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Purpose: To evaluate the efficacy and safety of dabrafenib-trametinib-131I for the treatment of radioactive iodine refractory metastatic differentiated thyroid cancer (DTC) with a BRAF p.V600E mutation. Patients and Methods: A prospective phase II trial including patients with RECIST progression within 18 months and no lesion > 3 cm. Following a baseline recombinant human (rh)TSH-stimulated diagnostic whole-body scan (dc1-WBS), dabrafenib and trametinib were given for 42 days. A second rhTSH-stimulated dc WBS (dc2-WBS) was done at day 28 and 131I (5.5 GBq–150 mCi after rhTSH) was administered at day 35. Primary endpoint was the 6-month RECIST objective response rate. In case of partial response (PR) at 6 or 12 months, a second treatment course could be given. Among 24 enrolled patients, 21 were evaluable at 6 months. Results: Abnormal 131I uptake was present on 5%, 65%, and 95% of the dc1-WBS, dc2-WBS, and post-therapy scans, respectively. At 6 months, PR was achieved in 38%, stable disease in 52%, and progressive disease (PD) in 10%. Ten patients received a second treatment course: one complete response and 6 PRs were observed at 6 months. The median progression-free survival (PFS) was not reached. The 12- and 24-month PFS were 82% and 68%, respectively. One death due to PD occurred at 24 months. Adverse events (AE) occurred in 96% of the patients, with 10 grade 3–4 AEs in 7 patients. Conclusions: Dabrafenib-trametinib is effective in BRAF p.V600E-mutated DTC patients for restoring 131I uptake with PR observed 6 months after 131I administration in 38% of the patients.

Purpose: We compared the mutational profile of second breast cancers (SBC) following first ipislateral hormone receptor–positive breast cancers of patient-matched tumors to distinguish new primaries from true recurrences. Experimental Design: Targeted next-generation sequencing using the Oncomine Tumor Mutation Load Assay. Variants were filtered according to their allele frequency ≥ 5%, read count ≥ 5X, and genomic effect and annotation. Whole genome comparative genomic hybridization array (CGH) was also performed to evaluate clonality. Results: Among the 131 eligible patients, 96 paired first breast cancer (FBC) and SBC were successfully sequenced and analyzed. Unshared variants specific to the FBC and SBC were identified in 71.9% and 61.5%, respectively. Paired samples exhibited similar frequency of gene variants, median number of variants per sample, and variant allele frequency of the reported variants except for GATA3. Among the 30 most frequent gene alterations, ARIDIA, NSD2, and SETD2 had statistically significant discordance rates in paired samples. Seventeen paired samples (17.7%) exhibited common variants and were considered true recurrences; these patients had a trend for less favorable survival outcomes. Among the 8 patients with available tissue for CGH analysis and considered new primaries by comparison of the mutation profiles, 4 patients had clonally related tumors. Conclusions: Patient-matched FBC and SBC analysis revealed that only a minority of patients exhibited common gene variants between the first and second tumor. Further analysis using larger cohorts, preferably using single-cell analyses to account for clonality, might better select patients with true recurrences and thereby better inform the decision-making process.

Cancer immunotherapy combinations have recently been shown to improve the overall survival of advanced mesotheliomas, especially for patients responding to those treatments. We aimed to characterize the biological correlates of malignant pleural mesotheliomas’ primary resistance to immunotherapy and antiangiogenics by testing the combination of pembrolizumab, an anti–PD-1 antibody, and nintedanib, a pan-antiangiogenic tyrosine kinase inhibitor, in the multicenter PEMBIB trial (NCT02856425). Thirty patients with advanced malignant pleural mesothelioma were treated and explored. Unexpectedly, we found that refractory patients were actively recruiting CD3+CD8+ cytotoxic T cells in their tumors through CXCL9 tumor release upon treatment. However, these patients displayed high levels of somatic copy-number alterations in their tumors that correlated with high blood and tumor levels of IL6 and CXCL8. Those proinflammatory cytokines resulted in higher tumor secretion of VEGF and tumor enrichment in regulatory T cells. Advanced mesothelioma should further benefit from stratified combination therapies adapted to their tumor biology. Significance: Sequential explorations of fresh tumor biopsies demonstrated that mesothelioma resistance to anti–PD-1 + antiangiogenics is not due to a lack of tumor T-cell infiltration but rather due to adaptive immunosuppressive pathways by tumors, involving molecules (e.g., IL6, CXCL8, VEGF, and CTLA4) that are amenable to targeted therapies. This article is highlighted in the In This Issue feature, p. 799

Although the role of epigenetic alterations in oncogenesis has been well studied, their prevalence in metastatic solid tumours is still poorly described. We therefore aimed at: (i) describing the presence of epigenetic gene alterations (EGA) - defined by an alteration in a gene encoding an epigenetic regulator; and (ii) evaluating their relationship with clinical characteristics and outcome in patients (pts) included in prospective molecular profiling trials.On-purpose tumour biopsies from pts with metastatic solid tumours enrolled in the Gustave Roussy-sponsored MOSCATO (NCT01566019) and MATCHR (NCT02517892) trials were molecularly profiled using whole exome sequencing (WES). Alterations in 176 epigenetic genes were assessed and classified as pathogenic variants (PV) or non-pathogenic variants by a molecular tumour board. Clinical characteristics and outcome were collected.Between Dec 2011 and Oct 2016, WES was successfully performed in 292 pts presenting various solid tumours. We found 496 epigenetic gene alterations in 134 patients (49%), including 237 pathogenic variants in 86 patients; 63 tumour samples (47%) presented ≥3 EGAs. The median number of previous treatment lines was 3 (1-10). The most frequently altered genes were KMT2D and KMT2C (16% each), ARID1A and SETD2 (10% each) and KMT2A (8%).; 31% of EGA co-occurred with a driver gene alteration (p amp;lt; 0.001). Outcome was not correlated with the presence of EGA.Epigenetic alterations occur frequently in metastatic solid tumours. With the current development of epigenetic modifiers, they increasingly represent actionable targets. Such genes should now be systematically analysed in molecular profiling studies.

Purpose: Targeted therapies (TT) and immune checkpoint blockers (ICB) have revolutionized the approach to non–small cell lung cancer (NSCLC) treatment in the era of precision medicine. Their impact as switch maintenance therapy based on molecular characterization is unknown. Patients and Methods: SAFIR02-Lung/IFCT 1301 was an open-label, randomized, phase II trial, involving 33 centers in France. We investigated eight TT (substudy-1) and one ICB (substudy-2), compared with standard-of-care as a maintenance strategy in patients with advanced EGFR, ALK wild-type (wt) NSCLC without progression after first-line chemotherapy, based on high-throughput genome analysis. The primary outcome was progression-free survival (PFS). Results: Among the 175 patients randomized in substudy-1, 116 received TT (selumetinib, vistusertib, capivasertib, AZD4547, AZD8931, vandetanib, olaparib, savolitinib) and 59 standard-of-care. Median PFS was 2.7 months [95% confidence interval (CI), 1.6–2.9] with TT versus 2.7 months (1.6–4.1) with standard-of-care (HR, 0.97; 95% CI, 0.7–1.36; P = 0.87). There were no significant differences in PFS within any molecular subgroup. In substudy-2, 183 patients were randomized, 121 received durvalumab and 62 standard-of-care. Median PFS was 3.0 months (2.3–4.4) with durvalumab versus 3.0 months (2.0–5.1) with standard-of-care (HR, 0.86; 95% CI, 0.62–1.20; P = 0.38). Preplanned subgroup analysis showed an enhanced benefit with durvalumab in patients with PD-L1 tumor proportion score (TPS) ≥1%, (n = 29; HR, 0.29; 95% CI, 0.11–0.75) as compared with PD-L1 Conclusions: Molecular profiling can feasibly be implemented to guide treatment choice for the maintenance strategy in EGFR/ALK wt NSCLC; in this study it did not lead to substantial treatment benefits beyond durvalumab for PD-L1 ≥ 1 patients.

Abstract MAPPYACTS (NCT02613962) is an international prospective precision medicine trial aiming to define tumor molecular profiles in pediatric patients with recurrent/refractory malignancies in order to suggest the most adapted salvage treatment. From February 2016 to July 2020, 787 patients were included in France, Italy, Ireland, and Spain. At least one genetic alteration leading to a targeted treatment suggestion was identified in 436 patients (69%) with successful sequencing; 10% of these alterations were considered “ready for routine use.” Of 356 patients with follow-up beyond 12 months, 107 (30%) received one or more matched targeted therapies—56% of them within early clinical trials—mainly in the AcSé-ESMART platform trial (NCT02813135). Overall, matched treatment resulted in a 17% objective response rate, and of those patients with ready for routine use alterations, it was 38%. In patients with extracerebral tumors, 76% of actionable alterations detected in tumor tissue were also identified in circulating cell-free DNA (cfDNA). Significance: MAPPYACTS underlines the feasibility of molecular profiling at cancer recurrence in children on a multicenter, international level and demonstrates benefit for patients with selected key drivers. The use of cfDNA deserves validation in prospective studies. Our study highlights the need for innovative therapeutic proof-of-concept trials that address the underlying cancer complexity. This article is highlighted in the In This Issue feature, p. 1171

Background: While studies have shown feasibility and reported preliminary evidence of utility, there is no evidence that multigene sequencing improves outcome in patients with metastatic cancer. The aim of the present study was to assess the clinical utility of multigene sequencing and DNA copy number analyses.. Methods: In SAFIR02-BREAST (NCT: 02299999) and SAFIR-PI3K (NCT: 03386162), open-label multicentric phase II randomized trials, patients were selected if they had a Her2-negative metastatic breast cancer eligible to 1st or 2nd line chemotherapy. Patients underwent a pre-treatment biopsy of metastatic disease when feasible, followed by genomic analysis by next generation sequencing and SNParray. After 6 to 8 cycles of induction chemotherapy, patients without progressive disease and presenting an actionable genomic alteration, were randomized between targeted therapies matched to genomic alterations or maintenance chemotherapy. The primary objective was to evaluate whether targeted therapies guided by genomics improves progression-free survival (PFS) as compared to maintenance chemotherapy, in a pooled analyses of SAFIR02-BREAST and SAFIR-PI3K populations. A hierarchical testing was applied. The efficacy of targeted therapies matched to genomic alterations was first tested in patients presenting an ESCAT I/II alteration (ESMO Scale of Actionability of Molecular Targets). If a p value Citation Format: Fabrice André, Anthony Gonçalves, Thomas Filleron, Florence Dalenc, Amélie Lusque, Mario Campone, Marie-Paule Sablin, Hervé Bonnefoi, Ivan Bieche, Ludovic Lacroix, Alicia Tran-Dien, Marta Jimenez, Alexandra Jacquet, Qing Wang, Etienne Rouleau, David Gentien, Isabelle Soubeyran, Alain Morel, Monica Arnedos, Thomas Bachelot. Clinical utility of molecular tumor profiling: Results from the randomized trial SAFIR02-BREAST [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 GS1-10.

