LAUSR

B-cell lymphomas that concurrently carry MYC, BCL2 and/ or BCL6 gene rearrangements – so-called double-hit or triple-hit lymphomas – are rare entities associated with poor prognosis. B-cell lymphomas with translocations involving MYC, BCL2, BCL6 and CCND1 genes (quadruple-hit) are extremely rare, with limited cases reported to date. Herein we report a case diagnosed at an early stage and remaining disease-free for 15 months after the initial diagnosis. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Soochow University. Written informed consent was obtained from the patient. The patient was a 76-year-old man suffering from pharyngeal obstruction for 2 months. A positron emission tomography scan showed multiple cervical lymphadenopathies (Fig 1A). The laboratory tests were unremarkable including a normal serum lactate dehydrogenase (LDH) level. Biopsy of the left cervical lymph node displayed effaced architecture by a diffuse infiltrate of medium to large lymphocytes. Tangible body macrophages were observed at lower magnification. At higher magnification, the tumour cells revealed a moderate amount of cytoplasm, round or slightly irregular nuclei, moderately dispersed chromatin and one or more small and occasionally prominent nucleoli (Fig 2A). By flow-cytometric immunophenotyping, 58 4% of the gated lymphoid events revealed a kappa immunoglobulin light chain restriction. These cells expressed pan-B markers such as CD19, CD20, and FMC7 with weak CD23 positivity. CD200, CD79b, CD5 and 10 were negative. By immunohistochemical (IHC) staining, the lymphoma cells were positive for CD20, CD79a, BCL6, cyclin D1, CD21, BCL2 and MYC. P53 was positive in occasional cells (Fig 2B–H). The Ki67 proliferative rate was 60% (Fig 2I). The tumour cells were negative for CD3, CD5, SOX11, MUM1, and EBER by in situ hybridisation. Examination of the bone marrow biopsy showed it to be negative for tumour infiltration. Given the over-expression of BCL6, MYC, cyclin D1, and BCL2, interphase fluorescence in situ hybridisation (FISH) analyses of these relevant genes were performed. Dual-color dual-fusion (D-FISH) probes (Beijing Jinpujia Medical Technologies, Beijing, China) were selected for MYC-IGH, CCND1-IGH and IHG-BCL2 rearrangements. In addition, a break-apart probe (BAP; Beijing Jinpujia Medical Technologies) was used for BCL6 rearrangement. The FISH analyses were performed on 4 lm paraffin-embedded tissue sections and it was considered positive if more than 15% of the tumour cells exhibited a fusion or break-apart signal. D-FISH of IGH-MYC (Fig 2J), IGH-CCND1 (Fig 2K) and IGH-BCL2 (Fig 2L) showed one fusion signal in 34% of 100 suspension cells, indicating MYC-IGH, CCND1-IGH and IGH-BCL2 rearrangements. Similarly, a BCL6 (Fig 2M) break was detected in 38% of 100 suspension cells, suggesting translocations of the genes. The FISH analyses were also repeated on tissue sections. Moreover, FISH analyses using BAP (Abbott Molecular, Des Plaines, IL, USA) of BCL2 (Figure S1A) and CCND1 (Figure S1B) revealed split signals on tissue sections, demonstrating a break of both genes. Therefore, according to the morphology, immunophenotype and FISH results, a final diagnosis of quadruple-hit pleomorphic mantle cell lymphoma (MCL) was favoured. Cytogenetic analysis was failed due to a lack of growth of metaphase cells after a short term of cell culture. However, we were able to perform Cytoscan 750k (Affymetrix, Santa Clara, CA, USA) single nucleotide polymorphism microarray (SNP-array) assay to assess genome-wide chromosomal copy number aberrations (CNAs). The SNP-array revealed apparent gains of whole chromosomes X, Y, 5 and 7, as well as segmental gains of chromosomes 1p36p23, 1q23q25, 1q25q31, 1q31q32, 1q41q42, 1q42q44, 2p25p16, 2p12p11, 2p11q12, 3p26q27, 6p25p12, 17q12q24, and 21q11q22 (Fig 2N). The genome-wide CNA pattern was suggestive for the presence of a complex hyperdiploid clone, with evidence of a chromothripsis-like pattern of 1q. Additionally, next-generation sequencing (NGS) was performed for somatic mutations with a targeted panel covering 222 genes, with an average sequencing depth of more than 1000 times. NGS revealed 12 nonsynonymous single nucleotide variants, one frameshift mutation, and one nonsense mutation, involving ARID1B, BCL2 (3), CCND1 (3), FOXO1, IL7R, MGA, PIM1 and SGK1 (3) genes (Table SI). No mutation was identified in the TP53 gene, which was consistent with IHC results. Deletions or mutations of the ARID1B gene have been observed in lymphoplasmacytic lymphoma. The Y24I mutation in FOXO1 gene has been identified in diffuse large B-cell lymphoma (DLBCL) and was associated with decreased overall survival. Mutations of the SGK1 gene were common in DLBCL and the PIMI gene mutation has been identified in DLBCL (leg type) and multiple myeloma. Interestingly, multiple mutations of BCL2 and CCND1 genes were also identified, which have rarely been reported to be co-existing with gene rearrangements. Of note, the CCND1 mutations have been correspondence