Clinicaltrials.gov, NCT02502656.

Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis.Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRASmutated allele fraction.Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors.Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

The BRAF mutation is reported in half of patients with Langerhans cell histiocytosis (LCH). This study investigated the detection of the BRAF allele in circulating cell-free (ccf) DNA in a paediatric LCH cohort. Children with BRAF -mutated LCH were investigated to detect ccf BRAF at diagnosis (n = 48) and during follow-up (n = 17) using a picolitre-droplet digital PCR assay. At diagnosis, ccf BRAF was positive in 15/15 (100%) patients with risk-organ positive multisystem (RO+ MS) LCH, 5/12 (42%) of patients with RO- MS LCH and 3/21 (14%) patients with single-system (SS) LCH (P < 0·001, Fisher's exact test). The positive BRAF load was higher for RO+ patients (mean, 2·90%; range, 0·04-11·4%) than for RO- patients (mean, 0·16%; range, 0·01-0·39) (P = 0·003, Mann-Whitney U test). After first-line vinblastine-steroid induction therapy, 7/7 (100%) of the non-responders remained positive for ccf BRAF compared to 2/4 (50%) of the partial-responders and 0/4 of the complete responders (P = 0·002, Fisher's exact test). Six children treated with vemurafenib showed a clinical response that was associated with a decrease in the ccf BRAF load at day 15. Thus, ccf BRAF is a promising biomarker for monitoring the response to therapy for children with RO+ MS LCH or RO- LCH resistant to first-line chemotherapy.

This prospective study showed that tumor-specific mutation and NPY methylation ddPCR assays performed on circulating DNA can be used for the follow-up of mCRC patients during treatment and could complement current follow-up methods. The analysis of NPY methylation is promising, as it has the additional advantage that no prior knowledge of tumor mutations is needed.

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In patients with metastatic colorectal cancer (mCRC), RAS and BRAF mutations are currently determined by tumor sample analysis. Here, we report BRAF mutation status analysis in paired tumor tissue and plasma samples of mCRC patients included in the AGEO RASANC prospective cohort study. Four hundred and twenty-five patients were enrolled. Plasma samples were analyzed by next-generation sequencing (NGS). When no mutation was identified, we used two methylated specific biomarkers (digital droplet PCR) to determine the presence or absence of circulating tumor DNA (ctDNA). Patients with conclusive ctDNA results were defined as those with at least one mutation or one methylated biomarker. The kappa coefficient and accuracy were 0.79 (95% CI: 0.67–0.91) and 97.3% (95% CI: 95.2–98.6%) between the BRAF status in plasma and tissue for patients with available paired samples (n = 405), and 0.89 (95% CI: 0.80–0.99) and 98.5% (95% CI: 96.4–99.5%) for those with conclusive ctDNA (n = 323). The absence of liver metastasis was the main factor associated to inconclusive ctDNA results. In patients with liver metastasis, the kappa coefficient was 0.91 (95% CI, 0.81–1.00) and accuracy was 98.6% (95% CI, 96.5–99.6%). We demonstrate satisfying concordance between tissue and plasma BRAF mutation detection, especially in patients with liver metastasis, arguing for plasma ctDNA testing for routine BRAF mutation analysis in these patients.

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor growth and aggressiveness. Endoplasmic Reticulum (ER) stress has been documented in most major cancers, and the ability to tolerate persistent ER stress through an effective unfolded protein response enhances cancer cell survival, angiogenesis, metastasis, drug resistance and immunosuppression. The ER stress sensor IRE1α contributes to tumor progression through XBP1 mRNA splicing and regulated IRE1α-dependent decay of mRNA and miRNA. The aim of this study was to perform a molecular characterization of series of tumor samples to explore the impact of intratumoral IRE1 signaling in non-small cell lung cancer characteristics. To monitor IRE1 splicing activity, we adopted a fragment length analysis to detect changes in the length of the XBP1 mRNA before and after splicing as a method for measuring sXBP1 mRNA levels in tumors because sXBP1 mRNA is not probed by standard transcriptomic analyses. We demonstrate for the first time that XBP1 splicing is a valuable marker of lung cancer aggressiveness, and our results support a model in which IRE1 downstream signaling could act as a regulator of Epithelial to Mesenchymal Transition (EMT). Our findings study highlights the role of IRE1α downstream signaling in non-small cell lung cancer and opens a conceptual framework to determine how IRE1α endoribonuclease activity shapes the EMT program.