Differential Outcomes in Codon 12/13 and Codon 61 NRAS-Mutated Cancers in the Phase II NCI-MATCH Trial of Binimetinib in Patients with NRAS-Mutated Tumors.

PURPOSE: Preclinical and clinical data suggest that downstream inhibition with an MEK inhibitor, such as binimetinib, might be efficacious for NRAS-mutated cancers. PATIENTS AND METHODS: Patients enrolled in the NCI-MATCH trial master protocol underwent tumor biopsy and molecular profiling by targeted next-generation sequencing. Patients with NRAS-mutated tumors, except melanoma, were enrolled in subprotocol Z1A, a single-arm study evaluating binimetinib 45 mg twice daily. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). A post hoc analysis examined the association of NRAS mutation type with outcome. RESULTS: In total, 47 eligible patients with a refractory solid tumor harboring a codon 12, 13, or 61 NRAS mutation were treated. Observed toxicity was moderate, and 30% of patients discontinued treatment because of binimetinib-associated toxicity. The ORR was 2.1% (1/47 patients). A patient with malignant ameloblastoma harboring a codon 61 NRAS mutation achieved a durable partial response (PR). A patient with NRAS codon 61-mutated colorectal cancer had an unconfirmed PR, and two other patients with NRAS codon 61-mutated colorectal had stable disease for at least 12 months. In an exploratory analysis, patients with colorectal cancer bearing a NRAS codon 61 mutation (n = 8) had a significantly longer OS (P = 0.03) and PFS (P = 0.007) than those with codon 12 or 13 mutations (n = 16). CONCLUSIONS: Single-agent binimetinib did not show promising efficacy in NRAS-mutated cancers. The observation of increased OS and PFS in patients with codon 61 NRAS-mutated colorectal cancer merits further investigation.

Tumor Sidedness, Recurrence, and Survival After Curative Resection of Localized Colon Cancer.

BACKGROUND: Right-sided primary tumor location is associated with worse prognosis in metastatic colon cancer, but the effect of sidedness on recurrence and prognosis for non-metastatic disease is less understood. The purpose of this study was to examine the relationship between sidedness, recurrence, and survival among patients with localized colon cancer. PATIENTS AND METHODS: Consecutive patients who underwent curative resection of colon cancer (2006-2013) were identified from a prospective database and retrospectively analyzed. Risk for recurrence, overall survival, and survival after recurrence (SAR) were compared between left- and right-sided tumors using the log-rank test, and multivariable Cox proportional hazards regression. RESULTS: We evaluated 673 patients (347 right-sided). There was no difference in overall recurrence rates (adjusted hazard ratio [HR], 0.92; 95% confidence interval [CI], 0.54-1.55; P = .75) or overall survival (HR, 1.22; 95% CI, 0.75-1.97; P = .42) between right- and left-sided primary tumors. However, right-sided tumors were more likely to develop multi-focal and poor prognostic site recurrence (P = .04). Among the 71 patients who developed recurrence, those with right-sided tumors had significantly lower SAR (HR, 3.88; 95% CI, 1.42-10.62; P = .008). CONCLUSIONS: Among patients with colon cancer who underwent curative resection, tumor sidedness was not associated with recurrence risk. However, among patients who developed recurrence, right-sidedness was associated with unique recurrence patterns and inferior SAR. For patients presenting with localized disease, treatment stratification should not be based on tumor sidedness alone.

Liquid Biopsies Using Plasma Exosomal Nucleic Acids and Plasma Cell-Free DNA Compared with Clinical Outcomes of Patients with Advanced Cancers.

Purpose: Blood-based liquid biopsies offer easy access to genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. Exosomal nucleic acids (exoNAs) originate from living cells, which can better reflect underlying cancer biology.Experimental Design: Next-generation sequencing (NGS) was used to test exoNA, and droplet digital PCR (ddPCR) and BEAMing PCR were used to test cfDNA for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations in 43 patients with progressing advanced cancers. Results were compared with clinical testing of archival tumor tissue and clinical outcomes.Results: Forty-one patients had BRAF, KRAS, or EGFR mutations in tumor tissue. These mutations were detected by NGS in 95% of plasma exoNA samples, by ddPCR in 92% of cfDNA samples, and by BEAMing in 97% cfDNA samples. NGS of exoNA did not detect any mutations not present in tumor, whereas ddPCR and BEAMing detected one and two such mutations, respectively. Compared with patients with high exoNA mutation allelic frequency (MAF), patients with low MAF had longer median survival (11.8 vs. 5.9 months; P = 0.006) and time to treatment failure (7.4 vs. 2.3 months; P = 0.009). A low amount of exoNA was associated with partial response and stable disease ≥6 months (P = 0.006).Conclusions: NGS of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared with clinical testing of archival tumor and testing of plasma cfDNA. Low exoNA MAF is an independent prognostic factor for longer survival. Clin Cancer Res; 24(1); 181-8. ©2017 AACR.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03).Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.

