Mutational analysis of KRAS codons 12 and 13 is standard for patients with metastatic colorectal cancer since mutations in these codons predict lack of response to anti-EGFR therapies. However, even among patients whose tumors are wildtype for KRAS codons 12 and 13, only a subset respond to therapy. Since additional activating mutations downstream of EGFR may also play a role in treatment resistance, we sought to establish the frequency of these mutations. We evaluated 2121 colorectal tumors for mutations in codons 12 and 13 of the KRAS gene. A subset of these samples, comprised of 513 samples wildtype for KRAS codons 12 and 13, were tested for mutations in codons 61 and 146 of KRAS, codon 600 of BRAF, and codons 12, 13, and 61 of NRAS. Mutation status was determined by targeted pyrosequencing. Mutations in KRAS codon 12 or 13 were identified in 900/2121 (42.4%) samples. Of the 513 wildtype samples tested for additional mutations, 78 samples were mutant for BRAF, 19 for KRAS codon 61, 17 for KRAS codon 146, and 26 for NRAS. In total, 140/513 (27.3%) tumors wildtype for KRAS codons 12 and 13 harbored a mutation in another of the RAS pathway genes. While further study is needed to determine the full therapeutic implications of mutations in these codons, mutational testing of these codons may be useful for identifying a significant proportion of patients who may also be resistant to anti-EGFR therapies.

BackgroundAbout half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal cancer. Study variables include clinical stage, tumor location and histologic grade.MethodsPlasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients.ResultsThe improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a 12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%.ConclusionsA sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who are unwilling or unable to undergo colonscopy.

• Targeted

Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided.

Purpose Adrenocortical carcinoma (ACC) is an endocrine malignancy with a poor prognosis. The association of adult-onset ACC with inherited cancer predisposition syndromes is poorly understood. Our study sought to define the prevalence of Lynch syndrome (LS) among patients with ACC. Patients and Methods One hundred fourteen patients with ACC were evaluated in a specialized endocrine oncology clinic and were prospectively offered genetic counseling and clinical genetics risk assessment (group 1). In addition, families with known mismatch repair (MMR) gene mutations that were recorded in the University of Michigan Cancer Genetics Registry were retrospectively reviewed for the presence of ACC (group 2). ACC tumors from patients with LS were tested for microsatellite instability and immunohistochemistry (IHC) to evaluate for MMR deficiency. Results Ninety-four (82.5%) of 114 patients with ACC underwent genetic counseling (group 1). Three individuals (3.2%) had family histories suggestive of LS. All three families were found to have MMR gene mutations. Retrospective review of an additional 135 MMR gene–positive probands identified two with ACC (group 2). Four ACC tumors were available (group 1, 3; group 2, 1). All four tumors were microsatellite stable; three had IHC staining patterns consistent with germline mutation status. Conclusion The prevalence of LS among patients with ACC is 3.2%, which is comparable to the prevalence of LS in colorectal and endometrial cancer. Patients with ACC and a personal or family history of LS tumors should be strongly considered for genetic risk assessment. IHC screening of all ACC tumors may be an effective strategy for identifying patients with LS.

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

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