CCND1 was rearranged with IG genes in all tumors, 12 cases with IGH and 1 with IGL. In 3 cases, the IG breaks had evidence of being mediated by RAG enzymes during V(D)J recombination (cases #1–3), whereas the IG breaks in the remaining 10 tumors involved AID-related mechanisms through aberrant CSR in 9 cases and SHM in 1, therefore, generated in a mature B-cell probably during a follicular germinal center reaction (Fig. 1A, Supplementary Tables 6, 7).

A Schematic representation of the location of the breakpoints found in the IG loci (IGH, top, IGL, bottom). CSR, class-switch recombination region. IGHJ genes (1–6) are shown as a single gene due to their small size and compactness for illustrative purposes. Genomic locations are shown on the x axis. Breakpoints are colored based on their underlying mechanism as AID-mediated through aberrant CSR/SHM or RAG-mediated during the initial J-D recombination. B Representation of the breakpoints found in chromosome 11. MTC major translocation cluster.

In the 3 cases with RAG-mediated rearrangements, the IG breaks occurred in IGHD and IGHJ genes, likely during the initial IGHD–IGHJ recombination. We identified the RAG recombination signal sequence (RSS) at the IGHD and IGHJ breakpoints with the addition of non-templated nucleotides (N-nucleotides) at both derivative junctions. The nine tumors with AID-mediated rearrangement had breakpoints in the IGHA1 and IGHM defined CSR region in two cases each, and IGHG3, IGHG1, IGHG2, IGHG4, and IGHE in one case, respectively. The remaining case had the IG breakpoint downstream of IGLJ1 generated by SHM mechanism (Fig. 1A).

Breakpoints on chromosome 11 (chr11) were found upstream of the 5’ of CCND1 in 12 tumors and in the 3’ region in one (case #6) (Fig. 1B). Only one tumor with RAG-mediated breakpoint had the 11q13 breakpoint in the previously recognized major translocation cluster (MTC) region in MCL whereas the breakpoints in the other 11 tumors were located upstream or downstream of this region (Fig. 1B). As previously described for MCL [17], most breaks on chr11 occurred near CpG sites and AID motifs (Supplementary Table 7). The G allele of SNP rs9344 has been associated with t(11;14) in Multiple Myeloma but not MCL [32]. We did not observe differences in the G and A allele in DLBCL with CCND1-R (Supplementary Table 8).

The three tumors with RAG-mediated translocations had IG rearrangements with 99.65, 97.89, and 95.14% identity (cases #1–3, respectively) with the germline IGHV sequences (Supplementary Fig. 1, Supplementary Table 9). The IGHV genes used were IGHV4-34 in 2 cases and IGHV3-23 in one, both commonly used in different types of lymphomas, including MCL [17]. The SMZL and the subsequent DLBCL (case #4) had the same IG rearrangement IGHV1-2*04–IGHD3-10*01–IGHJ6*02, frequently used in SMZL [28], with 97.22% identity. The transformed NMZL (case #9) had an IG rearrangement using IGHV4-34. In three DLBCL we observed the usage of IGHV4-34, frequently used in DLBCL. The uncommon rearranged genes in DLBCL IGHV1-3, IGHV1-46, IGHV3-48, and IGHV3-7 were observed in one case each (Supplementary Fig. 1) [27]. In one DLBCL (case #6) the IGHV gene rearrangement could not be properly identified, probably due to the high number of SHM. The identity with the germline in these tumors was very variable from 90.28 to 98.25% (Supplementary Fig. 1, Supplementary Table 9).

The results of the sequencing studies are summarized in Fig. 2 and Supplementary Tables 10–19. Tumor and paired germline DNA from cases #1 and #2 with CCND1-R mediated by RAG were studied by WGS and WES, respectively. Among all coding mutations identified, only TP53 mutations in both cases, and NOTCH1 truncating mutation in case #2 were previously described as recurrent in lymphoid neoplasms. Case #3 had an unusual genomic profile with MYC translocation and mutations in TP53, ID3, ARID1A, ARID1B, CCND3, SMARCA4, PCBP1 and P2RY8, among others (Fig. 2, Supplementary Fig. 2). The two cases (cases #4 and #9) diagnosed as transformed SMZL and NMZL had an AID-meditated CCND1-R and mutations in KLF2 and TNFAIP3, frequently described in these tumors (Fig. 2). The SMZL and its transformation shared the mutations in these genes and in KMT2D. The transformed tumor also acquired mutations in BCL10 and the translocated CCND1 allele (Fig. 2, and Supplementary Fig. 2). Of the other eight tumors with AID-mediated CCND1-R, 6 had mutations in CCND1, 3 in CARD11, and mutations of KMT2D, PIM1, KRAS, DDX3X, SYNE1, PRMD1, CD79B, and CREBBP were observed in two cases each. Mutated genes in one single case were MYD88, CD70, ATM, SMARCA4, and IRF4, among others (Fig. 2, Supplementary Fig. 2).

