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Introduction: Cell-free tumor DNA (ctDNA) is an emerging biomarker in head and neck squamous cell carcinoma (HNSCC) for disease staging, patients’ recurrence risk stratification and early detection of relapse. We aimed to compare variants identified in ctDNA versus surgical tumor specimen, and to study the evolution of the mutational landscape of ctDNA over time in HNSCC. Patients and Method: Forty-one HNSCC patients treated with curative-intent primary surgery from SCANDARE cohort (NCT03017573) were evaluated for longitudinal ctDNA-based NGS. Overall, 28 patients were treated with adjuvant (chemo)radiotherapy, and 31 experienced recurrence. Formalin-fixed paraffin-embedded tumor tissues at surgery were available for 41 patients. Serial contributive ctDNA were retrieved from all 41 patients at the date of surgery, 36 patients within 19 weeks after surgery, 20 patients at six months after surgery, and 22 patients at recurrence. Tissue DNA was personalized detected with a custom NGS panel of 571 genes (DRAGON) and ctDNA was sequenced using another personalized dedicated NGS panel including up to 15 genes (OncoFOLLOW). Results: Most frequently mutated genes in tissue included TP53 (15.9%), FAT1 (6.7%), NOTCH1 (5.5%) and PIK3CA (4.3%) with similar allelic ratio to ctDNA at baseline surgery. Higher prevalence of KRAS and TP53 mutations was found in ctDNA at recurrence in comparison with ctDNA and tissue, respectively, at baseline surgery (KRAS: 6.3% versus 1.6% and 0.6%; TP53: 31.2% versus 21.1% and 15.9%). Additional variants in NRAS, HRAS, TP53, JAK2 and SDHA were detected in 6 patients in ctDNA at surgery and were not found in tissue, suggesting spatial intratumor heterogeneity. Twenty-three/36 patients (64%) had detected ctDNA within 19 weeks after surgery among whom, 17/23 patients (74%) had disease recurrence. Eleven/20 patients (including 10 with adjuvant treatment) had detected ctDNA at six months after surgery among whom 6 patients (55%) had disease recurrence. Fifteen/22 patients (68%) had detected ctDNA at recurrence. Emerging pathogenic variants were found in patients with detected ctDNA after surgery (n=7/23; 30%), at six months after surgery (n=1/11; 9%) and at recurrence (n=4/15; 27%). Conclusion: Our study suggests spatial and longitudinal tumor heterogeneity and reports emerging mutations in ctDNA over time in HNSCC. Prognostic significance characterization of the ctDNA dominant clone allele frequency is ongoing. Citation Format: Grégoire Marret, Constance Lamy, Sophie Vacher, Mathieu Séné, Ladidi Ahmanache, Laura Courtois, Zakhia El Beiano, Jerzy Klijanienko, Charlotte Martinat, Nicolas Servant, Choumouss Kamoun, Linda Larbi Chérif, Thierry Bronzini, Cédric Balsat, Jean-François Laes, Aubray Prévot, Sébastien Sauvage, Maxime Lienard, Emmanuel Martin, Bérengère Genin, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Ivan Bièche, Maud Kamal, Christophe Le Tourneau. Spatial and longitudinal tumor heterogeneity in head and neck squamous cell carcinoma patients treated with primary surgery [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 3363.

