Chromosomal anomalies in primary cutaneous follicle center cell lymphoma do not portend a poor prognosis Alya Abdul-Wahab, BSc, MBBS, MRCP, Soo-Yong Tang, FRCPath, DPhil, Alistair Robson, FRCPath, DipRCPath, Stephen Morris, MRCP, FRCR, Nita Agar, MBBS (Hons), FACD, PhD, E. Mary Wain, MD, FRCP, Fiona Child, MRCP, MD, Julia Scarisbrick, FRCP, Michael Neat, BSc (Hons), and Sean Whittaker, MD, FRCP London, United Kingdom Background: The t(14;18)(q32;q21) chromosomal translocation is found in the majority of nodal follicular lymphomas but only rarely in primary cutaneous follicle center cell lymphomas (PCFCL). Recent studies have postulated that the translocation is more prevalent in PCFCL than previously described and that it might be a molecular prognostic marker. Objectives: The purpose of our study was to analyze cases of PCFCL for the presence of a t(14;18) translocation using fluorescence in situ hybridization to detect balanced translocations involving either the BCL2 or MALT1 loci and to correlate the results with growth pattern, immunophenotype, and clinical outcome. Method: In all, 57 patients with PCFCL were extracted from our cutaneous lymphoma database. Retrospective analysis of clinical parameters including lesion type, location, diagnostic stage, lactate dehydrogenase, initial treatment, relapse rate, and survival was performed. Results: In all, 57 patients with PCFCL were included in this study. We detected 1 BCL2 chromosomal amplification, 4 translocations of BCL2, and 1 IGH/MALT1 translocation. Limitations: This was a case series retrospective study. Conclusions: PCFCL has an excellent 5-year overall survival (100% disease-specific survival). Chromosomal abnormalities of either BCL2 or MALT1 were detected in 10% of cases but do not correlate with a specific immune pathology or clinical outcome. ( J Am Acad Dermatol 2014;70:1010-20.) Key words: B-cell lymphoma; BCL2 translocation; disease-free survival; fluorescence in situ hybridization; primary cutaneous follicle center cell lymphoma; t(14;18) translocation.
P
rimary cutaneous follicle center cell lymphoma (PCFCL) is an indolent cutaneous B-cell lymphoma (bcl) with an excellent prognosis.1 It is most common in males and presents as isolated or grouped plaques and nodules, usually on the scalp, forehead, or trunk. PCFCL characteristically shows a follicular, mixed follicular and diffuse, or diffuse infiltrate of centrocytes and centroblasts.2
From Guy’s and St Thomas’ National Health Service Foundation Trust. Supported by the United Kingdom National Institute for Health Research Biomedical Research Center based at Guy’s and St Thomas’ National Health Service Foundation Trust. Conflicts of interest: None declared. Accepted for publication January 9, 2014. Reprint requests: Alya Abdul-Wahab, BSc, MBBS, MRCP, St John’s Institute of Dermatology, Guy’s and St Thomas’ National Health
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Abbreviations used: bcl: B-cell lymphoma EORTC: European Organization for the Research and Treatment of Cancer FISH: fluorescence in situ hybridization PCFCL: primary cutaneous follicle center cell lymphoma PCR: polymerase chain reaction WHO: World Health Organization
Service Foundation Trust, London SE1 7EH, United Kingdom. E-mail:
[email protected]. Published online March 28, 2014. 0190-9622/$36.00 Ó 2014 by the American Academy of Dermatology, Inc. http://dx.doi.org/10.1016/j.jaad.2014.01.862
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PCFCL can be distinguished from primary cutaneous non-Hodgkin lymphoma but have not been marginal zone lymphoma by the absence of lymshown in cases of PCFCL. phoplasmacytoid differentiation and expression of germinal center markers CD10 and bcl-6.3,4 METHODS Characteristically, PCFCL do not express bcl-2 proPatient selection tein or may only show faint bcl-2 expression by a The study group consisted of 57 cases of PCFCL, the minority of tumor cells.5 histology of which was reviewed by a dermatopatholAlthough the morphoogist (A. R.) and classified logic features of PCFCL can according to the World CAPSULE SUMMARY resemble those of nodal Health Organization (WHO) follicular lymphoma, nodal European Organization for PCFCL is the most common variant of