Elizabeth Fabre

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.

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.

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.

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.

Traitements néoadjuvants : évaluation d’une pratique couramment indiquée en RCP

UNLABELLEDnThe management of localized non-small cell lung cancer (NSCLC) has been modified over the last decades, with induction therapies being increasingly recommended as a prerequisite to surgical resection. However, the relative impact of chemo- and chemoradiotherapy on tumours pathology and patients survival is still discussed.nnnMETHODSnWe set a retrospective study including every patient who underwent surgical resection for NSCLC in 2 French centres from 1980 to 2009. We then compared the tumours pathology and patients survival according to the use of induction chemotherapy (group 1) or induction chemoradiotherapy (group 2).nnnRESULTSnThere were 733 patients in group 1 and 126 patients in group 2. In group 1, 669 patients (91%) had platinum-based chemotherapy, for 2 to 3 cycles in 564 cases (77%). In group 2, chemoradiotheray was concomitant in 68 patients (54%), and sequential in 58 patients (46%). As compared with group 1, group 2 was characterized by younger age (mean 59.8±9.5 vs 56.4±9.6, respectively, P<.001), a higher rate of tumours deemed unresectable before induction treatment (25% vs 44%, P<.001), and a higher proportion of T4 (25% vs 44%, P<.001) or N2 diseases (56% vs 69%, P=.005). The type of resection, postoperative complications, and postoperative mortality were not significantly different between groups. On final pathologic report, as compared with group 1, there were more N0 and N1 disease in group 2 (N0: 43% vs 58%, P=.002; N1: 22% vs 10%, P=.002) while the rate of N2 disease was comparable (34% vs 32%, P=ns). The median, 5-, and 10-year survivals were 28 months, 35%, and 21% for group 1, and 29 months, 36%, and 23% for group 2, respectively (P=ns).nnnCONCLUSIONnAs compared with induction chemotherapy, induction chemoradiotherapy was performed in more advanced NSCLC, and resulted in better downstaging, similar postoperative course, and comparable long-term outcome after surgical resection.

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 (nxa0=xa01343). 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 (nxa0=xa040) and KRAS (nxa0=xa060) 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.

Les inhibiteurs des points de contrôle immunitaire dans le cancer bronchique non à petites cellules de stade avancé

IMMUNE CHECKPOINT INHIBITORS IN ADVANCED NON-SMALL CELL LUNG CANCER: T-cell-directed strategies represent currently a major advance in the treatment of advanced non-small-cell lung cancer, regarding their efficacy and tolerance. Nivolumab and pembrolizumab, two monoclonal antibodies targeting programmed cell death protein 1 (PD-1) have shown their efficacy in phase III studies. Several other drugs are developed and the benefit of association is being evaluated. In this article, we propose to summarize the clinical development of immune checkpoint inhibitors in patients with advanced non-small cell lung cancer.