Introduction: With the increasing complexity of current diagnostic investigations, the integration of clinical, pathological and molecular characteristics is crucial for the management of patients (pts) with cancers of unknown primary (CUP). A national multidisciplinary tumor board (NatCUPMTB) was created 2 years ago in France to discuss the diagnostic and therapeutic management of CUP pts. The objective of this study was to evaluate its diagnostic, prognostic and therapeutic impact after 2 years of activity. Methods: This was a multicenter retrospective study with prospective follow-up. All pts discussed at least once in the NatCUPMTB between June 2020 and August 2022 were included. Pts and tumors characteristics, pathological and molecular analyses including WGS, WES and RNAseq performed on SEQOIA and AURAGEN national large-scale sequencing platforms, multidisciplinary tumor board (MTB) conclusions, and follow-up after MTB were collected. Results: 76 pts for whom a long-term follow-up was available were included. The median age at diagnosis was 57 yo, 54% were female, and the median number of metastatic sites at diagnosis was 2. The median time between diagnosis and first MTB presentation was 3.8 months (0.2-55). MTB investigations enabled to identify a likely primary origin in 44/76 (58%) pts, and the MTB recommended a personalized therapeutic strategy in 50/76 patients (66%). MTB recommendations were based on the combination of clinical, pathological and molecular investigations in 55% of pts. After a median follow-up of 6.2 months, the median overall survival (OS) was 17.7 months from diagnosis and 11.0 months from the 1st MTB presentation. Pts for which the MTB had a diagnostic impact, and having received a treatment following MTB recommendation (based on putative origin or targetable alteration) had increased OS compared to pts with no diagnostic orientation (median OS 18.4 months vs 5.6 months, p=0.003) or having received other treatments (median OS 18.4 vs 4.4 months, p=0.0001). Conclusion: NatCUPMTB provides significant diagnostic and therapeutic benefit in pts with CUP. Early presentation of pts at NatCUPMTB as soon as CUP diagnosis is suspected should be recommended. Citation Format: Nicolas Jacquin, Maud Kamal, Ivan Bieche, Célia Dupain, Isabelle Guillou, Linda Larbi-Chérif, Etienne Rouleau, Julien Masliah Planchon, Isabelle Soubeyran, Christelle de la Fouchardière, Camille Tlemsani, Hélène Blons, Fabienne Escande, Michel Vidaud, Jennifer Wong, Pierre Saintigny, Sandrine Boyault, Adrien Buisson, Yves Allory, Anne Vincent-Salomon, Vincent Cockenpot, Janick Selves, Christophe Le Tourneau, Sarah Watson. National Multidisciplinary Tumor Board improves diagnostic stratification and therapeutic management in Cancers of Unknown Primary: the French Experience. [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 4534.

Background: Chordomas are considered as rare bone neoplasms characterized by a high level of recurrence and a poor long-term prognosis. Considering their chemo-radio-resistance, alternative treatment strategies are therefore strongly requested, but their development is limited by the paucity of relevant preclinical models. Mutations affecting genes of the SWI/SNF complex including PBMR1 are frequently found in chordomas, suggesting a potential therapeutic effect of epigenetic regulators in this indication. Methods: Thirty-eight chordoma specimens derived from primary patients’ tumors were grafted into nude mice. Relationship between grafted tumors and clinical, biological and therapeutic features of corresponding patients were investigated. We subjected the established PDX models to histopathological and genetic examination; finally, a therapeutic program focused on the EZH2-inhibition was performed in one PDX model. Results: Twelve PDX models were established and characterized on histological and biomolecular features. Patients whose tumors were able to grow into mice had a statistically significant lower progression-free survival than those whose tumors did not grow after in vivo transplantation (p=0.007). All PDXs maintained the same histopathological features as patients’ tumors. Homozygous deletions of CDKN2A/2B (58.3%) and PBRM1 (25%) variants were the most common genomic alterations found. In the tazemetostat treated PDX model harboring a PBRM1 variant, an overall survival of 100% was observed. Conclusion: Our panel of chordoma PDXs represents a useful preclinical tool for both pharmacologic and biological assessments. The first demonstration of a high antitumor activity of tazemetostat in a PDX model harboring a PBRM1variant supports further evaluation of EZH2-inhibitors in this subgroup of chordomas. Citation Format: Thibault Passeri, Ahmed Dahmani, Julien Masliah-Planchon, Adnan Naguez, Marine Michou, Rania El-Botty, Sophie Vacher, Rachida Bouarich, André Nicolas, Marc Polivka, Coralie Franck, Anne Schnitzler, Fariba Némati, Sergio Roman-Roman, Franck Bourdeaut, Homa Adle-Biassette, Hamid Mammar, Sébastien Froelich, Ivan Bièche, Didier Decaudin. Dramatic in vivo efficacy of the EZH2-inhibitor tazemetostat in PBRM1-mutated human chordoma xenograft [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 LB567.

