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This file provides supplementary methods for genomics, including TGS, CGH array, WES, RNAseq Analysis, IHC -MET.

Individual PFS2 vs PFS1 profile plot. Each patient has been aligned at time 0 of PFS2 and sorted by the sum of PFS1 and PFS2.

Genes that contain molecular alterations matched with therapies according to tumor type.

Supplementary Figure Legends

Purpose: This phase I study aimed to determine the recommended dose (RD), safety profile, and feasibility of a procedure combining intratumoral injection of hafnium oxide nanoparticles (NBTXR3; a radioenhancer) and external beam radiotherapy (EBRT) for preoperative treatment of adults with locally advanced soft tissue sarcoma (STS).Experimental Design: Patients had a preoperative indication of EBRT for STS of the extremity or trunk. Baseline tumor volume (TV) was calculated by MRI. NBTXR3 was injected percutaneously into tumors at 53.3 g/L. Dose escalation was based on four levels equivalent to 2.5%, 5%, 10%, and 20% of baseline TV. NBTXR3 was visualized in the tumor 24 hours postinjection, and EBRT was initiated (50 Gy over 5 weeks). Surgery was performed 6 to 8 weeks after EBRT completion.Results: Twenty-two patients completed NBTXR3 injection, EBRT, and surgery and were followed for a median 22 months (range, 6–40). At NBTXR3 20% of TV, two dose-limiting toxicities occurred: injection-site pain and postoperative scar necrosis. The RD was defined as 10%. No leakage of NBTXR3 into surrounding tissues occurred; intratumor NBTXR3 levels were maintained during radiotherapy. At the RD, median tumor shrinkage was 40% (range 71% shrinkage, 22% increase); median percentage of residual viable tumor cells was 26% (range, 10%–90%). Patients receiving 20% of TV demonstrated pathologic complete responses. Seven grade 3 adverse events occurred, which were reversible.Conclusions: A single intratumoral injection of NBTXR3 at 10% of TV with preoperative EBRT was technically feasible with manageable toxicity; clinical activity was observed. Clin Cancer Res; 23(4); 908–17. ©2016 AACR.

Techniques to tag the injected tumor

Supplementary Data from Transcriptional Analysis of an E2F Gene Signature as a Biomarker of Activity of the Cyclin-Dependent Kinase Inhibitor PHA-793887 in Tumor and Skin Biopsies from a Phase I Clinical Study

Angular error impact on accuracy according to tumor size and depth

Devices for direct intratumoral immunotherapy

Sensitivity Prediction Score ROC Curve of the logistic regression model.

Whole blood hafnium concentrations for each patient following NBTXR3 injection. Quantification of NBTXR3 in the blood was performed to evaluate passage into the systemic circulation. The hafnium component of NBTXR3 was quantified through inductively coupled plasma mass spectrometry (ICP-MS). Whole blood samples were collected on Day 1: before injection, immediately after the injection start, at the end of the injection, 5, 10, 15, 60, 120 and 240 minutes after completion of the injection; and on Day 2. Three additional blood samples were collected during radiotherapy, between the 11th and 20th radiotherapy sessions. One blood sample was also collected prior to surgery.

Human intratumoral immunotherapy (HIT-IT) is under rapid development, with promising preliminary results and high expectations for current phase III trials. While outcomes remain paramount for patients and the referring oncologists, the technical aspects of drug injection are critical to the interventional radiologist to ensure optimal and reproducible outcomes. The technical considerations for HIT-IT affect the safety, efficacy, and further development of this treatment option. Image-guided access to the tumor allows the therapeutic index of a treatment to be enhanced by increasing the intratumoral drug concentration while minimizing its systemic exposure and associated on-target off-tumor adverse events. Direct access to the tumor also enables the acquisition of cancer tissue for sequential sampling to better understand the pharmacodynamics of the injected immunotherapy and its efficacy through correlation of immune responses, pathologic responses, and imaging tumor response. The aim of this article is to share the technical insights of HIT-IT, with particular consideration for patient selection, lesion assessment, image guidance, and technical injection options. In addition, the organization of a standard patient workflow is discussed, so as to optimize HIT-IT outcome and the patient experience.

