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Relationship between JARID2 transcripts levels and classical clinical biological parameters in a series of 454 breast cancers.

Survival curves of two patients groups according to GPR126 overexpression status in the series of 59 MIBC. x-axes represents the time (months). y-axes represents A : Recurrence-free-survival ; B: Overall survival. p=value is the result of log-rank test.

Relationship between ANRIL transcripts levels and classical clinical biological parameters in a series of 456 breast cancers.

Relationship between SUZ12 transcripts levels and classical clinical biological parameters in a series of 456 breast cancers.

The ANRIL pathway.

Relationship between BMI1 transcripts levels and classical clinical biological parameters in a series of 456 breast cancers.

Supplementary Figure from Comprehensive Genome Profiling in Patients With Metastatic Non–Small Cell Lung Cancer: The Precision Medicine Phase II Randomized SAFIR02-Lung Trial

Purpose: EGFR is frequently overexpressed in cervical cancer, suggesting EGFR blockade as a promising treatment approach. Cetuximab, an anti EGFR antibody, used conjointly with radiochemotherapy, was feasible in first-line treatment of cervix carcinoma limited to the pelvis.Experimental Design: This randomized phase II trial enrolled 78 FIGO stage IB2–IIIB cervical cancer patients to either cisplatin-based radiochemotherapy alone (arm B, n = 38) or conjointly with a 6-week course of weekly cetuximab (arm A, n = 40). Brachytherapy was given to the pelvic mass. Primary endpoint was disease-free survival (DFS) at 2 years. EGFR expression and targeted sequencing were performed in 54 of 78 patients.Results: Cetuximab over a 6-week period did not improve DFS at 24 months. At 31 months median follow-up, DFS was not significantly different (P = 0.18). Complete response at 4 to 6 months was strongly predictive for excellent DFS (log-rank test; P < 0.001). PIK3CA, KRAS, and STK11 mutations were observed in 22%, 4%, and 2% of patients, respectively. No tumor with a PI3K pathway mutation showed complete response (0/8 in arm A and 0/6 in arm B), whereas 14 of 52 (27%) tumors without mutations did (P = 0.021). PI3K pathway-mutated tumors showed a trend toward poorer DFS (P = 0.06) following cetuximab (8/22) as compared with those following standard treatment only (6/18).Conclusions: Similar to patients with head and neck cancer, patients with cervical cancer showed no gain in DFS at 2 years following a combined treatment of cetuximab with radiochemotherapy. Although treatment tolerance and compliance were satisfactory, it remains to be demonstrated whether maintenance therapy with cetuximab could be beneficial in selected patient groups. Clin Cancer Res; 21(11); 2530–7. ©2015 AACR.

supplementary table 4. Multivariate analysis for Complete Clinical Response and Figo stage

Supplementary Data from Comprehensive Genome Profiling in Patients With Metastatic Non–Small Cell Lung Cancer: The Precision Medicine Phase II Randomized SAFIR02-Lung Trial

supplementary table 2. Protocol�defined treatment�related Adverse Events

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 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 n = 31; HR, 0.71; 95% CI, 0.31–1.60; Pinteraction = 0.036).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.

supplementary table 3. Response evaluation at 2 time points

supplementary table 1. Radiotherapy and Chemotherapy Treatment dose and Surgery

Supplementary Table from Comprehensive Genome Profiling in Patients With Metastatic Non–Small Cell Lung Cancer: The Precision Medicine Phase II Randomized SAFIR02-Lung Trial

Supplementary Figure Legends 1-2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Supplementary Tables 1-6 from Genome-Wide DNA Methylation Profiling of CpG Islands in Breast Cancer Identifies Novel Genes Associated with Tumorigenicity

Supplementary Figures 2-8 from Genome-Wide DNA Methylation Profiling of CpG Islands in Breast Cancer Identifies Novel Genes Associated with Tumorigenicity

Supplementary Figure 1 from Genome-Wide DNA Methylation Profiling of CpG Islands in Breast Cancer Identifies Novel Genes Associated with Tumorigenicity

Supplementary Figure 1 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Supplementary Figure and Table Legends from Genome-Wide DNA Methylation Profiling of CpG Islands in Breast Cancer Identifies Novel Genes Associated with Tumorigenicity