Context: Polymerase epsilon (POLE) gene missense hotspot mutations can generate pathogenic (p) proofreading defects resulting in hypermutated genomic profiles. Aim: Determine the prevalence, genomic consequences and immunotherapy sensitivity of advanced POLE mutated tumors according to mutation site, primary tumor and tumor mutational burden (TMB). Results: Pan-Cancer TCGA & MSKCC databases genomic analyses found a prevalence of non-pathogenic POLE mutations (POLEnp) of 3.4% with median TMB of 11 mutations/Megabase (mt/Mb, IQR 3-34). Pathogenic POLE mutations (POLEp) prevalence was 0.4% with median TMB of 215 mt/Mb (IQR 107-324), predominantly in colorectal and endometrial cancers. Prevalence dropped to 0.1% in metastatic cancers. We assessed prospectively the efficacy of PD-1 blockade in mismatch repair proficient advanced solid tumors harboring POLE missense mutations (phase II ASCe Nivolumab trial; NCT03012581). Variants were categorized prospectively by a molecular board as POLEp, POLEnp or Variants with Unknown significance (VUS). The primary endpoint was the Overall Response Rate (ORR) at 12 weeks according to RECIST 1.1, and secondary endpoints included survival analyses according to POLE variants pathogenicity. Among 61 screened patients, 21 were eligible and 20 received Nivolumab and 19 were assessable for response (table 1). The 12-week ORR was 37% for patients harboring POLEp and VUS and resulted in major survival improvement compared to POLEnp patients (HR=0.1 ; CI95% 0.02-0.7); see results in Table 1. Among patients POLEp tumors, while higher TMB was not predictive of response, higher proportion of POLE-related mutational signature correlated with improved benefit. In silico exonucleasic POLE domain analyses confirmed that all POLEp and 2 VUS clustered in the DNA binding or the Catalytic site. Recategorizing the VUS according to the location within the exonucleasic domain improved the prediction of survival outcomes. Impact: This study gives new insights on how DNA repair defects, mutational burden and signatures sensitize to PD-1 blockade and may offer emerging tumor agnostic biomarkers for benefit to checkpoint blockade. POLE variant pathogenicity All(N=21) POLEnp(N=5) VUS(N=4) POLEp(N=12) Age, years ± SD 57 ± 16 64 ± 10 56 ± 16 54 ± 17 Sex, Male (%) 12 (57) 5 (100) 2 (50) 5 (42) PS (ECOG)=1 (%) 16 (75) 4 (80) 2 (50) 10 (83) Primary tumor Colorectal 9 (43) 2 (40) 2 (50) 5 (42) Endometrial 6 (29) 0 (0) 0 (0) 6 (50) Gastric 2 (9) 2 (40) 0 (0) 0 (0) Glial 1 (5) 0 (0) 0 (0) 1 (8) Biliary tract 1 (5) 0 (0) 1 (25) 0 (0) Pancreas 2 (9) 1 (20) 1 (25) 0 (0) Number of previous treatments 2.4 ± 2 5 ± 2 1.8 ± 1 1.5 ± 1 TMB (mt/Mb, Min-Max)(N=16) 36.2 (2-385) 5 (4-9) 3 (2-4) 114 (25-385) ORR at 12 weeks (CR+PR) 37%(N=7/19) 0%(N=0/5) 50%(N=2/4) 46%(5/10) DCR at 12 weeks (CR+PR+SD) 58%(N=11/19) 0%(N=0/5) 75%(N=3/4) 80%(8/10) Median Progresssion-Free survival (months) 5.6 2.3 10.3vs POLEnp: HR=0.2 IC95% 0.1-0.7 Median Overall Survival (months) 9.1 5.0 Not Reachedvs POLEnp:HR=0.1 IC95% 0.02-0.7 Citation Format: Benoît Rousseau, Ivan Bieche, Eric Pasmant, Nadim Hamzaoui, Nicolas Leulliot, Lucas Michon, Aurelien de Reynies, Mike Foote, Julien Masliah-Planchon, Magali Svrcek, Romain Cohen, Victor Simmet, Paule Augereau, David Malka, Antoine Hollebecque, Damien Pouessel, Carlos Gomez-Roca, Rosine Guimbaud, Amandine Bruyas, Marielle Guillet, Muriel Duluc, Sophie Cousin, Christelle de la Fourchardiere, Frederic Rolland, Sandrine Hiret, Esma Saada-Bouzid, Olivier Bouche, Thierry Andre, Diane Pannier, Farid El Hajbi, Stephane Oudard, Christophe Tournigand, Jean-Charles Soria, Drew Gerber, Dennis Stephens, Michelle Lamandola-Essel, Steven B Maron, Bill Diplas, Guillem Argiles, Asha Krishnan, Neil Segal, Andrea Cercek, Nathalie Hoog-Labouret, Frederic Legrand, Clotide Simon, Assia Lamrani-Ghaouti, Luis A. Diaz, Pierre Saintigny, Sylvie Chevret, Aurelien Marabelle. PD-1 blockade in solid tumors with defects in polymerase epsilon [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 CT021.

