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International audience; The clinical actionability of circulating tumor DNA requires sensitive detection methods with a short turnaround time. In the PADA-1 phase 3 trial (NCT03079011), metastatic breast cancer patients treated with an aromatase inhibitor and palbociclib were screened every 2 months for activating ESR1 mutations in blood (bESR1mut). We report the feasibility of the droplet digital polymerase chain reaction (ddPCR) and cross-validation with next-generation sequencing (NGS). bESR1mut testing was centralized in two platforms using the same ddPCR assay. Results were reported as copies/mL of plasma and mutant allele frequency (MAF). We analyzed 200 positive ddPCR samples with an NGS assay (0.5-1% sensitivity). Overall, 12,552 blood samples were collected from 1017 patients from 83 centers. Among the 12,525 available samples with ddPCR results, 11,533 (92%) were bESR1mut-negative. A total of 267 patients newly displayed bESR1mut (26% patients/2% samples) with a median copy number of 14/mL (range: 4-1225) and a median MAF of 0.83% (0.11-35), 648 samples (20% patients/5% samples) displayed persistent bESR1mut, and 77 (

Background: Mutations of ESR1 (ESR1mut), the gene encoding for Estrogen Receptor (ER) alpha, have been functionally characterized as driving resistance to aromatase inhibitors (AI) given to in ER+ HER2- metastatic breast cancer (mBC). In the randomized multicenter phase 3 PADA-1 trial (NCT03079011), ER+ HER2- mBC patients treated with first line AI+Palbociclib were screened every two months for ESR1mut in blood (bESR1mut), by subjecting cell-free circulating DNA (cfcDNA) to a droplet digital PCR (ddPCR) assay. Upon the onset of a rising (i.e., increasing or appearing) bESR1mut, patients with no concomitant disease progression were randomized between keeping the same regimen (AI-Palbociclib) or switching to Fulvestrant-Palbociclib. We report herein the sample flow of this first-in-its-class trial, from April 2017 to March 2021, and the cross-validation of ddPCR results with NGS. Methods: At each time point, 20 mL of blood were drawn into BCT Streck® tubes. cfcDNA was extracted from 4 mL of plasma. bESR1mut testing was centralized in 2 platforms using the same ddPCR assay, which combines a drop-off ddPCR, targeting the clustered hotspot L536, Y537 and D538 mutations found in exon 8, with an unconventional ddPCR interrogating specifically the E380Q mutation, located in exon 5 (Jeannot et al, Oncogene 2020). Results were expressed as the number of ESR1mut copies detected per mL of plasma together with the Mutant Allele Frequency (MAF). As a control, we submitted 200 ESR1mut+ (by ddPCR) left-over samples to an amplicon-based Next Generation Sequencing (NGS) assay covering all ESR1 exons - with a nominal sensitivity of >0.5% MAF. Results: From 03/2017 to 03/2021, 1,017 MBC patients have been included in 83 centers and 12,552 blood samples have been collected. The median time of delivery to central platforms was 1 day (range: [0-11]). A median volume of 9.5 mL [2-20] of plasma was obtained after 2 centrifugations. bESR1mut results were notified to investigators with a median turnaround time of 13 days [1-813] (32 days in 2017, 9 days in 2019, 8 days in 2021). Among the 12,525 available ddPCR results: N=77 ( Citation Format: Céline Callens, François-Clément Bidard, Anais Curto-Taribo, Olfa Trabelsi-Grati, Samia Melaabi, Suzette Delaloge, Anne-Claire Hardy-Bessard, Thomas Bachelot, Florian Clatot, Thibault De La Motte Rouge, Jean-Luc Canon, Laurent Arnould, Fabrice André, Sandrine Marques, Marc-Henri Stern, Jean-Yves Pierga, Anne-Vincent Salomon, Emmanuelle Jeannot, Frederique Berger, Ivan Bieche, Anne Pradines. Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-01.