PURPOSE High-risk clonal hematopoiesis (CH) is frequently incidentally found in patients with solid tumors undergoing plasma cell–free DNA sequencing. Here, we aimed to determine if the incidental detection of high-risk CH by liquid biopsy may reveal occult hematologic malignancies in patients with solid tumors. MATERIALS AND METHODS Adult patients with advanced solid cancers enrolled in the Gustave Roussy Cancer Profiling study (ClinicalTrials.gov identifier: NCT04932525 ) underwent at least one liquid biopsy (FoundationOne Liquid CDx). Molecular reports were discussed within the Gustave Roussy Molecular Tumor Board (MTB). Potential CH alterations were observed, and patients referred to hematology consultation in the case of pathogenic mutations in JAK2, MPL, or MYD88, irrespective of the variant allele frequency (VAF), or in DNMT3A, TET2, ASXL1, IDH1, IDH2, SF3B1, or U2AF1 with VAF ≥ 10%, while also considering patient cancer-related prognosis. TP53 mutations were discussed case-by-case. RESULTS Between March and October 2021, 1,416 patients were included. One hundred ten patients (7.7%) carried at least one high-risk CH mutation: DNMT3A (n = 32), JAK2 (n = 28), TET2 (n = 19), ASXL1 (n = 18), SF3B1 (n = 5), IDH1 (n = 4), IDH2 (n = 3), MPL (n = 3), and U2AF1 (n = 2). The MTB advised for hematologic consultation in 45 patients. Overall, 9 patients of 18 actually addressed had confirmed hematologic malignancies that were occult in six patients: two patients had myelodysplastic syndrome, two essential thrombocythemia, one a marginal lymphoma, and one a Waldenström macroglobulinemia. The other three patients were already followed up in hematology. CONCLUSION The incidental findings of high-risk CH through liquid biopsy may trigger diagnostic hematologic tests and reveal an occult hematologic malignancy. Patients should have a multidisciplinary case-by-case evaluation.

PURPOSE To facilitate implementation of precision medicine in clinical management of cancer, the European Society of Medical Oncology proposed in 2018 a new scale to harmonize and standardize the reporting and interpretation of clinically relevant genomics data (ESMO Scale of Actionability of molecular Targets [ESCAT]). This study aims to characterize the clinical impact of matching targetable genomic alterations (GAs) in patients with advanced cancer according to ESCAT. MATERIAL AND METHODS Analysis of next-generation sequencing results from 552 patients is included in two prospective precision medicine studies at Gustave Roussy. End points included objective response rates, progression-free survival, and overall survival according to ESCAT. RESULTS Molecular data from 516 patients were available and discussed within a Molecular Tumor Board. The most common tumor types were GI (n = 164; 30%), lung (n = 137; 25%), and urologic tumors (n = 68; 13%). Overall, 379 GAs were considered as actionable targets according to ESCAT in 348 (67%) patients. In 31 (6%) patients, two concomitant actionable targets were identified. On the basis of ESCAT, GAs were considered to be classified as tier I in 120 patients (29%), II in 25 patients (5%), III in 80 patients (16%), and IV in 153 patients (30%). A total of 136 patients (27%) received a matched therapy. ESCAT was significantly associated with objective response rates and clinical benefit rates. The median progression-free survival was 6.5 months (95% CI, 4.2 to 8.9), 3 months (95% CI, 1 to not available), 3 months (95% CI, 2.2 to 3.8), and 4 months (95% CI, 2.8 to 6.3) for ESCAT I, II, III, and IV, respectively ( P = .0125). CONCLUSION Implementation of ESCAT classification for clinical decision making by Molecular Tumor Board is feasible and useful to better tailor therapies in patients with cancer.

International audience; Aberrant activation of RET is a critical driver of growth and proliferation in diverse solid tumours. Multikinase inhibitors (MKIs) showing anti-RET activities have been tested in RET-altered tumours with variable results. The low target specificity with consequent increase in side-effects and off-target toxicities resulting in dose reduction and drug discontinuation are some of the major issues with MKIs. To overcome these issues, new selective RET inhibitors such as pralsetinib (BLU-667) and selpercatinib (LOXO-292) have been developed in clinical trials, with selpercatinib recently approved by the Food and Drug Administration (FDA). The results of these trials showed marked and durable antitumour activity and manageable toxicity profiles in patients with RET-altered tumours. The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) launched a collaborative project to review the available methods for the detection of RET gene alterations, their potential applications and strategies for the implementation of a rational approach for the detection of RET fusion genes and mutations in human malignancies. We present here recommendations for the routine clinical detection of targetable RET rearrangements and mutations.

Purpose:The limited knowledge on the molecular profile of patients with BRAF-mutant non–small cell lung cancer (NSCLC) who progress under BRAF-targeted therapies (BRAF-TT) has hampered the development of subsequent therapeutic strategies for these patients. Here, we evaluated the clinical utility of circulating tumor DNA (ctDNA)-targeted sequencing to identify canonical BRAF mutations and genomic alterations potentially related to resistance to BRAF-TT, in a large cohort of patients with BRAF-mutant NSCLC.Experimental Design:This was a prospective study of 78 patients with advanced BRAF-mutant NSCLC, enrolled in 27 centers across France. Blood samples (n = 208) were collected from BRAF-TT–naïve patients (n = 47), patients nonprogressive under treatment (n = 115), or patients at disease progression (PD) to BRAF-TT (24/46 on BRAF monotherapy and 22/46 on BRAF/MEK combination therapy). ctDNA sequencing was performed using InVisionFirst-Lung. In silico structural modeling was used to predict the potential functional effect of the alterations found in ctDNA.Results:BRAFV600E ctDNA was detected in 74% of BRAF-TT–naïve patients, where alterations in genes related with the MAPK and PI3K pathways, signal transducers, and protein kinases were identified in 29% of the samples. ctDNA positivity at the first radiographic evaluation under treatment, as well as BRAF-mutant ctDNA positivity at PD were associated with poor survival. Potential drivers of resistance to either BRAF-TT monotherapy or BRAF/MEK combination were identified in 46% of patients and these included activating mutations in effectors of the MAPK and PI3K pathways, as well as alterations in U2AF1, IDH1, and CTNNB1.Conclusions:ctDNA sequencing is clinically relevant for the detection of BRAF-activating mutations and the identification of alterations potentially related to resistance to BRAF-TT in BRAF-mutant NSCLC.

PURPOSE Liquid biopsy specimen genomic profiling is integrated in non–small-cell lung cancer (NSCLC) guidelines; however, data on the clinical relevance for ALK /ROS1 alterations are scarce. We evaluated the clinical utility of a targeted amplicon-based assay in a large prospective cohort of patients with ALK/ROS1-positive NSCLC and its impact on outcomes. PATIENTS AND METHODS Patients with advanced ALK/ROS1-positive NSCLC were prospectively enrolled in the study by researchers at eight French institutions. Plasma samples were analyzed using InVisionFirst-Lung and correlated with clinical outcomes. RESULTS Of the 128 patients included in the study, 101 were positive for ALK and 27 for ROS1 alterations. Blood samples (N = 405) were collected from 29 patients naïve for treatment with tyrosine kinase inhibitors (TKI) or from 375 patients under treatment, including 105 samples collected at disease progression (PD). Sensitivity was 67% (n = 18 of 27) for ALK/ROS1 fusion detection. Higher detection was observed for ALK fusions at TKI failure (n = 33 of 74; 46%) versus in patients with therapeutic response (n = 12 of 109; 11%). ALK-resistance mutations were detected in 22% patients (n = 16 of 74) overall; 43% of the total ALK-resistance mutations identified occurred after next-generation TKI therapy. ALK G1202R was the most common mutation detected (n = 7 of 16). Heterogeneity of resistance was observed. ROS1 G2032R resistance was detected in 30% (n = 3 of 10). The absence of circulating tumor DNA mutations at TKI failure was associated with prolonged median overall survival (105.7 months). Complex ALK-resistance mutations correlated with poor overall survival (median, 26.9 months v NR for single mutation; P = .003) and progression-free survival to subsequent therapy (median 1.7 v 6.3 months; P = .003). CONCLUSION Next-generation, targeted, amplicon-based sequencing for liquid biopsy specimen profiling provides clinically relevant detection of ALK/ROS1 fusions in TKI-naïve patients and allows for the identification of resistance mutations in patients treated with TKIs. Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy.

Patients with cancer are presumed to be at increased risk of severe COVID-19 outcomes due to underlying malignancy and treatment-induced immunosuppression. Of the first 178 patients managed for COVID-19 at the Gustave Roussy Cancer Centre, 125 (70.2%) were hospitalized, 47 (26.4%) developed clinical worsening and 31 (17.4%) died. An age of over 70 years, smoking status, metastatic disease, cytotoxic chemotherapy and an Eastern Cooperative Oncology Group score of ≥2 at the last visit were the strongest determinants of increased risk of death. In multivariable analysis, the Eastern Cooperative Oncology Group score remained the only predictor of death. In contrast, immunotherapy, hormone therapy and targeted therapy did not increase clinical worsening or death risk. Biomarker studies found that C-reactive protein and lactate dehydrogenase levels were significantly associated with an increased risk of clinical worsening, while C-reactive protein and D-dimer levels were associated with an increased risk of death. COVID-19 management impacted the oncological treatment strategy, inducing a median 20 d delay in 41% of patients and adaptation of the therapeutic strategy in 30% of patients. Barlesi and colleagues describe the determinants of severe COVID-19 outcomes in patients with cancer managed at the Gustave Roussy Cancer Centre.

