Duplications of KIAA1549 and BRAF screening by Droplet Digital PCR from formalin-fixed paraffin-embedded DNA is an accurate alternative for KIAA1549-BRAF fusion detection in pilocytic astrocytomas

Pilocytic astrocytomas represent the most common glioma subtype in young patients and account for 5.4% of all gliomas. They are characterized by alterations in the RAS–MAP kinase pathway, the most frequent being a tandem duplication on chromosome 7q34 involving the BRAF gene, resulting in oncogenic BRAF fusion proteins. BRAF fusion involving the KIAA1549 gene is a hallmark of pilocytic astrocytoma, but it has also been recorded in rare cases of gangliogliomas, 1p/19q co-deleted oligodendroglial tumors, and it is also a common feature of disseminated oligodendroglial-like leptomeningeal neoplasm. In some difficult cases, evidence for KIAA1549-BRAF fusion is of utmost importance for the diagnosis. Moreover, because the KIAA1549-BRAF fusion constitutively activates the MAP kinase pathway, it represents a target for drugs such as MEK inhibitors, and therefore, the detection of this genetic abnormality is highly relevant in the context of clinical trials applying such new approaches. In the present study, we aimed to use the high sensitivity of Droplet Digital PCR (DDPCR™) to predict KIAA1549-BRAF fusion on very small amounts of formalin-fixed paraffin-embedded tissue in routine practice. Therefore, we analyzed a training cohort of 55 pilocytic astrocytomas in which the KIAA1549-BRAF fusion status was known by RNA sequencing used as our gold standard technique. Then, we analyzed a prospective cohort of 40 pilocytic astrocytomas, 27 neuroepithelial tumors remaining difficult to classify (pilocytic astrocytoma versus ganglioglioma or diffuse glioma), 15 dysembryoplastic neuroepithelial tumors, and 18 gangliogliomas. We could demonstrate the usefulness and high accuracy (100% sensitivity and specificity when compared to RNA sequencing) of DDPCR™ to assess the KIAA1549-BRAF fusion from very low amounts of DNA isolated from formalin-fixed paraffin-embedded specimens. BRAF duplication is both necessary and sufficient to predict this fusion in most cases and we propose that this single analysis could be used in routine practice to save time, money, and precious tissue

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population

Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation

BackgroundMany studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx.MethodsThis prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF).ResultsQuantification of total cfDNA was not correlated with the patient’s status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%).ConclusionWith the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries

Like a Rolling Stone: Sting-Cgas Pathway and Cell-Free DNA as Biomarkers for Combinatorial Immunotherapy

International audienceCombining immune checkpoint inhibitors with other treatments likely to harness tumor immunity is a rising strategy in oncology. The exact modalities of such a combinatorial regimen are yet to be defined, and most attempts have relied so far on concomitant dosing, rather than sequential or phased administration. Because immunomodulating features are likely to be time-, dose-, and-schedule dependent, the need for biomarkers providing real-time information is critical to better define the optimal time-window to combine immune checkpoint inhibitors with other drugs. In this review, we present the various putative markers that have been investigated as predictive tools with immune checkpoint inhibitors and could be used to help further combining treatments. Whereas none of the current biomarkers, such as the PDL1 expression of a tumor mutational burden, is suitable to identify the best way to combine treatments, monitoring circulating tumor DNA is a promising strategy, in particular to check whether the STING-cGAS pathway has been activated by cytotoxics. As such, circulating tumor DNA could help defining the best time-window to administrate immune checkpoint inhibitors after that cytotoxics have been given

Évolution du statut braf dans le melanome : mythe ou Réalité ?