Supplementary Figure 2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Acquired resistance to protein kinase C (PKC) modulators may explain the failure of clinical trials in patients with cancer. Herein, we established a human colon cancer cell line resistant to PEP005, a drug that inhibits PKCα and activates PKCδ. Colo205-R cells, selected by stepwise exposure to PEP005, were >300-fold more resistant to PEP005 than parental Colo205-S cells and were cross-resistant to phorbol 12-myristate 13-acetate, bryostatin, bistratene A, and staurosporine. No PKCα or PKCδ mutation was detected in Colo205-S and Colo205-R cells. Changes in Colo205-R cells were reminiscent of the epithelial-to-mesenchymal transition (EMT) phenotype. Accordingly, Colo205-R cells were more invasive than Colo205-S in Matrigel assays and in mouse xenografts. We also found an increased mRNA expression of several EMT genes, such as those encoding for transforming growth factor-β and vimentin, along with a decreased mRNA expression of genes involved in epithelial differentiation, such as CDH1 (E-cadherin), CLDN4 (claudin 4), S100A4, and MUC1, in Colo205-R compared with Colo205-S cells in vitro and in vivo. Interestingly, high expression of ET-1 was shown in Colo205-R cells and correlated with low sensitivity to PEP005 and staurosporine in a panel of 10 human cancer cell lines. Inhibition of the ET-1 receptor ETR-A with bosentan restored the antiproliferative effects of PEP005 in Colo205-R cells and decreased the invasive properties of this cell line. Exogenous exposure to ET-1 and silencing ET-1 expression using small interfering RNA modulated cell signaling in Colo205-S and Colo205-R. In summary, acquired resistance to PEP005 was associated with expression of EMT markers and activates the ET-1/ETR-A cell signaling. [Cancer Res 2009;69(10):4260–69]

Epigenetic profiling of tumor DNAs may reveal important new theranostic targets to improve prognosis and treatment of advanced cancer patients. In this study, we performed a genome-wide profile of DNA methylation patterns in sporadic breast tumors by using the HumanMethylation27 BeadChips to assess relationships between DNA methylation changes and patient tumor characteristics. The arrays identified 264 hypermethylated loci/genes present in genomic CpG islands. Hierarchical clustering based on methylation levels divided the specimens into three distinct groups, within which certain clinical features also clustered. Statistically significant differences were determined between overall methylation levels of these clusters and estrogen receptor and progesterone receptor (ER/PR) status (P = 0.001), tumor relapse (P = 0.035), and lymph node metastasis (P = 0.042). We identified several individual methylated genes associated with clinical features, including six genes (RECK, SFRP2, UAP1L1, ACADL, ITR, and UGT3A1) that showed statistical significance between methylation and relapse-free survival. Notably, the RECK gene in this group has been associated in other cancers with poorest prognosis. Among the leading relapse-associated genes and the genes associated with ER/PR status, we sequenced an independent set of paired normal/tumor breast DNA samples to confirm tumor specificity of methylation. Further, we carried out quantitative real-time reverse transcriptase PCR to confirm reduced expression in methylated tumors. Our findings suggest the utility for the DNA methylation patterns in these genes as clinically useful surrogate markers in breast cancer, as well as new molecular pathways for further investigation as therapeutic targets. Cancer Res; 71(8); 2988–99. ©2011 AACR.

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

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.

