Increased complexity of t(11;14) rearrangements in plasma cell neoplasms compared with mantle cell lymphoma

Abstract Plasma cell neoplasms (PCN) and mantle cell lymphoma (MCL) can both harbor t(11;14)(q13;q32) (CCND1/IGH), usually resulting in cyclin D1 overexpression. In some cases, particularly at low levels of disease, it can be morphologically challenging to distinguish between these entities in the bone marrow (BM) since PCN with t(11;14) are often CD20‐positive with lymphoplasmacytic cytology, while MCL can rarely have plasmacytic differentiation. We compared the difference in CCND1/IGH by fluorescence in situ hybridization (FISH) in PCN and MCL to evaluate for possible differentiating characteristics. We identified 326 cases of MCL with t(11;14) and 279 cases of PCN with t(11;14) from either formalin‐fixed, paraffin‐embedded tissue or fresh BM specimens. The “typical,” balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals, three total IGH signals, and two total fusion signals. Any deviation from the “typical” pattern was defined as an “atypical” pattern, which was further stratified into “gain of fusion” vs “complex” patterns. There was a significantly higher proportion of cases that showed an atypical FISH pattern in PCN compared with MCL (53% vs 27%, P < .0001). There was also a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL (47% vs 17%, P < .0001). We confirmed these findings using mate‐pair sequencing of 25 PCN and MCL samples. PCN more often have a complex CCND1/IGH FISH pattern compared with MCL, suggesting possible differences in the genomic mechanisms underlying these rearrangements in plasma cells compared with B cells.

(CCND1/IGH), occurring in >95% of cases of MCL 1 and in 15% of PCM. 3 This translocation is associated with cyclin D1 overexpression, a critical regulator of the cell cycle. [4][5][6] Detection of the t (11;14) in the evaluation of lymphoma is used to aid in establishing the diagnosis of MCL. In contrast, a panel of FISH probes including CCND1/IGH are evaluated in PCM for prognostic and therapeutic purposes. 3 PCM with t (11;14) has been reported to be associated with a favorable prognosis with a median overall survival of 7 to 10 years. 3 In addition, this translocation in the setting of PCM is associated with an increased incidence of developing plasma cell leukemia. 4 PCN with t (11;14) often show a lymphoplasmacytic cytology and express CD20, which can make these cases especially challenging to distinguish from low-grade B cell lymphomas with plasmacytic differentiation, which also involve the bone marrow and cause overlapping clinical features with monoclonal gammopathy of undetermined significance (MGUS) and PCM. 7 Plasmacytic differentiation has rarely been reported in MCL cases. [8][9][10][11][12][13] While usually easily distinguished from a PCN with t(11;14) based on pathologic features, at low level infiltrates in the BM there are occasional cases which may cause diagnostic uncertainty. This differentiation can also be particularly challenging in the setting of a small biopsy with crush artifact or poor fixation. After encountering rare diagnostically challenging cases in which overlap between a low-level MCL with plasmacytic differentiation and a true PCN with t (11;14), we sought to compare the difference in genomic patterns in PCN and MCL by fluorescence in situ hybridization (FISH) using the CCND1/IGH probe set with the goal to evaluate possible differentiating characteristics potentially to aid in diagnostically challenging cases. Thirty-four consult cases in which the final diagnosis was unavailable were reviewed by a hematopathologist (JCD) to confirm the pathologic diagnosis.

| Fluorescence in situ hybridization testing
Interphase FISH was performed on formalin-fixed, paraffin-embedded tissue (FFPE) or fresh BM specimens using standard FISH pretreatment, hybridization, and fluorescence microscopy protocols. 14 A dual-color, double fusion probe set for t(11;14) CCND1/IGH, a break-apart probe set for MYC (8q24.1) rearrangement, and an enumeration probe for TP53 deletion or monosomy 17 (centromere 17/TP53) were utilized (Abbott Molecular, Des Plaines, IL, for all probe sets). The IGH probe is labeled with Spectrum Green (Abbott Molecular) and the CCND1 probe is labeled with Spectrum Orange (Abbott Molecular); the imaging of the Orange fluorophore is herein referred to as Red. A total of 100 or 200 interphase nuclei were evaluated in each case, with 50 or 100 nuclei evaluated independently by two qualified clinical cytogenetic technologists and interpreted by a board-certified (American Board of Medical Genetics and Genomics) clinical cytogeneticist. The "typical," balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals (red), three total IGH signals (green), and two total fusion signals (yellow) or 1R1G2F ( Figure 1A). Any deviation from the "typical" pattern was defined as an "atypical" pattern, including both gain of fusion signals (1 or more; eg, 1R1G3F) and unbalanced/complex abnormalities ( Figure 1B-C).
The percentage of cases with a "typical" pattern vs "atypical" pattern and gain of fusion vs complex pattern were compared for each group.
The "atypical" patterns were further stratified into "gain of fusion" vs "complex" patterns. Groups were compared using two-tailed Fisher's F I G U R E 1 Representative FISH patterns in MCL and PCN. A, Mantle cell lymphoma case showing the "typical" balanced CCND1/IGH translocation pattern with three total CCND1 signals (red), three total IGH signals (green), and two total fusion signals (yellow). B, Plasma cell myeloma showing amplification of the fusion signal (yellow). This case additionally showed a TP53 deletion. C, Plasma cell myeloma showing atypical FISH pattern with 6 CCND1 signals (red), 4 IGH signals (green) and 2 fusion signals (yellow) exact statistical analysis and visualized using GraphPad Prism version 8.0.0 for Windows, GraphPad Software (San Diego, CA).

