Purpose: Personalized medicine strategies using genomic profiling are particularly pertinent for pancreas cancer. The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial was initially designed to exploit results from genome sequencing of pancreatic cancer under the auspices of the International Cancer Genome Consortium (ICGC) in Australia. Sequencing revealed small subsets of patients with aberrations in their tumor genome that could be targeted with currently available therapies. Experimental Design: The pilot stage of the IMPaCT trial assessed the feasibility of acquiring suitable tumor specimens for molecular analysis and returning high-quality actionable genomic data within a clinically acceptable timeframe. We screened for three molecular targets: HER2 amplification; KRAS wild-type; and mutations in DNA damage repair pathways (BRCA1, BRCA2, PALB2, ATM). Results: Tumor biopsy and archived tumor samples were collected from 93 patients and 76 were screened. To date 22 candidate cases have been identified: 14 KRAS wild-type, 5 cases of HER2 amplification, 2 mutations in BRCA2, and 1 ATM mutation. Median time from consent to the return of validated results was 21.5 days. An inability to obtain a biopsy or insufficient tumor content in the available specimen were common reasons for patient exclusion from molecular analysis while deteriorating performance status prohibited a number of patients from proceeding in the study. Conclusions: Documenting the feasibility of acquiring and screening biospecimens for actionable molecular targets in real time will aid other groups embarking on similar trials. Key elements include the need to better prescreen patients, screen more patients, and offer more attractive clinical trial options. Clin Cancer Res; 21(9); 2029–37. ©2015 AACR.
BRAFV600E mutation in microsatellite unstable (MSI) CRCs virtually excludes Lynch Syndrome (LS). In microsatellite stable (MSS) CRC it predicts poor prognosis. We propose a universal CRC LS screening algorithm using concurrent reflex immunohistochemistry (IHC) for BRAFV600E and MMR proteins. We compared BRAFV600E IHC to multiplex polymerase chain reaction (PCR) and MALDI-TOF spectrometry in 216 consecutive CRCs from 2011. Discordant cases were resolved with rt-PCR. BRAFV600E IHC was performed on 51 CRCs from the Australasian Colorectal Cancer Family Registry (ACCFR) which were fully characterised for BRAF mutation by allele-specific PCR, MMR status (MMR IHC and MSI), MLH1 promoter methylation and germline MLH1 mutation. We then assessed MMR and BRAFV600E IHC on 1403 consecutive CRCs. By MALDI-TOF 15 cases did not yield a BRAF result, while 38/201(19%) were positive. By IHC 45/216(20%) were positive. Of the 7 discordant cases, rt-PCR confirmed the IHC result in 6. In the 51 CRCs from the ACCFR, IHC was concordant with allele-specific PCR in 50 cases. BRAFV600E and MSI IHC on 1403 CRCs demonstrated the following phenotypes: BRAF-ve/MSS (1029 cases,73%), BRAF+ve/MSS (98,7%), BRAF+ve/MSI (183,13%), and BRAF-ve/MSI (93,7%). All 11/1403 cancers associated with proven LS were BRAF-ve/MSI. We conclude that BRAF IHC is highly concordant with two commonly used PCR-based BRAFV600E assays, performed well in identifying MLH1 mutation carriers from the ACCFR and identified all cases of proven LS out of 1403 CRCs. Reflex BRAFV600E and MMR IHC are simple cheap tests which facilitate universal LS screening and identify the poor prognosis BRAFV600E mutant MSS CRC phenotype.
We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.
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