E-cadherin, a

Background: ESR1 mutations are known drivers of resistance to first line aromatase inhibitors (AI)-based therapy in hormone receptor-positive (HR+) HER2- metastatic breast cancer (mBC) patients (pts), but their clinical actionability remains unknown. The randomized phase 3 PADA-1 trial aimed at evaluating the clinical benefit associated with a switch to fulvestrant-palbociclib upon the detection of a rising ESR1 mutation in blood (bESR1mut) in HR+ HER2- mBC pts treated by first line AI-palbociclib.. Methods: PADA-1 (NCT03079011), a multicenter, randomized, open-label, phase 3 trial, enrolled HR+ HER2- mBC pts with no prior therapy for mBC in the absence of AI-resistance. In the first step, pts were treated with AI-palbociclib as first line therapy and underwent centralized bESR1mut screening every two months. Rising bESR1mut+ pts with no clinical/imaging concomitant disease progression were included in the second step, in which they were randomized between continuing the same therapy (standard arm) or switching to fulvestrant-palbociclib (experimental arm). The third step consisted in an optional crossover to fulvestrant-palbociclib following tumor progression for patients randomized in the standard arm. PADA-1 co-primary endpoints were PFS in the second step, and global safety.. Results: Between 03/2017 and 01/2019, 1,017 pts have been included in the first step. After a median time of 15.6 months in the first step, 172 pts with rising bESR1mut and no synchronous disease progression were randomized to continuing AI-palbociclib (N=84 pts) or to fulvestrant-palbociclib (N=88 pts). Among the 172 randomized pts, N=136 PFS events have been observed in the second step after a median follow-up of 26 months. The median PFS was 5.7 months (95%CI[3.9;7.5]) in the AI-palbociclib arm and 11.9 months (95%CI[9.1;13.6]) in the fulvestrant-palbociclib arm (HR=0.63; 95%CI|0.45-0.88], p=0.007). Among the 70 patients who subsequently developed a disease progression in the AI-palbociclib arm, 47 were included in the optional crossover cohort. With a median follow-up of 14.7 months and 37 events, the median second-PFS observed in the cross-over cohort was 3.5 months (95%CI [2.7-5.1]). Treatment safety and validation of the ESR1mut assay are reported separately (poster session #1). Conclusion: PADA-1 reached its primary objective. This first-of-its-kind liquid biopsy-based trial demonstrates that targeting bESR1mut-associated resistance through a change in the endocrine partner of palbociclib is feasible and allows a doubling in the subsequent median progression free survival.. Funding: Pfizer Citation Format: François-Clément Bidard, Anne-Claire Hardy-Bessard, Thomas Bachelot, Jean-Yves Pierga, Jean-Luc Canon, Florian Clatot, Fabrice Andre, Thibault De La Motte Rouge, Barbara Pistilli, Florence Dalenc, Nadine Dohollou, Olivier Arsene, Thierry Petit, Cécilia Riedl, François Morvan, Adina Marti, Emma Lachaier, Mihaela Achille, Michel Gozy, Anne Escande, Dominique Mille, Fanny Trouboul, Laurent Arnould, Ivan Bieche, Anne Pradines, Jerome Lemonnier, Frederique Berger, Suzette Delaloge. Fulvestrant-palbociclib vs continuing aromatase inhibitor-palbociclib upon detection of circulating ESR1 mutation in HR+ HER2- metastatic breast cancer patients: Results of PADA-1, a UCBG-GINECO randomized phase 3 trial [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 GS3-05.

The use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy.

Missense mutations in the polymerase epsilon (POLE) gene have been reported to generate proofreading defects resulting in an ultramutated genome and to sensitize tumors to checkpoint blockade immunotherapy. However, many POLE-mutated tumors do not respond to such treatment. To better understand the link between POLE mutation variants and response to immunotherapy, we prospectively assessed the efficacy of nivolumab in a multicenter clinical trial in patients bearing advanced mismatch repair–proficient POLE-mutated solid tumors. We found that only tumors harboring selective POLE pathogenic mutations in the DNA binding or catalytic site of the exonuclease domain presented high mutational burden with a specific single-base substitution signature, high T-cell infiltrates, and a high response rate to anti–PD-1 monotherapy. This study illustrates how specific DNA repair defects sensitize to immunotherapy. POLE proofreading deficiency represents a novel agnostic biomarker for response to PD-1 checkpoint blockade therapy. Significance: POLE proofreading deficiency leads to high tumor mutational burden with high tumor-infiltrating lymphocytes and predicts anti–PD-1 efficacy in mismatch repair–proficient tumors. Conversely, tumors harboring POLE mutations not affecting proofreading derived no benefit from PD-1 blockade. POLE proofreading deficiency is a new tissue-agnostic biomarker for cancer immunotherapy. See related video: https://vimeo.com/720727355 This article is highlighted in the In This Issue feature, p. 1397

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.