Supplementary Figure 3: NF2 deleterious mutations and sensitivity to lorlatinib (corresponding to main Figure 4).

Purpose:Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting.Experimental Design:Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance.Results:Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial–mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors.Conclusions:This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.

Supplementary Figure 4: NF2 inhibition of mTORC1 and its canonical pathway.

mutations' allele frequencies in tDNA and cfDNA

Intratumoral injection techniques

Supplementary figures legends

Purpose: Liquid biopsies based on circulating cell-free DNA (cfDNA) analysis are described as surrogate samples for molecular analysis. We evaluated the concordance between tumor DNA (tDNA) and cfDNA analysis on a large cohort of patients with advanced or metastatic solid tumor, eligible for phase I trial and with good performance status, enrolled in MOSCATO 01 trial (clinical trial NCT01566019).Experimental Design: Blood samples were collected at inclusion and cfDNA was extracted from plasma for 334 patients. Hotspot mutations were screened using next-generation sequencing for 50 cancer genes.Results: Among the 283 patients with tDNA–cfDNA pairs, 121 had mutation in both, 99 in tumor only, 5 in cfDNA only, and for 58 patients no mutation was detected, leading to a 55.0% estimated sensitivity [95% confidence interval (CI), 48.4%–61.6%] at the patient level. Among the 220 patients with mutations in tDNA, the sensitivity of cfDNA analysis was significantly linked to the number of metastatic sites, albumin level, tumor type, and number of lines of treatment. A sensitivity prediction score could be derived from clinical parameters. Sensitivity is 83% in patients with a high score (≥8). In addition, we analyzed cfDNA for 51 patients without available tissue sample. Mutations were detected for 22 patients, including 19 oncogenic variants and 8 actionable mutations.Conclusions: Detection of somatic mutations in cfDNA is feasible for prescreening phase I candidates with a satisfactory specificity; overall sensitivity can be improved by a sensitivity score allowing to select patients for whom cfDNA constitutes a reliable noninvasive surrogate to screen mutations. Clin Cancer Res; 22(12); 2960–8. ©2016 AACR.

Supplementary Figure 1: EMT mediated lorlatinib resistance (corresponding to main Figure 2).

NBTXR3 Injection _ DCE US. Dynamic contrast-enhanced ultrasonography (DCE-US) was performed to examine treatment-related changes to the tumor

Supplementary Figure 2: ALK secondary mutations (corresponding to main Figure 3).

A transcriptional signature of the pan–cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors. Mol Cancer Ther; 9(5); 1265–73. ©2010 AACR.

Systemic immunotherapies such as immune checkpoint blockade targeted at PD(L)1 and CTLA4 have demonstrated their ability to provide durable tumor responses and long-term overall survival benefits for some patients in several solid tumor types. However, a majority of patients remain resistant to these treatments and a significant proportion of them develop severe autoimmune and inflammatory adverse events. Preclinical studies have demonstrated that intratumoral injections of immunostimulatory products (oncolytics, pattern recognition receptor agonists,…) that are able to trigger type I IFN release and enhance tumor antigen presentation on immune cells could generate a strong antitumor immunity and overcome the resistance to systemic immune checkpoint blockade therapies. The intratumoral immunotherapy strategies that are currently in clinical development offer a unique therapeutic and exploratory setting to better understand the immune contexture across tumor lesions of patients with metastatic cancer. Also these local therapeutic products could turn cold tumors into hot and improve the response rates to cancer immunotherapies while diminishing their systemic exposure and toxicities. Intratumoral immunotherapies could prime or boost the immunity against tumors and therefore radically change the combinatorial therapeutic strategies currently pursued for metastatic and local cancers to improve their long-term survival. We aimed to review and discuss the scientific rationale for intratumoral immunotherapy, the challenges raised by this strategy in terms of drug development within clinical trials and the current state-of-the-art regarding the clinical practice of this innovative approach.

