A Rapid and Robust Molecular Diagnostic Approach For Multiple Myeloma – Results From a Large Trial Cohort
Authors: Kaiser Martin F. , Begum Dil , Proszek Paula , Dahir Nasrin , Gonzalez de Castro David , Pawlyn Charlotte , Brioli Annamaria , Savola Suvi , Gregory Walter , Owen Roger G. , Jackson Graham H. , Davies Faith E , Walker Brian A , Morgan Gareth J 
Published: October 2019

Abstract

Introduction
Obtaining reliable information about the molecular subtype of myeloma is and will become ever more important in a number of clinical settings, such as alternative treatment strategies for high risk or ultra high risk disease (Boyd KD et al., Leukemia 2011), or patient selection for small molecule inhibitors, that are currently under development, targeting myeloma subtype specific proteins (e.g. MMSET or MAF). We report here our experience with a novel, highly applicable and high throughput diagnostic approach in a large sample set of 1016 myeloma presentation cases, using a combination of qRT-PCR and Multiplex Ligation-dependent Probe Amplification (MLPA) for molecular patient characterization of Ig loci translocations and well-defined copy number abnormalities.

Material and Methods
Recurrent translocations were assessed for 1016 presentation NCRI Myeloma XI trial cases and 41 matched relapse samples, using a previously published and interphase fluorescence in situ hybridization (iFISH)-validated in house qRT-PCR assay on purified bone marrow plasma cell material. The assay measures expression of translocation partner genes and their downstream effectors (e.g. CCND1, MMSET, FGFR3, MAF, MAFB, CCND2) with subsequent interpretation and categorization of results based on the translocation/cyclin D (TC) classification. This allows prediction of presence of the recurrent translocations with high sensitivity and specificity (Kaiser MF et al., Leukemia 2013) and evaluation of overexpression of potential drug targets independent of translocations (e.g. MAF). For selected cases, the myeloma specific SALSA MLPA assay (MRC-Holland) was performed, containing 46 probes that inform about prognostically relevant copy number alterations, such as del(1p), gain(1q), or del(17p). High correlation between MLPA and FISH results for clinically relevant copy number aberrations has been previously reported (Alpar D et al., Genes Chrom Canc 2013).

Results
The TC classification based translocation qRT-PCR assay worked reliably even for poor quality input RNA, providing results for >96% of analyzed samples. Predicted translocation frequencies among the 1016 evaluable cases were comparable to previously reported results [t(11;14): 16.6%; t(4;14): 12.6%, of which 21.1% lacked FGFR3 expression; t(14;16): 2.6%; t(14;20): 0.5%; t(6;14): 0.7%]. Relapse samples showed consistent results with matched presentation samples, with one t(4;14) case losing initial high FGFR3 expression at relapse. Correlation with clinical data will be available for presentation at the meeting. Measurement and analysis of the samples was performed by a single lab technician in a short time, demonstrating the high throughput capability of the method. This makes rapid analysis of very large sample collections possible, revealing novel findings. When the assayed group was split by median age, the younger group (22-66 years) contained relatively more t(4;14) [15.7% vs. 9.4%; p=0.003] cases than the older group (67-88 years), consistent with recent reports on iFISH data (Avet-Loiseau H, 2013). We also found a lower frequency of t(11;14) [13.6% vs. 19%;p=0.022] in the younger vs. the older group, which has not been reported.
MLPA results were generated for a subset of 30 samples for which iFISH and copy number array data were available. The previously reported high level of correlation with iFISH results (Alpar D et al., Genes Chrom Canc 2013) was confirmed and extended for copy number array data, with >85% sensitivity and >95% specificity for del(1p), gain(1q), del(13p) and del(17p). MLPA assessments will be extended in the coming months to include a large group of Myeloma XI cases, and results and their associations with qRT-PCR results and clinical features will be presented at the meeting.

Conclusion
Precision medicine approaches in myeloma require fast, robust and practicable molecular diagnostic tools. The current diagnostic standard iFISH doesn’t fulfill any of these criteria. Other approaches such as microarray analyses have never found acceptance outside of highly specialized centers due to practicability issues. With the approach presented here, clinically relevant molecular features can be assessed within 48 hours with standard molecular laboratory equipment. This approach is a suitable candidate for a novel standard for routine clinical molecular analysis of multiple myeloma.

Disclosures:
Savola: MRC-Holland: Employment.