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Supplementary Figure 4

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Supplementary Table 2

Vemurafenib-induced BRAF-CRAF dimerization (2 µM, 24h) detected by in situ PLA in SK-MEL2 cell line is not decreased by the adjunction of MEK inhibitor PD0325901 (1µM, 24h): each red dot represents a BRAF-CRAF heterodimer. The cells were counterstained with DAPI (blue).

BRAF inhibitors (BRAFi) elicit therapeutic responses in metastatic melanoma, but alarmingly, also induce the formation of secondary benign and malignant skin tumors. Here, we report the emergence and molecular characterization of 73 skin and extracutaneous tumors in 31 patients who underwent BRAFi therapy. The majority of patients presented with classic epidermal tumors such as verrucous papillomas, keratoacanthomas, and squamous cell carcinomas (SCC). However, 15 patients exhibited new or rapidly progressing tumors distinct from these classic subtypes, such as lymph node metastasis, new melanomas, and genital and oral mucosal SCCs. Genotyping of the tumors revealed that oncogenic RAS mutations were found in 58% of the evaluable tumor samples (38/66) and 49% of the control tumors from patients not treated with BRAFi (30/62). Notably, proximity ligation assays demonstrated that BRAF–CRAF heterodimerization was increased in fixed tumor samples from BRAFi-treated patients compared with untreated patients. Our findings reveal that BRAF–CRAF complex formation is significantly associated with BRAFi treatment, and may therefore serve as a useful biomarker of BRAFi-induced cutaneous and extracutaneous tumor formation. Cancer Res; 76(6); 1476–84. ©2016 AACR.

Supplementary Figure 1

Supplementary Data - text

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Supplementary Table 3

Tumoral and non tumoral biospies from patients treated with or without BRAF inhibitors: Mutational status, pERK level and BRAF-CRAF PLA score.

Supplementary Table 1

Proximity Ligation Assay principle. Two primary antibodies recognize the two antigens BRAF and CRAF. Specific secondary antibodies (Probes) bind to the primary antibodies and are bound to a unique short DNA. When the PLA probes are in close proximity, the DNA strands can interact. After joining they are amplified via rolling circle amplification (RCA) using a polymerase. The signal is easily made visible as a distinct brown or fluorescent dot after enzymatic conversion of a substrate.

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Supplementary Table 4

Cellular immune defects associated with suboptimal responses to SARS-CoV-2 mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyzed antibody, B cell, CD4+ and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CI). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses and enhances comparatively more Thelper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (TNFα/IL-2 skewing), while others (CCR6, CXCR6, PD-1 and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieve robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.

SUMMARYSpacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare in SARS-CoV-2 naïve donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype and function of CD4+ and CD8+ T cell responses at post-boost memory timepoints. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (P

International audience; The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

SUMMARYSpacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naïve and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in naïve participants only. While boosting did not increase magnitude of CD4+ T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.

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

Patients with anaplastic lymphoma kinase (Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in

