IDNetwork: A deep illness-death network based on multi-state event history process for disease prognostication.

Multi-state models can capture the different patterns of disease evolution. In particular, the illness-death model is used to follow disease progression from a healthy state to an intermediate state of the disease and to a death-related final state. We aim to use those models in order to adapt treatment decisions according to the evolution of the disease. In state-of-the art methods, the risks of transition between the states are modeled via (semi-) Markov processes and transition-specific Cox proportional hazard (P.H.) models. The Cox P.H. model assumes that each variable makes a linear contribution to the model, but the relationship between covariates and risks can be more complex in clinical situations. To address this challenge, we propose a neural network architecture called illness-death network (IDNetwork) that relaxes the linear Cox P.H. assumption within an illness-death process. IDNetwork employs a multi-task architecture and uses a set of fully connected subnetworks in order to learn the probabilities of transition. Through simulations, we explore different configurations of the architecture and demonstrate the added value of our model. IDNetwork significantly improves the predictive performance compared to state-of-the-art methods on a simulated data set, on two clinical trials for patients with colon cancer and on a real-world data set in breast cancer.

Intratumoral Immune Cell Densities Are Associated with Lung Adenocarcinoma Gene Alterations.

RATIONALE: Tumor-infiltrating immune cells affect lung cancer outcome. However, the factors that influence the composition and function of the tumor immune environment remain poorly defined and need investigation, particularly in the era of immunotherapy. OBJECTIVES: To determine whether the tumoral immune environment is related to lung adenocarcinoma mutations. METHODS: This retrospective cohort included 316 consecutive patients with lung adenocarcinoma (225 men; 258 smokers) studied from 2001 to 2005 in a single center. We investigated the association of densities of intratumoral mature dendritic cells (mDCs), CD8+ T cells, neutrophils, and macrophages with clinical and pathological variables and tumor cell mutation profiles obtained by next-generation sequencing. MEASUREMENTS AND MAIN RESULTS: In 282 tumors, we found 460 mutations, mainly in TP53 (59%), KRAS (40%), STK11 (24%), and EGFR (14%). Intratumoral CD8+ T-cell density was high in smokers (P = 0.02) and TP53-mutated tumors (P = 0.02) and low in BRAF-mutated tumors (P = 0.005). Intratumoral mDC density was high with low pathological tumor stage (P = 0.01) and low with STK11 mutation (P = 0.004). Intratumoral neutrophil density was high and low with BRAF mutation (P = 0.04) and EGFR mutation (P = 0.02), respectively. Intratumoral macrophage density was low with EGFR mutation (P = 0.01). Intratumoral CD8+ T-cell and mDC densities remained strong independent markers of overall survival (P = 0.001 and P = 0.02, respectively). CONCLUSIONS: Intratumoral immune cell densities (mDCs, CD8+ T cells, neutrophils, macrophages) were significantly associated with molecular alterations in adenocarcinoma underlying the interactions between cancer cells and their microenvironment.

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study.

BACKGROUND: Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive "real-time" biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). METHODS AND FINDINGS: We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01-3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30-3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66-6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations. CONCLUSIONS: In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.

Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA.

BACKGROUND: Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing. METHODS: We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer. RESULTS: Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90). CONCLUSIONS: Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.

[Lung cancer molecular testing, what role for Next Generation Sequencing and circulating tumor DNA]. / Place du NGS (Next Generation Sequencing) et de l'ADN tumoral circulant dans le testing moléculaire des cancers bronchiques.

