Seeking AL Amyloidosis Very Early: The SAVE Trial — Identifying Clonal Lambda Light Chain Genes in Patients with MGUS or Smoldering Multiple Myeloma
Authors: Zhou Ping , Kugelmass Adin , Toskic Denis , Warner Melissa , Lee Lisa X. , Fogaren Teresa , Varga Cindy , Comenzo Raymond L 
Published: October 2019

Abstract
INTRODUCTION : Currently patients with systemic AL amyloidosis (AL) are not identified until they are sick due to end-organ damage, usually a result of elevated clonal Ig free light chains (FLC) produced by λ clones in 75% of cases. Delays in diagnosis put patients at risk of developing irreversible organ damage making AL much harder to treat. Only about 25% of newly diagnosed AL patients are eligible for melphalan (MEL) based stem cell transplant (SCT). To extend the overall survival and quality of life of AL patients, early diagnosis is critical. A retrospective study of AL λ-type patients using the US Department of Defense serum repository showed that 100% of patients had a monoclonal FLC present for up to 4 years preceding diagnosis, 92% of whom had a difference between the pathologic and non-involved FLC (dFLC) of >23 mg/L (J Clin Oncol 2014;32:2699). Progression to AL from precursor states such as monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM) is well described but often not clinically appreciated. SAVE is an internet-based diagnostic trial (NCT02741999) for patients with λ MGUS or SMM with a κ:λ ratio < 0.26 and dFLC > 23mg/L. Using peripheral blood and, when available, marrow aspirates, we try to identify each patient's λ germline variable region (IGLV) donor gene. Five IGLV genes account for 75% of AL λ-type (IGLV1-44. 1-51, 2-14, 3-1, 6-57) (Blood 2017;129:299). Based on data in AL-Base, relative risk of AL versus myeloma with these clonal genes can be significantly high (7.3, 6-57; 2.5, 1-44), intermediate (1.6, 2-14; 1.2, 1-51) or low (0.8, 3-1) (Amyloid 2009;16:1).
MATERIAL S & METHODS : Patients provide written consent to be screened by medical records review. Eligible patients ship peripheral blood (PB) or marrow (BM) samples to us obtained only at times of routine clinical testing. We select CD138+ cells from PBMNC or BM for whole-cell RNA preparation (RNeasy Mini Kit, QIAGEN, Valencia, CA) and cDNA synthesis (ThermoScript RT-PCR System, Invitrogen Life Technologies, Carlsbad, CA) for storage at -80o C. Primer sets are degenerate FR1 and 5' CR primers for the Vλ1, Vλ2, Vλ3 and Vλ6 families (Blood 2001;98:714). Three RT-PCR products of appropriate size per clone are sent to our core facility for sequencing. We then use the sequences to search for the corresponding IGLV germline gene in IMGT (ImMunoGene-Tics, www.imgt.org). Patients and their MDs are notified of the results and, if the germline donor is associated with AL with high or intermediate risk, further evaluation for AL is discussed and pursued.
RESULTS : Twenty asymptomatic λ patients from 13 states thus far have consented to be screened for SAVE, and 19 enrolled (3M, 16F). Twenty-three PB and 4 BM specimens have been obtained. Median months from diagnosis to receipt of first specimen was 15, involved FLC 107mg/L and κ:λ ratio 0.09. Median PBMNC and CD34-selected cells were 8x106 (2.6-24) and 3x105 (0-6) respectively. Twelve patients had IGLV genes identified by PCR with the first specimen; 5 sent additional specimens including 3 BM. There were no differences in CD34-selected cell numbers or λ light chain levels between first-specimen PCR successes and failures. Seventeen patients have had IGLV genes identified: four 2-14, three 1-44, two 3-1 and eight others. Increased risk of AL was identified in 7 patients who were screened with abdominal fat pad studies. One of the 7 (with IGLV2-14) had a positive fat pad and was then diagnosed with AL λ-type with GI involvement. She was induced with 4 cycles of bortezomib-based therapy and is now 3 months status post an uncomplicated MEL 200 SCT. Two SMM patients (of 19) have proceeded to symptomatic myeloma requiring therapy.
CONCLUSIONS : The SAVE trial may enable early appreciation of risk of AL λ-type based on the variable region germline gene used by the clonal plasma cells. In λ MGUS and SMM patients with elevated lambda FLC, below normal ratios, and dFLC > 23mg/L, the clonal gene can be identified by RT-PCR with CD138-selected cells from a single peripheral blood specimen 70% of the time. Close follow up of patients at risk may reduce the likelihood of eventually being diagnosed with advanced organ involvement. Earlier diagnosis may permit prompt induction therapy if required and expanded application of MEL 200 SCT, a preferred treatment for patients with < 10% clonal marrow plasma cells at diagnosis or after induction (Biol Blood Marrow Transplant 2016;22:1729).

Disclosures
No relevant conflicts of interest to declare.