DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort

Background: Recognition of molecular abnormalities in acute myeloid leukaemia (AML) has improved our understanding of its biology. NPM1 and FLT3-ITD mutations are recurrent in AML and clinically significant. NPM1 mutations are associated with a favourable prognosis, while FLT3-ITD mutations are an independent poor prognostic factor in AML.Objective: This study described the prevalence and molecular characteristics of the NPM1 and FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa.Methods: The study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex polymerase chain reaction assay was optimised to screen patients for the respective mutations and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD mutations were determined, and mutation-specific characteristics were described in relation to patients’ demographic information and AML classifications.Results: The patients’ median age was 38.5 years, with 77.5% (n = 31) of patients being self-proclaimed Black Africans. AML with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (APL) was most common (40.0%; n = 16). None of the patients had the NPM1 mutation. FLT3-ITD was present in 37.5% (6/16) of APL patients and in one (20.0%) of five AML patients with a t(8;21) translocation. Most patients had an FLT3-ITD allele ratio of ≥ 50% and ITD lengths of 39 bp.Conclusion: FLT3-ITD mutations were mainly found in APL cases at a similar prevalence as reported in the literature. High FLT3-ITD allele ratios and long ITD lengths predominated. No NPM1 mutations were detected.

Prognostic impact of low allelic ratio FLT3-ITD and NPM1 mutation in acute myeloid leukemia

In the opinion of the European LeukemiaNet (ELN), nucleophosmin member 1 gene mutation (NPM1 mut)–positive acute myeloid leukemia (AML) with an fms-like kinase 3-internal tandem duplication (FLT3-ITD) allele ratio (AR) <0.5 (low AR) has a favorable prognosis, and allogeneic hematopoietic stem cell transplant (allo-HSCT) in the first complete remission (CR1) period is not actively recommended. We studied 147 patients with FLT3-ITD gene mutation–positive AML, dividing them into those with low AR and those with AR of ≥0.5 (high AR), and examined the prognostic impact according to allo-HSCT in CR1. Although FLT3-ITD AR and NPM1 mut are used in the prognostic stratification, we found that NPM1 mut–positive AML with FLT3-ITD low AR was not associated with favorable outcome (overall survival [OS], 41.3%). Moreover, patients in this group who underwent allo-HSCT in CR1 had a significantly more favorable outcome than those who did not (relapse-free survival [RFS] P = .013; OS P = .003). Multivariate analysis identified allo-HSCT in CR1 as the sole favorable prognostic factor (RFS P < .001; OS P < .001). The present study found that prognosis was unfavorable in NPM1 mut–positive AML with FLT3-ITD low AR when allo-HSCT was not carried out in CR1.

Genetic Contribution of Ningmai 9 Wheat to Its Derivatives Evaluated by Using SNP Markers

Founder parent usually plays an important role in wheat breeding. Ningmai 9 is a soft wheat variety with good performance in yield, quality, and resistance to wheat disease. Therefore it serves as an important commercial variety and founder parent in middle and lower Yangtze River of China. To date, 20 new cultivars have been developed from Ningmai 9 and released to wheat production in the last 10 years. In this study, the 90K iSELECT ILLUMINA chip was used to analyze the genotype of Ningmai 9 and its 17 derivatives. The genetic similarity coefficients between Ningmai 9 and its derivatives were more than 0.7 except for Yangfumai 4. Neighbor-Joining analysis showed that Yangfumai 4 had the largest genetic distance from Ningmai 9 in all derivatives. There was a great difference for the same allele ratio in either derivatives or chromosomes, though the average values of the same allele ratio in genomes A, B, and D were close to each other. The phenotypic difference in Ningmai 9, Ningmai 13, and Yangfumai 4 was consistent with their difference in genetic background by comparing previous reported QTLs. Some hot chromosome regions were found and might be used for marker assisted selection in wheat breeding.