Molecular screening has become a standard of care for patients with advanced cancers and impacts on how to treat a patient. Advances in genomic technologies with the development of high throughput sequencing methods will certainly improve the possibilities to access a more accurate molecular diagnosis and to go beyond the identification of validated targets as a large number of genes can be screened for actionable changes. Moreover, accurate high throughput testing may help tumor classification in terms of prognosis and drug sensitivity. Finally, it will be possible to assess tumor heterogeneity and changes in molecular profiles during follow-up using ultra-deep sequencing technologies and circulating tumor DNA characterization. The accumulation of somatic ADN alterations is considered as the main contributing factor in carcinogenesis. The alterations can occur at different levels: mutation, copy number variations or gene translocations resulting in altered expression of the corresponding genes or impaired protein functions. Genes involved are mainly tumor suppressors, oncogenes or ADN repair genes whose modifications in tumors will impinge cell fate and proliferation from tumor initiation to metastasis. The entire genome of various tumor types, have now been sequenced. In lung cancer, the average number of mutations is very high with more than 8.9 mutations/Mb (Network TCGAR, 2014) that is to say more than 10,000 mutations/genome. These alterations need to be classified, indeed, some are true drivers that directly impact proliferation and some are passenger mutations linked to genetic instability. The development of targeted therapies relies on the identification of oncogenic drivers. The identification of genotype-phenotype associations as in the case of EGFR-TKI (Epidermal growth factor receptor-tyrosine kinase inhibitor) and EGFR mutations in lung cancer led to the restriction of drugs to patients for which tumor genotype predicts efficacy. Tumor-molecular directed therapy based on validated targets (EGFR, ALK) is in the clinics, rapidly, with the developments of multi-targets or multi-drug assays there will be a need for tumor-molecular-profile directed therapy. Today, there are practical challenges to a successful implementation of NGS technologies for clinical applications. Broadly, some are linked to the tumor (heterogeneity), to the tissue (availability, storage, fixative), to the design of specific assays or set of genes, to the interpretation of non-driver mutations and to a possible access to drugs once a target is identified. Technical challenges are solved, NGS (at least targeted-NGS) plateforms have been validated by INCa labeled laboratories, in this context, we will address different questions: How, for whom, what kind of profiling and what can we expect?

Bleomycin-Induced Pneumonitis in the Treatment of Ovarian Sex Cord-Stromal Tumors: A Systematic Review and Meta-analysis.

OBJECTIVE: Adult ovarian sex cord-stromal tumors (SCSTs) are a rare histological subtype of ovarian cancer associated with a favorable prognosis. Bleomycin-containing regimens are standards of care, although pneumonitis may cause potentially fatal dose-limiting toxicity. We aimed to evaluate the safety of bleomycin in SCST treatment. METHODS: We performed a systematic literature review of all studies of bleomycin therapy for SCSTs that were referenced in MEDLINE (PubMed), EMBASE, and Cochrane Central Register of Controlled Trials and published from 1986 to 2014. RESULTS: Eight studies totaling 221 patients were included. Rates of pneumonitis (7.7%; 95% confidence interval, 4.2-11.2) and mortality (1.8%; 95% confidence interval, 0.1-3.6) related to bleomycin were significant. However, these results were very similar to those reported for men who were treated with bleomycin for a male germ cell tumor, suggesting that women with ovarian SCSTs are not particularly vulnerable to bleomycin lung toxicity. The main risk factors of bleomycin-induced pneumonitis are high cumulative bleomycin dose (>400 U or mg), age older than 40 years, and impaired renal function. Whether granulocyte colony-stimulating factor is a risk factor remains controversial. CONCLUSIONS: Bleomycin-induced pneumonitis frequently occurs in patients with SCSTs and lacks effective treatment. Prevention lies in limiting cumulative bleomycin dose, monitoring pulmonary function during treatment, discontinuing bleomycin at the onset of pulmonary symptoms or if pulmonary function is impaired, and avoiding bleomycin in older patients.

MS-CPFI: A model-agnostic Counterfactual Perturbation Feature Importance algorithm for interpreting black-box Multi-State models.

Multi-state processes (Webster, 2019) are commonly used to model the complex clinical evolution of diseases where patients progress through different states. In recent years, machine learning and deep learning algorithms have been proposed to improve the accuracy of these models' predictions (Wang et al., 2019). However, acceptability by patients and clinicians, as well as for regulatory compliance, require interpretability of these algorithms's predictions. Existing methods, such as the Permutation Feature Importance algorithm, have been adapted for interpreting predictions in black-box models for 2-state processes (corresponding to survival analysis). For generalizing these methods to multi-state models, we introduce a novel model-agnostic interpretability algorithm called Multi-State Counterfactual Perturbation Feature Importance (MS-CPFI) that computes feature importance scores for each transition of a general multi-state model, including survival, competing-risks, and illness-death models. MS-CPFI uses a new counterfactual perturbation method that allows interpreting feature effects while capturing the non-linear effects and potentially capturing time-dependent effects. Experimental results on simulations show that MS-CPFI increases model interpretability in the case of non-linear effects. Additionally, results on a real-world dataset for patients with breast cancer confirm that MS-CPFI can detect clinically important features and provide information on the disease progression by displaying features that are protective factors versus features that are risk factors for each stage of the disease. Overall, MS-CPFI is a promising model-agnostic interpretability algorithm for multi-state models, which can improve the interpretability of machine learning and deep learning algorithms in healthcare.

