Purpose Mutations in the and (mutations can lead to high levels

Purpose Mutations in the and (mutations can lead to high levels of 2HG circulating in the blood and whether serum 2HG can be used as a biomarker for mutational status and tumor burden in ICC. 2HG. Conclusions This study indicates that circulating 2HG may be a surrogate biomarker of or mutation status in ICC and that circulating 2HG levels may correlate directly with tumor burden. and (mutations confined primarily to a small number of cancer types which is then further limited when considering a relatively rare malignancy such as ICC. We have previously identified elevated levels of 2HG in the tumor tissue of or mutation. Accurate detection and quantification of serum 2HG could potentially serve as an efficient and less-invasive method of assessing a patient’s response to IDH-targeted therapies once promising drugs currently undergoing preclinical evaluation enter into clinical testing (17 18 In this study we therefore sought to characterize serum 2HG levels as a biomarker of mutational status and its association with tumor burden in gene at nucleotide positions c.394 and c.395 (amino acid position p.R132) were identified using a multiplexed mutational profiling platform that has been previously described and clinically implemented (6 19 Rare mutations that have been reported in other tumor types were not evaluated (20). Sanger sequencing was used to identify mutations in the gene at exon 4 (including mutations at codons p.140 and p.172) using methods and polymerase chain reaction primers that have been previously reported (6). Labeled PCR products were separated using an ABI PRISM 3730 DNA Analyzer and the data were interpreted with GeneMapper Analysis Software (Life Technologies/Applied Biosystems). Circulating 2-Hydroxyglutarate analysis Serum was isolated from whole blood aliquoted and stored at -80°C until IP1 analysis. Circulating levels of 2HG were measured at Agios Pharmaceuticals (Cambridge MA) using lLC-MS/MS analysis (AB Sciex 4000 Framingham MA) operating in bad electrospray mode. MRM data was acquired for each compound using the following transitions: 2HG (146.9/128.8 amu) BMS-794833 13 (151.9/133.8 amu) & 3HMG (160.9/98.9 amu). Chromatographic separation was performed using an ion-exchanged column (Bio-rad Fast Acid analysis 9 μm 7.8 mm × 100 mm; Bio-rad). The circulation rate BMS-794833 was 1 ml/min of 0.1% formic acid in water with a total run time of 4 minutes. 30 μl of sample was extracted by adding 30 μl of internal standard (ISTD) in water followed by 200 μl of acetonitrile. The sample was vortex combined centrifuged and 100 μL of supernatant transferred to a clean 96-well plate. The supernatant was diluted with 100 μl of deionized water and 25 μl injected on column. Statistical analysis The assessment of biomarkers with respect to mutational status or study site was performed using precise Mann-Whitney-Wilcoxon test. Median and interquartile ranges are provided as descriptive statistics. Correlations were quantified as Spearman’s correlation coefficients and tested with the Spearman’s test. P-values of <0.05 were considered statistically significant. Results Patient Characteristics The patient characteristics of the Screening and Validation cohorts are summarized in Table 1. A total of 31 diagnosed ICC individuals with clinically-determined and gene mutational status and with available banked whole blood comprised the Screening cohort. The median age of this group was 57 years and approximately 65% of these individuals presented with stage IV disease. These characteristics are consistent with individuals that normally undergo medical mutational profiling in an effort to identify alternate treatment programs after failing standard of care. Table 1 Characteristics of individuals with intrahepatic cholangiocarcinoma across cohorts. In order to increase analysis of ICC individuals a second Validation cohort consisting of 38 ICC individuals who underwent medical resection was then recognized and retrospectively genotyped to identify mutations. The age sex and CA19-9 blood levels of this Validation cohort were comparable to those in the Screening cohort (Table 1). However BMS-794833 the Validation group individuals spanned early disease phases including stage I (50%) stage II (~13%) and stage BMS-794833 III (37%) and did not include stage IV individuals. IDH1 and IDH2 mutational status In the Screening cohort mutations were found in 11 out of 31 individuals with ICC for an overall incidence of 35%. These included point mutations in p.R132C (n=7) p.R132L (n=3) and p.R132G (n=1) (Table 2 and Supplemental Table 1). No mutations were recognized. The ICC resected.

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