differentiate between PTGCs and NLPHL exists, and hematopathologists rely on combinations of morphologic, immunohistochemical, and clinical findings to render the correct diagnosis. 21 A single case of florid PTGCs has been reported to show a t(3;22)(q27;q11) translocation, likely affecting BCL6 and raising the possibility that a subset of PTGCs could represent a precursor lesion of NLPHL. 22 Similarly, an early intrafollicular stage of NLPHL has recently been proposed. 23 In our examination of cases with PTGCs, we did not detect BCL6 rearrangement or other abnormalities, although this does not exclude the possibility that examination of additional cases could uncover evidence of such a change and support a cytogenetic relationship between PTGCs or intrafollicular neoplasia and frank NLPHL. Given the available evidence, FICTION for BCL6 abnormalities could have clinical value in supporting a diagnosis of NLPHL in these types of challenging cases, especially those in which apparent PTGCs follow an 540 Arch Pathol Lab Med Vol 138, April 2014 Fluorescence Immunophenotyping Bakhirev et al

4 Figure 1. Nodular lymphocyte-predominant Hodgkin lymphoma is characterized by large popcorn cells with highly convoluted nuclear contours (black arrows) (hematoxylin-eosin, original magnification 3600). Figure 2. A, BCL6 rearrangement is indicated by separated red and green signals with the break-apart BCL6 probe (yellow arrows). B, Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) confirms positivity for CD20 as revealed by immunofluorescence (seen here as the central ring of blue staining) and ring of surrounding lymphocytes that do not label with CD20 (original magnification 31000). Figure 3. Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) detects multiple copies of BCL6. The signals are tightly clustered, consistent with gene amplification (original magnification 31000). Figure 4. Immunohistochemistry for BCL6 in a case of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) with BCL6 translocation identified by fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms FICTION (a) and in a case of NLPHL without BCL6 amplification or translocation (b) (original magnifications 3600). Arch Pathol Lab Med Vol 138, April 2014 Fluorescence Immunophenotyping Bakhirev et al 541

5 established diagnosis of NLPHL. T-cell and histiocyte rich large B-cell lymphoma may also enter into the differential diagnosis of some cases of NLPHL, particularly in cases with limited sampled tissue. 30,31 In this context, it is notable that BCL6 rearrangement has not been strongly associated with T-cell and histiocyte rich large B-cell lymphoma. 31 The clinical significance of BCL6 rearrangements remains uncertain. The literature reveals a wide spectrum of findings from good to poor prognoses in better-studied entities such as diffuse large B-cell lymphomas While NLPHL is generally considered responsive to therapy and rarely fatal, with a low rate of progression to large B-cell lymphoma, cases of transformation are described We did not formally assess survival or disease progression as a function of BCL6 rearrangement in our study population because of the few cases without BCL6 abnormality. Although some progress has been made in developing pharmacologic BCL6 antagonists, these have not yet been introduced into clinical practice. 1,39 In conclusion, we developed a FICTION protocol that includes tyramide signal amplification and used this technique to discover evidence of BCL6 abnormalities (including the novel observation of gene amplification) in more than three-quarters of the studied NLPHL cases. These changes were not seen in cases with only progressively transformed germinal centers. While at present FICTION is not widely used in the clinical setting, this approach could be useful in the diagnosis of NLPHL, particularly in difficult or borderline cases. Optimized CD20 FICTION might also be applicable to some types of diffuse large B-cell lymphoma in which neoplastic cells are more widely dispersed. This study was supported by the University of New Mexico Department of Pathology Foucar Endowment Fund. References 1. Wagner SD, Ahearne M, Ko Ferrigno P. 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