Responsiveness of FLT3-ITD+ AML to Kinase Inhibition Correlates with High Allele Ratio and Relapsed Disease but Not to Inhibition of Canonical Targets

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase and is the most commonly mutated gene in acute myeloid leukemia (AML). Second generation FLT3 inhibitors such as quizartinib (AC220) are clinically active in relapsed FLT3-ITD+ patients. However, not all patients respond and to date primary resistance has not been characterized. A previous study proposed that AML cells from patients with relapsed FLT3 mutant AML and samples with high allelic burden for FLT3 ITD are more sensitive to FLT3 inhibitor cytotoxicity (Pratz KW, et al Blood 2010). We performed studies to test this hypothesis and determine if cells with homodimeric FLT3 ITD are more heavily dependent on FLT3 ITD for growth and survival than cells expressing heterodimeric FLT3 WT:FLT3 ITD. 16 primary AML samples that contain FLT3 ITD mutations were incubated in increasing concentrations of the second generation FLT3 inhibitor crenolanib and assayed for survival in short term liquid culture assays. Only 6 samples demonstrated greater than 30% inhibition of survival in this culture system whereas 10 samples showed little or no cytotoxic response. Consistent with previous results (Pratz, et. al, Blood 2010), responding samples tended to be from relapsed patients and to have higher FLT3 ITD allelic ratios. We then analyzed FLT3 expression and phosphorylation levels as well as inhibition of and crenolanib inhibition of FLT3 phosphorylation, as well as the canonical downstream signal transduction pathways STAT5, ras/MAPK, and PI3K/AKT/mTOR in 13 FLT3-ITD+ primary AML samples. For 11/13 samples, crenolanib strongly inhibited phosphorylation of FLT3 kinase. However, neither FLT3 protein expression nor baseline phosphorylation level correlated with cytotoxic response in liquid culture assays. Crenolanib inhibited phosphorylation of STAT5, ribosomal S6 and ERK to varying degrees and inhibition of none of these pathways consistently correlated with cytotoxicity. Overall, these results are consistent with the hypothesis that FLT3 ITD mutant AML with high allele burden or relapsed samples are more addicted to FLT3 ITD. To further examine this topic, we studied AML cell lines that express only wild type FLT3 (THP1), both FLT3-ITD and WT FLT3 (MOLM14) or FLT3-ITD but not WT-FLT3 (Mv4;11, TF1-ITD). Growth of all three cell lines expressing FLT3-ITD but not THP1 cells was inhibited by crenolanib. Crenolanib inhibited tyrosine phosphorylation and activation of downstream signaling pathways in all three FLT3-ITD+ cell lines. Importantly, crenolanib was active at 10 nM in TF1-ITD cells but higher concentrations were required to inhibit signaling in Mv4;11 cells. This suggests that allele ratio alone does not determine sensitivity to FLT3 inhibitors. Interestingly, FLT3 ligand (FL) impairs inhibition of FLT3 by kinase inhibitor and, again, mutant cell lines are similarly responsive to FL in the presence of kinase inhibitor. In summary, these data demonstrate that AML cells have variable dependence on mutant FLT3 for survival. Samples with high allele ratio and relapsed samples behave in a manner consistent with oncogene addiction and are likely to show cytotoxicity to FLT3 inhibition. However, the basis for oncogene addiction is unclear and does not depend on activation of the canonical signal transduction pathways known to be downstream of FLT3. Interestingly, a recent unbiased screen of phosphorylated proteins in FLT3-ITD+ AML to predict clinical response to AC220 did not identify tyrosine phosphorylation of canonical FLT3 targets, but correlated clinical response with serine phosphorylation on EEPD1-S160, BCL11A-S630, and RANBP3-S333 (Schaab C, et al. Leukemia 2014). Analysis of the effects of FLT3 inhibitors upon these proteins in FLT3 mutant primary samples and cell lines is ongoing. Disclosures Perl: Arog pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy.