Loss of heterozygosity at 13q13 and 14q32 predicts BRCA2 inactivation in luminal breast carcinomas.

BRCA2 is the major high-penetrance predisposition gene for luminal (estrogen receptor [ER] positive) breast cancers. However, many BRCA2 mutant carriers lack family history of breast/ovarian cancers and do not benefit from genetic testing. Specific genomic features associated with BRCA2 inactivation in tumors could help identify patients for whom a genetic test for BRCA2 may be proposed. A series of ER-positive invasive ductal carcinomas (IDCs) including 30 carriers of BRCA2 mutations and 215 control cases was studied by single-nucleotide polymorphism (SNP) arrays. Cases and controls were stratified by grade and HER2 status. Independently, 7 BRCA2 and 51 control cases were used for validation. Absolute copy number and Loss of heterozygosity (LOH) profiles were obtained from SNP arrays by the genome alteration print (GAP) method. BRCA2 tumors were observed to display a discriminatively greater number of chromosomal breaks calculated after filtering out and smoothing <3 Mb variations. This argues for a BRCA2-associated genomic instability responsible for long-segment aberrations. Co-occurrence of two genomic features-LOH of 13q13 and 14q32-was found to predict BRCA2 status with 90% of sensitivity and 87% of specificity in discovery series of high-grade HER2-negative IDCs and 100% of sensitivity and 88% of specificity in an independent series of 58 IDCs. Estimated positive predictive value was 17.2% (confidence interval: 6.7-33.5) in the whole series. In conclusion, the simplified BRCA2 classifier based on the co-occurrence of LOH at 13q13 and 14q32 could provide an indication to test for BRCA2 mutation in patients with ER-positive IDC.

New insights into the management of renal cell cancer.

Kidney cancer is composed of several bio-histological entities. The most frequent type, clear-cell carcinoma, is not homogenous regarding gene mutations or transcriptomic profiles, but the biologic classifications are not yet mature. Therefore, biologically driven strategies of treatment have not yet been developed in the clinical setting. The choice of first-line agent currently depends on the prognostic criteria published by Motzer et al. [J Clin Oncol 1999;17:2530-2540] and recently by Heng et al. [J Clin Oncol 2009;27:5794-5799], with anti-vascular endothelial growth factor (VEGF) therapies for good- or intermediate-prognosis groups and anti-mammalian target of rapamycin (mTOR) for poor-risk patients. In the past years, biological changes leading to resistance to targeted agents have been widely investigated. Discoveries resulted in the development of second-generation VEGF receptor tyrosine kinase inhibitors, characterized by an improved potency and selectivity. Besides, co-inhibition of signalling pathways mediating resistance to anti-VEGF are being developed targeting fibroblast growth factor and c-Met. Dual mTOR/phosphatidylinositol 3-kinase inhibitors have greater efficacy than rapalogs in preclinical models and are being investigated in early clinical trials. In conclusion, the changing landscape in the biology and treatment of kidney cancer offers new opportunities for clinicians to treat patients, but, due to relatively high costs, the use of targeted therapies will likely be strongly controlled by health authorities.

Gemcitabine plus platinum-based chemotherapy in combination with bevacizumab for kidney metastatic collecting duct and medullary carcinomas: Results of a prospective phase II trial (BEVABEL-GETUG/AFU24).

