Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.
The results suggest a possible association between KRAS mutations and mucinous differentiation in endometrial carcinogenesis. KRAS status can help in assessing benign from precursor or malignant mucinous lesions as well as differentiate endometrial lesions from those of cervical origin.
PURPOSE Combining the oral AKT inhibitor ipatasertib with paclitaxel as first-line therapy for metastatic triple-negative breast cancer significantly improved progression-free survival (PFS) in the placebo-controlled, randomized, phase II LOTUS trial, with a more pronounced effect in patients with PIK3CA/AKT1/PTEN-altered tumors. We report findings from the extensive translational research program. PATIENTS AND METHODS Pretreatment plasma and tumor samples were evaluated for genetic alterations using FoundationACT and FoundationOne (Foundation Medicine, Cambridge, MA) hybrid capture next-generation sequencing assays, respectively. Prevalences of the most common mutations and PIK3CA/AKT1 mutation status were determined using both assays, and concordance was assessed. In longitudinal analyses, circulating tumor DNA (ctDNA) mutations were quantified in baseline and on-treatment (cycle 3, day 1 [C3D1]) samples. The relationship between outcomes and ctDNA fraction (CTF; highest variant allele frequency) and CTF ratio (C3D1 CTF to baseline CTF) was explored. RESULTS Among 89 patients evaluable for ctDNA sequencing, 81 patients (91%) had 149 detectable mutations. There was high agreement between plasma- and tissue-based sequencing for known or likely short variant mutations but not amplifications. There was 100% concordance between ctDNA and tissue sequencing in patients with activating PIK3CA or AKT1 mutations. High baseline CTF was associated with shorter PFS in both treatment arms. Longitudinal analyses showed more favorable outcomes with lower absolute CTF at C3D1 and, to a lesser extent, greater CTF decreases. CONCLUSION These results suggest that plasma ctDNA sequencing may allow reliable and convenient assessment of prognosis and identification of genetic markers associated with increased benefit from ipatasertib. On-treatment CTF showed a meaningful association with objective response and PFS.
Background New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10–20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. Methods In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (High performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for presence of KRAS proto-oncogene mutations at codon 12 and 13. Results Initially, sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations. Conclusion In these studies the analysis of three patients’ colorectal cancer tissue were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.
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