follicular lymphoma usually the Research and Treatment cutaneous B-cell lymphoma. presents with widespread of Cancer (EORTC) classificaTen percent of cases showed evidence of lymphadenopathy and tion for cutaneous lymchromosomal abnormalities either bone-marrow involvement. phomas.2 In all cases, the involving BLC2 or MALT1 using FISH Although nodal follicular tumor was limited to the skin analysis. lymphoma often follows an and there was no evidence of indolent course, transformed Presence of chromosomal abnormalities extracutaneous spread at diagfollicular lymphoma, was not correlated with clinical outcome. nosis. All patients included in composed largely of centrothis study had a bone-marrow blasts, typically requires aspirate or trephine and rituximab, cyclophosphamide, Adriamycin, vincriscomputerized tomography scan ruling out systemic tine, and prednisone chemotherapy. In contrast, lymphoma. From 1980, all patients referred to our PCFCL has an excellent prognosis and is usually multidisciplinary cutaneous lymphoma clinic have effectively treated with either surgery or local radiobeen entered onto a cutaneous lymphoma database therapy producing durable responses.2 (ICARSIS), which supports a cutaneous lymphoma The pathogenesis of PCFCL remains unclear research tissue bank (Ethics Code NRES:07/H10712/ and the t(14;18) translocation, present in approx106). A censure date was set at June 2010. Clinical data imately 90% of nodal follicular lymphoma,6 has were collated using this database and correlated with generally only been detected at very low fremedical records. In cases with multiple biopsy speciquency in PCFCL using polymerase chain reaction mens, the original diagnostic biopsy specimen was (PCR)-based methods, including in our original used for analysis. study.7-11 In contrast, the t(14;18) translocation has been detected in approximately 20% to 30% of cases of de novo diffuse large B-cell lymphoma Immunohistochemistry and rare cases of chronic lymphocytic leukemia.12 Immunophenotyping was performed on paraffin However, some PCR-based studies have reported sections using antibodies against CD20, CD79a, CD3, the presence of t(14;18) in a higher proportion of CD10, CD21, CD23, bcl-2, bcl-6, MUM-1, cyclinD1, PCFCL cases (18%-34%).3,13-16 Indeed, Yang et al17 and Ki67. Briefly, tissue sections were deparaffinized reported that 53% of PCFCL had bcl-2 protein and rehydrated by incubating twice in Citroclear expression, t(14;18), or both. Recently, Streubel (TCS Biosciences Ltd, Botolph Claydon, United et al18 reported t(14;18) in 41% of 27 cases of Kingdom), then in decreasing concentrations of PCFCL using fluorescence in situ hybridization alcohol (100%, 100%, 70%, 70%, 50%) at 5 minutes (FISH) but interestingly they did not find PCR each. Heat-induced antigen retrieval was applied by evidence of a BCL2 rearrangement. Prompted by boiling in 0.01 mol/L tris(hydroxymethyl) aminotheir findings, we reviewed 57 cases of PCFCL and methane EDTA (pH 9.0), in a decloaking chamber analyzed their histologic, immunophenotypic, and (Biocare Medical Inc, Concord, CA) for 2 minutes at molecular features with a particular focus on BCL2 operating pressure. The slides were then cooled to and MALT1 (mucosa-associated lymphoid tissue room temperature and washed in phosphatelymphoma translocations) rearrangements using buffered saline. FISH. MALT-1 rearrangements were also analyzed After antigen retrieval, endogenous peroxidase based on the rationale that that they involve the was blocked by means of a commercial peroxidasesame chromosomal breakpoint and would indiblocking reagent (Dako Denmark A/S, Glostrup, cate chromosomal instability. MALT1 translocaDenmark). Slides were then incubated for 30 mitions have been associated with low-grade B-cell nutes at room temperature with the primary d
d
d
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Fig 1. Fluorescence in situ hybridization images using the IGH-BCL2 dual-color dual-fusion probe set. IGH-BCL2 cells showing 2 copies of IGH ( green) and BCL2 (red ) with no IGHBCL2 fusions (A) and IGH-BCL21 cells (arrowed ) in a primary cutaneous follicular lymphoma showing the 2 fusion/1 red/1 green signal pattern indicative of IGH-BCL2 rearrangement (B).
antibody. Antibody binding was detected by the 2stage peroxidase-based EnVision (Dako Denmark A/ S) method, and sections were counterstained with hematoxylin before mounting with an aqueous mountant (Gurr, Chadwell Heath, United Kingdom). In line with previous publications, bcl-2 expression was detected on the basis of more than 25% of tumor cells.4
Fluorescence in situ hybridization We received 3-m unstained FFPE sections accompanied by an appropriate immunohistochemically stained slide (CD20 or CD79a) to enable areas of tumor to be clearly identified. The slides for FISH were warmed to 578C, washed in xylene then ethanol, and allowed to air dry. They were then treated with 0.2 mol/L hydrochloric acid and subsequently washed in sterile water and treated using the Spot-light tissue pretreatment kit (Life Technologies, Carlsbad, CA). The slides were then washed in sterile
water and allowed to air dry before hybridization with FISH probes. FISH was carried out according to manufacturer’s protocols using the IGH-BCL2 dualcolor fusion probe set (Abbott Molecular Inc, Des Plaines, IL). The FISH slides were correlated with the immunohistochemically stained slide and analyzed using an epifluorescence microscope (Zeiss Ltd, Cambridge, United Kingdom) equipped with singleand dual-band pass filters (Abbott Molecular Inc). Wherever possible 3 separate areas of tumor were selected by each of 2 appropriately trained cytogeneticists and a total of 100 nuclei were analyzed under high power (3100 objective) for each case, as described in previous publications.19-21 The nuclei had to be nonoverlapping and harbor sufficiently bright signals to be included in the analysis (Fig 1). MALT1 rearrangements were detected using a dual-color, break-apart FISH probe for MALT1 (Dako Denmark A/S) and the FISH ancillary kit
Fig 2. Clinical photographs. A, Tumors of primary cutaneous B-cell lymphoma on the upper aspect of arm. B, Infiltrated erythematous plaques over the scalp in a patient presenting with primary cutaneous follicle center cell lymphoma.
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Alive; under follow-up No
62
44
47
38
47
57
RESULTS
LDH, Lactate dehydrogenase; N/A, not available; WBC, white blood cell count. *IGH/MALT translocation.
Diffuse Negative T1a 7.2 439
6.4 398
6.9 304
N/A N/A 61 64 4* 36
Nodule; trunk Nodule; head and neck Papules; head and neck Nodule; head and neck Nodule; head and neck
N/A N/A
(Dako Denmark A/S) following the vendor’s instructions. Briefly the slides were dewaxed in Citroclear (TCS Biosciences Ltd) and rehydrated through decreasing concentrations of alcohol. They were then heated in a microwave oven (10 minutes, 750W) in pretreatment buffer and then rinsed in wash buffer twice for 3 minutes each time. After pepsin digestion for 3 minutes at 378C, the slides were rinsed twice in wash buffer, again for 3 minutes each time. They were then dehydrated in increasing concentrations of alcohol and allowed to air dry. The hybridization mixture containing FISH probe(s), water, and hybridization buffer (in 1:2:7 ratio) were then applied, cover-slipped, and sealed with vulcanizing rubber. When the rubber cement had dried, slides were placed in a hybridizer (Dako Denmark A/S) and denatured at 828C for 5 minutes, followed by overnight hybridization at 378C. The next day, the slides were washed in stringency buffer at 658C for 10 minutes and rinsed in wash buffer twice for 3 minutes. Slides were mounted using Vectorshield fluorescence mounting medium containing DAPI (Vector Laboratories Inc, Burlingame, CA) and viewed using a fluorescence microscope equipped with a dual-pass fluorescein isothiocyanate/rhodamine filter and an ultraviolet longpass filter (Chroma Technology Corp, Bellows Falls, VT).
