Next Generation Sequencing Identifies Light-Chain Amyloid (AL)-Related Igvl Genes in Patients with λ Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Multiple Myeloma (SMM)

Introduction: Eighty percent of AL patients have clonal free light chain (FLC) abnormalities for years prior to diagnosis (J Clin Oncol 2014;32). Strategies for early identification of AL are critically needed; 75% of AL cases are λ-type and 9/33 λ light-chain variable region genes account for 85% (Blood 2017;129; Amyloid 2009;16).

In two clinical studies (NCT02741999, NCT04615572) λ MGUS and SMM patients were eligible if the difference between involved and uninvolved FLC was > 23mg/L, the κ:λ ratio below normal and they had no prior evidence of amyloid (J Clin Oncol 2014;32; J Clin Oncol 2019;37). We identified clonal IGVL genes by next generation sequencing (NGS), screened for AL and followed for progression to MM or AL. The 9 IGVL genes we deemed AL-related were 6-57, 2-14, 1-44, 3-1, 1-51, 3-21, 3-19, 2-23 and 1-40. We now report results of these studies.

Methods: Eligible MGUS and SMM patients had PB or BM shipped and processed as previously described (J Clin Oncol 2019;37). We performed RNA extraction with CD138 cells and marrow mononuclear cells (MNC) and synthesized cDNA for NGS evaluation. PCR products were amplified using 9 λ IGVL and 2 λ constant region primers, fragmented, end-repaired, covalently linked to adapters using ligation and sequenced on a MISeq (Illumina, San Diego CA). Approximately 500,000 reads were obtained per sample. FASTA sequences were mapped with IMGT/HighV-QUEST to obtain CDR3-related reads (CDR3-reads) linked to an IGVL gene. GraphPad PRISM was used for statistical analyses.

Results: Forty-seven patients had specimens shipped for NGS, 21 on the pilot study NCT02741999 (06/2016-12/2020) and 26 on the multicenter study NCT04615572 (04/2021-04/2022).

In the pilot study (MGUS=2, SMM=19), NGS identified IGVL genes in 13/16 PB and in 5/5 BM specimens; the remaining 3 PB had genes identified by RT-PCR. AL-related genes were found in 1/2 MGUS and 13/19 SMM (68%). No MGUS had AL or progressed. Two women with SMM and 2-14 were found to have AL, aged 56 (GI) from Illinois with weight loss and 58 (cardiac) from North Carolina with palpitations (Table 1; patients #7 and #16); 5 patients with SMM progressed at a median 32.5 months (range, 23-46), 4 to active MM and 1 to surgery for a meningioma.

In the multicenter NCT04615572 study (MGUS=12, SMM=14), we use BM specimens only, make cDNA from CD138 cells and MNC and also document clonal cytogenetic findings (t(11;14), gain 1q (found in 75% of AL and 50% of SMM) (Blood 2016;128; J Clin Oncol 2013;31)). In the MGUS cases, a single IGVL gene was identified in MNC cDNA in 10, two genes were found in 1 (2-23 and 5-52 with different CDR3-reads), and three genes in another (ranked by reads 1-44>3-19>10-54). In the 14 SMM cases, amplification failed in 1 with both MNC and CD138 cell cDNA (2-11 by RT-PCR), a single IGVL gene was identified in MNC cDNA in 12, and both 1-44 and 1-47 were identified in MNC cDNA in 1 case with reads of 21683 for the former and 67886 for the latter and similar findings with CD138 cell cDNA. Unique CDR3-reads had a median of 31bp (range, 24-39). Twenty-five marrows had paired MNC and CD138 samples and IGVL genes were concordant while CDR3-reads were higher with CD138 cell cDNA, mean (+/- SD) 4.85 (+/- 2.80)x10⁴ vs MNC cDNA 3.39 (+/- 2.27)x10⁴ (p<0.01, paired t-test, r=0.88). In SMM cases median MNC cDNA CDR3-reads were 2.7x10⁴ (1-10.1). AL-related genes were found in 11/12 MGUS (92%) and 11/14 SMM cases (79%). Cytogenetic findings of t(11;14) and gain 1q were found in 4/12 MGUS (33%) and 9/14 SMM (64%). No MGUS had AL or progressed. Two men with t(11;14) SMM and 3-1 and 2-23 had AL, aged 70 (cardiac) from New York with genital ecchymosis and 60 (peripheral neuropathy) from Tennessee with LE numbness (Table 1; NCT04615572 patients #11 and #19); 1 patient progressed after SMM for 60 months to active MM.

All 4 patients found to have AL were successfully treated, achieved complete remissions and are alive and well. The distribution by diagnosis of IGVL genes in the 47 cases is shown in Table 2.

Conclusions: NGS reliably identified clonal IGVL genes in 30/31 (97%) BM MNC cDNA from λ MGUS and SMM patients. Four of 33 SMM patients had AL (12%) and were in the 24 SMM with AL-related IGVL genes (17%). A large study is planned evaluating a likelihood algorithm for AL using as parameters SMM (both κ and λ), the FLC screen, and having both IGVL genes and clonal cytogenetics that are AL-related. Establishing the likelihood of AL and risk of AL in SMM will enable early diagnosis and save lives.

Wong:Janssen: Research Funding; Fortis: Research Funding; GSK: Research Funding; Catalent Biologics: Consultancy; Genentech: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Dren Bioscience: Consultancy; Caelum: Research Funding; Bristol Myers Squibb: Research Funding; Patient Discovery: Research Funding. Tuchman:Shattuck Labs: Honoraria; Prothena: Honoraria; Janssen: Honoraria. Hoffman:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Seagen Inc.: Research Funding.