Synchronous bilateral breast cancer (sBBC) occurs after both breasts have been affected by the same germline genetics, reproductive life factors and environmental exposures for decades. It represents an opportunity to decipher the complex interplay between host, tumor, immune system and response to neoadjuvant chemotherapy (NAC). On a cohort of 17575 BCs treated between 2005 and 2012, sBBCs (n=404) were associated with less aggressive proliferative patterns and higher rates of luminal breast cancers (BCs) when compared with unilateral BCs (n=17171). The left and right tumors were concordant for the majority of clinical and pathological features. Tumor pairs of concordant BC subtype were more frequent than pairs of discordant BC subtype, with notably a particularly high frequency of pairs of luminal BCs. Intriguingly, both the levels of tumor infiltrating lymphocytes (TILs) and the response to NAC were modified by the subtype of the contralateral tumors. Whole exome sequencing and RNAseq analyses revealed that left and right tumors were independent from a somatic mutation and transcriptomic point of view, while primary tumors (PT) before NAC and specimens with residual disease (RD) after NAC were more closely related. The analysis of the TCR repertoire identified very little overlap between patients, while common clones were shared in bilateral tumors within each patient. After NAC, the TCR repertoire of RD was enriched and expanded with clones edited by the contralateral PT.

Gastric cancer (GC) is one of the major causes of cancer-related mortality worldwide. The vast majority of GC cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GCs, often associated with poor overall survival, has constantly increased in USA and Europe The molecular basis of diffuse GC aggressivity remains unclear. Using mRNA from diffuse and intestinal GC tumor samples of a Western cohort, this study reports the expression level of the immunomodulatory aryl-hydrocarbon receptor (AhR), and genes involved in immune suppression (PD1, PD-L1, PD-L2) and the early steps of tryptophan metabolism (IDO1, IDO2, TDO2). Strongly increased expression of IDO1 (p < 0.001) and PD1 (p < 0.003) was observed in the intestinal sub-type. The highest expression of IDO1 and PDL1 correlated with early clinical stage and absence of lymphatic invasion (×25 p = 0.004, ×3 p = 0.04, respectively). Our results suggest that kynurenine, produced by tryptophan catabolism, and AhR activation play a central role in creating an immunosuppressive environment. Correspondingly, as compared to intestinal GCs, expression levels of IDO1-TDO2 and PD-L1 were less prominent in diffuse GCs which also had less infiltration of immune cells, suggesting an inactive immune response in the advanced diffuse GC. Confirmation of these patterns of gene expression will require a larger cohort of early and advanced stages of diffuse GC samples.

Novel anti-EGFR therapies target resistance to standard-of-care anti-EGFR in patients with metastatic lung cancer. We describe tumors at progression versus at the initiation of novel anti-EGFR agents in patients with metastatic lung adenocarcinoma harboring EGFR mutation. This clinical case series reports the histological and genomic features and their evolution following disease progression under amivantamab or patritumab-deruxtecan in clinical trials. All patients had a biopsy at disease progression. Four patients harboring EGFR gene mutations were included. Three of them received anterior anti-EGFR treatment. Median delay to disease progression was 15 months (range: 4–24). At progression, all tumors presented a mutation in the TP53 signaling pathway associated with a loss of heterozygosis (LOH) of the allele in 75% (n = 3), and two tumors (50%) presented an RB1 mutation associated with LOH. Ki67 expression increased above 50% (range 50–90%) in all samples compared to baseline (range 10–30%), and one tumor expressed a positive neuroendocrine marker at progression. Our work reports the potential molecular mechanisms of resistance under novel anti-EGFR in patients with metastatic EGFR-mutated lung adenocarcinoma, with the transformation to a more aggressive histology with acquired TP53 mutation and/or the increase in Ki67 expression. These characteristics are usually found in aggressive Small Cell Lung Cancer.

Per literature, patients with epidermal growth factor receptor (EGFR) exon-20 insertions respond poorly to tyrosine kinase inhibitors (TKIs). This study analyzed real-world data to examine the prognostic and predictive value of these mutations.We conducted a retrospective cohort study using Czech TULUNG Registry data, with data on multiple mutation types, collected in 2011-2020.We analyzed 554 (95.85%) patients with EGFR exon-19 deletions or exon-21 L858R substitutions and 24 (4.15%) patients with exon-20 insertions who received first-line high-value therapies. We summarized clinical characteristics and outcomes in all patients and by cohort. The risk of progression was statistically significantly higher (86%) in the exon-20 insertion cohort compared to the cohort with other mutations. Although not statistically significant, the risk of death was 44% higher in patients with exon-20 insertions.Advanced NSCLC patients with rare EGFR exon-20 insertions have a high risk of progression.