Objective Tumors harboring a POLE pathogenic variant, associated with high tumor mutational burden, are good candidates for immunotherapy. However, POLE pathogenic variants are not currently screened in routine clinical practice. Can these tumors be identified by means of an already available test? Methods We describe seven tumors harboring a POLE pathogenic variant, among eight patients with tumors harboring multiple BRCA1/2 variants (from 4 to 20). All patients were managed at Institut Curie, Paris. Five patients were selected because of unexpected tumor BRCA testing results with multiple variants and another three patients were selected because of a POLE pathogenic variant detected by large tumor testing. We looked for other tumor variants by Next-Generation Sequencing in tumors harboring multiple BRCA1/2 variants, and for multiple BRCA1/2 variants in tumors harboring a POLE pathogenic variant. Results Four of the five tumors selected because of multiple BRCA1/2 variants exhibited a POLE pathogenic variant, and all three tumors selected for POLE pathogenic variants exhibited multiple BRCA1/2 variants. Conclusions Tumor BRCA testing could be a way to detect tumors harboring a highly mutagenic POLE pathogenic variant.

HER3, a member of the EGFR family of receptor tyrosine kinases coded by the ERBB3 gene, plays an important role in cancer, despite its lack of intrinsic kinase activity. As with genes coding for potential heterodimeric partners of HER3, EGFR, and HER2, oncogenic mutations of ERBB3 have been explored by several studies. In this review, we discuss the evidence presenting ERBB3 somatic mutations as potential tumoral drivers. We then show that ERBB3 mutations are not uncommon in many cancer types. Finally, we present the recent results of several studies evaluating different therapeutic approaches for treating patients with oncogenic ERBB3 mutations.

Introduction: In hormone receptor-positive breast cancer, ESR1 mutations have emerged as a key mechanism of resistance to endocrine therapy.Areas covered: Here, we review currently available data o...

About 2-3% of non-small-cell lung cancers (NSCLCs) harbor MET exon-14-skipping (METex14) mutations. Efficacy of the MET-inhibitor crizotinib has been reported, but progression-free survival (PFS) was very short. Immune-checkpoint inhibitors (ICIs) have become a cornerstone of NSCLC treatment but appear to be less effective in non-smokers and against tumors exhibiting oncogenic addiction. We describe 6 remarkable (PFS exceeding 18 months) and durable responses to ICIs of NSCLCs harboring a METex14 mutation.Each patient's clinical and biological characteristics, and tumor responses after ICIs were examined. Complete tumor-DNA sequencing was available after starting second-line ICIs, which followed first-line chemotherapy. Tumor-cell programmed cell-death protein-1 ligand-1 (PD-L1) expression on tumor cells was evaluated using antibody clone E1L3N (Cell Signaling Technology).Among 25 patients with METex14-mutated NSCLCs, 13 of whom were ICI-treated, 6 had prolonged responses: 5 women, 1 man; 57-80 years old; 3 never-smokers, 1 ex-smoker and 2 smokers; 5 adenocarcinomas, 1 sarcomatoid carcinoma; 5 received nivolumab, 1 pembrolizumab. No EGFR, BRAF or KRAS mutations (only 1 minority KRAS mutation), or ALK or ROS translocations were detected. No concurrent MET amplification was observed. Tumor-mutation burden was low (10 mutations/Mb) in 3 tested tumors. Four partial and 2 complete responses were obtained during the first 3 months for 5 patients, while pseudoprogression was initially observed in 1. Tolerance was excellent, with only 1 grade-3 immune-related adverse event. Response was maintained for 18-49 months.ICIs could be considered to treat patients whose NSCLCs harbor a METex14 mutation. More biological marker data are needed to identify which patients are most likely to benefit from ICIs.

BackgroundMicrosatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool.MethodsWe developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy.ResultsThe 3 ddPCR assays reached analytical sensitivity ConclusionsThis innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

T790M mutations inEGFR-mutated non-small cell lung cancer (NSCLC) account for nearly 50% of acquired resistance mechanisms to EGFR-TKIs. Earlier studies suggested that tumor T790M could also be detected in TKI-naïve EGFR-mutated NSCLC. The aim of the study is to assess the prevalence and clinical significance of quantification of tumor pre-treatment T790M subclones.We analyzed 366 EGFR-mutated NSCLC patients of the real-life IFCT Biomarkers France study with available pre-treatment formalin-fixed paraffin-embedded (FFPE) tumor DNA before treatment by first/second-generation EGFR-TKI. We used ultra-sensitive Droplet Digital Polymerase Chain Reaction (ddPCR) QX200 (BIO-RAD®, Hercules, CA, USA). All samples were tested in duplicate.ddPCR identified T790M in 19/240 specimens (8%). T790M-positive and T790M-negative populations were not different for clinical baseline characteristics. T790M Variant Allele Frequency (VAF) was 0.01% 0.1%, 0.1% 1%, 1% 10%, and10% in five (26.3%), six (31.6%), six (31.6%), and two (10.5%) patients, respectively. T790M VAF was0.1% in 11/13 (84%) patients with rapid (3 months) or usual progression (3-20 months) compared to 0/3 with low progression (20 months) (p = 0.02). In a Cox model, T790M mutation positivity was correlated with overall survival (OS) and progression-free survival (PFS) for 10%VAF1% (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.13-7.07, p = 0.03; HR=3.62, 95%CI 1.43-4.92, p = 0.007, respectively) and for VAF10% (HR = 19.14, 95%CI 4.35-84.26, p 0.001; HR = 17.89, 95%CI 2.21-144.86, p = 0.007, respectively).Ultra-sensitive detection of tumor T790M mutation concerned 8% of EGFR-mutated TKI-naïve NSCLC patients and has a negative prognostic value only for T790M VAF over 1%.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Background Metaplastic breast cancer (MBC) is a rare form of breast cancer characterized by an aggressive clinical presentation, with a poor response to standard chemotherapy. MBCs are typically triple-negative breast cancers (TNBCs), frequently with alterations to genes of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. The objective of this study was to determine the response to PI3K and MAPK pathway inhibitors in patient-derived xenografts (PDXs) of MBCs with targetable alterations. Methods We compared survival between triple-negative MBCs and other histological subtypes, in a clinical cohort of 323 TNBC patients. PDX models were established from primary breast tumors classified as MBC. PI3K-AKT-mTOR and RTK-MAPK pathway alterations were detected by targeted next-generation sequencing (NGS) and analyses of copy number alterations. Activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways was analyzed with reverse-phase protein arrays (RPPA). PDXs carrying an activating mutation of PIK3CA and genomic changes to the RTK-MAPK signaling pathways were treated with a combination consisting of a PI3K inhibitor and a MEK inhibitor. Results In our clinical cohort, the patients with MBC had a worse prognosis than those with other histological subtypes. We established nine metaplastic TNBC PDXs. Three had a pathogenic mutation of PIK3CA and additional alterations to genes associated with RTK-MAPK signaling. The MBC PDXs expressed typical EMT and stem cell genes and were of the mesenchymal or mesenchymal stem-like TNBC subtypes. On histological analysis, MBC PDXs presented squamous or chondroid differentiation. RPPA analysis showed activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. In vivo, the combination of PI3K and MAPK inhibitors displayed marked antitumor activity in PDXs carrying genomic alterations of PIK3CA, AKT1, BRAF, and FGFR4. Conclusion The treatment of metaplastic breast cancer PDXs by activation of the PI3K-AKT-mTOR and RTK-MAPK pathways at the genomic and protein levels with a combination of PI3K and MEK inhibitors resulted in tumor regression in mutated models and may therefore be of interest for therapeutic purposes.