| Mate-pair sequencing
Mate-pair sequencing (MPseq) was performed on a subset of 16 PCN and 9 MCL cases. DNA extraction and mate-pair library preparation methods have been previously described. [15][16][17] Briefly, DNA was isolated from plasma cells as described in Smadbeck et al 18 Figure 2A,B). In both FFPE and BM specimens, there was a significantly higher proportion of cases that showed an atypical CCND1/IGH FISH pattern in PCN compared with MCL (53% vs 27%, P < .0001) ( Table 1). We further divided the atypical category into those with a simple gain of CCND1/IGH fusion signal, and complex representing any other atypical FISH result. There was a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL (47% vs 17%, P < .0001) ( Table 1). One PCN FFPE specimen showed amplification of the fusion signal ( Figure 1B). These data demonstrate that cases of PCN were nearly 2 to 3-fold more likely to have an atypical or complex CCND1/IGH FISH result compared with MCL.
We evaluated whether CCND1/IGH FISH complexity was associated with an increased incidence of TP53 deletion, MYC rearrangement, or tetraploidy.TP53 deletion is associated with      Figure 2C). The distribution of TP53 deletion among the CCND1/IGH FISH subtypes (typical vs atypical) was not considered significant (P = .4182). MYC rearrangements can also occur as a secondary cytogenetic abnormality and contribute to progression in PCN 1,3,21,22 and are associated with a higher proliferation in MCL. 20 Figure 2C). Similar to TP53 deletion, the distribution of MYC rearrangement among the CCND1/IGH FISH subtypes (typical vs atypical) was also not considered significant (P = .6571). A tetraploid clone, reported to be more common in pleomorphic and blastoid variants of MCL, 23 was seen in 12 cases of MCL (3.7%) and
MPseq has been shown to be superior to FISH in characterizing rearrangement complexity. 15 (Table 3, Figure 3). In addition, 3/9 MCL samples were found more frequently to harbor an 11q deletion telomeric to This type of complexity was absent in all nine MCL samples analyzed by MPseq, which were more likely to be simple, balanced rearrangements as depicted in Figure 4B. Similar to our findings by FISH analysis, our MPseq data also show that PCN demonstrates a more complex t (11;14) pattern in comparison to MCL.

| DISCUSSION
We show that PCNs have a significantly higher propensity to have atypical and complex CCND1/IGH FISH patterns compared with MCL. However, the IGH breakpoint has been shown to be different in MCL vs PCN. 25,26 In MCL, the majority of breaks occur in the VDJ region of IGH and frequently involves the IGHV3-23 and IGHV4-59 genes. 25,27,28 In contrast, the breakpoints identified in PCN are variable and are usually located in an IGH switch region. 6 (14) and CCND1/IGH fusions on der (11). 26,31,32 Previous studies showed that IGH rearrangements in MCL appeared to be due to aberrant VDJ recombination (RAG1/2 mediated), while in PCN IGH rearrangements appeared to be due to aberrant class switch recombination (AID mediated). 29,30,33 However, there is evidence that AID can also cooperate with RAG to mediate t (11;14) in MCL, as evidenced by breaks near AID hotspots. 28 Open and active chromatin structure could allow AID accessibility to mediate the DNA breaks and subsequent translocation. 20,28 The gains of CCND1 and IGH observed in PCN cases could be described as templated insertions, a previously reported complex event found in about 20% of plasma cell myeloma cases. 34 In contrast, templated insertions do not appear to be a common feature in MCL. 20 The reason for these differences in the incidence of templated insertions between PCN and MCL requires additional investigation.
The single nucleotide polymorphism rs603965 (also known as rs9344) occurring at the splice site of cyclin D1 leading to the 870G > A polymorphism has been reported to be associated with a risk of t(11;14) PCM and AL amyloidosis. 35,36 In contrast, the rs603965 genotype showed no relationship with MCL risk. 35,37 These findings most likely reflect the different underlying mechanisms associated with the development of t (11;14) in PCN vs MCL.
We also evaluated whether the complex CCND1/IGH positive cases were more likely to have TP53 deletions or MYC rearrangements; however, our sample size was small and, our findings were not considered significant. We also investigated the presence of tetraploid clones; however, there was also no significant difference in the association of tetraploidy with the complex CCND1/IGH positive cases. In our cohort, only one PCN case with t(11;14) amplification was identified, while no MCL cases with amplification were observed. It is noted that previous studies have shown that amplification can also be seen in MCL, 20,38,39 and the lack of MCL cases with amplification in our cohort may represent sampling bias. In addition, increased complexity has been previously reported to be associated with the blastoid variant of MCL 38 ; however, we were unable to assess for this association in our cohort due to the absence of pathology reports for all cases.
Limitations of this study include the small sample size of cases