Assistant Professor University of Miami Miami, Florida, United States
Introduction: Ionizing radiotherapy (RT) is a routine treatment of multiple myeloma (MM). RT induces double-strand breaks (DSB) and deletions linked to the ID8 indel mutational signature, but its mutagenic impact on MM relapse patterns is underexplored.
Methods: We reworked mmsig for indel signatures (https://github.com/UM-Myeloma-Genomics/mmsig) and used 80x whole genome sequencing (WGS) to characterize 266 newly diagnosed (ND) and 98 relapsed/refractory (RR) MM (327 patients). For validation, we included 56 relapsed and 100 untreated large B-cell lymphomas (LBCL). Because chemoRT is often co-administered in patient samples, we validated the mutagenic impact of each individual therapy using treated single-cell-derived colonies.
Results: Of 514 total WGS, 29 and 16 samples were from MM and LBCL patients exposed to RT. Of 45 cases with prior RT exposure, 41 (91%) also had prior mutagenic chemo (platinum/melphalan). Indel burden was increased in RRMM compared to NDMM (median 596 vs 527, p< 0.001). We performed both indel and single base substitution (SBS) signatures analysis for all samples. SBS signatures include platinum- (SBS31, SBS35, E_SBS37) and melphalan-induced (SBS99) mutagenesis in exposed samples. We deconvoluted a total of 8 indel signatures including ID8. ID8 was seen in 18 relapsed samples (of 154 total, 11.6%) and not in any at baseline. Importantly, while presence of ID8 was strongly correlated with prior RT (p< 0.001), it was also observed in 10 unexposed samples. All of these RT-unexposed cases had evidence of platinum/melphalan mutagenesis.
As chemo-induced ID8 has not been reported, we performed a single cell expansion experiment wherein DHL-4 cells were exposed daily to platinum or RT. Single cells from days corresponding to a lethal dose and a half-lethal dose were expanded, and the resulting colony subjected to 60x WGS. An unexposed single cell expansion was performed as control (in triplicate, total 15 WGS). RT and platinum expansions had a higher indel burden (mean, 4247) compared to control (mean, 3065; p = 0.004). Where all RT expansions had evidence of ID8, only lethal dose platinum harbored ID8, implying a higher dose threshold must be met, but that DSB from chemotherapy can indeed cause ID8.
Finally, we focused on 2 ID8+ cases from patients with RT exposure, but no chemo signatures. One LBCL case had an irradiated neck node as a bridge prior to CART. At relapse, 9 months later, a biopsy from an inguinal site had ID8. One MM case had irradiation of a clavicular plasmacytoma and a relapse 7 years later with ID8 detected in a marrow biopsy. Given the requisite clonal expansion of unique RT-induced indels required for ID8 detection in WGS, this constitutes evidence that a single cell survived RT and later seeded systemic relapse.
Conclusions: ID8 is strongly associated with palliative RT in RRMM and LBCL, indicating surviving tumor cells from RT-exposed lesions can seed systemic relapse. Yet, ID8 is not solely the result of RT, and can be induced via DSB from chemo.