Neuroendocrine breast carcinomas represent a rare subtype of breast cancer. Their definition, prevalence, and prognosis remain controversial in the literature. The 2012 WHO classification of breast cancer categorizes neuroendocrine carcinomas into three morphologically distinct subtypes: well-differentiated neuroendocrine tumors, poorly differentiated neuroendocrine carcinomas, and invasive breast carcinomas with neuroendocrine differentiation. We aimed to gain insight into the clinical, morphologic, phenotypic, and molecular features of 47 neuroendocrine breast carcinomas. Targeted next-generation sequencing by an AmpliSeq 22 cancer gene hotspot panel and the Prosigna assay were performed on 42/47 and 35/47 cases, respectively. Average age at diagnosis was 69 years. All tumors were estrogen receptor-positive and the large majority expressed progesterone receptor (89%), GATA3 (98%), FOXA1 (96%), and CK8/18 (98%). There was an almost equal distribution of luminal A (52%) and B (48%) carcinomas. Almost half of the cohort (49%) displayed a high risk of recurrence score with the Prosigna test. Patients with a neuroendocrine carcinoma had a shorter disease-free survival compared with those affected by carcinomas of no special type matched for age, size, grade, and estrogen receptor status. No significant differences were observed in terms of overall survival. Stratification of neuroendocrine carcinomas using the 2012 WHO criteria did not reveal statistically significant differences among the distinct categories (well-differentiated neuroendocrine tumors, poorly differentiated neuroendocrine carcinomas, and invasive breast carcinomas with neuroendocrine differentiation), in terms of either progression-free or overall survival. Our targeted sequencing analysis found three cases (7%) harboring a PIK3CA mutation, and in three other cases (7%) TP53 mutations were detected. This study showed that neuroendocrine breast carcinoma is a distinct subtype of luminal carcinoma with a low rate of PIK3CA mutations and with an aggressive clinical behavior. An accurate identification of neuroendocrine differentiation may be useful to better tailor patient adjuvant therapy within luminal carcinomas.Modern Pathology advance online publication, 8 September 2017; doi:10.1038/modpathol.2017.107.

Background A randomised trial SHIVA01 compared the efficacy of matched molecularly targeted therapy outside their indications based on a prespecified treatment algorithm versus conventional chemotherapy in patients with metastatic solid tumours who had failed standard of care. No statistical difference was reported between the two groups in terms of progression-free survival (PFS), challenging treatment algorithm. The European Society for Medical Oncology (ESMO) Scale for Clinical Actionability of molecular Targets (ESCAT) recently defined criteria to prioritise molecular alterations (MAs) to select anticancer drugs. We aimed to retrospectively evaluate the efficacy of matched molecularly targeted agents (MTAs) given in SHIVA01 according to ESCAT tiers. Patients and methods MAs used in SHIVA01 were retrospectively classified into ESCAT tiers, and PFS and overall survival (OS) were compared using log-rank tests. Results One hundred fifty-three patients were treated with matched MTAs in SHIVA01. MAs used to allocate MTAs were classified into tiers II, IIIA, IIIB and IVA according to the ESCAT. Median PFS was 2.0 months in tier II, 3.1 in tier IIIA, 1.7 in tier IIIB and 3.2 in tier IVA (p = 0.13). Median OS in tier IIIB was worse than that in tiers II, IIIA and IVA (6.3 months versus 11.7, 11.2 and 12.1, p = 0.002). Conclusions Most MAs used to allocate therapy in SHIVA01 were shown to improve outcomes in other tumour types (tier IIIA). Worst outcome was observed in patients treated based on another type of alteration than the one reported to improve outcomes (tier IIIB), highlighting the crucial impact of the type of the alterations beyond the gene and the signalling pathway.