International audienceKnowledge of the BRAF mutational status has become essential for melanoma therapeutic management. B-Raf inhibitors are associated with significant overall survival in patients with BRAFV600-mutated metastatic melanoma. Although the BRAF mutation appears to be an early and driver mutation, some authors hypothesized that its expression was not stable during melanoma progression, suggesting a molecular heterogeneity. This argument is often used to explain discrepancy in molecular status among patients with melanoma, discrepancies that we occasionally met during our practice. We retrospectively compared BRAF mutational status on matched melanoma samples (primary & metastatic lesions), thus 150 samples from 56 patients were analysed through immunohistochemistry anti-BRAF, PCR-HRM and Sanger sequencing, Next Generation Sequencing (NGS) and digital PCR. Seven cases presented an apparent tumor heterogeneity. The analysis of these discrepancies by a technique of increasing sensitivity made it possible to identify 1 false-negative result for the immunohistochemistry, 1 false-negative result for the NGS sequencing and 5 (3%) false-negative results by PCR-HRM SANGER. Our results are consistent with the most recent data, demonstrating the stability of the BRAF mutation during the course of melanoma. Immunohistochemistry shows excellent sensitivity for detecting the main BRAF mutation. In our study, the mutational heterogeneity was actually misleading, a result of imperfect sensitivity of some older molecular approaches.La connaissance du statut mutationnel du gène BRAF est devenue essentielle dans la prise en charge du mélanome. Les inhibiteurs de B-Raf sont à l’origine d’une nette amélioration de la survie globale chez les patients présentant un mélanome métastatique muté BRAF V600. Bien que la mutation BRAF soit considérée comme un événement précoce et conducteur (driver), certains travaux ont émis l’hypothèse qu’elle n’était pas stable lors de l’évolution d’un mélanome, impliquant une hétérogénéité moléculaire. Cet argument est souvent utilisé pour expliquer les discordances de statut moléculaire observées chez certains patients atteints de mélanome, discordances occasionnellement rencontrées au cours de notre pratique. Nous avons rétrospectivement comparé le statut mutationnel du gène BRAF sur plusieurs prélèvements appariés de mélanome (primitif & localisations secondaires), soit 150 prélèvements pour 56 patients, en utilisant l’immunohistochimie anti-BRAF, la PCR-HRM et séquençage Sanger, le séquençage massivement parallèle (NGS) et la PCR digitale. Sept cas présentaient une apparente hétérogénéité tumorale. L’analyse de ces discordances par une technique de sensibilité croissante a permis d’identifier 1 résultat faux-négatif pour l’immunohistochimie, 1 résultat faux-négatif pour le séquençage NGS et 5 (3 %) résultats faux-négatifs par technique PCR-HRM SANGER. Nos résultats sont cohérents avec les données les plus récentes, témoignant de la stabilité de la mutation BRAF au cours de l’évolution d’un mélanome. L’immunohistochimie présente une sensibilité excellente pour détecter la mutation majoritaire de BRAF. Dans notre étude, l’hétérogénéité mutationnelle était en réalité factice, conséquence d’une sensibilité imparfaite de certaines approches moléculaires plus anciennes

High EGFR protein expression and exon 9 PIK3CA mutations are independent prognostic factors in triple negative breast cancers

International audienceBACKGROUND:Triple negative breast cancers (TNBC) are a more aggressive subset of breast cancer. A better understanding of its biology could allow the rational development of targeted therapies.METHODS:We extensively analyzed the EGFR/PI3K/PTEN axis in a large, homogeneous population of TNBC to help defining the putative role of anti-EGFR and -PI3K targeted therapies in this setting. EGFR gene amplification, EGFR protein expression, PIK3CA and PTEN gene alterations (two members of EGFR downstream pathways) and their clinicopathological and prognostic implications were analyzed in 204 TNBC samples from European patients.RESULTS:EGFR amplification was detected in 18 of the 204 TNBC specimens (8.9 %) and was significantly associated with higher EGFR protein levels. Fourteen PIK3CA mutations were identified in exon 9 (6.7 %), and 17 in exon 20 (8.3 %). PIK3CA mutations, especially in exon 9, were significantly associated with grade I-II tumors. PTEN deletions were detected in 43 samples (21.50 %) and were significantly associated with grade III tumors (p < 0.001). Univariate analysis showed a significant association between relapse-free survival (RFS), T and N stage and exon 9 PIK3CA mutations. Overall survival was significantly associated with T stage, N stage and adjuvant chemotherapy, which was administered to 70.3 % of patients. In multivariate analyses, T stage, N stage, presence of exon 9 PIK3CA mutations and high EGFR protein level were independent poor prognostic factors for RFS, while adjuvant chemotherapy was associated with a better outcome.CONCLUSIONS:High EGFR protein expression and exon 9 PIK3CA activating mutations are independent prognostic factors in TNBC. The efficacy of anti-PI3K targeted therapies needs to be evaluated in this setting

Regional molecular genetics centers in thoracic oncology: what and who should be tested?

International audienceManagement of NSCLC patients is more and more individualized especially on the base of bioguided treatments. In order to guarantee an access for all the patients too this type of strategy, the French NCI supports since 2006 a nationwide network of 28 regional genetics center. The financial support is based on public funds. The French NCI recommends today the assessment of seven biomarkers for all stage IV non squamous NSCLC patients. Due to financial and technical reasons, this recommendation must be followed. However, the molecular profiling of lung cancer patients would ideally be extended across all stages and all histological types of the disease in order to improve our knowledge in this field and provides the patient with an opportunity to access a bioguided treatment as frequently as possible