Introduction Liquid biopsy (LB) is increasingly used in non-small cell lung cancer (NSCLC) for molecular diagnosis or resistance detection. Due to its non-invasive nature, it is often preferred over tissue biopsies, especially when sequential biopsies are warranted. Here, we report the clinical utility of liquid biopsy in patients with advanced NSCLC and ALK, ROS1 or RET fusions and its relevance to detect resistance after targeted therapy. Methods Between December 2020 and June 2022, 597 patients with advanced lung cancer had at least one liquid biopsy assessed by Foundation One CDx Liquid (panel of 324 genes) in a single institution. Plasma collection was performed in treatment-naïve patients and/or at time of progression. Clinical and molecular data were collected from patients with known fusions (ALK, ROS1, RET). LB were defined as “positive” if the fusion or resistance mutations were identified and “negative” in the absence of circulating-tumor DNA. The clinical utility of LB was evaluated as the proportion of positive results. The clinical relevance for resistance detection was defined as the proportion of LB that identified a putative resistance mechanism after targeted therapy. Results A total of 68 patients (29 ALK+, 22 RET+ and 17 ROS1+) with 83 LB were included. Patients were females in 50% of cases, had no smoking history in 58% of cases and had adenocarcinoma in 91% of cases. LB was positive in 55/83 (66%) cases overall, in 14/15 (93%) treatment-naïve patients and in 39/66 (59%) pre-treated patients. Factors significantly associated with a negative LB were limited disease progression (brain- or thoracic-only, p Conclusion LB was able to detect resistance mechanisms in one third of NSCLC patients with ALK, RET or ROS1 fusions. However, LB frequently failed to detect circulating-tumor DNA especially in patients with limited disease progression or with ongoing treatment at time of sample collection. Citation Format: Mihaela Aldea, Arianna Marinello, Marco Tagliamento, Filippo Dall'Olio, Damien Vasseur, Arnaud Bayle, Anas Gazzah, Miruna Grecea, Claudio Nicotra, Ludovic Lacroix, Santiago Ponce, Luc Friboulet, Fabrice Barlesi, Fabrice Andre, David Planchard, Etienne Rouleau, Antoine Italiano, Benjamin Besse. Clinical utility of liquid biopsy for molecular characterization and resistance detection in patients with advanced NSCLC and ALK, ROS1 or RET fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1034.

Introduction: High LDH activity is associated with a poor prognosis in many cancer types, especially in melanoma where it is the strongest indicator of poor outcome even with the most potent anticancer immunotherapies or targeted therapies. Although high LDH blood activity is often associated with high tumor burden, this is not always the case and there is no definitive demonstration that blood LDH activity is directly derived from tumors cells. Our objectives were 1) to explore the biological impact of LDHA and LDHB independently of their metabolic function 2) to explore the respective prognostic values of the 5 LDH isoforms as well as the relationship between tumor and blood LDH isoforms repartition in melanoma patients. Material, Patients, Methods: A375 melanoma cells cultured in glucose or galactose (to evaluate glycolytic independent effects) were silenced for LDHA or LDHB expression using si-RNAs. RNAseq analysis of total RNAs and polysomal RNAs was performed to explore the transcriptional and translational impacts of LDHA and LDHB expression. In melanoma patients treated with immunotherapy, the enzymatic activities of each of the 5 LDH isoforms (encoded by LDHA and LDHB) were measured in tumors and blood samples. The prognostic values of the distinct isoforms as well as the correlation between the isoforms distribution in blood and tumors were evaluated. Results: In vitro, independently of their metabolic effect, both LDHA and LDHB had distinct transcriptional and translational impacts on the control of several key oncogenic pathways (adhesion, survival, proliferation, immunity) with a more important effect of LDHA vs LDHB silencing. In vivo, among 64 melanoma patients including 25% with high LDH, high LDH1 (B4) activity was significantly associated with response and overall survival (OS) whereas high LDH4 (A3B1) was inversely associated with response and OS. LDH1 and LDH4 were associated with survival even among patients with normal LDH levels. For patients with paired (tumor and blood) isoforms analysis, there was no correlation between isoforms repartition suggesting that blood LDH activity is not directly related to tumor LDH. Conclusion: In addition to their metabolic activity, LDH enzymes, particularly LDHA, control several key oncogenic pathways at the transcriptional level but also at the translational level for a set of genes involved in cancer biology. LDH1 and LDH4 isoforms blood levels are differentially correlated with response to immunotherapy and survival. They are more robust prognostic biomarkers than total blood LDH level used in our daily practice. Thus, LDH is much more than just a marker of tumor burden in melanoma and the origins, the significance and the roles of blood LDH isoforms need to be revisited. These results highlight the need to develop anticancer drugs targeting LDH, and especially LDHA. Citation Format: Laura Soumoy, Virginie Quidville, Lisa Fredeau, Giuseppina Claps, Caroline Pradon, Ludovic Lacroix, Emilie Routier, Djaouida Belkadi, Séverine Roy, Feras Chehade, Stephan Vagner, Caroline Robert. Roles of lactate dehydrogenase (LDH) in melanoma: an underestimated prognostic biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2177.

EGFRm represent 15% of advanced NSCLC in European patients. LB for molecular profiling offers a non-invasive alternative to tissue. cdPCR is a high-sensitive and low-cost LB to detect molecular alterations. We aimed to describe cdPCR clinical utility for EGFRm detection in advanced NSCLC.Prospective blood sample collection in patients with advanced NSCLC harbouring EGFRm either at diagnosis, under response or at PD between January 16 and September 20 at Gustave Roussy. LB was performed by cdPCR (Stilla): sensitizing (exon19; exon21 [p.L858R]) and exon 20 p.T790M resistance EGFRm. We defined high tumour burden (high-TB) as2 metastatic sites. We analysed EGFRm detection by cdPCR and its correlation with progression-free and overall survival (PFS, OS).A total of 252 blood samples were collected in 140 patients. At baseline (n=25), sensitizing EGFRm were detected in 64% of samples, 88% in patients with high-TB (n=8) and 40% among those with intracranial/intrathoracic isolated lesions (n=5). At PD to tyrosine-kinase inhibitors (TKI) (n=117), detection rate (sensitizing EGFRm) was 56%; 30% in patients with intracranial/thoracic isolated lesions (n=37) vs. 67% in those with high-TB (n=63). At PD to first/second generation TKI (n=81), the p.T790M mutation was found in 22% (18/81); detection rate was 9% for intracranial/thoracic (n=23) vs. 32% for high-TB (n=41) cases. The clearance of EGFRm allelic frequency was correlated with radiological response. The absence of EGFRm detection at TKI-failure was associated with longer OS (39.1 vs. 18.4 months; P=.02).cdPCR is a sensitive LB for sensitizing and resistance EGFRm detection. cdPCR positivity was more likely observed in systemic PD cases with high-TB. It is a low-cost EGFRm detecting approach to guide treatment in NSCLC, however metastatic profile should be taken into account.

Background We aimed to determine the safety and efficacy of nintedanib, an oral anti-angiogenic tyrosine kinase inhibitor, in combination with pembrolizumab, an anti-PD1 immunotherapy, in patients with advanced solid tumors (PEMBIB trial; NCT02856425). Methods In this monocentric phase Ib dose escalation cohort, we evaluated escalating doses of nintedanib (Dose level 1 (DL1) = 150 mg bid [bis in die, as twice a day]; DL2 = 200 mg bid, oral delivery) in combination with pembrolizumab (200 mg Q3W, IV). Patients received a 1-week lead-in dose of nintedanib monotherapy prior starting pembrolizumab. The primary objective was to establish the maximum tolerated dose (MTD) of the combination based on dose limiting toxicity (DLT) occurrence during the first 4 weeks. Secondary objectives were to assess the anti-tumor efficacy and to identify the associated immune and angiogenic parameters in order to establish the recommended nintedanib dose for expansion cohorts. Flow cytometry (FC), Immuno-Histo-Chemistry (IHC) and electrochemiluminescence multi-arrays were prospectively performed on baseline & on-treatment tumor and blood samples to identify immune correlates of efficacy. Results A total of 12/13 patients enrolled were evaluable for DLT (1 patient withdrew consent prior receiving pembrolizumab). Three patients at 200 mg bid experienced a DLT (grade 3 liver enzymes increase). Four patients developed grade 1–2 immune related adverse events (irAE). Eight patients died because of cancer progression. Median follow-up was 23.7 months (95%CI: 5.55–40.5). Three patients developed a partial response (PR) (ORR = 25%) and five patients (42%) had durable clinical benefit (DCB), defined as PR or stable disease (SD) ≥ 6 months. At baseline, patients with DCB had higher plasma levels of Tie2, CXCL10, CCL22 and circulating CD4+ PD1+ OX40+ T cells than patients without DCB. Patients with DCB presented also with more DC-LAMP+ dendritic cells, CD3+ T cells and FOXP3+ Tregs in baseline tumor biopsies. For DCB patients, the nintedanib lead-in monotherapy resulted in higher blood CCL3, Tregs and CCR4+ CXCR3+ CXCR5− memory CD4 T cells. After the first pembrolizumab infusion, patients with DCB showed lower IL-6, IL-8, IL-27 plasma levels. Conclusion Nintedanib 150 mg bid is the recommended dose for combination with pembrolizumab and is currently investigated in multiple expansion cohorts. Early tumoral and circulating immune factors were associated with cancer outcome under nintedanib & pembrolizumab therapy. Trial registration ClinicalTrials.gov, NCT02856425. Registered August 4, 2016 — Prospectively registered.