BACKGROUND: Renal medullary carcinoma (RMC) and collecting duct carcinoma (CDC) are rare entities with a poor outcome. First-line metastatic treatment is based on gemcitabine + platinum chemotherapy (GC) regimen but retrospective data suggest enhanced anti-tumour activity with the addition of bevacizumab. Therefore, we performed a prospective assessment of the safety and efficacy of GC + bevacizumab in metastatic RMC/CDC. METHODS: We conducted a phase 2 open-label trial in 18 centres in France in patients with metastatic RMC/CDC and no prior systemic treatment. Patients received bevacizumab plus GC up to 6 cycles followed, for non-progressive disease, by maintenance therapy with bevacizumab until progression or unacceptable toxicity. The co-primary end-points were objective response rates (ORRs) and progression-free survival (PFS) at 6 months (ORR-6; PFS-6). PFS, overall survival (OS) and safety were secondary end-points. At interim analysis, the trial was closed due to toxicity and lack of efficacy. RESULTS: From 2015 to 2019, 34 of the 41 planned patients have been enroled. After a median follow-up of 25 months, ORR-6 and PFS-6 were 29.4% and 47.1%, respectively. Median OS was 11.1 months (95% confidence interval [CI]: 7.6-24.2). Seven patients (20.6%) discontinued bevacizumab because of toxicities (hypertension, proteinuria, colonic perforation). Grade 3-4 toxicities were reported in 82% patients, the most common being haematologic toxicities and hypertension. Two patients experienced grade 5 toxicity (subdural haematoma related to bevacizumab and encephalopathy of unknown origin). CONCLUSION: Our study showed no benefit for bevacizumab added to chemotherapy in metastatic RMC and CDC with higher than expected toxicity. Consequently, GC regimen remains a therapeutic option for RMC/CDC patients.

NF1 mutations identify molecular and clinical subtypes of lung adenocarcinomas.

The tumor suppressor gene neurofibromin 1 (NF1) is a major regulator of the RAS-MAPK pathway. NF1 mutations occur in lung cancer but were not extensively explored. We hypothesized that NF1-mutated tumors could define a specific population with a distinct clinical and molecular profile. We performed NF1 sequencing using next generation sequencing (NGS) in 154 lung adenocarcinoma surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status, to evaluate the molecular and clinical specificities of NF1-mutated lung cancers. Clinical data were retrospectively collected, and their associations with molecular profiles assessed. In this series, 24 tumors were NF1 mutated (17.5%) and 11 were NF1 deleted (8%). There was no mutation hotspot. NF1 mutations were rarely associated with other RAS-MAPK pathway mutations. Most of patients with NF1 alterations were males (74.3%) and smokers (74.3%). Overall survival and disease-free survival were statistically better in patients with NF1 alterations (N = 34) than in patients with KRAS mutations (N = 30) in univariate analysis. Our results confirm that NF1 is frequently mutated and represents a distinct molecular and clinical subtype of lung adenocarcinoma.

TP53, STK11, and EGFR Mutations Predict Tumor Immune Profile and the Response to Anti-PD-1 in Lung Adenocarcinoma.

Purpose: By unlocking antitumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non-small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed.Experimental Design: We performed in-depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry, and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and PD-L1 expression, and a public clinical database was used to validate the results obtained.Results: We showed that distinct combinations of STK11, EGFR, and TP53 mutations were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations (TP53-mut/STK11-EGFR-WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T-cell density and PD-L1 expression. In this tumor subtype, pathways related to T-cell chemotaxis, immune cell cytotoxicity, and antigen processing were upregulated. Finally, a prolonged progression-free survival (PFS: HR = 0.32; 95% CI, 0.16-0.63, P < 0.001) was observed in anti-PD-1-treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression.Conclusions: Our study reveals that different combinations of TP53, EGFR, and STK11 mutations, together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Clin Cancer Res; 24(22); 5710-23. ©2018 AACR.

Validity of Targeted Next-Generation Sequencing in Routine Care for Identifying Clinically Relevant Molecular Profiles in Non-Small-Cell Lung Cancer: Results of a 2-Year Experience on 1343 Samples.