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: The androgen receptor axis inhibitors (ARi) (e.g, enzalutamide, abiraterone acetate) are administered in daily practice for men with metastatic castration-resistant prostate cancer (mCRPC). However, not all patients respond, and mechanisms of both primary and acquired resistance remain largely unknown. Methods: In a prospective trial MATCH-R (NCT02517892), 55 mCRPC patients underwent whole exome sequencing (WES) (n=45) and RNA-sequencing (RNA-seq) (n=52) of metastatic biopsies before starting ARi. Also, 16 mCRPC patients underwent biopsy at time of resistance (WES=14, RNA-seq = 14). The objectives were to identify genomic alterations associated with resistance to ARi as well as to describe clonal evolution. Primary resistance was determined at 4 months of treatment using composite criteria for progression that included serum prostate specific antigen measurements, bone scan, CT imaging and symptom assessments. Acquired resistance was defined by occurrence of progressive disease after initial response or stable disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using Wilcoxon and Fisher's exact tests. Results: At 4 months, 22/55 patients in the cohort had disease progression (primary resistance). No genomic alterations from WES analysis were significantly associated with primary resistance. Analysis of sequential biopsies suggests that mCRPC follows mainly a parallel evolution model and involve DNA-repair related mutational processes. At time of acquired resistance to ARi, most tumors acquired new drivers affecting AR pathway (e.g, AR, NCOR1/2) or lineage switching (e.g, RB1, PTEN, TP53). Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis to identify pathways whose activity state correlated with resistance. AR gene alterations and AR expression were similar between responding and non-responding patients. Transcriptional analysis demonstrated that multiple specific gene sets — including those linked to low AR transcriptional activity, stemness program, RB loss and homologous repair deficiency — were activated in both primary and acquired resistance. Conclusion: Resistance to AR axis inhibitors results from multiple transcriptional programs already activated in pre-treatment samples. Clonal evolution analysis along with RNA-seq data indicate the role of genomic instability and lineage switching in driving acquired resistance Citation Format: Naoual Menssouri, Loic Poiraudeau, Carole Helissey, Ludovic Bigot, Jonathan Sabio, Tony Ibrahim, Claudio Nicotra, Maud Ngocamus, Lambros Tselikas, Thierry De Baere, Etienne Rouleau, Ludovic Lacroix, Anne Chaucherau, Luc Friboulet, Ronan Flippot, Giulia Baciarello, Laurence Albiges, Emeline Colomba, Pernelle Lavaud, Stefan Michiels, Aline Maillard, Antoine Italiano, Fabrice Barlesi, Jean-Charles Soria, Jean-Yves Scoazec, christophe Massard, Benjamin Besse, Fabrice André, Karim Fizazi, Daniel Gautheret, Yohann Loriot. A prospective study of prostate cancer metastases identifies an androgen receptor activity-low, stemness program associated with resistance to androgen receptor axis inhibitors and unveils mechanisms of clonal evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 358.

