Longitudinal Fluorescence in Situ Hybridization at Primary Diagnosis and Relapse Reveals Clonal Evolution after Autologous Stem Cell Transplantation in Multiple Myeloma
Authors: Merz Maximilian , Hielscher Thomas , Seckinger Anja , Hose Dirk , Bertsch Uta , Neben Kai , Raab Marc S , Blau Igor , Lindemann Hans Walter , Schmidt-Wolf Ingo GH , Scheid Christof , Haenel Mathias , Weisel Katja , Goldschmidt Hartmut , Jauch Anna , Hillengass Jens 
Published: October 2019

Abstract
Introduction:
Multiple myeloma is a heterogeneous disease with survival ranging from months to more than 10 years. Cytogenetic abnormalities (CA) detected by fluorescence in situ hybridization (FISH) are of major prognostic significance, since e.g. patients with del(17p), t(4;14) or gain 1q21 show dismal outcome. We evaluated CA at primary diagnosis and relapse to investigate clonal evolution in patients treated with upfront autologous stem cell transplantation (ASCT).
Methods:
We identified 128 patients with paired samples at primary diagnosis before the start of therapy (1st FISH) and at relapse after ASCT (2nd FISH). Forty-four patients were initially treated within the GMMG HD4 trial which compared 3 cycles of conventional induction chemotherapy (Arm A) with a bortezomib-based induction therapy (Arm B) followed by tandem ASCT and thalidomide (Arm A) or bortezomib (Arm B) maintenance. Eighty-four non-study patients (NSP) treated outside clinical trials were included who had received comparable induction therapies (bortezomib: n=45, thalidomide: n=11, other: n=28) before ASCT. FISH was performed on purified plasma cells using probes for 1q21, 5p15, 5q35, 8p21, 9q34, 11q23, 13q14, 15q22, 17p13 and 19q13, immunoglobulin H (IgH) translocations, t(11;14), t(4;14) and t(14;16). McNemar`s test was used to assess differences between FISH assessments. Kaplan-Meier method and Cox regression were used to analyze survival differences between patients without CA or with CA only at 1st, 2nd or both FISH assessments. Last follow-up for the whole cohort was performed in 06/2016.
Results:
Median time to first progression for the whole cohort was 27.0 months (HD4: 29.8 months; NSP: 25.5 months). There were no significant differences in CA as well as remission after induction therapy or ASCT between the HD4 and NSP cohort. The number of patients with high-risk CA was significantly higher after relapse (odds ratio (OR): 6.33; 95% confidence interval (CI): 1.86, 33.42; p<0.001). This was due to an increase of patients with del(17p) (OR: 3.4 [1.2, 11.79]; p=0.02) and gain 1q21 (OR: 16 [2.49, 670.96]; p<0.001) since none of the patients developed de novo t(4;14) after relapse. Also for t(11;14) and t(14;16) no changes between 1st and 2nd FISH were found. IgH translocations involving an unknown partner occurred more frequently at 2nd FISH only (8.4%). Hyperdiploidy (HD) was observed in 45 patients (44.6%) at both time points, while 7 patients lost their HD karyotype at relapse and 2 patients developed HD during follow-up. To investigate whether de novo high-risk CA evolve as major clone at relapse, we analyzed percentages of plasma cells carrying del(17p) or gain1q21. For del(17p), 10 out of 17 patients and for gain 1q21, 5 out of 16 patients carried the respective aberration in less than 60% of plasma cells after progression. Patients with gain 1q21 at both time points (n=48) showed in 35.4% (n=17) changes in copy number and/or clone size after relapse. We could not identify clinical risk factors (age, sex, type of induction therapy, remission before/after ASCT, initial ISS) associated with de novo high-risk CA. However, patients with t(11;14) at baseline developed more often high-risk CA at relapse. When analyzing impact of CA on overall survival (OS) we found dismal outcome regardless whether high-risk CA were present at baseline (hazard ratio/HR: 3.53 [1.53, 8.14]; p=0.003) or developed at relapse (HR 3.06 [1.09, 8.59]; p=0.03) in multivariable Cox regression. OS analysis from different landmarks (date of progression and date of 2nd FISH) confirmed this effect.
Conclusion:
We demonstrate clonal evolution and a higher incidence of high-risk CA at relapse after ASCT in a homogeneously treated group of patients. The most common translocations in myeloma are highly conservative. High-risk CA can emerge on a subclonal level during disease progression or might evolve from initially present subclones which results in similar dismal outcome

Disclosures
Merz: Celgene: Other: Travel grant; Janssen: Other: Travel grant. Hose:Sanofi: Research Funding; EngMab: Research Funding; Celgene: Other: personal fees outside this work. Bertsch:Janssen: Research Funding; Celgene: Research Funding; Chugai: Research Funding. Schmidt-Wolf:Janssen: Research Funding; Novartis: Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Weisel:Amgen: Consultancy, Honoraria; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria. Goldschmidt:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Chugai: Consultancy, Research Funding; Onyx: Consultancy; Millenium: Consultancy; BMS: Other: fees outside this work. Hillengass:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; Amgen: Consultancy, Honoraria; BMS: Honoraria; Celgene: Honoraria; Novartis: Research Funding.