Theranostic assays are based on single-gene testing, but the ability of next-generation sequencing (NGS) to interrogate numerous genetic alterations will progressively replace single-gene assays. Although NGS was evaluated to screen for theranostic mutations, its usefulness in clinical practice on large series of samples remains to be demonstrated. NGS performance was assessed following guidelines. TaqMan probes and NGS were compared for their ability to detect EGFR and KRAS mutations, and NGS mutation profiles were analyzed on a large series of non-small-cell lung cancers (n = 1343). The R2 correlation between expected and measured allelic ratio, using commercial samples, was >0.96. Mutation detection threshold was 2% for 10 ng of DNA input. κ Scores for TaqMan versus NGS were 0.99 (95% CI, 0.97-1.00) for EGFR and 0.98 (95% CI, 0.97-1.00) for KRAS after exclusion of rare EGFR (n = 40) and KRAS (n = 60) mutations. NGS identified 693 and 292 mutations in validated and potential oncogenic drivers, respectively. Significant associations were found between EGFR and PI3KCA or CTNNB1 and between KRAS and STK11. Potential oncogenic driver mutations or gene amplifications were more frequent in validated oncogenic driver nonmutated samples. This work is a proof of concept that targeted NGS is accessible in routine screening, including large screening, at reasonable cost. Clinical data should be collected and implemented in specific databases to make molecular data meaningful for direct patients' benefit.

Epithelial-to-Mesenchymal Transition and MicroRNAs in Lung Cancer.

Despite major advances, non-small cell lung cancer (NSCLC) remains the major cause of cancer-related death in developed countries. Metastasis and drug resistance are the main factors contributing to relapse and death. Epithelial-to-mesenchymal transition (EMT) is a complex molecular and cellular process involved in tissue remodelling that was extensively studied as an actor of tumour progression, metastasis and drug resistance in many cancer types and in lung cancers. Here we described with an emphasis on NSCLC how the changes in signalling pathways, transcription factors expression or microRNAs that occur in cancer promote EMT. Understanding the biology of EMT will help to define reversing process and treatment strategies. We will see that this complex mechanism is related to inflammation, cell mobility and stem cell features and that it is a dynamic process. The existence of intermediate phenotypes and tumour heterogeneity may be debated in the literature concerning EMT markers, EMT signatures and clinical consequences in NSCLC. However, given the role of EMT in metastasis and in drug resistance the development of EMT inhibitors is an interesting approach to counteract tumour progression and drug resistance. This review describes EMT involvement in cancer with an emphasis on NSCLC and microRNA regulation.

Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker.

PURPOSE: Despite recent therapeutic advances, prognosis of patients with pancreatic adenocarcinoma remains poor. Analyses from tumor tissues present limitations; identification of informative marker from blood might be a promising alternative. The aim of this study was to assess the feasibility and the prognostic value of circulating tumor DNA (ctDNA) in pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: From 2011 to 2015, blood samples were prospectively collected from all consecutive patients with pancreatic adenocarcinoma treated in our center. Identification of ctDNA was done with next-generation sequencing targeted on referenced mutations in pancreatic adenocarcinoma and with picoliter droplet digital PCR. RESULTS: A total of 135 patients with resectable (n = 31; 23%), locally advanced (n = 36; 27%), or metastatic (n = 68; 50%) pancreatic adenocarcinoma were included. In patients with advanced pancreatic adenocarcinoma (n = 104), 48% (n = 50) had ctDNA detectable with a median mutation allelic frequency (MAF) of 6.1%. The presence of ctDNA was strongly correlated with poor overall survival (OS; 6.5 vs. 19.0 months; P < 0.001) in univariate and multivariate analyses (HR = 1.96; P = 0.007). To evaluate the impact of ctDNA level, patients were grouped according to MAF tertiles: OS were 18.9, 7.8, and 4.9 months (P < 0.001). Among patients who had curative intent resection (n = 31), 6 had ctDNA detectable after surgery, with an MAF of 4.4%. The presence of ctDNA was associated with a shorter disease-free survival (4.6 vs.17.6 months; P = 0.03) and shorter OS (19.3 vs. 32.2 months; P = 0.027). CONCLUSIONS: ctDNA is an independent prognostic marker in advanced pancreatic adenocarcinoma. Furthermore, it arises as an indicator of shorter disease-free survival in resected patients when detected after surgery. Clin Cancer Res; 23(1); 116-23. ©2016 AACR.

Different prognostic impact of STK11 mutations in non-squamous non-small-cell lung cancer.