Background: Recent advances in the biology of prostate cancer have identified aggressive variants of metastatic castration-resistant prostate cancer [mCRPC] (e.g, CRPC with neuroendocrine (NE) features, or microsatellite instability [MSI], or BRCA2 mutations). However, few preclinical models have been successfully established to study these aggressive mCRPC variants owing to their historically low establishment success and to the difficulty in accessing such clinical samples Methods: Fresh tumor biopsy specimens were obtained prospectively from patients with mCRPC through a prospective single-institution clinical trial (MATCH-R, NCT02517892). Patient-derived xenografts (PDX) in NOD Scid Gamma mice were developed and characterized as well as PDX-derived organoids derived from the same PDX (PDXO). Whole-exome sequencing, RNA sequencing and immunohistochemistry were performed on human samples and their corresponding PDX and PDXO. The primary aim was to successfully derive PDX and PDXO models reproducing the clinical features of mCRPC aggressive variants. Results: As of November 2019, 83 tumor biopsies were obtained from 61 mCRPC patients, 16 biopsies from 13 patients were successfully engrafted with an overall success rate of 26% (16/61). In addition, 16 PDXO were developed with a success rate of 100%. Overall, we developed 4 PDX and 4 PDXO from 2 patients with germline BRCA2 mutation, 2 PDX and 2 PDXO from 1 patient with MSI-high CPRC, 10 PDX and 6 PDXO from 10 patients with NE mCPRC. Molecular profiling revealed a high concordance between PDX, PDXO and human tumor samples for histological phenotype, driver mutations and transcriptomic phenotypes. The two models harbouring BRCA2 mutation were highly sensitive to both carboplatin and olaparib (in PDX and PDXO) reflecting the clinical scenario observed in the patients. BRCA2-mutated PDX models were treated in vivo to derive olaparib-resistant mCRPC models. Genetic analysis identified secondary mutations restoring the reading-frame of the gene which reversed the sensitivity of the PDX to carboplatin and olaparib. Preclinical models derived from mCRPC with NE features (including the case of small cell carcinoma) globally reproduced the clinical patterns seen in human samples. Results from high-throughput organoid drug screening suggest good concordance with patients clinical response Conclusion: The study demonstrated the feasibility of establishing preclinical models (PDX and PDXO) derived from aggressive mCRPC variants. Overall, our models reproduce the phenotypic, molecular and pharmacological characteristics of their initial human samples and may represent a unique preclinical platform modeling BRCA2 mutated, MSI and neuroendocrine prostate cancers. Citation Format: Ludovic Bigot, Jonathan Sabio, Tony Ibrahim, Naoual Menssouri, Loic poiraudeau, Carole Helissey, Jean-Yves Scoazec, Zahira Merabet, Thierry De Baere, Frederic Deschamps, Maud Ngocamus, Claudio Nicotra, Etienne Rouleau, Ludovic Lacroix, Olivier Deas, Luc Friboulet, Gilles Vassal, Eric Solary, Jean-Charles Soria, Karim Fizazi, Fabrice André, Christophe Massard, Benjamin Besse, Yohann Loriot. Novel preclinical models of aggressive variants of castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1114.

Background: Molecular alterations involving FGFR family genes (FGFR 1-4) are emerging driver events in a variety of solid tumors, mainly represented by urothelial carcinoma (UC) and intrahepatic cholangiocarcinoma (CC). Several tyrosine kinase inhibitors (TKI) are in clinical development to counteract FGFR-driven diseases, being especially active against activating gene mutations and rearrangements. Progression on these targeted agents eventually appears and the understanding of molecular mechanisms of resistance is crucial to develop novel strategies. Methods: In the MATCH-R prospective study (NCT02517892), patients with unresectable or metastatic cancer are included upon acquired resistance to targeted therapies or immunotherapy, defined as progressive disease after complete/partial response or stable disease for six months. Serial blood samples are collected and tumor biopsy is performed upon progression. Targeted NGS, CGH, WES and RNAseq are performed on the tissue samples. PDX models and patient-derived cell lines are developed to fully investigate the underlying mechanisms of resistance. Only patients receiving TKI for FGFR-mutated or -rearranged tumors were included (i.e. FGFRamplifications were excluded) in the analysis. Results: From June 2015 to November 2018, 113 patients treated with a TKI were included in the MATCH-R study, of which 17 (15%) had received an FGFR inhibitor. Tumor types and corresponding molecular aberrations were as follows: 8 CC (n=6 FGFR2-rearranged, n=1 FGFR2:C383R, n=1 FGFR3:S249C), 7 UC (n=5 FGFR3:S249C, n=1 FGFR3:R248C, n=1 FGFR3:Y373C), 1 breast (FGFR3-rearranged) and 1 ovarian (FGFR2-rearranged) cancers. Evaluable tumor biopsies were taken upon progression to treatment with erdafitinib (n=12), pemigatinib (INCB54828) (n=3) or TAS-120 (n=4). Two patients underwent multiple biopsies as progressing on sequential FGFR inhibitors. Resistance mechanisms consisted of polyclonal secondary mutations (n=5), bypass pathways activation (n=3) and the remaining nine cases are still under investigation. PDX models/patient-derived cell lines were obtained in eight cases and extensively characterized in three. Adaptive treatment with novel FGFR TKI or combinatorial strategies aiming to block the bypass pathways allowed to restore sensitivity in both cell lines (readouts: IC50 and Western Blots) and PDX (readout: median tumor growth). Novel mutations potentially implicated in resistance to FGFR TKI were characterized by infecting Ba/F3 cells with respective lentiviral vectors, as well as the inhibitory potential of the differential FGFR inhibitors. Conclusions: Novel mechanisms of resistance to FGFR inhibitors in solid tumors were identified and consequent treatment strategies allowed to regain sensitivity in both patient-derived cell lines and PDX. Updated results will be presented at the Meeting. Citation Format: Francesco Facchinetti, Rastislav Bahleda, Antoine Hollebecque, Yohann Loriot, Gonzalo Recondo, Ludovic Bigot, Ken A. Olaussen, Gilles Vassal, Stefan Michiels, Rosa L. Frias, Justine Galissant, Tony Sourisseau, Claudio Nicotra, Maud Ngo-Camus, Linda Mahjoubi, Ludovic Lacroix, Etienne Rouleau, Catherine Richon, Aurélie Abou-Lovergne, Olivier Deas, Nathalie Auger, Thierry De Baere, Frederic Deschamps, Eric Solary, Jean-Yves Scoazec, Eric Angevin, Alexander Eggermont, Fabrice André, Benjamin Besse, Jean-Paul Thiery, Jean-Charles Soria, Christophe Massard, Luc Friboulet. Mechanisms of acquired resistance to FGFR inhibitors in molecularly-selected solid tumors: A prospective cohort from the MATCH-R study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 318.