BackgroundReinvigoration of anti-tumor immunity via immune checkpoint blockade (ICB) has transformed outcomes in a-NSCLC. However, a majority of patients are innately resistant to ICB, and a better understanding of the resistance mechanisms may guide the development of new treatment strategies and therapies for patients.MethodsBiopsies performed immediately before treatment with single agent ICB in patients with a-NSCLC (MATCH-R trial [NCT02517892]) were analyzed. The stromal microenvironment and immune context were characterized via an integrated analysis of whole transcriptome (RNA-seq), whole exome sequencing (WES), and immunohistochemistry (IHC) of CD3, CD8, FOXP3 and PDL1. Specifically, the immune context and the relative abundance of 10 immune and stromal cell types were assessed with integrated IHC and Cell Populations-counter (MCP-counter) [1] analysis of the RNA-seq. Somatic mutations and Tumor Mutation Burden (TMB) were evaluated. The transcriptional state of the tumor and its microenvironment were assessed by GSVA analysis [2] of the MSigDB collection [3]. Patient‘s outcome was associated to molecular data. Primary resistance to ICB was defined as PD (progressive disease) in the first radiological examination, or a median PFS inferior to 3 months.ResultsFifty-two patients with NSCLC were enrolled (43 adeno, 6 squamous, and 3 other carcinoma): Median age was 61 (34–93), 18 were female, 46 were smokers, 22 were responders, and 30 were non-responders. Median tumor cellularity was 60% (30%–90%).Patients may be divided into two groups (HIGH and LOW) at baseline based on their degree of immune infiltration as assessed by RNAseq or IHC. A hallmark of the HIGH infiltration group is an increase in Interferon Gamma (IFN-γ) pathway signature [4]. In contrast, patients in the LOW infiltration group (relative to the HIGH infiltration group) exhibit a decrease in IFN-γ pathway signaling and concomitantly an increase in hypoxia and gluconeogenic pathway signatures. Response rates to ICB were not associated to immune infiltration groups at baseline, but an analysis within each infiltration group revealed that high TMB is only associated to response in the HIGH infiltration group. Furthermore, only in the LOW infiltration group was increased the transforming growth factor (TGF-β) pathway signature associated to ICB response.ConclusionsThis study suggests that the tumor and its microenvironment influence baseline immune infiltration. Tumors with LOW baseline infiltration show altered metabolism such as gluconeogenic activation and hypoxia activation. In contrast, factors such as TMB are not associated with baseline infiltration

α-Selective phosphatidylinositol 3-kinase (PI3K) inhibitors improve outcome in patients with PIK3CA-mutated, hormone receptor-positive (HR+)/Her2- metastatic breast cancer (mBC). Nevertheless, it is still unclear how to integrate this new drug family in the treatment landscape.A total of 649 patients with mBC from the SAFIR02 trial (NCT02299999), with available mutational profiles were selected for outcome analysis. PIK3CA mutations were prospectively determined by next-generation sequencing on metastatic samples. The mutational landscape of PIK3CA-mutated mBC was assessed by whole-exome sequencing (n = 617). Finally, the prognostic value of PIK3CA mutations during chemotherapy was assessed in plasma samples (n = 44) by next-generation sequencing and digital PCR.Some 28% (104/364) of HR+/Her2- tumors and 10% (27/255) of triple-negative breast cancer (TNBC) presented a PIK3CA mutation (P0.001). PIK3CA-mutated HR+/Her2- mBC was less sensitive to chemotherapy [adjusted odds ratio: 0.40; 95% confidence interval (0.22-0.71); P = 0.002], and presented a worse overall survival (OS) compared with PIK3CA wild-type [adjusted hazard ratio: 1.44; 95% confidence interval (1.02-2.03); P = 0.04]. PIK3CA-mutated HR+/Her2- mBC was enriched in MAP3K1 mutations (15% versus 5%, P = 0.0005). In metastatic TNBC (mTNBC), the median OS in patients with PIK3CA mutation was 24 versus 14 months for PIK3CA wild-type (P = 0.03). We further looked at the distribution of PIK3CA mutation in mTNBC according to HR expression on the primary tumor. Some 6% (9/138) of patients without HR expression on the primary and 36% (14/39) of patients with HR+ on the primary presented PIK3CA mutation (P0.001). The level of residual PIK3CA mutations in plasma after one to three cycles of chemotherapy was associated with a poor OS [continuous variable, hazard ratio: 1.03, 95% confidence interval (1.01-1.05), P = 0.007].PIK3CA-mutated HR+/Her2- mBC patients present a poor outcome and resistance to chemotherapy. Patients with PIK3CA-mutated TNBC present a better OS. This could be explained by an enrichment of PIK3CA mutations in luminal BC which lost HR expression in the metastatic setting.SAFIR02 trial: NCT02299999.

Breast cancer remains the first cause of death in women worldwide even if the average 10-year survival rate is 83%. It remains difficult to accurately evaluate the risk of recurrence, and consequently the need of chemotherapy, in estrogen receptor (ER)-positive, HER2-negative early breast cancers, at the individual level, leading to overtreatment for some patients. In that context, multigene prognostic scores are proposed in combination with clinicopathological parameters, to guide adjuvant treatment decisions. In our study, patients diagnosed with ER-positive, Her2-negative breast cancer, analyzed with the 12-gene EndoPredictTM test are prospectively included in the national SiMosein registry. The decision for Endopredict testing is determined by an oncological tumor board in expert breast cancer centers. The test is locally performed (10 national laboratories, evaluated by a national quality control exchange program). Clinicopathological parameters are recorded and the treatment decision before and after the genomic test is requested. The aim of the study is to prospectively evaluate the real-life use of the EndopredictTM test and the impact of the recurrence risk on the adjuvant treatment decision. A total of 3153 patients were included between May 2016 and December 2018. Patients are 75.6% post-menopausal (median age: 57.55y) and are mostly diagnosed as invasive ductal carcinoma not otherwise specified (81.2%) with SBR grade 2 (74.02%) and positive Ki67 staining (>14%, 66.4%). Tumor sizes are pT1ab, pT1c or pT2 in respectively 12.1%, 54.7% and 31.4% of the cases. Lymph node involvement is negative (pN0) in 70.5% of the cases. However, 29.5% of cases are pN1-3 with 5.2% of pN1mi. All cases are ER-positive, HER2-negative early breast cancers and 74.8% are progesterone receptor (PR)-positive (PR>10%) and 76.43% of the cases are of luminal B-like subtype. The population evaluated in this prospective study is in accordance to the intermediate prognostic subpopulation of luminal breast cancers where genomic signatures could contribute to guide the adjuvant treatment decision. The risk of relapse at 10 years, as reflected by the EPclin score, is of 13.16% on average, with 55.18% of high risk (positive threshold 10%). Treatment decisions before and after the test were recorded for 599 patients. The decision remains unmodified after the test in 54.59% of the cases. Conversely, change in adjuvant treatment decision was made from hormone therapy (HT) to adjunction of chemotherapy (HT+CT) in 18.20% of the cases and from (HT+CT) to (HT) leading to chemotherapy withdrawn in 27.21%. Finally, the national SiMosein registry, evaluated the utility of the test. The use of EndopredictTM is mostly restricted to the intermediate prognostic subgroup of the early luminal breast subtype as requested by the French health authority (HAS). The test is routinely used in France and clinicians need mainly the test for treatment decision. The EPclin score leads to 45.41% of modification of treatment with a majority of avoidance of chemotherapy (59.93%). Citation Format: Jacqueline Lehmann-Che, Jennifer Wong, Lucie Tixier, Ludovic Lacroix, Magali Triki-Lacroix, Pierre-Jean Lamy, Agnes Garnier, Véronique Quillien, Florence Godey, Isabelle Soubeyran, Gaëtan MacGrogan, Laurent Arnould, Juliette Haudebourg, Anne Tallet, Linda Dainese, Caroline Hugonin, Frédérique Penault-Llorca. Simosein registry, the French national real-life prospective evaluation of the impact of Endopredict® (EPclin) on treatment decision in ER-positive, HER2-negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-25.

Purpose: Patients with anaplastic lymphoma kinase (ALK)–rearranged non–small-cell lung cancer (NSCLC) inevitably develop resistance to ALK inhibitors. New diagnostic strategies are needed to assess resistance mechanisms and provide patients with the most effective therapy. We asked whether single circulating tumor cell (CTC) sequencing can inform on resistance mutations to ALK inhibitors and underlying tumor heterogeneity in ALK-rearranged NSCLC. Experimental Design: Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (n = 14) or lorlatinib (n = 3). Three strategies including filter laser-capture microdissection, fluorescence activated cell sorting, and the DEPArray were used. One hundred twenty-six CTC pools and 56 single CTCs were isolated and sequenced. Hotspot regions over 48 cancer-related genes and 14 ALK mutations were examined to identify ALK-independent and ALK-dependent resistance mechanisms. Results: Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (EGFR, KRAS, BRAF genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored ALK compound mutations. CTC-1 harbored the ALKG1202R/F1174C compound mutation virtually similar to ALKG1202R/F1174L present in the corresponding tumor biopsy. CTC-10 harbored a second ALKG1202R/T1151M compound mutation not detected in the tumor biopsy. By copy-number analysis, CTC-1 and the tumor biopsy had similar profiles, whereas CTC-10 harbored multiple copy-number alterations and whole-genome duplication. Conclusions: Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in ALK-rearranged patients. Single CTC sequencing may be a unique tool to assess heterogeneous resistance mechanisms and help clinicians for treatment personalization and resistance options to ALK-targeted therapies.

Hormone receptor-positive (HR+) and human epidermal growth factor receptor 2 negative (HER2−) early breast cancer (BC) is the most prevalent BC subtype with substantial biological heterogeneity. Although clinicopathological (CP) characteristics have a clear prognostic value, additional biomarkers could refine survival prediction and guide treatment decision. Copy number aberrations and somatic driver mutations were obtained with OncoScan CGH array and sequencing of 36 genes on HR+/HER2− node-positive early BC patients treated with chemotherapy from the PACS04 trial. We built a two-gene genomic score (GS) associated with distant disease-free survival (DDFS), whose prognostic value was assessed on the external METABRIC data (n = 1413) using overall survival (OS) and breast cancer-specific survival (BCSS). In the PACS04 trial (n = 327), the median follow-up for DDFS (65 events) was 9.6 years. FGFR1 amplifications ($${\text{HR}}_{\text{Amplification}}$$ = 2.44, 95% CI [1.25; 4.76], p = 0.009) and MAP3K1 mutations ($${\text{HR}}_{\text{Mutation}}$$ = 0.10, [0.01; 0.78], p = 0.03) were associated with DDFS beyond CP characteristics. A prognostic GS combining FGFR1 amplifications and MAP3K1 mutations added more information to CP model ($$\chi_{\text{DDFS}}^{2}$$ = 12.97, $$p_{\text{DDFS}}$$