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.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient’s outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Background Endocrine therapies targeting oestrogen signalling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signalling, oestrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumour response in vivo.Methods The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analysed their anti-proliferative effects on 6 ERα + and 3 ERα- PDX models. Genomic and non-genomic oestrogen signalling were assessed by measuring ERα/PI3K interaction by PLA or the expression of key oestrogen target genes by RT-QPCR, respectively.Results We confirmed that ERα/Src and especially ERα/PI3K interaction were associated with a trend to poorer survival. In ERα + tumours, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status or ERα/PI3K targeting. Interestingly, in some ERα- models, fulvestrant alone impacted tumour growth and this was associated with a decrease in ERα/PI3K interaction.Conclusions Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant was clearly associated with a lack of ERα/PI3K targeting in the cytoplasm.

The impact of single-agent antibodies against programmed death-ligand 1 (PD-L1) as maintenance therapy is unknown in patients with metastatic breast cancer. The SAFIR02-BREAST IMMUNO substudy included patients with human epidermal growth factor receptor type 2 (Her2)-negative metastatic breast cancer whose disease did not progress after six to eight cycles of chemotherapy. Patients (n = 199) were randomized to either durvalumab (10 mg kg

Purpose Functional studies have demonstrated that some mutations of ERBB3, which encodes for human epidermal growth factor receptor (HER) 3, are oncogenic via activation of the ErbB family signaling pathway. Significant clinical activity of anti-HER2 therapies (trastuzumab plus lapatinib combination or afatinib) has been reported in patients with ERBB3-mutated cancers. This study was designed to report the rate of activating ERBB3 mutations in small bowel adenocarcinoma (SBA), a rare tumor type in which we previously reported a high rate (12%) of ERBB2-activating mutations. Materials and Methods DNA from 74 SBAs, previously characterized for ERBB2 mutations and mismatch repair status, was submitted for sequencing of ERBB3 exons 3, 6, 7, 8, and 23. Orthogonal validation by targeted next-generation sequencing was performed. Results Four of 74 SBAs (5.4%) displayed ERBB3-activating mutations, including three p.V104M mutations (c.310 G>A) in exon 3 and one p.E928G mutation (c.2783 A>G) in exon 23. No mutations were detected in exons 6, 7, and 8. ERBB3-activating mutations were associated with microsatellite instability ( P = .002) and the presence of ERBB2-activating mutations ( P = .002). Two SBAs with co-occurrence of ERBB2 and ERBB3 mutations were further analyzed by targeted next-generation sequencing. Mutant allelic frequencies suggested that both mutations were shared by the same clone rather than being harbored by mutually exclusive tumor subclones. Conclusion SBAs display a high rate of ERBB3-activating mutations, which have been shown to be targetable by anti-HER2 therapies. Strikingly, ERBB3 was frequently comutated with ERBB2, suggesting a strong oncogenic addiction of these SBAs to the HER2 pathway.

The tumor suppressor gene neurofibromin 1 (NF1) is a major regulator of the RAS‐MAPK pathway. NF1 mutations occur in lung cancer but were not extensively explored. We hypothesized that NF1‐mutated tumors could define a specific population with a distinct clinical and molecular profile. We performed NF1 sequencing using next generation sequencing (NGS) in 154 lung adenocarcinoma surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status, to evaluate the molecular and clinical specificities of NF1‐mutated lung cancers. Clinical data were retrospectively collected, and their associations with molecular profiles assessed. In this series, 24 tumors were NF1 mutated (17.5%) and 11 were NF1 deleted (8%). There was no mutation hotspot. NF1 mutations were rarely associated with other RAS‐MAPK pathway mutations. Most of patients with NF1 alterations were males (74.3%) and smokers (74.3%). Overall survival and disease‐free survival were statistically better in patients with NF1 alterations (N = 34) than in patients with KRAS mutations (N = 30) in univariate analysis. Our results confirm that NF1 is frequently mutated and represents a distinct molecular and clinical subtype of lung adenocarcinoma.