// Eric Deutsch 1, 2, 3, 4 , Christine Haie-Meder 1 , Mohamed Amine Bayar 5 , Michele Mondini 4 , Melanie Laporte 6 , Renaud Mazeron 1 , Julien Adam 6 , Andrea Varga 2 , Gilles Vassal 7 , Nicolas Magne 8 , Cyrus Chargari 1, 4 , Emilie Lanoy 5, 9 , Patricia Pautier 2 , Antonin Levy 1, 2, 4 , Jean-Charles Soria 2, 3 1 Department of Radiation Oncology, Gustave Roussy Cancer Campus, Paris-Sud University, Villejuif, France 2 Drug Development Department (DITEP), Gustave Roussy Cancer Campus, Paris-Sud University, Villejuif, France 3 Paris-Sud University, Kremlin-Bicetre Medical University, DHU TORINO, SIRIC SOCRATES, LABEX LERMIT, Le Kremlin-Bicetre, France 4 INSERM U1030 Molecular Radiotherapy, Gustave Roussy Cancer Campus, Villejuif, France 5 Biostatistics and Epidemiology Unit, Gustave Roussy Cancer Campus, Villejuif, France 6 Department of Medical Biology and Pathology, Translational Research Laboratory and Biobank (UMS3655 CNRS/US23 INSERM), INSERM Unit U981, Villejuif, France 7 Department of Clinical Research, Gustave Roussy Cancer Campus, Paris-Sud University, Villejuif, France 8 Department of Radiation Oncology, Institut de Cancerologie de la Loire-Lucien Neuwirth, Saint-Priest en Jarez, France 9 Inserm U1018 Centre for Research in Epidemiology and Population Health, Paris-Sud University, Villejuif, France Correspondence to: Eric Deutsch, email: Eric.DEUTSCH@gustaveroussy.fr Keywords: phase I, HPV, Cidofovir, radiotherapy, cervix cancer Received: October 12, 2015 Accepted: March 06, 2016 Published: March 21, 2016 ABSTRACT Purpose: This phase I trial aimed to assess the safety and determine the recommended Phase II dose (RP2D) of Cidofovir combined with chemoradiotherapy in patients with stage IB2-IVA cervical cancer. Experimental design: Incremental doses (1, 2.5, 5 and 6.5 mg/kg) of IV Cidofovir were administered weekly for two weeks, and then every 2 weeks from the start of chemoradiotherapy to the initiation of utero-vaginal brachytherapy. Biological expression of HPV was analyzed during treatment and tumor response was assessed according to RECIST v1.0 criteria. Results: A total of 15 patients were treated with Cidofovir. Dose-limiting toxicities occurred in 2/6 patients at the 6.5 mg/kg dose level (G3 proteinuria, and G3 acute pyelonephritis with G3 febrile neutropenia). No toxicity occurred at the 5 mg/kg dose level, but only 3 patients received this dose due to trial interruption because of low accrual. The most frequent G3-4 adverse effects observed during the trial were: abdominal pain (n=3), infection (n=2), leuckoneutropenia (n=2), and others (n=6). No toxic death or major renal side effect occurred. The best response was that 8/9 evaluable patients achieved a complete response (89%). In the intention to treat population, the 2-year overall and progression-free survival rates were 93% and 76%, respectively. Biological monitoring of HPV-related markers (decreased p16 expression, and increased p53 and pRb levels) was possible on sequential tumor biopsy samples. The genomic alterations identified were PIK3CA (n=5; one also had a KRAS mutation), and HRAS (n=1) mutations. Conclusion: Cidofovir at a dose of 5mg/kg combined with chemoradiotherapy appeared tolerable and yielded tumor regressions. Due to early trial interruption, the RP2D was not confirmed.

BRAF inhibitors (BRAFi) elicit therapeutic responses in metastatic melanoma, but alarmingly, also induce the formation of secondary benign and malignant skin tumors. Here, we report the emergence and molecular characterization of 73 skin and extracutaneous tumors in 31 patients who underwent BRAFi therapy. The majority of patients presented with classic epidermal tumors such as verrucous papillomas, keratoacanthomas, and squamous cell carcinomas (SCC). However, 15 patients exhibited new or rapidly progressing tumors distinct from these classic subtypes, such as lymph node metastasis, new melanomas, and genital and oral mucosal SCCs. Genotyping of the tumors revealed that oncogenic RAS mutations were found in 58% of the evaluable tumor samples (38/66) and 49% of the control tumors from patients not treated with BRAFi (30/62). Notably, proximity ligation assays demonstrated that BRAF–CRAF heterodimerization was increased in fixed tumor samples from BRAFi-treated patients compared with untreated patients. Our findings reveal that BRAF–CRAF complex formation is significantly associated with BRAFi treatment, and may therefore serve as a useful biomarker of BRAFi-induced cutaneous and extracutaneous tumor formation. Cancer Res; 76(6); 1476–84. ©2016 AACR.

