Treatment Research for Multiple Myeloma (MM)

Treatment Research for Multiple Myeloma (MM)ABSTRACT inst everyationMultiple myeloma (MM) is a hematological neoplasm that appears after transformation and uncontrolled proliferation of plasma cells. MM is characterized by a heterogeneous genetic aberrations and very(prenominal) different clinical knocked out(p)comes (Avet-Loiseau, . Boyd KD). Although treatments for MM aim increased by flummox new and much sophisticated therapeutics drugs such an immunomodulatory drugs (IMiDs) and protea or so inhibitors (PIs)( Shaji K. Kumar, Raghupathy, Antonio Palumbo) the marked variability of responses direct that larger studies will be required.Currently, identification of cytogenetic abnormalities is performed by ceremonious karyotyping and fluorescence in situ hybridization. However, these disorders are not sufficient to explain the malignant phenotype granted that are excessively observed in premalignant states of MM such a monoclonal gammopathy of unknown significance (MGUS) or sm oldering myeloma(SM)(Bergsagel, Hideshima). This finding justifies the strike for a comprehensive screening of genetic abnormalities in MM patients, which has not been incorporated in the medical workup yet. Recently, the introduction of massive sequencing of patient genome exploitation next-generation sequencing (NGS) technologies has considerably increased the understanding of the biological features of MM. Many works stand described the complex and heterogeneous pas seulal profile of MM patients(bolli nuevo, walker). altogether Exome Sequency (WES) studies in saucily diagnosis MM patients have confirmed that more than 50 genes are mutated in the first manifestation of disease (walker). However, but few genes have been detected recurrently mutated at diagnosis, including KRAS, NRAS, BRAF, DIS3, TP53 and FAM46C, and only TP53 mutations have been recurentelly associated with poor natural selection. In addition, other studies have assessed the clonal evolution oer cartridge h older, pointing out that systemic treatment with chemotherapy may affect the livelihood of some subclones more than others, and thus may influence the tumor evolution everyplace time(Egan JB, bolli, Keats JJ, ) The introduction of targeted studies eachows the detection of mutations even with very low allele frequencies at an affordable price, allowing the incorporation of extensive genetic studies to the clinical workup. In the dying old age, several groups have applied this approach in order to achieve a better patients stratification and prognosis prediction. Although many studies have highlighted the splendour of the subclonal landscape in MM and many efforts have been undertaken to stratify patients and predicts their responses, on that point is no clear relation between sensitives or refractories clones to treatment, and more learning about the prognostic impact of this subclonal profile in series of homogeneously treated MM patients is needed. A large turn of events o f clinical streak are being carried out with this aim, unify treatments in order to field of operation more effectively the impact of genetic alterations in prognosis.In this work, freshly diagnosis MM patient homogenously treated have been genetically characterized utilise a combination of the most recent techniques, including FISH and ultra-deep targeted sequencing. We applied the highest postulate depth described in the literature with the aim 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.Patient samplesSamples were taken from the available 79 newly diagnosed MM patient enrolled in the clinical trial GEM10MAS65 (registered at www.clinicaltrials.gov as NCT01237249). This is a phase III trial where patients older than 65 years were randomly assigned between two treatment s arms sequential melphalan/prednisone/Velcade (MPV) followed by Revlimid/low dose dexamethasone (Rd) versus alternating melphalan/prednisone/Velcade (MPV) with Revlimid/low dose dexamethasone (Rd). Progression free survival (PFS) and overall survival (OS) were measured from the starting date of the treatment. The median(a) time to progression was 26.4 months with a median follow up of 31.5 months.Targeted sequencing and mutation tradingPositive plasma cells CD138 were isolated from bone marrow aspirates and DNA was extracted utilize AllPrep DNA/RNA mini kit (Quiagen). Only 20 ng of DNA were used to prepare libraries using Ampliseq Library Kit 2.0. We also sequenced DNA from the 15 available CD138 negative fractions in order to try out potential artifacts and corroborate detection sentivity. Samples were sequenced using Ion alcoholic weapons platform (IonProton, Thermofisher, Carlsbad, CA, USA) using the M3P gene panel (Mayo Clinic, Arizona). This panel spreads out over 77 