Background: ALK rearrangements occur in 3-6% of patients (pts) with lung adenocarcinoma. Lorlatinib, is a novel third generation ALK tyrosine kinase inhibitor (TKI) with proven efficacy for patients previously treated with second generation ALK TKI. Methods: The MATCH-R study is a prospective single-institution trial aiming to identify mechanisms of resistance to targeted agents and immunotherapy in pts with advanced cancer (NCT02517892). Patients that achieve an initial partial or complete response or stability of disease for at least 6 months with selected agents are included upon disease progression. Tumor biopsies are performed and serial blood samples are collected. Extensive molecular profiling with panel next-generation sequencing (NGS), whole exome sequencing (WES) and RNA sequencing (RNAseq) is performed on tumor samples. Patient-derived xenografts (PDX) in NOD scid gamma (NSG) or nude mice and patient-derived cell lines are developed. We report mechanisms of resistance in a cohort of pts with ALK-rearranged lung cancer treated with lorlatinib. Results: From June 29th 2015 to November 15th 2018, 113 pts treated with a TKI were included in the MATCH-R study, of which 14 (12%) received treatment with ALK TKI, 6 pts treated with lorlatinib and with adequate tumor biopsies for molecular analysis were included. Tumor types studied were lung adenocarcinoma (n=4), anaplastic thyroid carcinoma (ATC, n=1) and myofibroblastic inflammatory tumor (MIT, n=1). An NF2 frame-shift deletion was detected by NGS in the ATC sample and a TNIK Q674 missense mutation was detected in the MIT sample. In the four pts with lung cancer treated with lorlatinib, we identified novel ALK G1202R/F1174L compound mutations from the tumor biopsy in one case and characterized them with Ba/F3 models (ctDNA analysis will be presented). Induction of epithelial mesenchymal transition (EMT) with lorlatinib exposure was responsible for resistance in one patient-derived model and susceptible to combined ALK/SRC inhibition. This cell line also had ALK C1156Y/G1269A compound mutations, not contributing to lorlatinib resistance. In a third case, double deleterious events in NF2 were identified in temporo-spatial distinct tumor biopsies on progression to lorlatinib. We further validated the effect of these events in patient-derived cell lines developed from two different biopsies. Downstream mTOR pathway activation conferred resistance to lorlatinib, and was reversible with mTOR inhibitors. We performed NF2 knockout in H3122 cells using Crispr-Cas9 gene editing to validate these findings. The resistance mechanism to lorlatinib treatment is yet to be elucidated in one patient-derived model. Conclusions: Mechanisms of resistance to lorlatinib can be diverse and complex, involving compound mutations, EMT and bypass activation. The present evidence could provide new insights for the development of tailored treatments for patients. Citation Format: Gonzalo Recondo, Laura Mezquita, David Planchard, Anas Gazzah, Francesco Facchinetti, Ludovic Bigot, Ahsan Z. Rizvi, Jean-Paul Thiery, Jean-Yves Scoazec, Rosa L. Frias, Tony Sourisseau, Linda Mahjoubi, Justine Galissant, Aurelie Abou-Lovergne, Gilles Vassal, Rastislav Bahleda, Antoine Hollebecque, Claudio Nicotra, Maud Ngocamus, Stefan Michiels, Ludovic Lacroix, Catherine Richon, Nathalie Auger, Thierry De Baere, Frederic Deschamps, Eric Solary, Ken A. Olaussen, Eric Angevin, Alexander Eggermont, Fabrice André, Christophe Massard, Jean-Charles Soria, Benjamin Besse, Luc Friboulet. Diverse biological mechanisms drive resistance to Lorlatinib in ALK-rearranged Lung Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 311.

