Toward accurate high-throughput SNP genotyping in the presence of inherited copy number variation

<p>Abstract</p> <p>Background</p> <p>The recent discovery of widespread copy number variation in humans has forced a shift away from the assumption of two copies per locus per cell throughout the autosomal genome. In particular, a SNP site can no longer always be accurately assigned one of three genotypes in an individual. In the presence of copy number variability, the individual may theoretically harbor any number of copies of each of the two SNP alleles.</p> <p>Results</p> <p>To address this issue, we have developed a method to infer a "generalized genotype" from raw SNP microarray data. Here we apply our approach to data from 48 individuals and uncover thousands of aberrant SNPs, most in regions that were previously unreported as copy number variants. We show that our allele-specific copy numbers follow Mendelian inheritance patterns that would be obscured in the absence of SNP allele information. The interplay between duplication and point mutation in our data shed light on the relative frequencies of these events in human history, showing that at least some of the duplication events were recurrent.</p> <p>Conclusion</p> <p>This new multi-allelic view of SNPs has a complicated role in disease association studies, and further work will be necessary in order to accurately assess its importance. Software to perform generalized genotyping from SNP array data is freely available online <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Genotyping Cancer-Associated Genes in Chordoma Identifies Mutations in Oncogenes and Areas of Chromosomal Loss Involving CDKN2A, PTEN, and SMARCB1

The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity

Tumor mutational burden and PTEN alterations as molecular correlates of response to PD-1/L1 blockade in metastatic triple-negative breast cancer

Purpose: Few patients with metastatic triple-negative breast cancer (mTNBC) benefit from immune checkpoint inhibitors (ICI). On the basis of immunotherapy response correlates in other cancers, we evaluated whether high tumor mutational burden (TMB) ≥10 nonsynonymous mutations/megabase and PTEN alterations, defined as nonsynonymous mutations or 1 or 2 copy deletions, were associated with clinical benefit to anti-PD-1/L1 therapy in mTNBC. Experimental design: We identified patients with mTNBC, who consented to targeted DNA sequencing and were treated with ICIs on clinical trials between April 2014 and January 2019 at Dana-Farber Cancer Institute (Boston, MA). Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were correlated with tumor genomic features. Results: Sixty-two women received anti-PD-1/L1 inhibitors alone (23%) or combined with targeted therapy (19%) or chemotherapy (58%). High TMB (18%) was associated with significantly longer PFS (12.5 vs. 3.7 months; P = 0.04), while PTEN alterations (29%) were associated with significantly lower ORR (6% vs. 48%; P = 0.01), shorter PFS (2.3 vs. 6.1 months; P = 0.01), and shorter OS (9.7 vs. 20.5 months; P = 0.02). Multivariate analyses confirmed that these associations were independent of performance status, prior lines of therapy, therapy regimen, and visceral metastases. The survival associations were additionally independent of PD-L1 in patients with known PD-L1 and were not found in mTNBC cohorts treated with chemotherapy (n = 90) and non-ICI regimens (n = 169). Conclusions: Among patients with mTNBC treated with anti-PD-1/L1 therapies, high TMB and PTEN alterations were associated with longer and shorter survival, respectively. These observations warrant validation in larger datasets

Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of meningiomas1 but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas exhibited simple genomes, with fewer mutations, rearrangements, and copy-number alterations than reported in other adult tumors. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers among an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (E17K) and SMO (W535L) and exhibited immunohistochemical evidence of activation of their pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets

Colorectal Cancers from Distinct Ancestral Populations Show Variations in BRAF Mutation Frequency

It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap") to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents