The Complex Structural Variant Chromothripsis Can be Defined on Targeted Sequencing Panels, Allowing Direct Clinical Translation in Order to Improve Multiple Myeloma Prognostication

Introduction: the complex structural variant (SV) chromothripsis is an independent predictor of progression-free (PFS) and overall survival (OS) in newly diagnosed multiple myeloma (NDMM); predictive in multivariate analysis when considering ISS, IgH translocations and TP53 status (Rustad BCD 2020). Chromothripsis is not included in current prognostic scores, despite a prevalence of 24% in NDMM. While gold-standard definition requires whole genome sequencing (WGS), we’ve previously shown that copy-number (CN) signatures are also able to accurately define chromothripsis in whole exome sequencing (WES, Maclachlan Nat Com 2021). As neither WGS nor WES are currently used in clinical medicine, we applied CN signatures to targeted sequencing, aiming for direct clinical translation of this key biological feature into clinical practice. Memorial Sloan Kettering (MSK) now offers the MSK-Heme-IMPACT targeted sequencing panel to all MM patients having a bone marrow biopsy (BMBx), as well as offering retrospective analysis for current patients having leftover DNA from prior BMBx.

Methods: We analyzed 420 samples having either myTYPE or MSK-Heme-IMPACT targeted sequencing at MSK prior to July 2023. We excluded those with whole genome duplication, applied CN signatures, then for samples from NDMM performed multivariate analysis for PFS prediction by backwards stepwise regression.

In samples with CN signatures predictive for chromothripsis and with DNA available from the same BMBx, we performed parallel WGS in order to check for chromothripsis. WGS sequencing was at 60-80x for tumor, 30-40x for normal, with analysis including Battenberg for CN analysis, with SvABA, BRASS and GRIDSS for SV calling. Additional parallel WGS is in progress for 45 samples with NDMM prior to DVRd or DKRd, in order to examine chromothripsis prediction and PFS impact in the context of highly effective induction.

Results: From 420 samples, 48 were collected at the precursor stage, 205 with NDMM, and 167 with relapsed disease. 39 patients had 2 assays performed, while the remainder were unique patient samples. Within NDMM, the range of induction regimens received included lenalidomide and dexamethasone with bortezomib (VRd), carfilzomib (KRd) or daratumumab (DRd), along with quadruplet therapy (DVRd or DKRd).

Targeted sequencing data detected all 6 key CN features observed in WGS and WES analysis. Similar to WES, there was a lower contribution from very high breakpoint counts per 10mB and longer lengths of oscillating CN segments, due to the lower resolution of targeted sequencing data. De novo extraction of CN signatures defined 1 signature containing multiple features consistent with chromothripsis; high breakpoint count per 10mB, more jumps between adjacent CN segments, longer lengths of oscillating CN segments and a predominance of small CN segments.

When examining samples from NDMM, contribution from this signature was predictive of PFS in multivariate analysis when considering age, ISS, t(4;14), TP53 status and gain1q21 (hazard ratio > 4, p=0.003).

33 samples had a predictive probability for chromothripsis > 0.6 (the cut-off in WGS / WES having the highest sensitivity and specificity for prediction), with also having further DNA available. 30/33 (91%) were confirmed to harbor chromothripsis on manual inspection of WGS data. 19/30 (63%) with chromothripsis were BMBx from NDMM, while 11/30 (37%) were from relapsed disease.

Discussion: the complex SV chromothripsis is a key genomic risk factor predicting for poor PFS and OS in MM, which is currently not being captured in routine practice or in clinical trials. Here, we demonstrate that chromothripsis can be predicted from targeted sequencing data, confirmed by gold-standard WGS analysis. Ongoing studies are expanding our cohort of NDMM with concurrent targeting sequencing and WGS, to define the sensitivity and specificity of targeted-sequencing-based CN signature prediction of chromothripsis. The additional cohort largely comprises patients treated with daratumumab-quadruplet induction therapy, aiming to confirm that chromothripsis and CN signatures retain prognostic significance in the context of this therapy. With this data is in hand, we will develop a CN signature fitting tool, similar to mmsig for mutational signatures (Rustad Comm Biol 2021), to allow easily accessible incorporation of chromothripsis assessment into clinical practice.