Mutations in the KRAS gene, especially affecting the codon 12 and 13 region of the Kras protein, have implications in the treatment of certain cancer types. Because of the current potential for the FDA to oversee laboratory developed procedures (LPDs), it is imminently important that the validation of such tests be published to ensure that it is shown that these LPDs are properly reviewed and properly tested within the CLIA guidelines, thus keeping LPDs within the CLIA regulatory domain. We have developed a pyrosequencing assay that can detect eleven mutations in the codon 12 and 13 position of the KRAS gene. Our validation consisted of a sensitivity study in which a purified mutant PCR product was introduced into a wildtype DNA background and diluted down to undetectable levels. This sensitivity test was done on six different KRAS codon 12/13 mutants to determine the detection limit of the assay. Intra- and inter-assay precision and assay accuracy was determined by comparing assay results of over 40 total samples, both mutant and wild type, over five days carried out by two technicians. The results will show that this KRAS pyrosequencing assay falls within the acceptance criteria for sensitivity, accuracy and precision. Citation Format: Matthew L. Poulin, Ann Meyer, E. Andrew Mead, Jessica Xu, Ryan Drennan, Liyin Yan. The validation of a pyrosequencing KRAS mutation detection assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1829.
Based on research funded by The American Institute for Cancer Research, evidence is clear that weight can affect your risk of developing cancer. It is estimated that close to 1/3 of cancers that occur every year in the U.S. could be prevented by maintaining a healthy weight, being physically active and eating a nutritious diet. The body mass index (BMI) is defined as the body mass divided by the square of the body height and is typically expressed in kg/m2. BMI between 18.5 and 25 are considered normal while BMI of 25 to 30 is overweight and greater than 30 is considered obese. We have analyzed the DNA methylation levels of 18 individuals with BMI >30, and compared them to 7 individuals with BMI between 18.5 and 25 utilizing three targeted Next-Gen Bisulfite Sequencing (tNGBS) panels that we have developed as well as with individual pyrosequencing assays. The three panels, FOXP3, Immunology and Cancer Panels, cover regulatory regions of 44 genes that include over 400 CpG sites in addition to a global methylation assay to the LINE1 promoter. The results show that seven gene regulatory regions and the LINE1 global methylation assay show significant differential methylation between the obese and the normal BMI samples. ESR1, PAPR1, EN1, LXH2, TFF1, FOXP3 and LINE1 were shown to be hypermethylated and CD3G was hypomethylated, with p-values ranging between 0.08 and 0.005. Larger sample cohorts can now be examined more thoroughly with individual pyrosequencing assays to further validate the findings. Citation Format: Ryan Drennan, Nicolas Culhane, Matthew L. Poulin, Zhenhua Liu, Liying Yan. Differential methylation observed in cancer related genes between DNA samples from normal and obese individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5326.
Aberrant DNA methylation (e.g., global reduction in DNA methylation and differential methylation of promoter regions) is a hallmark of cancer and may function in various ways to influence transcription. We developed a panel of bisulfite specific methylation sequencing assays in order to assess the methylation status of large number of genes using pyrosequencing. By combining these pyrosequencing assays into a targeted NextGen bisulfite sequencing panel, we can screen both a large number of genes and a large number of samples simultaneously. We first assembled the 40 pyrosequencing assays and sensitivity and reproducibility were analyzed on pilot DNA samples isolated from FFPE tissue. Assays were specifically designed to work in highly fragmented and cross-linked DNA from formalin-fixed, clinical samples. Initial pyrosequencing on these pilot samples showed that 24 of the 40 assays gave quality validation and sample results. Reasons that some assays were not successful may be due to the A/T or G/C content within the amplicon, PCR Bias, low amplification efficiency, or strong cross-linking from fixation within the assay to name a few. The initial 40 assays were next assembled in a combination of 7 multiplex PCRs in which gradient PCRs were performed and analyzed for optimal multiplex amplification using a bioanalyzer. Final assay multiplexing combinations were established and PCR bias testing was performed by tNGBS on a sample set of high and low methylated DNA mixed at different ratios. Assays with an R-square of less than 0.9 between the expected and generated methylation from this mixing were removed, leaving us with 21 validated assays in our panel (9 of 24 assays that were previously validated in pyrosequencing and 12 of 19 assays that could not be validated in pyrosequencing). In the present study we looked at DNA methylation changes in breast cancer development using paired adjacent normal, invasive and in situ tissue from a 30 patients with breast cancer as well as 11 various normal tissues other than breast, and 12 cell lines for a total of 83 samples in a panel of 21 genes. The results of the tNGBS were compared with the pyrosequencing results. This process of panel development, where small amounts of bisulfite treated DNA can be amplified in a target specific manner, prior to sequencing, makes investigating a large number of samples on a large panel of genes possible in a small amount of time. We observed a high correlation between the pyrosequencing and tNGBS sequencing results, and we show that that there are several genes that are either hypermethylated or hypomethylated in tumor tissue when compared to adjacent normal tissue. Citation Format: Matthew L. Poulin, Ryan Drennan, Andrew Miller, Andrew Miller, Ann Meyer, Garth H. Rauscher, Liying Yan. Establishment and validation of a breast cancer panel using targeted next generation bisulfite sequencing (tNGBS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2017-4364
Continuing research demonstrates an ever increasing number of genes with measurable dysregulation in their DNA methylation state.We have developed a panel of targeted NextGen bisulfite sequencing amplicon assays (TGBS) centered on cancer genes with altered DNA methylation. This cancer panel includes tumor suppressor genes (BRCA1, RASSF1, RUNX3), DNA methyltransferases (DNMTs and MGMT), DNA repeats (LINE1 and Sat2), oncogenes (MYC,ERBB2), receptor genes (EGFR, ESRRA, NR2E1), Homeobox genes (HOXA10, HOXA11, EN1) and detoxification genes (GSTM1, GSTM2) among others. A total of 80 genes covering over 1500 CpG sites were selected and assays for these regions were developed and validated using PCR/Pyrosequencing. 10 pairs of DNA samples (tumor vs adjacent normal) for four cancer types (ovarian cancer, breast cancer, cervical cancer, and colorectal cancer) were sequenced using this panel on an Ion Torrent PGM™ system. We have used this data to characterize the general behavior of these cancer samples as compared to normal in three specific ways. First, we have assayed the change in overall methylation level between normal and cancer samples using these multiplexed amplicons, using these values for both an unsupervised hierarchical clustering and principal component analyses. We have then attempted to establish a per tissue normal methylation profile, and measured the distance to this normal profile for each of the cancer samples. As has been discussed in previous work, cancer has more of an anti-normal profile than a characteristic pattern per se. Finally, we have examined the methylation patterns within individual reads, and assessed the measured methylation entropy or randomness of these patterns in normal versus cancer samples Citation Format: Jessica Alexander, Ryan Drennan, Ann Meyer, Jessica Xu, Matthew L. Poulin, Winston Timp. Characterization of methylation patterns in cancer tissue shown by targeted Next-Gen bisulfite sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2015-1057
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