CERVOXY CLIN; International audience; IntroductionMET proto-oncogene (MET) exon 14 splice site (METex14) mutations were recently described in NSCLC and has been reported to correlate with efficacy of MET tyrosine kinase inhibitors. High diversity of these alterations makes them hard to detect by DNA sequencing in clinical practice. Because METex14 mutations induce increased stabilization of the MET receptor, it is anticipated that these mutations are associated with MET overexpression. We aim to determine whether NSCLC with high MET overexpression could define a subset of patients with a high rate of METex14 mutations.MethodsFrom The French Cooperative Thoracic Intergroup PREDICT.amm cohort of 843 consecutive patients with a treatment-naive advanced NSCLC who were eligible for a first-line therapy, 108 NSCLC samples with high MET overexpression defined by an immunochemistry score 3+ were tested for METex14 mutations using fragment length analysis combined with optimized targeted next-generation sequencing. MET copy number analysis was also derived from the sequencing data.ResultsMETex14 mutations were detected in two patients (2.2%) who also displayed a TP53 mutation and a PIK3CA mutation, respectively. An MET gene copy number increase was observed in seven additional patients (7.7%). Next-generation sequencing analysis revealed inactivating mutations in TP53 (52.7%) and PTEN (1.1%), and oncogenic mutations in KRAS (28.6%), EGFR (7.7%), PIK3CA (4.4%), BRAF (4.4%), NRAS (2.2%), GNAS (1.1%), and IDH1 (1.1%).ConclusionsThe rate of METex14 mutations in NSCLC with high MET overexpression was similar to that found in unselected NSCLC. Moreover, we observed a high frequency of driver alterations in other oncogenes. Consequently these findings do not support the use of MET immunohistochemistry as a surrogate marker for METex14 mutations.

BackgroundActivating mutations in the estrogen receptor 1 (ESR1) gene are recurrent mechanisms of acquired resistance to aromatase inhibitors (AI), and may be the target of other selective estrogen receptor down-regulators. To assess the clinical utility of monitoring ESR1 resistant mutations, a droplet digital PCR (ddPCR)-based assay compatible with body fluids is ideal due to its cost-effectiveness and quick turnaround.MethodsWe designed a multiplex ddPCR, which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with another pair of probes interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S or D538G mutations. Validation of the assay was performed on plasma samples from a prospective study and compared to next generation sequencing (NGS) data.ResultsThe multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency depending on the mutation tested. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them with perfect concordance (and higher sensitivity) to NGS data obtained in parallel. Additionally, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of ctDNA using this technique during treatment follow-up predicts the radiological response to palbociclib-fulvestrant.ConclusionThe multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations and is compatible with plasma samples. This method is thus suitable for real-time ESR1 mutation monitoring in large cohorts of patients.Statement of translational relevanceExons 5 and 8 mutations in ESR1 are recurrent mechanisms of resistance to aromatase inhibitors (AI) in estrogen receptor (ER)-positive metastatic breast cancer and may be targeted by selective ER down-regulators. We implemented a novel droplet digital PCR, which allows for the detection of the most frequent ESR1 mutations in circulating cell-free DNA. In prospectively collected plasma samples, ESR1 mutations were found in 29% of AI-resistant patients, with excellent concordance and higher sensitivity to next generation sequencing. Moreover, circulating ESR1 mutations appear to be reliable markers for ctDNA monitoring in order to predict treatment response. Ultimately, the short turnaround time, high sensitivity and limited cost of the ESR1-ddPCR are compatible with repeated samplings to detect the onset of resistance to AI before the radiological progression. This opens a window of opportunity to develop new clinical strategies for breast cancer hormone therapy, as tested in an ongoing phase 3 trial.List of abbreviationsAIAromatase InhibitorcfDNACell-free DNActDNACirculating tumor DNAddPCRDroplet digital PCRER+ HER2-MBCER+ HER2-negative Metastatic Breast CancerEREstrogen ReceptorER+Estrogen Receptor positiveLOBLimit of blankLODLimit of detectionMAFMutant Allele FrequencyPBMCPeripheral blood mononuclear cellsPDProgressive diseaseSDStandard deviationToPTime of progressionWTWild typeHuman genesESR1: Estrogen Receptor 1HER2: Human Epidermal Growth Factor Receptor 2EGFR: Epithelial Growth Factor ReceptorKRAS: KRAS proto-oncogene, GTPaseBRAF: B-Raf Proto-Oncogene, Serine/Threonine kinase