Introduction: Leptomeningeal metastases (LMs) are associated with dismal prognosis in NSCLC. Optimal management remains unknown in patients with EGFR-mutated NSCLC after initial tyrosine kinase inhibitor (TKI) failure.Methods: We conducted a multicenter retrospective study including patients with EGFR-mutated NSCLC and LM. TKI failure was defined as diagnosis of LM on TKI, or progression of known LM on TKI.Results: Ninety-two patients were included, median age of 60 years, predominantly female (68%), never-smokers (74%). EGFR mutations included L858R (45%), exon 19 deletions (28%), or other mutations (14%). Median time to LM diagnosis was 18.5 months after initial diagnosis of advanced NSCLC. LM was diagnosed after a median of 2 (range: 0-9) systemic therapies. Median overall survival from LM diagnosis was 6.1 months (95% confidence interval [ CI]: 4.2-7.6 months). Among 87 patients with TKI failure, patients rechallenged with TKI (n = 50) had a median LM overall survival of 7.6 months (95% CI: 5.7-10.9) compared to 4.2 months (95% CI: 1.6-6.7) in patients without further therapy. Overall, 60% of patients rechallenged with TKI experienced clinical benefit (clinical response or stable disease >2 months), and 23% were treatment failure-free at 6 months. Clinical benefit was reported in 11 of 20 (55%) patients treated with erlotinib after afatinib or gefitinib. Strategies based on increasing dose intensity (n = 17) yielded clinical benefit in 59% of patients. All four patients who received osimertinib after first-and second-generation TKI experienced clinical benefit.Conclusions: TKI rechallenge strategies, including dosing intensification, may improve clinical outcomes of patients with LM from EGFR-mutated NSCLC after initial TKI failure. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

A phase I open-label multicentre study was initiated to evaluate the association of tremelimumab with gefitinib in EGFR-mutant NSCLC patients who progressed after first-generation EGFR-TKI. Here we provide the efficacy data from the entire cohort.Patients with advanced EGFR-mutant NSCLC with progression after response to EGFR-TKI were enrolled. Study treatment was gefitinib 250 mg daily and tremelimumab at 3 dose levels: 3, 6 and 10 mg/kg IV Q4W for 6 cycles followed by Q12W until progression or unacceptable toxicity. The primary objective was safety and tolerability, and to establish a RP2D.Between January 2014 and July 2015, 27 patients (21 in the escalating dose cohort and 6 in expansion cohort) received at least one dose of tremelimumab. DLTs occurred in 4 patients: 1 at 3 mg/kg (one grade 3 diarrhoea), 1 at 6 mg/kg (one grade 3 diarrhoea) and 2 at 10 mg/kg (one grade 3 diarrhoea and one grade 3 AST/ALT increase) of tremelimumab. Grade 3 TRAE occurred in 22 patients (81%), most frequently diarrhoea (30%) and ALT/AST increase (15%). Stable disease was the best overall response in 72% patients, with median PFS of 2.2 months (95% CI, 1.8-4.2). All patients discontinued treatment, most frequently due to disease progression (63% of patients).The recommended dose of tremelimumab in combination with gefitinib in EGFR-mutant NSCLC patients was 3 mg/kg. The gastrointestinal toxicity and the limited efficacy data prevented further evaluation of this combination. (GEFTREM; clinical trial number NCT02040064).

International audience; Aim: Arms E and F of the AcSé-ESMART phase I/II platform trial aimed to define the recommended dose and preliminary activity of the dual mTORC1/2 inhibitor vistusertib as monotherapy and with topotecan-temozolomide in a molecularly enriched population of paediatric patients with relapsed/refractory malignancies. In addition, we evaluated genetic phosphatidylinositol 3-kinase (PI3K)/AKT/ mammalian (or mechanistic) target of rapamycin (mTOR) pathway alterations across the Molecular Profiling for Paediatric and Young Adult Cancer Treatment Stratification (MAPPYACTS) trial (NCT02613962). Experimental design and results: Four patients were treated in arm E and 10 in arm F with a median age of 14.3 years. Main diagnoses were glioma and sarcoma. Dose escalation was performed as per the continuous reassessment method, expansion in an Ensign design. The vistusertib single agent administered at 75 mg/m2 twice a day (BID) on 2 days/week and vistusertib 30 mg/m2 BID on 3 days/week combined with temozolomide 100 mg/m2/day and topotecan 0.50 mg/m2/day on the first 5 days of each 4-week cycle were safe. Treatment was well tolerated with the main toxicity being haematological. Pharmacokinetics indicates equivalent exposure in children compared with adults. Neither tumour response nor prolonged stabilisation was observed, including in the 12 patients whose tumours exhibited PI3K/AKT/mTOR pathway alterations. Advanced profiling across relapsed/refractory paediatric cancers of the MAPPYACTS cohort shows genetic alterations associated with this pathway in 28.0% of patients, with 10.5% carrying mutations in the core pathway genes. Conclusions: Vistusertib was well tolerated in paediatric patients. Study arms were terminated because of the absence of tumour responses and insufficient target engagement of vistusertib observed in adult trials. Targeting the PI3K/AKT/mTOR pathway remains a therapeutic avenue to be explored in paediatric patients. Clinical trial identifier: NCT2813135.

Simple Summary In this study, we measured the plasmatic concentration of Kinase inhibitors (KI) among a population with non-small cell lung cancer (NSCLC) harboring driver genetic alterations. They received erlotinib, gefitinib, osimertinib, crizotinib, or dabrafenib (with or without trametinib) for at least three months. The results were measured by ultra-performance liquid chromatography coupled with tandem mass spectrometry and compared to previously published data. Between November 2013 and February 2019, fifty-one samples were analyzed. The main outcome was the rate of samples with suboptimal KI plasma concentrations. Suboptimal plasma concentrations were observed in 51% (26/51) of cases and might contribute to treatment failure. Abstract Kinase inhibitors (KI) have dramatically improved the outcome of treatment in patients with non-small cell lung cancer (NSCLC), which harbors an oncogene addiction. This study assesses KI plasma levels and their clinical relevance in patients chronically exposed to KIs. Plasma samples were collected in NSCLC patients receiving erlotinib, gefitinib, osimertinib, crizotinib, or dabrafenib (with or without trametinib) for at least three months between November 2013 and February 2019 in a single institution. KI drug concentrations were measured by ultra-performance liquid chromatography coupled with tandem mass spectrometry and compared to published data defining optimal plasma concentration. The main outcome was the rate of samples with suboptimal KI plasma concentrations. Secondary outcomes included its impact on T790M mutation emergence in patients receiving a first-generation epidermal growth factor receptor (EGFR) KI. Fifty-one samples were available from 41 patients with advanced NSCLC harboring driver genetic alterations, including EGFR, v-Raf murine sarcoma viral oncogene homolog B (BRAF), anaplastic lymphoma kinase (ALK) or ROS proto-oncogene 1 (ROS1), and who had an available evaluation of chronic KI plasma exposure. Suboptimal plasma concentrations were observed in 51% (26/51) of cases. In EGFR-mutant cases failing first-generation KIs, EGFR exon 20 p.T790M mutation emergence was detected in 31% (4/13) of samples in optimal vs. none in suboptimal concentration (0/5). Suboptimal plasma concentrations of KIs are frequent in advanced NSCLC patients treated with a KI for at least three months and might contribute to treatment failure.

9535 Background: Molecular characterization of metastatic lung adenocarcinomas is mandatory but might be hampered by the quantity of tissue, restricted access to molecular platforms or limited economical resources. Our aim was to develop a tool supported by the hypothesis that radiological patterns of pts could help predict the rate of positivity of the most common oncogenic drivers. Methods: We defined an algorithm based on a molecularly defined cohort of 656 pts with stage IV lung adenocarcinoma. Two radiologists centrally reviewed the baseline imaging. Clinical data were retrospectively collected. There were 135 EGFR mutations, 81 ALK fusions, 47 BRAF mutations, 141 KRAS mutations, and 146 pan-negative tumors for these 4 oncogenic drivers. Univariate correlation analyses were performed to define an algorithm predicting the molecular testing positivity based on the metastatic pattern. Subsequently, an online tool was developed. This study was approved by our institutional review board. Results: Metastatic patterns correlated with the genomic drivers when compared to the pan-negative group. In the EGFR group, pleural metastases were more frequent (32% vs. 20%; p = 0.021), whereas adrenal and node metastases less frequent (6% vs.23%; p < 0.001 and 11% vs. 23% respectively; p = 0.011). In the ALK group, there were more brain and lung metastases (respectively 42% vs. 29%; p = 0.043 and 37% vs. 24% respectively; p = 0.037). In the BRAF group, pleural and pericardial metastases were more common (47% vs. 20%; p < 0.001 and 11% vs. 3% respectively; p = 0.04) and bone metastases less common (21% vs. 42%; p = 0.011). Lymphangitis was more frequent in EGFR, ALK and BRAF groups (6%, 7% and 15% vs. 1%; p = 0,016, p = 0,009 and p < 0,001 respectively). A free online access to the algorithm is now available after registration at http//tactic-ct.fr. Physicians enter age, sex, smoking status and the sites of metastases at diagnosis (present/absent/unknown). A mutation score is calculated, reflecting the % of chance to find an oncogenic driver. On the website, contributors can also enter new cases and an artificial intelligence will refine the algorithm and expand the number of oncogenic drivers. Conclusions: Our free access tool allows establishing a hierarchy in the molecular testing based on simple clinical and radiological information. Continual learning from new cases entered in the database will increase the sensitivity of the tool. This tool might save time, tumor tissue, economical resources and accelerate access to personalized treatment.

Introduction Molecular profiling is considered a standard of care in advanced NSCLC. A comprehensive next-generation sequencing panel can discover somatic or germline BRCA1/2 mutations that are new druggable molecular alterations. However, the phenotypic and potential therapeutic relevance of BRCA1/2 mutation in NSCLC remains poorly defined. Methods From April 2014 to March 2017, 600 newly diagnosed, EGFR/ALK negative patients with advanced NSCLC were enrolled in the SAFIR02-Lung trial. Molecular profiling was done at study entry on archival tissue or frozen tissue collected from a new biopsy specimen before the third cycle of platinum-based chemotherapy. The prevalence of BRCA1/2 variants and its biological relevance were assessed. A homologous recombinant deficiency (HRD) score was based on the copy number variation data, and the germline status was determined by blood analysis. The BRCA Share database and the French CGG consortium were the references for the variant classification. Results Of 379 patients with a molecular profile discussed in a tumor molecular board, BRCA1/2 variants were identified in 20 patients (5.3%), including eight patients (2.1%) with a confirmed pathogenic BRCA mutation. Two patients (0.5%) harbored a germline BRCA2 mutation, and for six others, a somatic BRCA mutation was identified (1.6%). All were men and mainly smokers (88%). The overall response rate to chemotherapy was 13%. BRCA variants of unknown significance were detected in 12 patients (3.2%), achieving an 8.3% overall response rate with chemotherapy. One-third of tumors carrying pathogenic BRCA mutations or variants of unknown significance had biallelic inactivation and high HRD score. Overall survival of this cohort was 12.8 months. Conclusions Pathogenic BRCA1/2 mutations occur in 2.1% of patients with advanced NSCLC. The predictive role of BRCA mutation for making treatment decisions in NSCLC seems limited based on clinical response (low platinum sensitivity) and molecular features (discrepancy between biallelic inactivation and high HRD score).