Oncoprint showing the morphology, SOX11 status, presence/absence of IG::CCND1, IGHV identity, NGS and FISH studies performed, and putative driver gene mutations found in the studied samples. Cases are grouped based on the mechanism underlying the IG::CCND1 rearrangement. *, Cryptic BCL6 rearrangement only identified by WGS.

In addition to coding driver mutations, genome-wide mutations also differ between RAG-mediated and AID-mediated CCND1-R cases. Case #1 (RAG-mediated) carried 7,074 single nucleotide variants, while cases #6 and #7 harbored 64,415 and 12,086 mutations, respectively. In terms of mutational signatures previously identified in MCL and DLBCL [17, 33], case #1 carried mutations explained by signatures 1 (spontaneous or enzymatic deamination of 5-methylcytosine to thymine; clock-like), 5 (unknown; clock-like) and 8 (unknown), cases #6 and #7 also had signatures 2 (APOBEC activity), 9 (polymerase eta SHM activity), 17b (unknown), and/or 18 (damage by reactive oxygen species) (Supplementary Fig. 3).

In terms of structural alterations, BCL2 and BCL6 rearrangements were not observed in RAG-mediated CCND1-R cases, while 6/10 cases with an AID-mediated CCND1-R carried a BCL6 rearrangement demonstrated by FISH/WGS. Of note, one of the latter cases carried a cryptic BCL6 rearrangement due to an inversion detected only by WGS (Supplementary Fig. 4). MYC rearrangement was only observed in case #3 (RAG-mediated), while no MYC rearrangements were observed in AID-mediated cases (Fig. 2). Case #8 showed an IRF4::IGH that was concordant with the mutations observed in the 5’ region of the gene (Fig. 2). Two IGH rearrangements, IGH::CCND1 and IGH::IRF4, were detected by FISH in the same cells, confirming the co-occurrence of the two alterations (Supplementary Fig. 5).

Regarding copy number alterations (CNA) identified by WGS/WES analyses, cases with a CCND1-R mediated by RAG carried alterations frequently observed in MCL [17], such as gains in 3q, 4q, 8q (MYC) and 18q, as well as deletions in 6q, 9p (CDKN2A/B), 9q, 10q, 13q and 17p (TP53). In contrast, cases with an AID-mediated CCND1-R harbored CNA recurrently detected in DLBCL [34], including gains in 1q, focal 2p affecting REL and BCL11A, 11q, 18q, and trisomies of chromosomes 3, 7, and X (Supplementary Fig. 6). Additionally, WGS analyses reveal in case #1 (RAG-mediated) the presence of a complex genomic profile including chromothripsis-like patterns in chromosome 15 and breakage-fusion bridge cycles frequently seen in MCL [17], as well as CD274 (PD-L1) alterations truncating the 3’UTR region in two cases with a CCND1-R mediated by AID (cases #6 and #7). These two cases had strong PD-L1 protein expression, whereas it was negative in the other 5 cases studied (Supplementary Fig. 7). CNA was also studied in case #8 using OncoScan assay (Thermo Fisher Scientific) detecting gains in 10p15.3-q26.3, 17p11.2-q25.3 and 21q11.1-q22.3 and losses in 8p22-p21.3, 17p13.3-p11.2/TP53 and 19p13.3-p13.2/CD70/TNFSF9. Additionally, this case had a 1p36.33-p11.2 CN neutral loss of heterozygosity.

Tumors carrying RAG-mediated CCND1-R had blastoid (case #1 and #2) or high-grade morphology (case #3) (Table 1 and Supplementary Table 1). Cases #1 and #2 were composed of medium to large-sized cells, with round nuclei, fine disperse chromatin and high number of mitosis. In addition, smaller atypical cells with irregular nuclei, dense chromatin, and scarce cytoplasm were also seen (Fig. 3). These cases were positive for CD20, CD5 and TP53 (Table 1). One case expressed MYC and both were negative for SOX11, CD10, BCL6 and IRF4. Cyclin D1 was positive in virtually all cells, including small cells. These cases were diagnosed as SOX11-negative blastoid MCL based on the morphology of the cells, and the presence of small cells with strong expression of cyclin D1 and CD5-positivity. The RAG-mediated CCND1-R and the genomic alterations were consistent with this diagnosis.

A The tumor cells are predominantly medium to large size with round nuclei and blastoid chromatin. Smaller cells with irregular nuclei are also present (H&E stain, original magnification ×400). B Cyclin D1 immunohistochemical staining shows expression in all range of tumor cells (immunoperoxidase, original magnification ×400).

Case 3 had two biopsies, one from a cervical mass and a subsequent gastric sample, both with similar features. The tumor was diagnosed as HGBL, NOS. The tumor cells were positive for CD20, CD10, BCL6, MYC and TP53 and negative for CD5 and IRF4/MUM1. Ki67 was positive in virtually all cells.