A transcriptional signature of the pan–cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors. Mol Cancer Ther; 9(5); 1265–73. ©2010 AACR.

Background Precision-medicine initiatives are driven by the molecular analysis of tumor samples (fresh or FFPE material). Such an approach is limited by the availability of the tumor material and the challenges related to on-purpose tumor biopsies. A very appealing alternative to advance precision-medicine initiatives is the development of liquid biopsies using cfDNA. Methods We investigated the use of NGS on tumor biopsy and plasma, and evaluated the consistency between the tissue biopsy (tDNA) and cfDNA analysis on a prospective cohort of patients with metastatic or locally advanced solid tumors enrolled in the MOSCATO 01 trial (NCT01566019). Blood samples were collected at inclusion before tumor biopsy and cfDNA was extracted from 500μl plasma. Hot-spot mutations from 50 genes were screened with Ampliseq CHP2 panel (IonTorrent, Life Technologies, Dramstadt). Only variants reported by the Torrent Suite Variant Caller v4.2 were retained for the analysis. Paired results in tumor and plasma were described using Cohen's Kappa agreement coefficient (κ). Results From November 2011 to May 2014, among the 516 patients enrolled in the MOSCATO 01 trial, 190 patients (37%) were analyzed for tDNA and cfDNA. In addition, cfDNA was evaluated in 43 patients for whom no tumor analysis was performed because of low cellularity (< 10% of tumor cells). Patient characteristics were as follows: median age at biopsy: 57 years (range, 18-78); main tumor types: lung (19%), ENT (14%), colorectal (10%), breast (10%); median of 3 previous lines of treatment. Overall, 325 mutations were identified in the tDNA of 184 patients: 146 mutations were identified both in tumor and plasma and 179 in tumor, but not in plasma. 15 mutations were only found in cfDNA, thereby providing additional information. The κ.was 59% (95%CI, 0.54-0.64). The sensibility of using NGS for 50 targeted hot-spot genes analysis in cfDNA compared to tDNA was 44.9% and the specificity was 99.8%, with a positive predictive value of 90.7% and negative predictive value of 98.1%. The ten most frequent pathogenic mutations found in the tDNA or cfDNA (>5 cases each) included KRAS (33 cases), PIK3CA (22 cases) and TP53 genes (22 cases). When considering only these ten most frequent mutations, 77 mutations were identified in 71 patients: 39 in tumor and plasma, 36 in tumor, but not in plasma and 2 mutations only in plasma, with a κ of 66% (95%CI, 56-76%). The p.H1047R PIK3CA mutation, was found only in tumor (5 cases); for the other nine mutations, the κ coefficient varied from 56% to 89%, with a median of 75%. The cfDNA analysis of the 43 patients without tDNA analysis revealed at least one mutation in 24 patients (56%), including 11 pathogenic variants of therapeutic interest. Conclusion The analysis of cfDNA using NGS represents an attractive and noninvasive alternative to tumor biopsies, and can be used as a surrogate method to screen for mutations. Further prospective validation is warranted. Citation Format: Ecaterina Ileana, Cécile Jovelet, Marie-Cécile Le Deley, Christophe Massard, Nelly Motté, Antoine Hollebecque, Amélie Boichard, Charles Ferté, Sophie Postel-Vinay, Silvia Rosellini, Maud Ngo-Camus, Thierry De Baere, Philippe Vielh, Catherine Richon, Mélanie Laporte, Siham Gouissem, Yohann Loriot, Rastilav Bahleda, Anas Gazzah, Andrea Varga, Gilles Vassal, Alexander Eggermont, Fabrice André, Jean-Charles Soria, Ludovic Lacroix. Circulating cell-free tumor DNA (cfDNA) analysis of 50-genes by next-generation sequencing (NGS) in the prospective MOSCATO trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2015-2401

