Interferon regulatory factor 4 (IRF4) rearrangement is a common cytogenetic anomaly reported in some B-cell neoplastic lymphoproliferative disorders, including myeloma, follicular lymphoma, and diffuse large B-cell lymphoma (LBL), and also in some T-cell neoplastic lymphoproliferative disorders including primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, and peripheral T-cell lymphoma not otherwise specified. IRF4 is a 19.7-kb gene located at the 6p25.3 locus and encodes for the IRF4 protein belonging to the interferon regulatory factor (IRF) family of transcription factors controlling the B-cell proliferation and differentiation and the proliferation of mitogen-activated T cells. Large B-cell lymphoma with IRF4 rearrangement described in 2011 by Salaverria et al1 constitutes a novel provisional entity recently included in the 2016 classification of lymphoid tumors.2 This rare entity is mainly associated with a young age and a cervical localization and defined by the presence of a rearrangement of the IRF4 gene.
We report a case of B-cell lymphoma in a 19-year-old patient presenting with chronic cryptic tonsillitis. Based on the clinical presentation, histologic analysis, and immunohistochemical profile, a detection of IRF4 rearrangement was performed using fluorescence in situ hybridization (FISH) analysis with a break-apart probe allowing the diagnosis of this rare entity. In this case report, we will present the approach of the pathologist to identify this specific entity and the process of FISH analysis for the detection of the gene rearrangement.
MATERIALS AND METHODS
We report the case of a 19-year-old female patient without any relevant medical history who presented with repeated tonsillitis treated by antibiotics for several years. The diagnosis of chronic tonsillitis was made, and bilateral tonsillectomy was performed in July 2017 without any hemorrhagic or infectious complication.
Immunohistochemical study was performed on formalin-fixed, paraffin-embedded tissue sections using the Dako AutostainerLink 48 (Agilent Technologies Canada Inc, Mississauga, ON). The antibodies used are as follows: CD5, CD10, CD20. CD21, CD23, CD43, CD138, BCL2, BCL6, IgD, MYC, and MIB-1.
Fluorescent in Situ Hybridization
Fluorescence in situ hybridization analysis was performed on formalin-fixed, paraffin-embedded tissue sample according to the local procedure previously described,3 using the LSI BCL2 break-apart probe (Abbott Molecular, Des Plaines, Ill), the LSI BCL6 break-apart probe (Abbott Molecular), and the LSI IRF4 break-apart probe KI-10613; Leica Biosystems (Leica Biosystems Inc, Concord, Ontario, Canada) probes.
As the identification of the partner gene of IRF4 may affect the diagnosis, a complementary FISH analysis was performed on the IRF4 slide after a washing procedure using 2X SSC and Carnoy solutions using a LSI IGH break-apart probe Cytocell LPH014 (Cytocell Ltd, Oxford GeneTechnology, Cambridge). We randomly selected 8 areas on the slide after the hybridization with the IRF4 probe and analyzed the same areas after the washing procedure and the hybridization with the IGH probe. Photographs were manually taken on a Leica DM microscope (Leica Microsystems Inc, Concord, Ontario, Canada).
Diagnosis and Follow-up
The diagnosis of LBL with IRF4 rearrangement was made according to the criteria cited in the 2016 World Health Organization (WHO) classification. A positron emission tomography scan prior to therapy was performed with no findings of residual abnormal metabolism and no other abnormal uptake. The patient started 6 cycles of chemotherapy (R-CHOP [combination of rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate and prednisone]) on October 2017. After the third cycle, she described signs of neuropathy leading to a vincristine dose adjustment.
Histologic and Immunohistochemical Observations
The histologic analysis of the right tonsil demonstrated a diffuse infiltration of the tonsil by a monomorphic lymphoid proliferation showing follicular growth pattern and composed of medium-sized cells positive for CD20, BCL6, and MUM1 and negative for CD5, CD10, BCL2, and MYC.
At low magnification, tonsil shows partially altered architecture composed of huge, coalescent, and monotonous follicles. Neoplastic cells are medium to large with centroblastic morphology. Few tangible body macrophages are seen. Sparse and small tumoral necrosis foci are visualized associated with numerous apoptotic cells and mitotic figures. Residual interfollicular zones show a majority of mature small T lymphocytes.
Neoplastic cells have a mature B-cell phenotype (CD20+) and express BCL-6, MUM1, and IgD. Only 5% of tumor cells express MYC, and mitotic index reaches 80% to 90% in the most proliferative areas.
Molecular Cytogenetic Findings
Neither BCL2 nor BCL6 rearrangement was observed, but the hybridization using the IRF4 Leica probe demonstrated a rearrangement of the 6p25 locus on 80% of the cells, with a typical pattern of signals.