Background: Circulating tumor DNA (ctDNA) sequencing is a promising approach for testing gene alterations and tailoring therapy in cancer patients given, its limited invasiveness, high sensitivity and potential to comprehensively represent tumor heterogeneity. Here, we report the results from a single-center study conducted at Gustave Roussy (Villejuif, France) where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors. Methods: Genomic analysis was performed using the Foundation One Liquid CDx Assay (324 genes, tumor mutational burden [TMB], microsatellite instability status). Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB) dedicated to precision medicine, attended by experts in clinical oncology, molecular biology, and clinical genetics. Actionable targets were defined by the MTB according to the existing level of evidence (classified by ESCAT tier), and molecular-based treatment suggestions were proposed where possible. Results: Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 theragnostic alterations including : high blood TMB (> 16 mutations/Mb) (N= 243, 13%), alteration of the DNA damage repair response pathway (N=336, 18%), PIK3CA mutations (N=150, 8%), FGFR alterations (N= 67, 4%), MET activations (N=13, 0.7%), ERBB family pathway alterations (N=127, 7%) and PTEN mutations (N=95, 5%). Overall, the MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (N= 639, 78%), off label/compassionate use (N=81, 10%), drug with a market authorization (N=51, 6%) and drug within an early access program (N=48, 6%). MTB did not recommend treatment for 462 patients (44%) with targetable molecular alterations for the following reasons: no clinical trial (N=421, 65%), matched treatment was already received (N=169, 26%), worsening of performance status (N= 49, 8%). Conclusions: This large-scale study demonstrates that liquid biopsy with a large NGS ctDNA panel is an efficient approach to match patients to genomically directed clinical trials/targeted therapies. Outcomes of patients treated with matched therapy will be presented at the meeting. Citation Format: Arnaud Bayle, Laila Belcaid, Miha Aldea, Florent Peyraud, Patricia Martin Romano, Félix Blanc-Durand, Rebecca Clodion, Santiago Ponce, Claudio Nicotra, Antoine Hollebecque, Yohann Loriot, Benjamin Besse, Damien Vasseur, Ludovic Lacroix, Etienne Rouleau, Jean-Charles Soria, Fabrice Barlesi, Geoffrey R. Oxnard, Fabrice Andre, Antoine Italiano. Clinical utility of circulating tumor DNA sequencing with a large panel: The experience of Gustave Roussy/National Center for Precision Medicine (PRISM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3413.

Background: Genomic profiling with tissue sequencing is still considered as the gold standard despite several limitations including screening failures due to limited tissue availability, and inability to capture intratumor spatial and temporal heterogeneity, which may impair accurate treatment selection. Several studies have demonstrated the potential of circulating tumor DNA (ctDNA) to detect genomic alterations at high accuracy compared with tissue analysis. However, no studies have comprehensively evaluated differences between tissue and ctDNA by using a large panel in the same cohort. Methods: Genomic analysis was performed for each patient by using the Foundation One Liquid CDx Assay and the Foundation One CDx Assay (324 genes, tumor mutational burden [TMB], microsatellite instability). Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB). Actionable targets were defined by the MTB according to the existing level of evidence (ESCAT tiers) and molecular-based treatment suggestions were proposed where possible. Results: Between Dec 2020 and Nov 2021, 1021 patients (median age: 62 years) with advanced cancer underwent both tissue and ctDNA NGS. Five most frequent tumor types were colorectal (N=137,13%), NSCLC (N=130,13%), breast (N=120, 12%), prostate (N=82, 8%) and pancreas (N=65, 6%). Median time elapsed between request and assay results was 12 days for ctDNA and 46 days for tissue. Testing failure was 15% for tissue and 3.9% for ctDNA. Overall, 824 (81%) patients had evaluable results for both tissue and liquid. Total number of cancer-related alterations and variants of unknown significance were 4704 and 11673 vs 4645 and 7481 for ctDNA and tissue, respectively. Proportion of patients with a higher number of cancer alterations identified in ctDNA compared with tissue increased in parallel with the time elapsed between the tissue and ctDNA sampling (45% vs 33% for a delay > 26 months or < 8 months). MSI and TMB status were concordant for 71% and 64% of patients, respectively. MSI status was evaluable for 97% of patients through ctDNA vs 90% through tissue. Number of actionable alterations was similar in 346 (42%) of cases, whereas it was higher in tissue for 289 (35%) and in liquid for 189 (23%) patients. ctDNA profiling allowed the identification of an ESCAT I/II or III or IV alteration not present in tissue for 74 (9%), 113 (14%) and 52 (6%) patients, respectively. Overall, MTB recommended a matched therapy for 430 patients (52%). Such a recommendation would not have been made without the results of ctDNA for 120 patients (15%). Conclusion: This systematic comparison of ctDNA vs tissue sequencing demonstrates the capacity of ctDNA for capturing clinically relevant alterations to guide therapy in cancer patients with high accuracy and rapid turnover results. Citation Format: Arnaud Bayle, Florent Peyraud, Laila Belcaid, Maxime Brunet, Miha Aldea, Rebecca Clodion, Paul Dubos, Damien Vasseur, Claudio Nicottra, Santiago Ponce, Isabelle Soubeyran, Emmanuel Khalifa, Yohann Loriot, Benjamin Besse, Ludovic Lacroix, Etienne Rouleau, Geoffrey Oxnard, Fabrice Barlesi, Fabrice Andre, Antoine Italiano. Systematic comparison of ctDNA vs tissue sequencing with a large panel to guide therapy in patients with advanced cancer: A study from the French National Center for Precision Medicine (PRISM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3414.

Liquid biopsies are a minimally invasive sampling approach to overcome the heterogeneity of tumors and represent a valuable source of circulating tumor DNA (ctDNA) for oncological biomarker analysis. ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. Digital PCR emerged as a powerful technology for the next-generation analysis of liquid biopsies. Stilla Technologies' 6-color naica® system is an ultrasensitive, easy-to-use digital PCR platform capable of simultaneously and precisely quantifying high numbers of biomarkers in a single reaction. Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated in NSCLC. A handful of anti-EGFR Tyrosine Kinase Inhibitor therapies are approved by the FDA; however, most patients develop resistance over time to these treatments. Both monitoring primary EGFR mutations to predict treatment response and precociously detecting resistance mutations to adapt treatments promptly through ctDNA analysis stand to improve the effectiveness of NSCLC patient management. Stilla has developed a highly multiplexed 6-color Crystal Digital PCR EGFR kit detecting more than 90% of EGFR mutations described in NSCLC from ctDNA. The assay detects 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, including both activating and resistant mutations. In this work, we evaluated the sensitivity, precision and specificity of the EGFR 6-color Crystal Digital PCR assay highly specific and sensitive for the detection of EGFR mutations, with a Limit of Detection in a high background of wild-type DNA ranging from 0.30 to 0.46 cp/μL (with observed MAFs ranging from 0.06 to 0.09%). Moreover, a high concordance was observed between those EGFR results obtained for NSCLC ctDNA samples using 6-color Crystal Digital PCR and other technologies. With a rapid time to results and straightforward ctDNA analysis workflow, Crystal Digital PCR on the naica system promises ultrasensitive, highly multiplexed 6-color mutation detection, maximizing the information obtained from precious samples. Citation Format: Cécile Jovelet, Myrtille Remy, Noémie Pata-Merci, Damien Vasseur, Ludovic Lacroix, Claudia Labrador-Rached, Allison C. Mallory. Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 75.

3016 Background: Despite the effectiveness of the various targeted therapies currently approved in solid tumors, acquired resistance remains a persistent problem that limits the ultimate effectiveness of these treatments. Polyclonal resistance to targeted therapy has been described in multiple solid tumors through high throughput analysis of multiple tumor tissue samples from a single patient. However, biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We used here sequencing of circulating tumor DNA (ctDNA) to characterize the landscape of secondary resistance mechanisms in a large cohort of patients with solid tumors. Methods: This study enrolled patients with advanced cancer from two institutional molecular profiling program STING (NCT04932525, sponsor: Gustave Roussy) or BIP (NCT02534649sponsor: Institut Bergonié). Genomic analysis was performed for each patient by using the Foundation One Liquid CDx Assay (324 genes, tumor mutational burden [TMB], microsatellite instability status). Results: 3435 patients with metastatic disease entered the study. Among them 992 patients (29%) received a targeted therapy matched to a specific molecular alteration before ctDNA. The main tumor types were: prostate cancer (349, 35%), luminal breast cancer (236, 24%), oncogene-addicted non-small cell lung cancer (129, 13%), KRAS-wild type colorectal cancer (126, 13 %). The most frequent class of targeted agents were androgen receptor pathway inhibitor (n = 350, 35%), aromatase inhibitor (236, 24%), anti- EGFR monoclonal antibodies (166, 17%), anti- EGFR tyrosine kinase inhibitors (83, 8%). ctDNA sequencing revealed DNA aberrations involved in secondary resistance in 308 patients (31%). The most frequent aberrations were AR mutations/amplifications, ESR1 point mutations, KRAS point mutations, EGFR point mutations. Among patients with resistance mutation, polyclonal aberrations were identified in 123 patients (40%). The median number of polyclonal aberrations per patient was 2 (range: 2-16). Polyclonal aberrations involved at least 2 different genes in 32 patients (10%). Preliminary results suggest that patients with polyclonal aberrations had worse outcome in comparison with patients with one or no detected aberration and final data will be presented at the time of the congress. Conclusions: We report here the first comprehensive landscape of genomic aberrations in ctDNA involved in resistance to targeted therapies in cancer patients. Polyclonal secondary genomic aberrations represent a frequent clinical resistance mechanism that may explain the poor rate of sustained complete remission observed with targeted therapies and must guide the development of future combinatorial strategies.

