Assessment of clinical outcomes with immune checkpoint inhibitor therapy in melanoma patients with CDKN2A and TP53 pathogenic mutations.

BACKGROUND: CDKN2A and TP53 mutations are recurrent events in melanoma, occurring in 13.3% and 15.1% of cases respectively and are associated with poorer outcomes. It is unclear what effect CDKN2A and TP53 mutations have on the clinical outcomes of patients treated with checkpoint inhibitors. METHODS: All patients with cutaneous melanoma or melanoma of unknown primary who received checkpoint inhibitor therapy and underwent genomic profiling with the 50-gene Mayo Clinic solid tumor targeted cancer gene panel were included. Patients were stratified according to the presence or absence of mutations in BRAF, NRAS, CDKN2A, and TP53. Patients without mutations in any of these genes were termed quadruple wild type (QuadWT). Clinical outcomes including median time to progression (TTP), median overall survival (OS), 6-month and 12-month OS, 6-month and 12-month without progression, ORR and disease control rate (DCR) were analyzed according to the mutational status of CDKN2A, TP53 and QuadWT. RESULTS: A total of 102 patients were included in this study of which 14 had mutations of CDKN2A (CDKN2Amut), 21 had TP53 mutations (TP53mut), and 12 were QuadWT. TP53mut, CDKN2Amut and QuadWT mutational status did not impact clinical outcomes including median TTP, median OS, 6-month and 12-month OS, 6-month and 12-month without progression, ORR and DCR. There was a trend towards improved median TTP and DCR in CDKN2Amut cohort and a trend towards worsened median TTP in the QuadWT cohort. CONCLUSION: Cell cycle regulators such as TP53 and CDKN2A do not appear to significantly alter clinical outcomes when immune checkpoint inhibitors are used.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

S768I Mutation in EGFR in Patients with Lung Cancer.

INTRODUCTION: Epidermal growth factor receptor gene (EGFR) mutations are relatively common oncogenic drivers in non-small cell lung cancer (NSCLC). The outcomes of patients who present with less common EGFR mutations or more than one EGFR mutation are uncertain. We reviewed our experience with the S768I mutation of exon 20 of EGFR to provide insight into the clinical significance of this mutation. METHODS: We used a natural language search program to search our electronic medical record system and every EGFR mutation analysis of patients with NSCLC treated at Mayo Clinic that was performed in our Department of Molecular Genetics to identify patients with EGFR S768I mutation. Relevant clinical and laboratory data were abstracted for selected cases, including evaluation of response after treatment with tyrosine kinase inhibitors. RESULTS: A total of 1527 patients with NSCLC who underwent EGFR testing were reviewed. The S768I mutation was present in nine patients (0.59%), four of whom were female. Only three had an isolated S768I mutation, four had a concurrent G719 mutation, and two had a concurrent L858R mutation. Among patients with stage IV disease treated with erlotinib (n = 4), one had an isolated S768I mutation and three had additional mutations (two patients with G719 and one patient with L858R). The tumor response to erlotinib of patients with stage IV disease was highly variable (progression-free survival ranged from 3 to 30 months and overall survival ranged from 5 to more than 51 months). CONCLUSIONS: S768I mutations in exon 20 of the EGFR gene are rare and are typically seen in conjunction with sensitizing EGFR mutations. Because of this mutation's rarity and the variability of responses of treated cases, its exact prognostic and predictive role is not fully understood. In our experience, S768I mutations in isolation do not necessarily confer sensitivity to erlotinib, but in conjunction with sensitizing EGFR mutations, S768I mutations do not restrict efficacy.

Genetic alterations associated with progression from pancreatic intraepithelial neoplasia to invasive pancreatic tumor.

BACKGROUND & AIMS: Increasing grade of pancreatic intraepithelial neoplasia (PanIN) has been associated with progression to pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that control progression from PanINs to PDAC are not well understood. We investigated the genetic alterations involved in this process. METHODS: Genomic DNA samples from laser-capture microdissected PDACs and adjacent PanIN2 and PanIN3 lesions from 10 patients with pancreatic cancer were analyzed by exome sequencing. RESULTS: Similar numbers of somatic mutations were identified in PanINs and tumors, but the mutational load varied greatly among cases. Ten of the 15 isolated PanINs shared more than 50% of somatic mutations with associated tumors. Mutations common to tumors and clonally related PanIN2 and PanIN3 lesions were identified as genes that could promote carcinogenesis. KRAS and TP53 frequently were altered in PanINs and tumors, but few other recurrently modified genes were detected. Mutations in DNA damage response genes were prevalent in all samples. Genes that encode proteins involved in gap junctions, the actin cytoskeleton, the mitogen-activated protein kinase signaling pathway, axon guidance, and cell-cycle regulation were among the earliest targets of mutagenesis in PanINs that progressed to PDAC. CONCLUSIONS: Early stage PanIN2 lesions appear to contain many of the somatic gene alterations required for PDAC development.

Characterization of hMLH1 and hMSH2 gene dosage alterations in Lynch syndrome patients.