Not specified Excision
Alive; under follow-up Radiotherapy No Monoclonal Diffuse Negative T1a
Follicular Monoclonal T2a
Negative
Radiotherapy No
Alive; under follow-up Alive; recurrence treated with radiotherapy Alive; under follow-up elsewhere Radiotherapy No Excision Yes Follicular Monoclonal Follicular Monoclonal Negative Positive T2b T1a
Clinical recurrence Treatment at diagnosis Clonality Growth pattern Stage at bcl-2 ) presentation expression 9
LDH WBC (240-480 IU/L) (4.0-11.0 3 10 Clinical (lesion; location) Case no. Age, y
Table I. Characteristics of patients with primary cutaneous follicle center cell lymphoma with IGH/BCL2 or IGH/MALT1 translocations
Outcome
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Clinical features In all, 57 patients (26 female, 31 male) were assessed retrospectively. The median age at diagnosis was 52.8 years (range, 38-83 years). The initial skin lesions were localized to the head and neck region (n = 28), trunk (n = 14), or the upper extremities (n = 6). One patient presented with disease in the groin. Multifocal disease was only present in 14% (n = 8). Clinically lesions included papules, nodules, plaques, and tumors (Fig 2). Three patients presented with subcutaneous nodules confirmed to represent disease arising in the subcutis rather than secondary nodal deposits as extensive staging investigations were normal.22 Based on the International Society for Cutaneous Lymphomas EORTC staging proposal for nonmycosis fungoides/Sezary syndrome cutaneous lymphomas, 25 (45%) patients were staged as T1a, 4 (7%) as T1b, 17 (30%) as T2a, 3 (5%) as T2b, 7 (12%) as T3a, and 1 (1%) as T3b at diagnosis. No nodal or visceral disease was detected in any patients with normal staging computerized tomography scans and bone-marrow biopsy specimens at diagnosis. Hematologic investigations showed no evidence of lymphocytosis in any case but serum lactate dehydrogenase was elevated in
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Fig 3. Kaplan-Meier curves. A, Overall and disease-specific survival for cohort of patients with primary cutaneous follicle center cell lymphoma (PCFCL). B, Disease-free survival (time to development of first disease recurrence) in PCFCL cohort.
77% cases. Borrelia serology was negative in all cases. Protein electrophoresis was normal in all cases. The clinical features of patients within the cohort with a translocation or amplification in BCL2 or MALT1 are shown in Table I. The median age of diagnosis was 61.5 years and 5 of 6 lesions were localized to the head and neck region. Four cases were stage T1a, 1 was T2a, and 1 was T2b. All cases were treated with either radiotherapy or surgical excision and only 1 case developed a clinical recurrence. The recurrence developed 2 years after initial diagnosis and was treated successfully with superficial electron beam radiotherapy.
Clinical outcomes The preferred treatment modality for localized disease is radiotherapy, with a dose of at least 30 Gy and a margin of clinically uninvolved skin of at least 1 to 1.5 cm.23 In our cohort treatment at diagnosis was mainly superficial electron beam radiotherapy (n = 39) (8 Gy in 2 fractions or 15 Gy in 5 fractions). Other treatment modalities consisted of: surgical excision (n = 12); chlorambucil (n = 3); rituximab in combination with radiotherapy (n = 1); potent topical steroid (n = 1); and rituximab, cyclophosphamide, Adriamycin, vincristine, and prednisone (n = 1). A total of 27 patients (40%) relapsed with cutaneous recurrence
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Fig 4. A, Histologic specimen of primary cutaneous follicle center cell lymphoma (PCFCL) showing a prominent follicular growth pattern. B, Histologic specimen of PCFCL. C, bcl-6 immunostaining. D, bcl-2 immunostaining. (Hematoxylin-eosin stain; original magnifications: A, 320; B, C, and D, 340.)
