BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics

Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population. Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR). Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects. Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects.

Identification of a Clinical Cutoff Value for Multiplex KRASG12/G13 Mutation Detection in Colorectal Adenocarcinoma Patients Using Digital Droplet PCR, and Comparison with Sanger Sequencing and PNA Clamping Assay

KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRASG12/G13 mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRASG12/G13 mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff (mutant allele frequency of 7.9%), ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRASG12/G13 mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.

The Allele Frequency of the HFE gene mutation H63D (rs1799945) and Its Relationship to a Hereditary Hemochromatosis Diagnosis in Metabolic Nutrition Students at Virginia Tech

Hereditary hemochromatosis (HH) is a disease that causes excess iron absorption from the diet. This excess iron can be stored in the liver, skin, heart, pancreas, and joints, and then can lead to other health conditions, as the human body has no way of actively excreting iron. The human hemochromatosis protein (HFE protein) is encoded by the HFE gene, and mutations in this gene can lead to a dysfunction of the protein resulting in HH or iron overload later in adulthood. The objective of this study was to analyze the mutant allele frequency and the penetrance of the H63D mutation (SNP rs1799945) of the HFE gene in a cohort of Virginia Tech students. This study had a total of 69 participants. Fifty-two participants provided saliva samples, genomic data from 23andMe®, and surveys with phenotypic information. Of these, 6 were genotyped using the RFLP technique and served as controls for genotype confirmation. An additional 17 participants provided saliva samples, but did not provide 23andMe® data; genomic DNA from these participants were genotyped using the RFLP technique. Our results showed that although none of the participants had been diagnosed with HH, the mutant allele frequency of this population was 13.04%. In conclusion, as HH is usually diagnosed in older adults, we could not identify any students with a phenotype of HH, even though we could detect the mutant allele. This data suggests that affordable and accessible genetic ancestry and health kits such as the 23andMe® kit, could provide an efficient way to identify, prevent, and manage HH and other genetic diseases before symptoms arise. KEYWORDS: Hereditary Hemochromatosis; Iron Absorption; Single Nucleotide Polymorphism; Restriction Fragment Length Polymorphism; College Student Population; 23andMe®; Population Analysis; Survey Results

The association of mutant allele frequency of TP53 with response of chemotherapy in advanced gastric cancer patients.

42 Background: The tumor suppressor gene TP53 is one of the most commonly altered genes in advanced gastric cancer (AGC). However, the predictive role of mutant allele frequency (AF) of TP53 on response of chemotherapy in AGC patients remains unclear. This study aims to explore the predictive value of mutant allele frequency of TP53 on efficacy of chemotherapy in AGC. Methods: We obtained a new biopsy using gastroscope and collected blood samples at baseline and at time of 3 cycles of chemotherapy (2 weeks each cycle) in 8 patients with AGC, and the circulating tumor DNA (ctDNA) and tumor tissue DNA were analyzed using next generation sequencing (NGS). The efficacy was evaluated at time of 3 cycles of chemotherapy by CT imaging according to the RECIST’s standard. Results: We selected 8 patients with advanced gastric adenocarcinoma confirmed pathologically, including 5 female, 3 male. Their Median age is 53 (32-78) years old. The sequencing depths of tissue samples and blood samples were greater than 1000x and 10000x, respectively. Of 8 patients, except for one patient with no mutation of TP53, 7 AGC patients had the mutations in the different sites of TP53 gene. The results showed that 5 patients had partial response (PR), and 2 patients revealed stable disease (SD) after chemotherapy. Five patients with PR showed more than 10% of mutant AF of TP53, but two patients with SD had less than 3% of the mutant AF. Of 5 AGC patients with PR, two patients showed that the mutant AF of TP53 had become from 77.0%, 20.20% to negative in the blood samples, three patients exhibited the decrease of mutant AF of TP53 from 36.99%, 21.61% and 10.04% to 15.77%, 0.40% and 0.69%, respectively. In two patients with SD, one patient revealed that the mutant AF of TP53 had decreased from 2.20% to negative, another one from 1.91% to 1.11%, respectively. Conclusions: Our results suggested that the decrease of mutant allele frequency of TP53 in blood sample can predict response to chemotherapy in patients with advanced gastric cancer. The AGC patients with more than 10% of mutant AF of TP53 maybe have a good outcome for chemotherapy but needs to be further explored.