BACKGROUND & AIMS: A significant proportion of Lynch syndrome cases are believed to be due to large genomic alterations in the mismatch repair genes hMLH1 and hMSH2. However, previous studies have not adequately identified the frequency and scope of such mutations, and routine clinical Lynch syndrome testing often does not include analysis for these mutations. Our aim was to characterize hMLH1 and hMSH2 genomic rearrangements in a large population of suspected Lynch syndrome patients. METHODS: A total of 365 samples from probands referred for genetic testing for Lynch syndrome were analyzed for the presence of large genomic alterations in hMLH1 or hMSH2 by using a combination of techniques. Samples with a deletion in exons 1-6 in hMSH2 were further characterized by polymerase chain reaction to establish the presence of the hMSH2 American founder deletion. RESULTS: An hMLH1 or hMSH2 mutation was identified in 153 cases, and, of these, 12 of 67 (17.9%) and 39 of 86 (45.3%) had a large genomic alteration in hMLH1 and hMSH2, respectively. Overall, 6 different hMLH1 and 12 different hMSH2 deletions/duplications, including 10 novel mutations, were identified. Analysis of the hMSH2 exon 1-6 deletion samples showed that 13 of 18 (72.2%) had the American founder deletion. CONCLUSIONS: These data show a high frequency and diverse spectrum of large genomic alterations in hMLH1 and hMSH2 in suspected Lynch syndrome patients. Thus, a comprehensive mutation identification strategy that includes the ability to detect large genomic rearrangements is imperative for the clinical genetic identification of Lynch syndrome patients and families.

Genotypic differences of MCAD deficiency in the Asian population: novel genotype and clinical symptoms preceding newborn screening notification.

PURPOSE: In contrast to its high prevalence in Caucasians, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is reported to be an extremely rare metabolic disorder in the Asian population. The common MCAD gene (ACADM) mutation 985A>G (p.K329E), accounting for the majority of cases in Caucasians, has not been detected in this ethnic group, and the spectrum of ACADM mutations has remained unknown. METHOD: Biochemical genetic testing including plasma acylcarnitine and urine acylglycine analyses, as well as sequencing of ACADM was performed in a Korean family with a newborn who had an elevated octanoyl (C8) carnitine concentration by newborn screening (NBS). Genotyping of 50 Korean newborns with normal NBS results was performed. RESULT: We report the identification of the first Korean patient with MCAD deficiency, caused by a novel missense mutation in ACADM, 843A>T (R281S), and a 4-bp deletion, c.449_452delCTGA. The patient became symptomatic before notification of the abnormal NBS result. Both the father and a brother who were identified as carriers for the 4-bp deletion had mildly elevated plasma C8 and C10:1 carnitine concentrations, whereas the acylcarnitine profile was normal in the mother who carries the missense mutation. CONCLUSION: The 4-bp deletion may represent a common Asian ACADM mutation, considering that it recently has also been found in two of the three Japanese patients in whom genotyping was performed. Greater availability of MCAD mutation analysis is likely to unravel the molecular basis of MCAD deficiency in the Asian population that might differ from Caucasians.

Analysis of hMLH1 and hMSH2 gene dosage alterations in hereditary nonpolyposis colorectal cancer patients by novel methods.

A significant fraction of hereditary nonpolyposis colorectal cancer cases with defective mismatch repair (ie, Lynch syndrome) have large genomic deletions or duplications in the mismatch repair genes, hMLH1 and hMSH2, which can be challenging to detect by traditional methods. For this study, we developed and validated a novel Southern blot analysis method that allows for ascertainment of the extent of the dosage alterations on an exon-by-exon basis and compared this method to a second novel technique, multiplex ligation-dependent probe amplification (MLPA). From a total of 254 patients referred for Lynch syndrome testing, 20 of the 118 MLH1 cases and 42 of the 136 MSH2 cases had large genomic alterations, as detected by Southern blot. MLPA and Southern blot results were concordant with the exception of three major discrepancies: one because of a lack of MLPA probes for the region altered, another because of a point mutation near the MLPA probe ligation site, and another that was unexplained. Compared to Southern blot, MLPA has a shorter turn-around time, the analysis is less costly, less time-consuming, and less labor-intensive, and results are generally clear and unambiguous. However, concerns with MLPA include the presence of false-negatives and -positives because of positioning of probes and DNA variants near the probe ligation site. Overall, both Southern blot and MLPA provide important tools for the complete evaluation of patients with Lynch syndrome.

BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.

Frequent BRAF mutations were reported recently in a variety of human malignancies, including colorectal cancer. In this study, we screened 293 colorectal cancers for mutations in exons 11 and 15, two regions containing hotspots for BRAF mutation. Of the 293 cancers, 170 had normal mismatch repair, and 123 had defective mismatch repair (originating from both somatic as well as germ-line mutations in several of the mismatch repair genes). A total of 63 exonic mutations (22%) were detected, 60 of which were V599E, and one each of D593G, G468E, and D586A. Of the tumors with defective mismatch repair, 34% (42 of 123) had a mutation in BRAF, whereas only 12% (21 of 170) of tumors with proficient mismatch repair demonstrated a mutation (P < 0.0001). Interestingly, BRAF mutations were found most often in cases with an hMHL1 abnormality (35 of 60) and rarely in cases with an hMSH2 abnormality (1 of 39; P < 0.0001). More interestingly, of the 31 hMLH1 cases with a BRAF mutation, 30 occurred in tumors known to have hypermethylation of hMLH1 promoter. Only 1 of the 15 cases with a germ-line mutation in hMLH1 had a mutation in BRAF. In this series, BRAF mutations occurred rarely in tumors with defective mismatch repair attributable to the presence of germ-line mutation in either hMLH1 or hMSH2. Furthermore, BRAF mutations were strongly associated with the epigenetic alteration of hMLH1. Overall, these data suggest that BRAF mutations are not a consequence of defective mismatch repair per se.