From the 10 tumors carrying an AID-mediated CCND1-R, nine had large cell morphology, whereas one was blastoid (case #8). Two tumors (cases #4 and #9) were transformed from a SMZL and a NMZL, respectively (Table 1 and Supplementary Table 1). The initial SMZL of case #4 was diagnosed in the splenectomy specimen and had typical features of this tumor with negative cyclin D1 expression and CCND1-R by FISH. The cytogenetic study found a subclonal del(7)(q31q35) and del(6)(q12q16). The subsequent transformed DLBCL was diagnosed in a cervical lymph node 14 years later with the same IGH clonal rearrangement. A subpopulation of the tumor cells was positive for cyclin D1 (Fig. 4) and CCND1-R was detected by FISH in around 20% of the cells. The transformed NMZL (case #9) was diagnosed in a core needle biopsy showing areas with small cells with clear cytoplasm and areas where large cells were predominant. The tumor cells in both MZL were positive for CD20 and BCL2 and negative for CD5, CD10, BCL6, IRF4/MUM1 and MYC.

A Splenic marginal zone lymphoma with expansion of the marginal areas of the white pulp (H&E stain, original magnification ×100). B Cyclin D1 staining was negative (immunoperoxidase, original magnification ×200). C Lymph node with diffuse large B-cell lymphoma obtained 14 years later. Both tumors had the same clonal IGHV1-2*04 - IGHD3-10*01 - IGHJ6*02 rearrangement (H&E stain, original magnification ×200). D The transformed tumor was positive for cyclin D1 in the large cells but also in occasional atypical smaller cells (immunoperoxidase, original magnification ×200).

The remaining eight tumors had a diffuse growth pattern with only one (case #6) having focal nodular areas with a meshwork of follicular dendritic cells suggestive of a transformed follicular lymphoma (Table 1 and Supplementary Table 1). Two cases (cases #6 and #8) had a GC phenotype with expression of CD10 and BCL6, but case #8 had a blastoid morphology and strong expression of IRF4/MUM1 (Fig. 5). Six cases had a non-GCB phenotype by Hans algorithm including the three with a testicular mass, and two nodal and one duodenal tumors. CD5 was only positive in one testicular tumor. Ki67 was very high in all these tumors with >80% positive cells.

A Computed tomography (CT)-scan demonstrates a large mass in the stomach that was biopsied. B Panoramic view of the gastric biopsy obtained at relapse with a diffuse lymphoid infiltration in one of the fragments. (hematoxylin and eosin (H&E) stain, original magnification, ×50). C Higher magnification shows that the tumor is composed or medium to large-sized lymphoid cells with irregular nuclei, blastic chromatin, inconspicuous nucleoli and clear cytoplasm. (H&E stain, original magnification ×400). D Giemsa stain highlights the blastic cytology of the lymphoid cells (Giemsa stain, original magnification, ×400). E The tumor cells are positive for CD20, CD10 (F), BCL6 (G), MUM1/IRF4 (H), and cyclin D1 (I). (immunoperoxidase, original magnification ×400). J The MIB1/Ki67 stain shows a proliferation index of 100%. K FISH analysis using an IRF4 break-apart assay demonstrates a signal constellation of one colocalized signal (inset, yellow arrow) and one split signal (inset, red and green arrows) consistent with gene rearrangement. L FISH analysis using an CCND1 break-apart assay shows a signal constellation of one colocalized signal (inset, yellow arrow) and one split signal (inset, red and green arrows) consistent with gene rearrangement.

The clinical features, treatment, and follow-up of the patients are summarized in Table 1 and Supplementary Table 20. The three patients carrying tumors with RAG-mediated CCND1-R were males with a median age of 64 years (59–84 years). The initial manifestation of the disease was generalized lymphadenopathy (case #1), isolated enlarged tonsil (case #2), or an explosive disseminated disease (case #3). Two of the patients (cases #1 and #3) had bone marrow and peripheral blood involvement. The disease did not respond to therapy in these two patients, and they died 2 and 6 months after diagnosis. The disease in patient #2 had an initial complete response but relapsed 5 months after diagnosis and the patient died 16 months later.

Patients with tumors carrying an AID-mediated CCND1-R were 7 males and 3 females with a median age of 61 years (45–86 years) (Supplementary Table 20). The two patients with transformed MZL had been initially diagnosed with a SMZL and NMZL 14 and one year before, respectively. The transformed SMZL LBCL was localized in a cervical lymph node and responded to Ibrutinib, being in complete remission 26 months later. The other eight patients had a presentation with lymphadenopathy in 3, testicular mass in 4, duodenal lesion in a patient with anemia, one patient had a mediastinal mass with also gastric involvement, and one patient had bone lesions in addition to generalized lymphadenopathy and a testicular mass. Patient 8 with an additional IGH::IRF4-translocation was a 76-year-old man with a bulky mediastinal mass and infiltration of the right bronchus. PET-CT scan demonstrated, in addition, small lymph nodes in the inguinal region (Fig. 5). The patient received 6 cycles of R-CHOP-14 achieving complete remission. One year later, the tumor recurred with orbital and gastric involvement. The patient received local radiotherapy to the orbital mass and R-bendamustine with poor response and the patient died 3 months after the recurrence. The clinical presentation and evolution of this patient suggest that this tumor does not correspond to the large B-cell lymphoma with IRF4 rearrangement observed in children and young adults.