gen es frequently mutated in MM, which are related to critical pathophysiological passs, associated to drug metro or targetable with molecular drugs paper mayo kortum etal. Quality filter and alignments was performed using Torrent Suit software (Life Technologies) Single nucleotide variants were calling and annotated using Ion newsperson software applying in-house modifications in call variants process. Variants listed in Single Nucleotide Polimorphism database (dbSNP, http//www.ncbi.nlm.nih.gov/SNP/) were excluded from samples without germline available, as well as variants that were detected in multiple samples. In addition, to try out the ability of the workflow previously described, we applied a novel bioinformatics line developed by Spanish National Cancer Research contract (CNIO). All reported mutations were detected by both bioinformatics approaches. The integrative genomic spectator pump (IGV) was used to visualize the read alignments, single variants and correct sequenci ng errors due to homopolymer regions.statistical analysisAll statistical analysis was performed using the statistical purlieu R. Correlation coefficients between mutated genes and cytogenetic aberrations was assessed and plotted using corrplot (https//cran.r-project.org/web/packages/corrplot/). Differences in survival were tested using the log-rank test. Cox proportional hazard reversion was employed to obtain hazards ratios (HR) and evaluated at 5% of significance level. A gage approach called LASSO (least absolute shrinkage and selection operator) was performed to detect applicable variables among clinical, cytogenetic and mutated genes. Further details can be ground in Data Supplement.RESULTSMutated genes and altered pathways (cambiar ttulo)We sequenced 79 tumor samples with a stiff coverage depth of 1600X. The minimum coverage of the detected variants was 60X and the average coverage 370X. We identified 170 nonsynonymous missense/nonsense/stoploss single variants, 81 of them (48%) were predicted pathogenic by Sift and Polyphen and 61 (36%) have been described in COSMIC data base. 85% of patients harbored at least 1 mutation with a median of 2.1 mutations per patient. We detected mutations in 53 genes (Figure1), although 6 genes accounted the 39% of the total number of mutations KRAS= 21.5%, DIS3= 19%, NRAS= 16.5%, BRAF= 10.1%, TP53= 8.8% and ATM= 7.6% of the patients. 48% of patients (38/79) presented at least one mutation in genes envolved in RAS/MAPK pathway, being the most frequently mutated pathway. 72 and 100% of variants in KRAS and NRAS respectively were detected in the hotspot codons 12, 13 and 61, and the targetable V600E BRAF mutation was detected in 1 patient. (Figure 2 supplemental?). NFKB pathway was the second most frequently mutated in our cohort, accounting for the 15% of all mutations distributed in 25 % of the patients (19/79). This pathway included TRAF3 (5 mutations, one nonsense and 4 missense) and TRAF2 (3 missense mutation) a ll of them predicted pathogenic by Sift and Polyphen. Other pathways importantly altered in the cohort were MYC in 11% of patients (9/79), cereblon and ciclyn both in 9% of patients (7/79).Multiple mutations within the same gene were observed in 11 patients 4 of whom harbored 2 and 3 mutations within DIS3 (patient 1-24= Glu501Lys and Phe120Leu at 8 and 53 % of VRF, patient 2-236= Asp487His and Asp479Glu at 4 and 21 of VRF respectively, patient 3 321= Tyr753Asn and Glu126Lys at 2 and 58 % of VRF respectively and patient 4-42 = Arg820Trp, Gly249Glu and at 14, 20 and 24% of VRF respectively). Other 2 patients showed 2 and 3 mutation in KRAS (patient 5-168= Gly13Asp and Gln61His at 9 and 13 %of VRF and patient 6-269 = Tyr71Asp, UTR3 in exon 6 and Gln61Glu at 3, 15 and 37 % of VRF respectively 2 patients with 2 mutation in NRAS(patient 7-177= .Gln61Lys and Gly12Ala at 5 and 12% of VRF respectively and patient 8-257= Gln61Glu and Gly12Ala at 5 and 6% of VRF respectively), one patient wit h 3 mutations in MAX (patient 9-190= Arg36Lys , Arg35Leu and Glu32Val at 10, 20 and 26 %of VRF), one patient with 3 mutations in TRAF3 (patient 10-40 = Lys453Asn, His136Tyr and Phe445Leu 8, 11 and1 3% of VRF) and one patient with 2 mutation in TP53 (patient 11-40 = Asp208Val and Glu204Ter at 35 and 36% of VRF respectively).Variant Read Frequency study The VRF found in our cohort were diverse. We detected 50% of variants (85/170) below 25% of VRF and 27% (46/170) below 10% (Figure 2). KRAS (n=5) DIS3 (n=5), BRAF (n=4), NRAS (n=4), and TP53 (n=3) were the genes that harbored the largest number of low frequency mutations (Figure 2).KRAS, NRAS, BRAF and TP53 mutations were, in all cases, lower than 50% of VRF while DIS3 showed mutations in a broad rove (from 2 to 85%)(Fig3). ). Most of DIS3 mutations with VRF