The Mutagenic Impact of Radiotherapy in Multiple Myeloma

Introduction The use of ionizing radiotherapy (RT) to palliate bone disease is a treatment staple of multiple myeloma (MM). RT introduces simultaneous double-strand breaks and, as shown for solid tumors, cells which survive RT acquire multiple small deletions linked to the ID8 indel mutational signature (Kocakavuk et al., 2021, Nat Gen). However, in contrast to chemotherapy mutational signatures (e.g. melphalan, SBS-MM1; and platinum, SBS31), the ID8 mutational process has additional intrinsic components and can be found in RT-naïve tumors with defects of DNA double-strand break repair by non-homologous DNA end-joining and with mutations in topoisomerase TOP2A (Alexandrov et al. Nature 2020). Despite its widespread use, the mutagenic impact of RT as well as the landscape of indel signatures have not yet been explored in MM.

Methods Using whole genome sequencing (WGS), we characterized the indel landscape of CD138-enriched MM cells from 265 newly diagnosed (NDMM) and 75 relapsed/refractory MM (RRMM) patients from the University of Heidelberg. As a validation group, we included WGS from 58 NDMM patients treated with Carfilzomib/Lenalidomide/Dexamethasone +/- Daratumumab (Landgren et al., JAMA Onc, 2021; Korde et al., JAMA Onc 2015). Indels were called and curated with Platypus. De novo signature extraction was performed with SigProfiler. Signature deconvolution was performed with a pairwise expectation maximization algorithm (https://github.com/UM-Myeloma-Genomics/Signature-Assignment) and fitting was performed using a reworked version of mmsig for indel signatures (https://github.com/UM-Myeloma-Genomics/mmsig).

Results Overall, 211 patients had high-risk disease (62%) as defined by having at least one of t(4;14), t(14;16), del17p, and gain1q. 63 of the 75 RRMM patients had high-risk disease and 22 (29%) received RT prior to relapse sample collection with a median latency of RT to sample collection of 3.5 years (IQR 1.6-5.0). In line with the recently published link between ID8 and radiotherapy in solid tumors, samples from RRMM with prior exposure to RT showed marked enrichment for ID8 (21 of 22 patients; 96%) as compared to NDMM without RT exposure (70 of 263 patients; 27%) (p<0.001). Importantly, 4 of 4 patients with both a pre- and a post-RT sample showed the ID8 signature only at relapse. In addition to the association with RT, ID8 was also enriched in NDMM with high-risk status (46/148, 31%; p=0.027). The enrichment of ID8 in patients with high-risk status could be confirmed in the validation group: 11/21 (52%) NDMM patients with high-risk disease had a detectable ID8 signature as compared to 8/37 with standard risk (22%; p = 0.02).

Importantly, the association between RT and ID8 held when restricting the analysis to only high-risk patients (p=0.002), further supporting the impact of RT on the landscape of indels. For RT unexposed patients, there was no appreciable increase in samples bearing ID8 at relapse (p=0.7), suggesting that the intrinsic contribution of the ID8 mutational process likely shapes tumor development prior to clinical diagnosis. Furthermore, ID8-positive cases without RT exposure had lower ID8 mutational contribution than those with ID8 post-RT (p= 0.007).

Finally, because all samples were collected from the bone marrow, the emergence of ID8 at relapse implicates that a single exposed cell from an RT-palliated lesion may survive and seed another anatomical site, contributing to the subsequent systemic relapse. This is in parallel to what has been observed with both platinum and melphalan-induced mutagenesis (Pich et al., Nat Gen, 2019; Landau et al., Nat Comm, 2020) but is striking due to the focal nature of RT as opposed to the systemic mutagenesis of chemotherapy.

Conclusion ID8 is seen here to be strongly associated with palliative RT in RRMM, indicating that surviving tumor cells from RT-exposed lesions can contribute to systemic relapse. Yet, ID8 is not solely the result of RT, as we see an enrichment of ID8 indels in unexposed patients with high-risk disease, suggesting that indel signatures that may aide in prognostication.

Fig 1: ID8 contribution to MM indel burden. Comparisons computed with Wilcoxon Rank Sum test. SR, Standard Risk; HR, High Risk; ND, Newly Diagnosed; RR, Relapsed/Refractory; RT, Radiotherapy.

Diamond:Sanofi: Honoraria; Medscape: Honoraria; Janssen: Honoraria. Usmani:Amgen, BMS, Janssen, Sanofi: Speakers Bureau; Abbvie, Amgen, BMS, Celgene, EdoPharma, Genentech, Gilead, GSK, Janssen,Oncopeptides, Sanofi, Seattle Genetics, SecuraBio, SkylineDX, Takeda, TeneoBio: Consultancy; Amgen, Array Biopharma, BMS, Celgene, GSK, Janssen, Merck, Pharmacyclics, Sanofi, Seattle Genetics, SkylineDX, Takeda: Research Funding. Davies:Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Landgren:Riney Foundation: Research Funding; Leukemia & Lymphoma Society: Research Funding; Tow Foundation: Research Funding; Rising Tide Foundation: Research Funding; Merck & Co., Inc.: Other: Independent Data Monitoring Committee (IDMC) member for clinical trials; Janssen: Honoraria, Other: Independent Data Monitoring Committee (IDMC) member for clinical trials, Research Funding; Aptitude Health: Honoraria; MMRF: Honoraria; NCI/NIH: Research Funding; Theradex: Other: Independent Data Monitoring Committee (IDMC) member for clinical trials; Pfizer Inc: Consultancy; Amgen: Honoraria, Research Funding. Raab:Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.