Little is known about mutational landscape of rare breast cancer (BC) subtypes. The aim of the study was to apply next generation sequencing to three different subtypes of rare BCs in order to identify new genes related to cancer progression. We performed whole exome and targeted sequencing of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular BCs. Micropapillary BCs exhibit a profile comparable to common BCs: PIK3CA, TP53, GATA3, and MAP2K4 were the most frequently mutated genes. Metaplastic BCs presented a high frequency of TP53 (78 %) and PIK3CA (48 %) mutations and were recurrently mutated on KDM6A (13 %), a gene involved in histone demethylation. Pleomorphic lobular carcinoma exhibited high mutation rate of PIK3CA (30 %), TP53 (22 %), and CDH1 (41 %) and also presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. Next generation sequencing methods applied on rare cancer subtypes can serve as a useful tool in order to uncover new potential therapeutic targets. Sequencing of pleomorphic lobular carcinoma identified a high rate of alterations in PYGM. These findings emphasize the role of glycogen metabolism in cancer progression.

12038Background: Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. ALK-rearranged NSCLC patients develop resistance ...

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: Rare cancers sometimes present clonal evolution. Genomic characterisation of these rare entities could lead to identify new genes involved in the progression of more frequent cancers. In the present study, we have performed whole exome sequencing of three rare and aggressive histological subtypes in order to identify new targets that could subsequently be validated in common breast cancers. Methods: Whole exome sequencing was applied in ten pleiomorphic lobular carcinoma, seven micropapillary BC and eight metaplastic BC. Each cancer sample was matched with normal DNA from the same patient. Overall 50 samples were therefore sequenced. Tumor samples were selected to present >30% cancer cells. Histological diagnosis was confirmed by a BC pathologist. Whole exome sequencing (WES) was performed by BGI (Bejing Genomic Institute, Hong-Kong, China) using the Illumina Hiseq2000 platform (90bp paired-end reads, depth ≥80x). Genes recurrently mutated (with somatic mutations in >2 cases) were selected for validation. The validation by IonTorrent target sequencing was performed on additional formalin-fixed paraffin-embedded (FFPE) tumor samples (>30% of tumor cells). Results: Complete results of both WES and targeted resequencing validation are available for pleiomorphic lobular carcinoma. Nine of the 10 DNA pairs were eligible for WES. Genes already implicated in BC were significantly mutated: PIK3CA (3/9), TP53 (3/9) and CDH1 (3/9). Novel significantly mutated genes were identified: PYGM (2/9), EMR1 (2/9), ALDH1A2 (2/9), and DCLK1 (2/9). The validation using target gene sequencing was performed on the same 9 cases and on 19 additional confirmed pleiomorphic lobular BC cases (n = 28). For 8 out of the 9 cases with WES data, the results were reproducible on FFPE samples. Validation set confirmed recurrent mutations on PYGM genes (25%). PYGM is involved in glycogen metabolism. Further analyses on gene expression array revealed that PYGM is downregulated in most of the common BC samples, as compared to normal tissue, suggesting a role for this gene in the metabolism of common BC. WES data are available for micropapillary and metaplastic BC. HSPA8 (coding for a Heat shock protein) was found mutated in 3/7 of micropapillary samples. Validation on additional FFPE samples is ongoing and will be presented. WES of metaplastic BC revealed a high frequency of TP53 and PIK3CA mutations (50% in both cases). Conclusions: A novel mutated gene was identified in PLBC. PYGM is a gene involved in glycogen metabolism and was found downregulated in most of the common breast cancer. WES of micropapillary cancers suggests mutations on HSPA8, a gene encoding for a chaperon protein. Validation is ongoing. Deeper sequencing is ongoing for metaplastic BC that may help identifying new mutated genes in this multiclonal disease. Results will be presented. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-4.