The cell(s) of origin of AT/RTs remain(s) unknown. We previously developed a mouse model consisting of tamoxifen inducible system in a Smarcb1Flox/Flox;Rosa26-CreERT2 background. We obtained two molecular subgroups of intracranial tumors, one with neuronal and the other with non-neuronal features, consistent with the diversity observed in human AT/RTs. To investigate the potential cell(s) of origin of these various AT/RTs, we first explored whether different time points of Smarcb1 inactivation correlated with anatomic location and/or molecular subgroups. We observed that the neuronal group, primarily developing from the subventricular zone and the spinal cord, was almost exclusively obtained with the earliest inactivation time points (E6-E7). In contrast, the non-neuronal group emerged upon Smarcb1 inactivation at any time point (E6-E10) and showed intracranial but extra-parenchymal/meningeal origins. High-resolution analysis of anatomic distribution of 55 human AT/RT and molecular subgroups is in progress. In order to more specifically identify the cell(s) of origin for the neuronal group, we next generated developmental stage-specific conditional knockout mice carrying Smarcb1 inactivation by restricting Cre expression with promoters characteristic for various neural stem cells/progenitors. While Smarcb1Flox/Flox;Atoh1CreERT2 showed ataxia but failed to give rise to any tumor at any embryonal time point, Smarcb1Flox/Flox;Ascl1CreERT2 did not show any phenotype. Targeting Nestin-expressing cells led to tumors with morphologic rhabdoid features; these tumors again showed molecular diversity as observed in human AT/RTs. In conclusion, we show that deletion of Smarcb1 determines mouse AT/RT subtypes depending on spatial and temporal factors. Our new mouse models not only give insight into the cell(s) of origin, but also provide interesting preclinical models of AT/RTs. Citation Format: Zhi-Yan Han, Mamy Andrianteranagna, Maria Jesus-Lobon-Iglesias, Arnault Tauziède-Espariat, Kevin Beccaria, Paul Fréneaux, Joshua J. Waterfall, Julien Masliah-Planchon, Christine Bourneix, Gaelle Pierron, Amaury Leruste, Céline Chauvin, Didier Surdez, Pascale Varlet, Christelle Dufour, Olivier Delattre, Volodia Dangouloff-Ros, Franck Bourdeaut. Spatial and temporal conditions for Smarcb1 deletion determines mouse AT/RT (atypical teratoid/rhabdoid tumor) subtype [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B68.

Renal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in young patients or children with sickle cell trait or disease (SCD). Although BAF47 staining is constantly negative in these tumors, the genetic mechanism underlying this phenotype remains unknown, since all cases reported so far show SMARCB1 hemizygous deletion only. By investigating a series of five RMC by gene expression profiling, array-CGH, and RNA and whole-exome sequencing, we demonstrated that SMARCB1 inactivation is related to an original mechanism of inter-chromosomal balanced translocations that disrupt SMARCB1 sequence and thus contribute to its inactivation. RMC were also characterised by a simple genome, with a low mutation rate and a lack of recurrent molecular alterations other than SMARCB1 deficiency, emphasizing its potency as single genetic event to drive oncogenesis. Finally, gene expression profiling revealed that RMC share common oncogenic pathways with pediatric malignant rhabdoid tumors, another lethal tumor subtype characterised by SMARCB1 deficiency Citation Format: Julien Calderaro, Julien Masliah-Planchon, Wilfrid Richer, Laetitia Planque, Pascale Maille, Alexandre de la Taille, Helene Boussion, Olivier Delattre, Yves Allory, Franck Bourdeaut. Balanced translocations disrupting SMARCB1 are hallmark recurrent genetic alterations in renal medullary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A29.