STK11 is commonly mutated in lung cancer. In light of recent experimental data showing that specific STK11 mutants could acquire oncogenic activities due to the synthesis of a short STK11 isoform, we investigated whether this new classification of STK11 mutants could help refine its role as a prognostic marker. We conducted a retrospective high-throughput genotyping study in 567 resected non-squamous non-small-cell lung cancer (NSCLC) patients. STK11 exons 1 or 2 mutations (STK11ex1-2) with potential oncogenic activity were analyzed separately from exons 3 to 9 (STK11ex3-9). STK11ex1-2 and STK11ex3-9 mutations occurred in 5% and 14% of NSCLC. STK11 mutated patients were younger (P = .01) and smokers (P< .0001). STK11 mutations were significantly associated with KRAS and inversely with EGFR mutations. After a median follow-up of 7.2 years (95%CI 6.8-.4), patients with STK11ex1-2 mutation had a median OS of 24 months (95%CI 15-57) as compared to 69 months (95%CI 56-93) for wild-type (log-rank, P = .005) and to 91 months (95%CI 57-unreached) for STK11ex3-9 mutations (P = .003). In multivariate analysis, STK11ex1-2 mutations remained associated with a poor prognosis (P = .002). Results were validated in two public datasets. Western blots showed that STK11ex1-2 mutatedtumors expressed short STK11 isoforms. Finally using mRNAseq data from the TCGA cohort, we showed that a stroma-derived poor prognosis signature was enriched in STK11ex1-2 mutated tumors. All together our results show that STK11ex1-2 mutations delineate an aggressive subtype of lung cancer for which a targeted treatment through STK11 inhibition might offer new opportunities.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

[What future for circulating tumor DNA? Current data and prospects in colorectal, non-small cell lung and pancreatic cancers]. / Quel avenir pour l'ADN tumoral circulant? État des lieux et perspectives dans les cancers colorectaux, pulmonaires non à petites cellules et pancréatiques.

Ten years after the discovery of the predictive value of KRAS status for anti-EGFR antibodies, other genes involved in oncogenesis and therapeutic responses were identified and are now systematically sought. Molecular diagnosis often requires invasive procedures, sometimes iatrogenic, and is limited by feasibility problems, quantity and quality of samples. Identifying these mutations from blood biomarkers would reduce costs and diagnostic delay. The circulating tumor DNA (ctDNA) is one of the most promising blood biomarkers. In this review, we report and discuss the latest results obtained with ctDNA in colorectal cancer, non-small cell lung cancer, and adenocarcinoma of the pancreas. If the methods highlighting appear very heterogeneous, the correlation between mutations found in tumor and those identified in the blood exceeds 95 % specificity in numerous studies. The detection sensitivity is in turn strongly related to tumor stage patients. The presence of ctDNA appears as a prognostic factor for progression-free survival and overall survival. Finally, recent studies have shown that the changing rate ctDNA during systemic treatments had a predictive value for therapeutic efficacy. These results allow to consider the use of ctDNA in monitoring patients to identify early recurrence or progression.

Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.

PURPOSE: Approximately 1% of lung adenocarcinomas are driven by oncogenic ROS1 rearrangement. Crizotinib is a potent inhibitor of both ROS1 and ALK kinase domains. PATIENTS AND METHODS: In the absence of a prospective clinical trial in Europe, we conducted a retrospective study in centers that tested for ROS1 rearrangement. Eligible patients had stage IV lung adenocarcinoma, had ROS1 rearrangement according to fluorescent in situ hybridization, and had received crizotinib therapy through an individual off-label use. Best response was assessed locally using RECIST (version 1.1). All other data were analyzed centrally. RESULTS: We identified 32 eligible patients. One patient was excluded because next-generation sequencing was negative for ROS1 fusion. Median age was 50.5 years, 64.5% of patients were women, and 67.7% were never-smokers. Thirty patients were evaluable for progression-free survival (PFS), and 29 patients were evaluable for best response. We observed four patients with disease progression, two patients with stable disease, and objective response in 24 patients, including five complete responses (overall response rate, 80%; disease control rate, 86.7%). Median PFS was 9.1 months, and the PFS rate at 12 months was 44%. No unexpected adverse effects were observed. Twenty-six patients received pemetrexed (either alone or in combination with platinum and either before or after crizotinib) and had a response rate of 57.7% and a median PFS of 7.2 months. CONCLUSION: Crizotinib was highly active at treating lung cancer in patients with a ROS1 rearrangement, suggesting that patients with lung adenocarcinomas should be tested for ROS1. Prospective clinical trials with crizotinib and other ROS1 inhibitors are ongoing or planned.