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Background ALK gene rearrangement in non-small cell lung cancer (NSCLC), results from fusion with another partner gene, mainly EML4 gene, and is observed in less than 5% of adenocarcinoma tumor. Patients usually manifest response to ALK tyrosine kinase inhibitors (TKI). Unfortunately, acquired resistance ineluctably occurs and has been associated with emergence of secondary mutations (1). Herein, we report the identification of an ALK resistance mutation in a patient with ALK-TKI-naive multi-metastatic-NSCLC.

ESR1 mutation is frequently encountered in hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC), especially after aromatase inhibitor (AI) therapy, as a mechanism of resistance to endocrine therapy. Circulating tumor DNA-based detection of ESR1 mutation in plasma has been demonstrated as a prognostic and predictive factor for poor outcomes in subsequent AI therapy. In this case report, for the first time, we describe the detection of ESR1 mutation (p.Tyr537Ser) only in the cerebrospinal fluid (CSF) and not in the plasma of a patient with isolated leptomeningeal progression who was treated with AI for HR-positive, HER2-negative MBC (bone metastasis only). Circulating tumor DNA levels also appeared to be correlated with clinical evolution. We suggest that in the presence of isolated leptomeningeal metastasis and when tamoxifen or AI has been prescribed for HR-positive MBC, CSF should be screened for ESR1 mutations to potentially adjust systemic treatment.

Background High throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs. Patients and methods We included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA. Results 736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5–168). Conclusions The implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy.

BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

Introduction Les inhibiteurs de points de controle immunologiques, dont le nivolumab, sont un traitement cle pour les CBNPC mais sont reputes peu efficaces chez les non-fumeurs et en cas d’addiction oncogenique (patients mutes pour EGFR ou transloques pour ALK). Les mutations induisant un epissage de l’exon 14 du gene MET, recemment decrites, sont retrouvees chez 3 % des adenocarcinomes (adk) et la reponse a l’immunotherapie dans cette situation n’a pas ete rapportee. Methodes Nous presentons 4 cas de CBNPC mutes pour MET (induisant un epissage de l’exon 14), survenus chez des patientes (A, B, C, D) traitees par nivolumab en deuxieme ligne ( Tableau 1 ). Elles etaient non fumeuses, sans antecedent particulier sauf un cancer du sein traite il y a 20 ans pour B et 15 ans pour D. Cette mutation de MET a ete recherchee au cours de ce traitement, les mutations EGFR, KRAS et BRAF et la translocation ALK etant negatives. Resultats Pour A, le nivolumab est debute apres une progression surrenalienne et permet apres 4 cures une excellente reponse partielle thoracique et surrenalienne. Le traitement est arrete apres la cinquieme cure pour diarrhee grade 3 et acidocetose diabetique attribuees au traitement. Apres un controle tumoral pendant 6 mois sans traitement, une radiotherapie (RT) thoracique est proposee sur la masse residuelle, uniquement thoracique, fixant au TEP scanner. Le nivolumab est repris 5 mois plus tard pour une recidive surrenalienne bilaterale et permet une reponse complete sans aucune toxicite. La patiente B subit d’abord l’exerese d’une metastase cerebrale, puis une RT stereotaxique postoperatoire, recoit une chimiotherapie puis une RT thoracique de cloture. Elle recidive au niveau thoracique (nodules bilateraux et pleuresie) et cerebrale. La patiente recoit 4 cures de nivolumab qui entrainent une reponse thoracique maintenue a 8 cures (en cours actuellement). La patiente C est en reponse complete apres 25 cures de nivolumab en deuxieme ligne. D a progresse apres 2 cures de carboplatine pemetrexed. Elle presente une aggravation clinique et radiologique majeure apres 4 cures de nivolumab. Elle est alors incluse dans l’essai ACSE et presente une excellente reponse sous crizotinib maintenue plus de 6 mois (en cours). Nous n’avons le niveau d’expression de PDL1 au niveau des cellules tumorales que pour A (70 %) et B (20 %). Conclusion Pour 3 des 4 patientes mutees pour MET, le nivolumab a permis une excellente reponse. Il peut donc etre une alternative therapeutique dans cette addiction oncogenique.