Background: The widespread use of high-throughput molecular techniques has allowed the identification of recurrent and actionable molecular traits across various tumor types. Translating these approaches to bedside may guide the decision-making for cancer patients candidates to early clinical trials. Methods: Patients with advanced solid tumors, refered to our early drug development department (DITEP), were prospectively enrolled in a prospective molecular screening program at Gustave Roussy (France). CT-Scan or ultrasound-guided biopsies were performed in metastatic or primary tumor sites to carry out a comprehensive molecular characterization. DNA was extracted from fresh tumor samples and analyzed by comparative genomic hybridization (CGH) (≥ 30% tumor cells required) and by Next Generation Sequencing (NGS) for up to 74 target genes (≥ 10% tumor cells required). A weekly molecular tumor board reviewed all the profiles to identify actionable traits for which the most relevant targeted therapy may be available through early clinical trials or marketed drugs. Treatment efficacy was evaluated by RECIST 1.1 criteria. Results: From November 2011 to December 2013, 420 heavily pretreated patients were included in the MOSCATO 01 trial. Out of them, a tumor biopsy could be performed in 368 patients (87%). CGH and NGS profiles were assessed in 284 (77%) and 315 (85%) of biopsied patients, respectively, with 283 patients (76% of biopsied patients) being profiled for both CGH and NGS. The median time for delivering results of 20 days. Actionable molecular aberrations were found in 161 patients (44%). Among them, 81 patients (50%) were matched to a targeted therapy and enrolled in ongoing phase I clinical trials. The most relevant genomic aberrations of interest were FGFR or FGF ligand amplification (n=24), PTEN/PI3K/AKT pathway activation (n=22), HER2 amplification (n=11), KRAS/NRAS/HRAS mutation (n=5), BRAF mutation (n=4), cyclin dependent kinase amplification or deletion (n=8), EGFR amplification or mutation (n=7), MET amplification (n=3), androgen receptor amplification (n=2), ALK translocation (n=2), and KIT amplification, MDM2 amplification, IGF1R amplification, and ROS1 rearrangement (all n=1). Out of the 81 patients profiled and treated according to the genomic profiles, we observed at the first tumor evaluation: 1 (1%) complete response (CR), 9 (11%) partial responses (PR), 52 (64%) stable disease (SD) and 14 (17%) progressive disease (PD). Whole exome analyses on selected patients are ongoing (currently n=24) and will be presented at the meeting. Conclusions: High throughput genomic analysis is feasible in daily practice and allows biological-orientation of patients in early clinical trials. The enrichment of phase I trials with patients harboring specific molecular traits leads to promising antitumor activity and is likely to exert a major influence on early drug development. Citation Format: Charles Ferté, Christophe Massard, Ecaterina Ileana, Antoine Hollebecque, Ludovic Lacroix, Samy Ammari, Maud Ngo-Camus, Rastislav Bahleda, Anas Gazzah, Andrea Varga, Sophie Postel-Vinay, Yohann Loriot, Nathalie Auger, Valerie Koubi-Pick, Bastien Job, Thierry De Baere, Frederic Deschamps, Philippe Vielh, Vladimir Lazar, Marie-Cécile Le Deley, Catherine Richon, vincent ribrag, eric deutsch, eric angevin, gilles vassal, Alexander Eggermont, Fabrice André, Jean-Charles Soria. Molecular screening for cancer treatment optimization (MOSCATO 01): a prospective molecular triage trial; Interim analysis of 420 patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT240. doi:10.1158/1538-7445.AM2014-CT240