1. Survival analysis and correlation study
Ultra-Deep Targeted Sequencing Does Not Identify MM Patients with Different Prognosis: Results from a Randomized Phase II Clinical Trial
LOGO
Yanira Ruiz-Heredia1*, Jose Carlos Martínez-Ávila1*, Beatriz Sanchez-Vega, PhD1*, Inmaculada Rapado, PhD1*, Cristina Jimenez, BSc2*, Esteban Braggio, PhD3, Rafael Martinez, MD PhD4*, Laura Rosiñol5*, EnriqueM. Ocio, MD, PhD2*, M Asuncion Echeveste6*, Jaime Perez de Oteyza7*, Albert Oriol8*, Joan Bargay, MD9*, Mercedes Gironella10*, Jesus Martin11*, Rosa M. Ayala, MD, PhD1, Joan Bladé5, Keith Stewart, M.B., Ch.B.3, Maria-Victoria Mateos, MD, PhD2, Jesus San Miguel, MD PhD12, Xabier Aguirre12*, Ramon Garcia-Sanz2*, Juan José Lahuerta, MD PhD1* , Joaquín Martínez-Lopez 1.
1Hospital Universitario 12 de Octubre-H12CNIO, Madrid, Spain; 2Hospital Universitario de Salamanca-IBSAL, Salamanca, Spain; 3Division of Hematology and Oncology, Mayo Clinic, Scottsdale, AZ; 4Hospital Universitario San Carlos, Madrid, Spain; 5Hematología, Hospital Clinic Barcelona, Barcelona, ESP; ;6Hospital Universitario Donostia, San Sebastian, Spain; 7Hospital Universitario Sanchinarro, Madrid, Spain; 8Hospital Germans Trias i Pujol, Barcelona, Spain; 9Hematology Department, Hospital de Son Llàtzer, Palma de Mallorca, Spain; 10Hospital Universitari Vall d'Hebron, Barcelona, Spain; 11Hospital Universitario Virgen del Rocío, Sevilla, Spain; 12CIMA/UNAV/IDISNA, Pamplona, Spain; 12Centro de Investigación Médica Aplicada (CIMA), IDISNA, Pamplona, Spain.
RESULTS
ABSTRACT
Patients with at least one high-risk cytogenetic aberration (del17p, t4;14 and t14;16) showed a trend toward shorter SLP (P-value = ). Finally, we investigated pairwise associations between clinical variables, cytogenetic aberrations and gene mutations and we did not find significant correlations( Fig 4).
Next-generation sequencing (NGS) has revealed new insight into the complexity of clonal and subclonal architecture of multiple myeloma (MM). The introduction of targeted studies allows the detection of mutations with very low variant read frequencies (VRF) at affordable price. However, there is few information about the prognostic impact of this mutational profile in series of homogeneously treated MM patients.
Mutated genes and altered pathways
We identified 164 gene mutations in the 79 patients, 152 were missense (93%), 5 nonsense(3%), 2 stoploss (1%) and 4 in Utr or splicing region (3%). 54 mutations (33%) were predicted pathogenic by Sift and PoliPhen and 65 (40%) have been described in COSMIC database. 85% of patients harbored at least 1 mutation. .
The most frequently mutated genes were KRAS (22%), DIS3 (18%), NRAS (15%), BRAF (10%), and TP53 (9%) of the patients, accounting for the 44% of the total number of mutations. The most frequently mutated pathways were RAS and NFKB in 47% and 27% of the patients respectively.
OBJECTIVES
In this study, we analyzed the most frequently mutated genes in MM from 79 patients included in a randomized clinical trial, applying ultra high read depth . The aim of this work is to detect minority subclones ignored to date. We also integrated these data with the clinical features to find out new patterns of behavior, relate them with survival and reveal new insight into the complexity of clonal and subclonal architecture of MM.
Figure 1: Most frequently mutated pathways.
Variant Allele Frequency study
We detected 46 mutations (28%) with VRF ≤10%, showing the complex subclonal landscape of MM patients. VRF for the most recurrently mutated genes (Fig 2) KRAS, BRAF and TP53 was, in all cases, lower than 50% while DIS3 showed mutations in a broad range (from 2 to 85%). Only one patient presented one mutation in NRAS at 73 % of allele frequency. 3 patients showed more than 1 genes of Ras pathway simultaneously mutated (6.3%), in all cases were KRAS and BRAF. By contrast, NRAS always appeared exclusively mutated. 2 patients with mutations in IRF4 gene a very low VRF (3 and 4%) underwent an aggressive progression with a premature death.
METHODS
Figure 3: Impact of TP53 mutations in OS and SLP in MM patients.
Figure 4: Correlation between cytogenetics aberrations and mutated genes.
We used Ampliseq Library Kit 2.0 to target 77 genes (M3Pv2.0 panel) related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs in MM. We sequenced DNA (15ng) from CD138+ purified plasma cells from 80 MM patients at diagnosis included in GEM2010MAS65 clinical trial, (registered at as #NCT ). An average of 1500x depth sequencing coverage was generated per base using Ion Proton sequencer (Thermo Fisher, USA, PaloAlto, CA). We used Ion Reporter software applying in-house modifications in the call variants process. We also sequenced DNA from CD138 negative cells in 30% of patients in order to filter out potential artifacts and establish conditions for excluding germline variants. Mutations were consider for analysis when were called with a minimal coverage of 10x. For samples without available germline counterpart, we excluded those that were present in dbSNP or 1000Genomes with more than 1% of minor VRF. Survival was estimated using Kaplan-Meier method and compared between conditions by cox-proportional hazard regression model. We used a second approach to predict impact in survival using a penalized regression LASSO model.
CONCLUSIONS
Ultra-deep targeted sequencing strategy is able to detect mutations in most of patients, at very low allele frequency and using a minimal amount of DNA. Despite of the huge capacity to detect mutations, the clinical significance of them remains unclear in MM patients treated with highly efficient therapies. This study again confirms the complexity of the genomic landscape of MM and the great heterogeneity between patients. The role of theses mutations at relapse as well as in the subsequent treatment effectiveness is still unknown and should be preferably investigated in larger cohorts of homogeneously treated MM patients.
Figure 2: VRF of most frequently mutated genes.
REFERENCES
Survival analysis and correlation study
We evaluated the impact on survival of demographics and clinical variables and, as might be expected, we did not find any impact in survival. We also evaluated the impact of each mutated gene individually and grouping according to their pathways. Only TP53 (Fig 3) had a significant and negative impact in OS (P-value = ) but not in SLP (P-value = 0.307).
1. Heterogeneity of genomic evolution and mutational profiles in multiple myeloma, Nature Communications 2014.
2. Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients. Blood 2016.
Journal Article, Name of Journal
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