Rapid, Robust and Accessible Molecular Profiling of Biologically and Clinically Relevant Copy Number Alterations in Multiple Myeloma from Small Amounts of Tumor DNA

Background Structural aberrations like copy number alterations (CNA) and translocations play a central role in multiple myeloma tumor biology. The analysis of CNA for complete molecular profiling of myeloma retains clinical significance in the context of recent next-generation sequencing (NGS) results which highlight the highly heterogeneous landscape of single nucleotide mutations, which are often sub-clonal. Detection of CNAs remains the domain of array-based technologies, as demonstrated by the use of copy number arrays by the TCGA consortium and others for CNA detection in NGS projects. High cost and infrastructure attached to CNA analysis by array limit access to this technology in many molecular diagnostic laboratories. We present here a robust and accessible method that combines two multiplex ligation-dependent probe amplification (MLPA) assays for the relevant CNAs in myeloma using low input amounts of purified tumor DNA. Methods Bone marrow myeloma cells from patients at presentation and at relapse from the Myeloma IX and Myeloma XI trials were purified to >95% purity by immunomagnetic separation (Miltenyi Biotech) and DNA was extracted using AllPrep columns (QIAGEN) for analysis by MLPA. A novel myeloma specific probemix was designed and technically validated by MRC-Holland (Amsterdam, The Netherlands), complementing the established myeloma specific probemix P425 (Alpar et al., Gen Chrom Cancer 2013). Results In total 130 purified myeloma cell DNA samples were successfully analyzed by MLPA, interrogating 43 different loci in the following regions: 6p22 - 6p12 [incl. CDKN1A]; 6q12 - 6q26 [incl. TNFAIP3, WTAP, PARK2]; 8p23 – 8p11 [incl. TNFRSF10A/B]; 8q12 – 8q24 [incl. MYC]; 11q13 – 11q25 [incl. CCND1, BIRC2, BIRC3]; 17p [all exons of TP53]; 22q11 – 22q13 [incl. HIRA, EP300]. The assay contains one probe for the detection of BRAF V600E, including determination of mutant allele ratio. This probemix complements another MLPA assay [P425] which interrogates prognostically and biologically relevant regions such as 1p32, 1q21-1q23, 13q14, 16q12-16q23, 17p13 and chr 5, 9 and 15 for detection of hyperdiploidy (HRD). The assay returned good quality results using as little as 25 ng DNA input material without modification of assay conditions. In this group of cases which were enriched with cases known to carry specific CNAs and mutations, frequent copy number aberrations encompassed loss of chr(6q), in the majority of cases (16%) affecting all genes investigated in the region 6q23 – 6q26. In addition, five cases (4%) with an isolated heterozygous deletion of TNFAIP3 at 6q23 and two cases with deletion of TNFAIP3 and WTAP were detected. Gains of MYC at 8q24 were observed in 13 cases (10%) of which 5 showed gains of other regions on 8q as well. BIRC3 and BIRC2 were both lost in 4 cases, including 1 homozygous deletion of both genes. All coding exons of TP53 were heterozygously deleted in 16% of cases in this group containing specifically selected relapsed cases. Gain of chromosome 11 was frequent (29%) and mostly associated with HRD, but gain(11q) without HRD was observed, including 3 cases with isolated gain of the CCND1locus at 11q13.3. Of 11 cases with a known BRAF V600E mutation identified by SSCA, 10 (91%) were detected by MLPA with the remaining mutation showing a signal just above detection limit by SSCA. Calculated median V600E mutant allele ratio in comparison to an artificial plasmid mix mimicking a heterozygous mutation was 39% (range 9%-77%), confirming our previous observation that this mutation is mostly heterozygous and sub-clonal in myeloma. Further cases at presentation from the Myeloma XI trial are currently being analyzed by MLPA and results will be presented at the meeting, including a comparison with matched CNA array data for a selection of cases as well as correlation with clinical data for trial cases. Discussion The combination of this panel with another MLPA probemix [P425], which has been extensively validated in over 1200 myeloma cases by our group, allows assaying of biologically and clinically relevant CNAs in myeloma as well as BRAF V600E mutation status. The tests can be run on standard laboratory equipment and require 25 ng of DNA as input material, making this method suitable for exploring the clinical impact of myeloma specific CNAs in large clinical trials with a potential of transferring the method to the routine diagnostic setting. Disclosures Walker: Onyx: Consultancy. Savola:MRC-Holland: Employment.

CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection

Background: Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes. Methods: We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a CNVphaser (copy number variation phaser). The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals. Results: Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population. Conclusions: Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.