// Jordan Madic 1, * , Cecile Jovelet 2, * , Julien Lopez 1 , Barbara Andre 1 , Jean Fatien 3 , Isabelle Miran 2 , Aurelie Honore 2 , Laura Mezquita 5 , Benjamin Besse 5 , Ludovic Lacroix 2, 4, * and Magali Droniou 1, * 1 Stilla Technologies, 1 Mail du Professeur Georges Mathe, Villejuif, France 2 Plateforme de Genomique, Module de Biopathologie Moleculaire et Centre de Ressources Biologiques, AMMICa, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, France 3 Ecole Polytechnique, Route de Saclay, Palaiseau, France 4 Departement de Biologie et Pathologie Medicales, Institut Gustave Roussy, Villejuif, France 5 Departement d’Oncologie Medicale, Gustave Roussy, Villejuif, France * These authors have contributed equally to this work Correspondence to: Magali Droniou, email: magali.droniou@stilla.fr Keywords: EGFR ; multiplexing; monitoring; NSCLC; digital PCR Received: June 21, 2018 Accepted: November 26, 2018 Published: December 21, 2018 ABSTRACT Background: Detection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated. Methods: We developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and evaluated this assay on 82 tumor and plasma samples from NSLC patients. Results: Limits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wild-type DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease. Conclusion: This 6-color Crystal digital PCR assay could represent a robust solution using digital PCR for the monitoring of EGFR mutations.

Purpose To evaluate efficacy, complications and preprocedural risk factors for percutaneous image-guided core needle biopsy of malignant tumours for genomic tumour analysis. Materials and methods Procedural data for core biopsies performed at a single centre for the MOSCATO-01 clinical trial were prospectively recorded between December 2011 and March 2016. Data assessed included patient demographics, tumour characteristics, procedural outcomes and complications. Results A total of 877 biopsies were performed under computed tomography (38.4%) or ultrasound guidance (61.6%) for tumours in the liver (n = 363), lungs (n = 229), lymph nodes (n = 138), bones (n = 15) and other miscellaneous sites (n = 124). Each biopsy harvested a mean 4.4 samples [1–15], with adequate tumour yield for genomic analysis in 95.3% of cases. Procedural complications occurred in 89 cases (10.1%), with minor grade I complications in 59 (66.3%); grade II in 16 (18%) and grade III in 14 (15.7%). No grade IV complications and no procedure-related death occurred. The most common complications were pneumothorax (51/89, 57.3%), haemorrhage (24/89, 27%) and pain (8/89, 8.9%). Predictive factors for complications by univariate analysis included biopsied organ (lung vs other), sample number, prone position, lesion size, lesion depth and biopsy approach. By multivariate analysis, only pulmonary biopsy was a significant risk factor (odds ratio = 27.23 [4.93–242.76], p Conclusion Percutaneous image-guided core needle biopsy in cancer patients provides an effective method to obtain molecular screening samples, with an overall low complication rate. Lung mass biopsies present a higher risk of complication, although complications are manageable by minimally invasive techniques without prolonged sequelae.

Anaplastic thyroid carcinoma (ATC) is a rare and undifferentiated form of thyroid cancer. Its prognosis is poor: the median overall survival (OS) of patients varies from 4 to 10 months after diagnosis. However, a doubling of the OS time may be possible owing to a more systematic use of molecular tests for targeted therapies and integration of fast-track dedicated care pathways for these patients in tertiary centers. The diagnostic confirmation, if needed, requires an urgent biopsy reread by an expert pathologist with additional immunohistochemical and molecular analyses. Therapeutic management, defined in multidisciplinary meetings, respecting the patient’s choice, must start within days following diagnosis. For localized disease diagnosed after primary surgical treatment, adjuvant chemo-radiotherapy is recommended. In the event of locally advanced or metastatic disease, the prognosis is very poor. Treatment should then involve chemotherapy or targeted therapy and decompressive cervical radiotherapy. Here we will review current knowledge on ATC and provide perspectives to improve the management of this deadly disease.

Introduction: Diffuse gastric cancer is associated with poor prognosis. We report a patient with metastatic gastric linitis plastica harboring human epidermal growth factor receptor 2 ( HER2) activating mutation and HER2 amplification. Case description: The patient received 5-fluorouracil/folinic acid and oxaliplatin combined with trastuzumab/pertuzumab, resulting in disease control for 8 months. Second-line therapy with nivolumab and trastuzumab/pertuzumab was well-tolerated, with macroscopic peritoneal response. Following ovarian progression and surgical resection of ovarian metastases, immunohistochemistry of PD-L1 was negative; proteomics demonstrated normal expression of HER2 and absence of PD-L1, while genomics showed HER2 amplification, suggesting mechanisms of escape to dual HER2 blockade by downregulation of HER2 and to nivolumab by the absence of PD-L1. Based upon this and nonexpression of biomarkers of taxane resistance, therapy was changed to paclitaxel. Two and a half years after diagnosis, the patient is undergoing treatment, with excellent performance status. Conclusions: Molecular analysis and personalized therapy can help optimize treatment in difficult-to-treat cancers.

Background While deregulation of the cyclin D1-CDK4/6-retinoblastoma pathway is common in hormone receptor positive (HR+) breast cancer, Rb is usually intact in HR+ breast cancer, and targeted CDK 4/6 inhibitors that act upstream of Rb, are routinely being utilized in clinical practice. However, factors that can lead to clinical resistance to CDK 4/6 inhibitors are not known. Patients and methods We identified patients who had pre- and post-genotyping in tissue and peripheral blood samples after receiving CDK 4/6 inhibitors. Genotyping was carried out in tumor tissue or blood collected before start of CDK 4/6 inhibitor and after disease progression on CDK 4/6 inhibitor, covering more than 90% of the coding region in RB1. Results We identified detectable acquired RB1 mutations in circulating tumor DNA (ctDNA) after exposure to CDK4/6 inhibitor (palbociclib, palbociclib, ribociclib) for 5, 8, and 13 months, respectively, in three patients. The RB1 mutations included substitution in donor splicing site of exon 8 of the RB1 gene in patient #1; substitution in donor splicing site of exon 22 of RB1 gene, exon 19 deletion, exon 3 insertion in patient #2; and RB1 exon 16 H483Y mutation in patient #3. None of these RB1 mutations were present in the pre-CDK 4/6 specimen highlighting these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor potentially confering therapeutic resistance. Conclusion This is the first clinical report to describe the emergence of somatic RB1 mutations after exposure to palbociclib or ribociclib, in patients with metastatic breast cancer. Further research is needed to validate these findings, identify how these mutations temporally emerge under selective pressure of CDK 4/6 inhibitor, and develop rational therapeutic strategies.

Purpose: There is a need to identify which patients present a high risk of relapse after optimal adjuvant therapies, in order to better define which population of early breast cancer patients is eligible to new drugs. Previous studies have shown that gene copy numbers drive breast cancer progression. In the BIG1-98 trial, FGFR1 (8p11) and ZNF217 (20q13) amplifications were associated with poor outcome in patients with HR+/Her2- early breast cancers. In the present study, we aim to validate these findings using retrospective analysis of samples prospectively collected in a randomized trial (PACS04). Patients and methods: Tumor DNA was extracted from FFPE samples obtained from patients included in PACS04 trial. This trial compared FEC to ED75 in patients with node positive, early breast cancer and included 3000 patients between 2001 and 2004. The biomarker study focuses on HR+/Her2- breast cancer. 500 samples were collected and hybridization was performed on 390. DNA copy numbers were assessed on 900 genes using Oncoscan FFPE assay kit. Prognostic value of FGFR1 (8p11) and ZNF217 (20q13) copy numbers was assessed in a Cox model that included prognostic parameters. Prognostic value was assessed using CNA as continuous variable, and with a predefined cut-off (CN>=3). Metastases-free survival (MFS) was chosen as endpoint for this biomarker study. Results: Tumor copy numbers alterations were obtained in 390 patients with HR+/Her2-, node positive, early breast cancer. 31% of the patients presented more than 3 lymph node involved, and 25% a poorly differenciated tumor. The median tumor size was 20 mm. 120 (30%) and 112 (28%) patients presented FGFR1 and/or ZNF217 gene gain with a cut-off for amplification predefined at 3 copies. When assessed as continuous variable, FGFR1 (HR: 1.02, 95%CI:1.007-1.04, p=0.0045) and ZNF217 (HR: 1.011, 95%CI:1.003-1.01, p=0.006) copy numbers were associated with MFS. The 10 years MFS rates were 68% (95%CI: 59-78%) and 85% (95%CI: 81-91%) in patients with FGFR1-gained and FGFR1-normal tumors respectively (HR: 2.51, 95%CI:1.56-4.01, p=0.0001). The 10 years MFS rates were 72% (95%CI:65%-83%) and 83% (95%CI: 77-87%) in patients with ZNF217-gained and ZNF217-normal tumors (HR: 1.79, 95%CI:1.12-2.86, p=0.013). In a multivariate analysis, FGFR1-gain (HR: 2.45, 95%CI:1.42-4.22, p=0.0012) and ZNF217-gain (HR:1.78, 95%CI:1.00-3.17, p=0.049) were associated with a higher likelihood of metastases and/or death. The 10 MFS rate was 88% (95%CI: 83-94%) for patients who presented FGFR1- and ZNF217-normal tumor (n=191), and 71% (95%CI: 66-76%) in patients presenting FGFR1 and/or ZNF217 gene gain. Conclusion: Using samples prospectively collected in a randomized trial, this study validates the prognostic value of FGFR1- and ZNF217- copy numbers in patients who present HR+/Her2- early breast cancer. Exploratory analyses on copy number alterations, together with targeted sequencing are ongoing and will be presented. Citation Format: Andre F, Penault Llorca F, Carene D, Roche H, Delaloge S, Lacroix L, Scott V, Richon C, Lacroix-Ticrit M, Lemonnier J, Michiels S. FGFR1 / ZNF217 copy numbers and outcome in patients with node positive, HR+/Her2- early breast cancer: A genomic analysis of PACS04 trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-10.