Background: Microsatellite instability (MSI) is a hypermutator phenotype occurring in gastrointestinal, endometrial and colorectal tumors, and more rarely, in urinary tract, ovarian, breast, prostate, lung, head and neck, liver and glioblastoma tumors. The diagnosis of MSI phenotype has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune-checkpoint blockade therapy, making its precise identification primordial for treatment decisions and disease monitoring. Current diagnosis of MSI is performed by multiplex PCR of five microsatellite markers, followed by capillary electrophoresis to detect shifts in allele size. An important limitation of this method is its low sensitivity preventing its use when tumor content is below 10%. Methods: We designed ddPCR assays examining 3 microsatellites. Analytical performance was assessed in vitro. Clinical performance was evaluated in a series of FFPE and body fluid DNA samples obtained from patients with Stage IV colorectal or endometrial carcinomas, previously classified as MSI-H or MSS using the standard pentaplex PCR method. Mutant allele frequencies (MAF) quantified with the ddPCR MSI assay were also compared with the MAFs reported by ddPCR assays targeting specific BRAF or PIK3CA mutations. Results: The ddPCR assays for the 3 microsatellites were found to have high specificity and reached a limit of detection of Conclusion: This technique allows a cost-effective, sensitive and large scale screening of the MSI phenotype, as well as the follow-up of MSI patients using liquid biopsies. Citation Format: Marc-Henri Stern, Amanda B. Silveira, Ivan Bieche, Samia Melaabi, Luc Cabel, Bruno Buecher, Jean-Yves Pierga, Francois-Clement Bidard, Charlotte Proudhon. Detecting MSI phenotype in circulating blood DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4599.

Introduction La presence d’une mutation T790 M du gene EGFR est retrouvee dans pres de 50 % des cas de resistance acquise aux ITK-EGFR. Des etudes anterieures realisees sur de petits effectifs suggerent que la mutation T790 M peut etre detectee avant la mise sous traitement. Nous utilisons dans cette etude une technique ultra-sensible et quantitative qui est la PCR digitale pour analyser la frequence et les correlations cliniques de la detection au diagnostic de la mutation T790 M dans une grande cohorte de patients porteurs d’un CBNPC avec mutation de l’EGFR. Methodes Nous avons analyse 256 patients mutes EGFR de la cohorte IFCT Biomarqueurs France pour lesquels de l’ADN etait encore disponible en quantite suffisante et traites par ITK-EGFR. La PCR digitale etait effectuee sur un systeme QX200 (Bio-Rad®, Hercules, Etats-Unis). Tous les echantillons ont ete testes en double. Une cohorte d’ADN de cancers colorectaux a servi de controle negatif. Resultats La PCR digitale a permis la mise en evidence d’une mutation T790 M pre-therapeutique chez 23/256 (9 %) patients. L’abondance fractionnelle (AF) de la mutation T790 M etait > 10 %, > 1 %, 0,1 %, 0,03 %, 10 % (HR 15,43, IC95 % : 5–47, p 24 mois) (n = 3/20, 15 %) (p = 0,02). Dans les cas de progression rapide, l’AF de la mutation T790 M etait de plus de 1 % chez 7/9 (77 %) patients. Conclusion La detection par PCR digitale de la mutation T790 M pre-therapeutique est rapportee chez 9 % des patients CBNPC mutes EGFR et a une valeur de mauvais pronostic, surtout quand l’AF est superieure a 10 %.