Mutation-Enrichment Next-Generation Sequencing for Quantitative Detection of KRAS Mutations in Urine Cell-Free DNA from Patients with Advanced Cancers.

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRASExperimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer.Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03).Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657-66. ©2017 AACR.

BRAF mutant colorectal cancer as a distinct subset of colorectal cancer: clinical characteristics, clinical behavior, and response to targeted therapies.

Despite new and more effective cytotoxic chemotherapy, limitations to conventional agents have been reached in a subset of patients with advanced colorectal cancer (CRC). The identification of novel prognostic and predictive biomarkers to guide individualized treatment plans is critical to overcoming therapeutic resistance. Mutation of the BRAF proto-oncogene is linked to a variety of cancers and is increasingly being used as a prognostic tool and therapeutic target. This paper is a comprehensive review of the literature that summarizes the clinical, pathologic, and molecular features of BRAF mutated CRC that support the hypothesis that BRAF mutant cancers represent a distinct subset of CRC with its own clinical implications with regard to prognosis, treatments and emerging therapeutic strategies.

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

Erratum: Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies.

The Abstract is incorrect in PubMed. The corrected Abstract is provided here.

BRAF mutation testing with a rapid, fully integrated molecular diagnostics system.

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction-based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation-detecting tests.

Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies.

Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 - 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71- 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 - 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 - 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with 1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with

Hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors.

PURPOSE: We used a clinical next-generation sequencing (NGS) hotspot mutation panel to investigate clonal evolution in paired primary and metastatic tumors. EXPERIMENTAL DESIGN: A total of 265 primary and metastatic tumor pairs were sequenced using a 46-gene cancer mutation panel capable of detecting one or more single-nucleotide variants as well as small insertions/deletions. Mutations were tabulated together with tumor type and percentage, mutational variant frequency, time interval between onset of primary tumor and metastasis, and neoadjuvant therapy status. RESULTS: Of note, 227 of 265 (85.7%) tumor metastasis pairs showed identical mutation calls. Of the tumor pairs with identical mutation calls, 160 (60.4%) possessed defining somatic mutation signatures and 67 (25.3%) did not exhibit any somatic mutations. There were 38 (14.3%) cases that showed at least one novel mutation call between the primary and metastasis. Metastases were almost two times more likely to show novel mutations (n = 20, 7.5%) than primary tumors (n = 12, 4.5%). TP53 was the most common additionally mutated gene in metastatic lesions, followed by PIK3CA and SMAD4. PIK3CA mutations were more often associated with metastasis in colon carcinoma samples. CONCLUSIONS: Clinical NGS hotspot panels can be useful in analyzing clonal evolution within tumors as well as in determining subclonal mutations that can expand in future metastases. PIK3CA, SMAD4, and TP53 are most often involved in clonal divergence, providing potential targets that may help guide the clinical management of tumor progression or metastases.

Postoperative chemotherapy use after neoadjuvant chemoradiotherapy for rectal cancer: Analysis of Surveillance, Epidemiology, and End Results-Medicare data, 1998-2007.

BACKGROUND: Neoadjuvant chemoradiotherapy followed by tumor resection and postoperative chemotherapy is the standard of care for patients with clinical stage II or III adenocarcinoma of the rectum. Significant variation exists in the receipt of postoperative chemotherapy after resection in this population. The objective of this study was to determine the demographic and clinicopathologic factors associated with the initiation of postoperative chemotherapy in elderly patients with rectal cancer and to identify potential targets for reducing treatment variation. METHODS: A retrospective cohort study was performed of patients with rectal cancer ages 66 to 80 years who received neoadjuvant chemoradiotherapy and underwent radical resection in the Surveillance, Epidemiology, and End Results-linked Medicare database (1998-2007). Multivariate logistic regression was used to assess chemotherapy use in relation to patient, tumor, and treatment response characteristics. RESULTS: Among 1492 patients who met the study criteria, 61.5% received adjuvant therapy with 5-fluorouracil. Pathologic stage was the strongest determinant of whether patients received postoperative chemotherapy (48.3% of patients with stage I disease, 59.6% of patients with stage II disease, and 77.6% of patients with stage III disease). Increasing age and postoperative readmission also were associated significantly with a decreased rate of adjuvant therapy initiation. CONCLUSIONS: Although standard treatment guidelines for locally advanced rectal cancer include postoperative chemotherapy for all patients after neoadjuvant chemoradiotherapy and radical resection, greater than 1 in 3 patients failed to receive adjuvant therapy. Despite the absence of established evidence, treatment decisions appear to be influenced by the findings at surgical pathology.