only and were treated with: radiotherapy (n = 23); cyclophosphamide, Adriamycin, vincristine, and prednisone (n = 1); potent topical steroid (n = 1); and rituximab (n = 2) either with chlorambucil or adjuvant radiotherapy. Of patients, 47% developed a cutaneous recurrence with a relapse-free interval of 5.9 years (1-13 years). The median time to first recurrence was 0.92 years (0.06-12.54 years) and the median follow-up was 49.12 months (4-160 months). Only 4 of the 39 (10%) patients initially treated with radiotherapy relapsed. There were 5 deaths (cases 1, 11, 19, 29, and 45), which were unrelated to lymphoma. None of the patients died as a result of disease. The overall 5-year survival for the group was 90% with 100% 5-year diseasespecific survival (Fig 3). Histologic, immunophenotypic, and molecular findings Skin biopsy specimens showed dermal lymphoid infiltrates with a mixture of follicular (n = 20), mixed follicular and diffuse (n = 10), and purely diffuse (n = 27) growth patterns, with centrocytes and variable proportions of centroblasts (Fig 4). In the 27 tumors with a diffuse pattern, immunostaining for CD21 failed to demonstrate any residual follicular dendritic structures. In the remaining cases, a follicular growth
pattern could be identified at least focally. The tumor cells were a mixture of centrocyte-like cells and variable numbers of centroblasts. In addition, the cytology of PCFCL often shows spindled cells with a straplike morphology that have the appearance of crush artifact, but possibly reflect an abnormal chromatin structure. In all cases tumor cells expressed pan-B-cell markers (CD79a, CD20) and the germinal center marker bcl-6. In contrast to previous studies, tumor CD10 expression was only seen in 26% (n = 20; 9/20 follicular, 4/20 mixed, and 7/20 diffuse growth patterns) and expression did not correlate with a specific growth pattern. In all cases tumor cells were negative for CD5, CD23, and cyclinD1, thus excluding small lymphocytic lymphoma and mantle cell lymphoma. In 51 of 57 cases (89%) tumor cells were bcl-2 (Table II). There was no statistically significant difference between disease-free survival in bcl-21 and bcl-2 cases (P = .32). No correlation was found between bcl-2 expression and growth pattern. MUM-1 expression was negative in all cases. In summary, the majority of samples showed a diffuse growth pattern (49%, n = 28/57) with a characteristic CD201, CD79a1, bcl-61 phenotype and bcl-2 expression was positive in 11% of cases, similar to previous reports.15,16
Presentation
1
Diffuse
Not specified
2 3 4
Mixed Mixed Follicular
Nodule Not specified Nodule
5
Diffuse
6 7 8 9 10
Follicular Mixed Diffuse Mixed Follicular
Nodule Plaque Not specified Plaque Plaque Nodule Papule
11 12 13 15 16 17 18 19
Diffuse Diffuse Follicular Follicular Diffuse Diffuse Diffuse Mixed
Not specified Not specified Not specified Nodule Nodule Not specified Not specified Not specified
Growth pattern
Presentation
20 21 22 23
Follicular Follicular Diffuse Follicular
24 25 27 28 29 30 32 33
Diffuse Mixed Follicular Diffuse Mixed Follicular Diffuse Mixed
Nodule Nodule Nodule Subcutaneous nodule Nodule Nodule Patches/nodules Nodules Nodules Nodules Nodules Papule/nodule
Case no.
