Disrupted BMP-9 Signaling Impairs Pulmonary Vascular Integrity in Hepatopulmonary Syndrome.

Rationale: Hepatopulmonary syndrome (HPS) is a severe complication of liver diseases characterized by abnormal dilation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced BMP-9 (bone morphogenetic protein-9) in maintaining pulmonary vascular integrity. Objectives: This study aimed to investigate the involvement of BMP-9 in human and experimental HPS. Methods: Circulating BMP-9 levels were measured in 63 healthy control subjects and 203 patients with cirrhosis with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation with cirrhosis and long-term partial portal vein ligation without cirrhosis. Additionally, the therapeutic effect of low-dose BMP activator FK506 was investigated, and the pulmonary vascular phenotype of BMP-9-knockout rats was analyzed. Measurements and Main Results: Patients with HPS related to compensated cirrhosis exhibited lower levels of circulating BMP-9 compared with patients without HPS. Patients with severe cirrhosis exhibited consistently low levels of BMP-9. HPS characteristics were observed in animal models, including intrapulmonary vascular dilations and an increase in the alveolar-arterial gradient. HPS development in both rat models correlated with reduced intrahepatic BMP-9 expression, decreased circulating BMP-9 level and activity, and impaired pulmonary BMP-9 endothelial pathway. Daily treatment with FK506 for 2 weeks restored the BMP pathway in the lungs, alleviating intrapulmonary vascular dilations and improving gas exchange impairment. Furthermore, BMP-9-knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. Conclusions: The study findings suggest that portal hypertension-induced loss of BMP-9 signaling contributes to HPS development.

Circulating cell-free BRAFV600E as a biomarker in children with Langerhans cell histiocytosis.

The BRAFV600E mutation is reported in half of patients with Langerhans cell histiocytosis (LCH). This study investigated the detection of the BRAFV600E allele in circulating cell-free (ccf) DNA in a paediatric LCH cohort. Children with BRAFV600E -mutated LCH were investigated to detect ccf BRAFV600E at diagnosis (n = 48) and during follow-up (n = 17) using a picolitre-droplet digital PCR assay. At diagnosis, ccf BRAFV600E 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 BRAFV600E 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 BRAFV600E 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 BRAFV600E load at day 15. Thus, ccf BRAFV600E is a promising biomarker for monitoring the response to therapy for children with RO+ MS LCH or RO- LCH resistant to first-line chemotherapy.

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker.

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.

Circulating tumor DNA is a prognostic marker of tumor recurrence in stage II and III colorectal cancer: multicentric, prospective cohort study (ALGECOLS).

BACKGROUND: In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. METHOD: The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. RESULTS: Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. CONCLUSION: This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques.

High expression of spliced X-Box Binding Protein 1 in lung tumors is associated with cancer aggressiveness and epithelial-to-mesenchymal transition.

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.

BRAF Mutation Status in Circulating Tumor DNA from Patients with Metastatic Colorectal Cancer: Extended Mutation Analysis from the AGEO RASANC Study.

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.

Mutation and Methylation Analysis of Circulating Tumor DNA Can Be Used for Follow-up of Metastatic Colorectal Cancer Patients.

BACKGROUND: Targeted therapies, although contributing to survival improvement in metastatic colorectal cancer (mCRC), are expensive and may cause adverse effects. Therefore, confirming that patients are responding to these therapies is extremely important. Currently, follow-up is performed using radiographic evaluation, which has its limitations. Liquid biopsies, reflecting real-time tumor characteristics, hold great potential in monitoring tumor disease. PATIENTS AND METHODS: Blood samples were collected at different time points during treatment of 24 mCRC patients. Mutation and NPY methylation picoliter droplet-based digital PCR (ddPCR) assays were performed on circulating DNA to investigate whether these assays can be used for disease monitoring. RESULTS: The results of the mutation and methylation assays were correlated with each other and corresponded with the results of radiographic evaluation. There was a steep decrease in circulating tumor DNA levels immediately after treatment initiation. Furthermore, circulating tumor DNA levels were increased in progressive samples and were undetectable in patients undergoing curative surgery. CONCLUSION: 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.

Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy.

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 KRAS-mutated 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.