A targeted therapy is recommended in case of ERBB2 alteration for breast and gastric carcinomas, but miscellaneous other tumor types are ERBB2-altered at low prevalence. Broadening the administration of HER2 inhibitors across tumor types and genomic alterations could benefit to patients with refractory metastatic tumors. Targeted next-generation-sequencing (tNGS) and comparative genomic hybridization array (CGH) have been performed on fresh tumor biopsies of patients included in the MOSCATO-01 and ongoing MOSCATO-02 trials to administrate HER2 inhibitors in case of ERBB2 pathogenic mutation of amplification. Between December 2011 and January 2017 a molecular analysis was performed for 934 patients (759 CGH and 912 tNGS). A novel ERBB2 alteration has been found in 4.7% (n = 44/934), including 1.5% (n = 14/912) ERBB2 mutations, and 4% (n = 30/759) ERBB2 amplifications. A matched HER2 inhibitor was administrated to 70% (31/44) of patients and consisted in trastuzumab plus chemotherapy for 90% of them (28/31). On the 31 evaluable patients, 1 complete response (CR), 10 partial response (PR) and 2 stable disease (SD) >24 weeks were observed accounting for a clinical benefit rate (CBR) of 42% (n = 13/31, 95% CI 25–61%). Besides breast and oesogastric carcinomas, 19 patients affected by 8 different tumor types had a CBR of 25% for ERBB2 mutations (n = 2/8, 95% CI 3%–65%, with 2 PR) and 64% for ERBB2 amplifications (n = 7/11, 95% CI 31%–89%; with 1 CR, 4 PR, 2 SD). ERBB2 genomic alterations were diffuse across metastatic tumor types and signs of efficacy emerged for HER2 targeted treatments, especially in case of ERBB2 amplifications or a p.S310Y ERBB2 mutation.

Comprehensive genomic profiling using high-throughput sequencing brings a wealth of information, and its place in the clinical setting has been increasingly prominent. This review emphasizes the utility of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) in patient care and clinical research, based on published reports as well as our experience with the MOSCATO-01 (MOlecular Screening for CAncer Treatment Optimization) molecular triage trial at Gustave Roussy Cancer Center. In this trial, all contributive samples of patients with advanced solid tumors were analyzed prospectively with targeted gene sequencing (TGS) and comparative genomic hybridization. In addition, 92 consecutive metastatic patients with contributive biopsies were sequenced for WES and RNAseq and compared with TGS and comparative genomic hybridization. Whole-exome sequencing allowed the reporting of additional variants in relevant genes in 38% of patients. Mutation detection sensitivity of WES was 95% compared with TGS. Additional information derived from WES and RNAseq could influence clinical decision, including fusion transcripts, expression levels, allele-specific expression, alternate transcripts, RNA-based pathogen diagnostic, tumor mutation load, mutational signatures, expression signatures, HLA genotyping, and neoepitope prediction. The current challenge is to be able to process the large-scale data from these comprehensive genome-wide technologies in an efficient way.

Background Recommended treatments of patients with advanced biliary tract cancer (aBTC) are limited to one chemotherapy doublet. Nevertheless, efficacy of treatment personalisation for aBTCs is supported by accumulating evidences but remains to be evaluated. Patients and methods Patients with aBTCs included in the prospective clinical trial MOSCATO-01 were treated by at least one previous systemic treatment, had an ECOG performance status of 0–1, and at least one tumour site accessible to biopsy. Multiple high-throughput molecular analysis was performed on biopsies to drive the administration of molecular targeted agents (MTAs). Results From November 2011 to March 2016, 43 patients (4%) of the 1035 adult patients included in MOSCATO-01 had aBTCs with a majority of intrahepatic localisation (67%). Successful biopsy procedures and DNA extractions led to molecular portraits for 34 patients (79%). Orientation to an appropriate early clinical trial or accessible MTA(s) was possible for 23 of these patients (68%), and 18 (53%) have received matched MTA(s). Among them, the overall response rate was 33% and the disease control rate was 88%. A PFS ≥6 months was observed in 37% and the PFS ratio was >1.3 for 50% of the patients. These patients had a lower risk for death as compared to the 20 patients not orientated to a matched MTA (HR, 0.29; 95% CI, 0.11–0.76; p = 0.008). Conclusions Within the MOSCATO-01 trial, patients with aBTCs had the highest rate of orientation to matched MTAs and derived a clear clinical benefit. A broader evaluation of these findings may improve future treatments strategies for aBTCs. Trial Registration: NCT01566019 .

Background LKB1/STK11 ( STK11 ) is among the most inactivated tumor-suppressor genes in non-small cell lung cancer (NSCLC). While evidence concerning the biologic role of STK11 is accumulating, its prognostic significance in advanced NSCLC has not been envisaged yet. Materials and methods This retrospective analysis included consecutive NSCLC patients with available STK11 information who underwent a platinum-based chemotherapy. STK11 mutational status was correlated to clinico-pathological and mutational features. Kaplan–Meier and Cox models were used for survival curves and multivariate analyses, respectively. Results Among the 302 patients included, 267 (89%) were diagnosed with stage IIIB/IV NSCLC and 25 (8%) harbored a STK11 mutation ( STK11mut ). No statistical differences were observed between STK11 status and clinico-pathological variables. We detected a significant correlation between STK11 and KRAS status (p = 0.008); among the 25 STK11mut patients, 13 (52%) harbored a concomitant KRAS mutation. Overall survival (OS) was shorter for STK11mut (median OS = 10.4 months) compared to wild-type patients ( STK11wt ; median OS = 17.3 months) in univariate analysis (p = 0.085). STK11 status did not impact upon OS in multivariate analysis (p = 0.45) and non-significant results were observed for progression-free survival. The co-occurrence of KRAS and STK11 mutations suggest a trend toward detrimental effect in OS (p = 0.12). Conclusions In our cohort enriched for advanced NSCLC patients who received platinum-based chemotherapy, STK11 mutations were not specifically associated with clinico-pathological features and they did not impact upon survival. We confirm the positive correlation between STK11 and KRAS mutations. The co-occurrence of KRAS and STK11 mutations could label a more aggressive molecular subtype of NSCLC.

High-throughput genomic analyses may improve outcomes in patients with advanced cancers. MOSCATO 01 is a prospective clinical trial evaluating the clinical benefit of this approach. Nucleic acids were extracted from fresh-frozen tumor biopsies and analyzed by array comparative genomic hybridization, next-generation sequencing, and RNA sequencing. The primary objective was to evaluate clinical benefit as measured by the percentage of patients presenting progression-free survival (PFS) on matched therapy (PFS2) 1.3-fold longer than the PFS on prior therapy (PFS1). A total of 1,035 adult patients were included, and a biopsy was performed in 948. An actionable molecular alteration was identified in 411 of 843 patients with a molecular portrait. A total of 199 patients were treated with a targeted therapy matched to a genomic alteration. The PFS2/PFS1 ratio was >1.3 in 33% of the patients (63/193). Objective responses were observed in 22 of 194 patients (11%; 95% CI, 7%–17%), and median overall survival was 11.9 months (95% CI, 9.5–14.3 months). Significance: This study suggests that high-throughput genomics could improve outcomes in a subset of patients with hard-to-treat cancers. Although these results are encouraging, only 7% of the successfully screened patients benefited from this approach. Randomized trials are needed to validate this hypothesis and to quantify the magnitude of benefit. Expanding drug access could increase the percentage of patients who benefit. Cancer Discov; 7(6); 586–95. ©2017 AACR. See related commentary by Schram and Hyman, p. 552. This article is highlighted in the In This Issue feature, p. 539

Background Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by theT790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib whenT790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advancedEGFR-mutant NSCLC patients. Material and methods ctDNAT790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. ProgressingT790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage ofT790M positive inctDNA. Results ThectDNAT790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4–NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s) ctDNA from liquid biopsy can be used as a surrogate marker forT790M in tumour tissue.

Introduction Despite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression. Methods Patients with EGFR-mutated metastatic NSCLC (N = 62) with tissue and plasma biopsies at EGFR TKI progression between January 2015 and June 2019, at a French hospital and optionally before progression, were identified from the prospective MATCH-R study (NCT02517892). Postprogression biopsy samples were analyzed for gene fusions using targeted gene panel sequencing, whole-exome sequencing, RNA sequencing, and comparative genomic hybridization array. Results Six gene fusions were detected in tumor progression biopsies under an EGFR TKI from 62 consecutive patients (9.7%) with EGFR-mutated advanced NSCLC. Among 31 patients progressing to first- or second-generation EGFR TKIs, one (3%) had an Eukaryotic translation initiation factor 4 gamma 2–GRB2 associated binding protein 1 (EIF4G2-GAB1) fusion. Among 31 patients progressing to the third-generation osimertinib, five (16%) presented oncogene fusions of fibroblast growth factor receptor 3–transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3) (n = 2), kinesin family member 5B–Ret proto-oncogene (KIF5B-RET) (n = 1), striatin–anaplastic lymphoma kinase (STRN-ALK) (n = 1), and zinc finger DHHC-Type palmitoyltransferase 20–Thr790Met (ZDHHC20-BRAF) (n = 1) transcripts. Out of two patients that received osimertinib at first-line, one acquired an FGFR3-TACC3 fusion at progression. In all patients, fusions co-occurred with the original activating EGFR mutation; however, among four patients with an acquired T790M mutation, three (75%) lost the T790M mutation. Conclusions Oncogenic fusions at the time of EGFR TKI resistance were identified at a relatively high frequency, mainly after the third-generation TKI osimertinib. Patients progressing to EGFR TKIs may have a new opportunity for targeted therapy when oncogenic fusions are identified.

Unravelling the biological processes driving tumour resistance is necessary to support the development of innovative treatment strategies. We report the design and feasibility of the MATCH-R prospective trial led by Gustave Roussy with the primary objective of characterizing the molecular mechanisms of resistance to cancer treatments. The primary clinical endpoints consist of analyzing the type and frequency of molecular alterations in resistant tumours and compare these to samples prior to treatment. Patients experiencing disease progression after an initial partial response or stable disease for at least 24 weeks underwent a tumour biopsy guided by CT or ultrasound. Molecular profiling of tumours was performed using whole exome sequencing, RNA sequencing and panel sequencing. At data cut-off for feasibility analysis, out of 333 inclusions, tumour biopsies were obtained in 303 cases (91%). From these biopsies, 278 (83%) had sufficient quality for analysis by high-throughput next generation sequencing (NGS). All 278 samples underwent targeted NGS, 215 (70.9%) RNA sequencing and 222 (73.2%) whole exome sequencing. In total, 163 tumours were implanted in NOD scid gamma (NSG) or nude mice and 54 patient-derived xenograft (PDX) models were established, with a success rate of 33%. Adverse events secondary to invasive tumour sampling occurred in 24 patients (7.6%). Study recruitment is still ongoing. Systematic molecular profiling of tumours and the development of patient-derived models of acquired resistance to targeted agents and immunotherapy is feasible and can drive the selection of the next therapeutic strategy.