Positive phenotypes
Negative phenotypes
BCL2, CD10
BCL2 CD10
CD10 CD10
BCL2 BCL2 BCL2, CD10 BCL2, CD10 BCL2 BCL2 BCL2 BCL2, CD10 BCL2 BCL2, CD10
BCL2, CD10 BCL2 BCL2, CD10 BCL2 BCL2 BCL2, CD10 BCL2 Positive phenotypes Negative phenotypes
BCL2-FISH
IGH-FISH
10% with 3 copies of BCL2, no translocation Normal Normal Normal
Normal
Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Normal Normal Translocated IGH/MALT11 Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
MALT1-FISH
3 copies of MALT1, no translocation Normal Normal Translocated IGH/MALT11 Normal Normal Normal Normal Failed Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
BCL2-FISH
IGH-FISH
Clonality
CD10 CD10 CD10 CD10
BCL2 BCL2 BCL2 BCL2
Normal Normal Normal Normal
Normal Normal Normal Normal
Monoclonal Monoclonal Not specified Polyclonal
CD10 CD10 BCL2
BCL2 BCL2
Normal N/A Normal Normal N/A Normal Normal Normal
Normal N/A Normal Normal N/A Normal Normal Normal
Polyclonal Polyclonal Polyclonal Equivocal Polyclonal Polyclonal Polyclonal Polyclonal
CD10
CD10
BCL2, CD10 BCL2 BCL2 BCL2, CD10 BCL2
Clonality
Not specified
Polyclonal Monoclonal Monoclonal Monoclonal Monoclonal Monoclonal Polyclonal Monoclonal Monoclonal Monoclonal Polyclonal Monoclonal Polyclonal Not specified Monoclonal Polyclonal Monoclonal
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Growth pattern
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Table II. Clinical, histologic, phenotypic, and molecular findings within the full cohort of patients
50 51 52 53 54 55 56 57 58 59 60
Follicular Diffuse Follicular Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Follicular Diffuse
Excluded patients (reclassified) 26 Follicular
Diffuse
Secondary cutaneous disease; nodule Not specified
Patient 31 excluded as no available histopathology sample. FISH, Fluorescence in situ hybridization, N/A, not available.
BCL2 BCL2
BCL2 BCL2 BCL2 BCL2, CD10
Normal Inconclusive IGH/BCL21 Normal IGH/BCL21 N/A Normal Normal Normal Normal N/A Normal Normal IGH/BCL21 N/A Normal
Normal Inconclusive IGH/BCL21 Normal IGH/BCL21 N/A Normal Normal Normal Normal N/A Normal Normal IGH/BCL21 N/A Normal
Polyclonal Monoclonal Monoclonal Monoclonal Polyclonal Polyclonal Monoclonal Monoclonal Polyclonal Polyclonal Monoclonal Polyclonal Monoclonal Monoclonal Polyclonal Monoclonal
BCL2 BCL2, CD10 BCL2 BCL2 BCL2 BCL2 CD10 BCL2 BCL2, CD10 BCL2, CD10 BCL2
N/A Normal Normal Normal Normal Normal Normal IGH/BCL21 N/A Normal Normal
N/A Normal Normal Normal Normal Normal Normal IGH/BCL21 N/A Normal Normal
Polyclonal Monoclonal Not specified Not specified Polyclonal Monoclonal Not specified Not specified Monoclonal Monoclonal Monoclonal
BCL2, BCL6, CD10, CD20, CD79A
MUM-1, CD15, CD30, CD58
IGH/BCL21
IGH/BCL21
CD20, CD79A, MUM-1
BCL2, BCL6, CD10, CD21, CD3, CD5
Normal
Normal
BCL2 CD10 CD10 CD10
CD10 BCL2, CD10 CD10
CD10
BCL2 CD10
BCL 2 BCL2 BCL2 BCL2, CD10 BCL2 BCL2, CD10 BCL2 BCL2
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14
Thick plaques Nodule Nodule Nodule (extensive) Papules Papule Nodule Nodule Nodule Nodule Patch/plaque Nodule Nodule Nodule Nodule Subcutaneous nodules Nodules Plaque/nodule Nodules Nodules Nodules Nodules Nodules Nodule Nodule Subcutaneous Nodule
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Diffuse Mixed Follicular Follicular Follicular Mixed Diffuse Diffuse Diffuse Diffuse Follicular Follicular Follicular Diffuse Follicular Diffuse
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34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
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Molecular analysis of IGH, BCL2, and MALT1 Of the 57 cases of PCFCL examined, 8 were unsuitable for FISH analysis as they were referral specimens with insufficient tissue material for further staining. Nineteen of these cases were also studied with a dual-color, break-apart probe for MALT1 rearrangements. In the 49 cases studied, only 4 revealed reciprocal translocations involving immunoglobulin heavy locus (IGH) and BCL2 (8%). These 4 cases had no correlation with bcl-2 expression. An additional case (case 1), with a diffuse growth pattern, showed additional copies of BCL2 in about 10% of cells without evidence of translocation, suggesting gene amplification. A further case (case 4) showed a translocation of IGH but the BCL2 gene was not rearranged. Subsequent studies confirmed that this translocation involved IGH and the MALT1 gene adjacent to BCL2 on chromosome 18. Overall, 10% (n = 5/49) of cases had a rearrangement of either the BCL2 or MALT1 genes. None of these patients died because of disease (Table I) and there was no correlation between disease-free survival and the presence of these chromosomal anomalies (P = .53).