Target-based therapeutic matching in early-phase clinical trials in patients with advanced colorectal cancer and PIK3CA mutations.

Target-matched treatment with PI3K/AKT/mTOR pathway inhibitors in patients with diverse advanced cancers with PIK3CA mutations have shown promise. Tumors from patients with colorectal cancer were analyzed for PIK3CA, KRAS, and BRAF mutations. PIK3CA-mutated tumors were treated, whenever feasible, with agents targeting the PI3K/AKT/mTOR pathway. Of 194 patients analyzed, 31 (16%) had PIK3CA mutations and 189 (97%) were assessed for KRAS mutations. Patients with PIK3CA mutations had a higher prevalence of simultaneous KRAS mutations than patients with wild-type PIK3CA (71%, 22/31 vs. 43%, 68/158; P = 0.006). Of 31 patients with PIK3CA mutations, 17 (55%) were treated with protocols containing PI3K/AKT/mTOR pathway inhibitors [median age, 57 years; median number of prior therapies, 4; mTORC1 inhibitors (11), phosphoinositide 3-kinase (PI3K) inhibitors (5), or an AKT inhibitor (1)]. None (0/17) had a partial or complete response (PR/CR) and only 1 [6%, 95% confidence interval (CI), 0.01-0.27] had stable disease 6 months or more, which was not significantly different from a stable disease ≥6 month/PR/CR rate of 16% (11/67; 95% CI, 0.09-0.27) in patients with colorectal cancer without PIK3CA mutations treated with PI3K/AKT/mTOR pathway inhibitors (P = 0.44). Median progression-free survival was 1.9 months (95% CI, 1.5-2.3). In conclusion, our data provide preliminary evidence that in heavily pretreated patients with PIK3CA-mutant advanced colorectal cancer, protocols incorporating PI3K/AKT/mTOR inhibitors have minimal activity. PIK3CA mutations are associated with simultaneous KRAS mutations, possibly accounting for therapeutic resistance.

GWAS-identified colorectal cancer susceptibility loci associated with clinical outcomes.

Recent genome-wide association studies (GWAS) have identified several common susceptibility loci associated with the risk of colorectal cancer (CRC). However, whether these loci affect clinical outcomes of CRC is not clear. In this study, we genotyped 26 single nucleotide polymorphisms (SNPs) in 10 GWAS-identified CRC susceptibility regions and evaluated their associations with survival and recurrence in 285 stage II and III patients receiving fluorouracil-based adjuvant chemotherapy. Only one SNP, rs10318 (15q13.3), was significantly associated with recurrence for patients with stage II disease. Three SNPs: rs10749971 (11q23.1), rs961253 (20p12.3) and rs355527 (20p12.3) in two regions were significantly associated with recurrence for patients with stage III disease. Five SNPs: rs961253 (20p12.3), rs355527 (20p12.3), rs4464148 (18q21.1), rs6983267 (8q24.21) and rs10505477 (8q24.21) in three regions were significantly associated with survival for patients with stage III disease. Cumulative effects of multiple unfavorable genotypes were observed for recurrence and survival in patients with stage III CRC. Our results suggest that cancer susceptibility loci may also affect the prognosis of CRC patients receiving fluorouracil-based adjuvant chemotherapy.

Endothelin-1 as a target for therapeutic intervention in prostate cancer.

The endothelins, a family of potent vasoconstricting peptides, have been implicated in the pathophysiology of advanced prostate cancer. Two endothelin receptors, ET-A and ET-B are found in normal prostate tissue. Malignant prostate cells are notable for the loss of ET-B receptors and increased levels of endothelin-1 [ET-1]; this distortion of the endothelin system may be a significant factor in the progression of prostate cancer. Proposed roles for endothelin in prostate cancer include growth promotion, apoptosis inhibition, bone formation, and stimulation of nociceptive receptors. ET-1 can act alone as a mitogen, but its effects are greatest as a comitogen with a variety of growth factors, including basic fibroblast growth factor, insulin-like growth factors, and platelet derived growth factor. Although their exact functions are unclear, ET-1, in conjunction with vascular endothelial growth factor, appears to play a major role in tumor angiogenesis. By a variety of methods, ET-1 alters the balance of osteoblast and osteoclasts to the favor new bone formation that is characteristic of metastatic disease. Several studies indicate that the refractory pain of metastatic cancer is related to the direct nociceptive effects ET-1. These findings suggest that ET receptors are promising therapeutic targets for pharmacologic intervention. Early clinical trials indicate that the ET-A receptor antagonist used in prostate cancer is reasonably well tolerated with mild but pervasive symptoms related to ET-1's vasoconstrictive effects. Results of ongoing clinical trials are eagerly awaited in order to see if the hypothetical promise of ET antagonism will result in clinical success.