The analysis of the IRF4 slide after using the IGH probe confirmed the presence of an IRF4-IGH rearrangement as the pattern of the split signals was comparable with both probes on the cells of the areas selected (Figs. 1–3).
A provisional specific subgroup of LBL with IRF4 rearrangement has recently been included in the WHO classification based on the publication of Salaverria et al.1 The case reported here was the first case in our laboratory since the publication of the 2016 WHO classification. The description of the case raised some questions concerning the difficulty to make such diagnosis and about how to demonstrate the IRF4 rearrangement.
Regarding the first question, 4 main differential diagnoses may be discussed. First, LBL must be distinguished from follicular hyperplasia; however, even with a follicular growth pattern, back-to-back growth of large follicles will raise suspicion for malignancy. The presence of an aberrant immunophenotype as coexpression of germinal center markers (CD10, BCL6) associated with expression of MUM1, the presence of monoclonal B-cell population demonstrated by molecular studies, and the demonstration of IRF4 rearrangement by cytogenetic studies will confirm the diagnosis. Second, pediatric-type follicular lymphoma must be considered in the differential diagnosis because it shows important clinical overlap with LBL as both entities are similar regarding disease localization and age at onset. Fortunately, pediatric-type follicular lymphoma can be ruled out because of usual LBL immunophenotype and cytogenetic anomalies. Third, known for a much more aggressive clinical course than LBL, CD10−/MUM1+ follicular lymphoma represents one of the most important pitfalls when facing LBL, because convincing immunophenotype overlap can be observed, mostly with CD10-negative LBL. Obviously, there is significant clinical heterogeneity between those 2 diagnoses, but identifying IRF4 rearrangement is of great help to make the correct diagnosis. Finally, diffuse LBL not otherwise specified should also be considered in the differential diagnosis of LBL, mostly when the latter shows diffuse growth pattern. When facing this situation, combination of clinical data (young age and localized disease) and cytogenetic anomalies (IRF4 rearrangement, in the absence of BCL2 and MYC rearrangement, which would preclude LBL diagnosis) will lead to the correct diagnosis.4,5 We think that three criteria should lead the pathologist to suspect LBL with IRF4 rearrangement: the young age of the patient, the location, and the characteristic immunohistochemical profile including the positivity of CD10, BCL6, and MUM1.
Regarding the “how” question, because IRF4 is described as a multipartner gene (including IGH, IGL, and IGK), the most sensitive technique to detect the rearrangement remains in situ hybridization using a break-apart probe, which allows the identification of the 6p25 locus rearrangement irrespective of the partner gene. Some companies started to distribute an IRF4 probe, all using a break-apart strategy. It remains unclear if the partner gene should be identified using complementary FISH or polymerase chain reaction (PCR) analysis. In this case, we confirmed the presence of IRF4-IGH rearrangement using 2 successive hybridizations on the same slide. In 2011, Salaverria et al1 used a combination of FISH and long-distance reverse transcriptase PCR approaches to identify the different types of IRF4 rearrangements; among the 23 cases reported with IRF4 rearrangement, 17 cases revealed IRF4-IGH rearrangement, 2 cases IRF4-IGL, and 1 case IGK-IRF4 rearrangement.1 Three cases notably demonstrated an atypical pattern of signals and were excluded from the study. Otherwise, in neoplastic T-cell lymphoproliferative disorders, alternative alterations of the IRF4 locus have been reported, including extra copies of IRF4 and extra copies of the proximal part of IRF4.6 In this context, it may be relevant to confirm the presence of an IG gene rearrangement, using a FISH (double fusion probe or successive hybridizations) or a reverse transcriptase PCR approach, to support the diagnosis.
We present a case of LBL with IRF4 rearrangement. The clinical, histological, and genetic features reported were consistent with the description proposed in the 2016 WHO classification. Systematic diagnostic approach and focused ancillary studies (immunohistochemical and cytogenetic studies) have led to the correct diagnosis while excluding other pathologies from the differential diagnoses, as discussed before. Fluorescence in situ hybridization approach using an IRF4 break-apart probe was useful in this diagnosis; the confirmation of the IG partner gene may be required in cases revealing atypical signal patterns.
1. Salaverria I, Philipp C, Oschlies I, et al. Molecular Mechanisms in Malignant Lymphomas Network Project of the Deutsche Krebshilfe; German High-Grade Lymphoma Study Group; Berlin-Frankfurt-Münster-NHL Trial Group. Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults. Blood
2. Pittaluga S, Harris NL, Siebert R, et al. Large B-cell lymphoma with IRF4 rearrangement. In: Swerdlow S, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues
. Lyon, France: IARC; 2016:280–281.
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