DISCUSSION Our study results confirmed the predicted prognosis for PCFCL with a 90% 5-year overall survival and 100% 5-year disease-specific survival. These results indicate that PCFCL has an excellent prognosis regardless of growth pattern, bcl-2 expression, or the presence of localized or multifocal disease. We detected a translocation involving IGH and either BCL2 or MALT1 in 5 of 49 patients (10%) with a t(14;18) involving BCL2 detected in 4 patients using FISH techniques. In addition, 1 patient showed an amplification of BCL2 in a proportion of cells with no translocation. Only 1 of the 6 cases with bcl-21 expression had evidence of a BCL2 chromosomal amplification or translocation. The t(14;18) translocation appeared to occur in a younger age group but this was not associated with a specific growth pattern or different clinical outcome. Although other studies have suggested that patients with bcl-2 protein expression and t(14;18) have a lower grade of disease,14 we would contend that the overall excellent prognosis and the small numbers in all these studies preclude any meaningful statistically significant correlation with clinical outcome. Indeed, in nodal follicular lymphoma the presence or absence of t(14;18) is not associated with different clinical outcomes. Several techniques can be used to detect chromosomal translocations such as conventional cytogenetics, Southern blot analysis, FISH with locus-specific probes, and PCR with a variety of
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primers. Although there is no gold standard, PCR and locus-specific FISH methods are most commonly used.24 Most previous studies of PCFCL have used PCR to detect the t(14;18) translocation.8,13-17 However, recent studies have shown that FISH, using a commercial dual-color fusion locus-specific probe in paraffin-embedded samples, has a higher detection rate for the IgH/BCL2 translocation compared with PCR techniques.24-26 In 2007, Deghiedy et al27 reported a sensitivity of 77.8% compared with 61.1% with PCR techniques. This is almost certainly because FISH probes cover a larger chromosomal region, thus enabling a higher proportion of potential IGH and BCL2 breakpoint regions to be identified. Our study suggests that the prevalence of a t(14;18) translocation in PCFCL is low compared with other FISH studies (range, 41%-52%).17,18 There are several possible reasons for this variable detection rate including geographic location, which has been reported previously for the detection of BCL2 translocations in nodal follicular lymphoma in Western and Asian populations.28 Of our cohort the majority were of white European origin with only 1 patient from the Indian subcontinent. Intriguingly, the prevalence of t(14;18) has been reported in up to 34% of the healthy Indian population.29 There are conflicting reports regarding the prevalence and frequency of this translocation within the healthy European population.28,30 Schmitt et al31 conducted a large study in Germany and detected the t(14;18) translocation in 24% (39 of 204) of healthy individuals using PCR. There have been no equivalent studies using FISH analysis. Subtle variation in the diagnostic criteria used for PCFCL in different studies may also be an explanation as both BCL2 and MALT1 translocations are known to occur in primary cutaneous marginal zone lymphoma32,33 and amplifications of BCL2/MALT1 have been reported in primary cutaneous diffuse large B-cell lymphoma.34 Our study of PCFCL is the only study to include samples with diffuse, follicular, and mixed growth patterns, as defined by the WHO EORTC classification35 and accurately reflects current diagnostic practice. In conclusion, our data suggest that chromosomal abnormalities involving either the BCL2 or MALT1 genes have a role in the pathogenesis of only a minority of PCFCL cases although this does not have any clinical relevance as patients have an excellent prognosis and invariably respond to radiotherapy as the standard treatment of choice. These findings confirm that in the majority of patients, PCFCL has a distinct pathogenesis to nodal follicular lymphoma.
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