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Purpose: This retrospective analysis of the largest available clinico-genomic database used de-identified patient-level electronic health record–derived real-world data (RWD) combined with FoundationOne comprehensive genomic profiling (CGP) to characterize patients with metastatic urothelial carcinoma (mUC) treated in the real-world setting, detect potential biomarkers, and develop a bladder immune performance index (BIPI). Experimental Design: Patients with mUC who started front-line single-agent immune checkpoint inhibitors (ICI) and an unmatched group treated with front-line platinum-based chemotherapy between January 1, 2011, and September 30, 2019, were selected. Clinical and genomic data were correlated with overall survival (OS). A novel BIPI predicting outcome with ICIs was developed using machine learning methods and validated using data from a phase II trial (NCT02951767). Results: In ICI-treated patients (n = 118), high tumor mutational burden (≥10 mutations/megabase) was associated with improved OS (HR, 0.58; 95% CI, 0.35–0.95; P = 0.03). In chemotherapy-treated patients (n = 268), those with high APOBEC mutational signature had worse OS (HR, 1.43; 95% CI, 1.06–1.94; P = 0.02). Neither FGFR3 mutations nor DNA damage–repair pathway alterations were associated with OS. A novel BIPI combining clinical and genomic variables (nonmetastatic at initial diagnosis, normal or above normal albumin level at baseline, prior surgery for organ-confined disease, high tumor mutational burden) identified ICI-treated patients with longest OS and was validated in an independent dataset. Conclusions: Contemporary RWD including FoundationOne CGP can be used to characterize outcomes in real-world patients according to biomarkers beyond PD-L1. A validated, novel clinico-genomic BIPI demonstrated satisfactory prognostic performance for OS in patients with mUC receiving front-line ICI therapy.

Summary of analyzed NK receptors.

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF-mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell–mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582–93. ©2017 AACR.

Phenotype and degranulation potential of IL-15 activated NK cells

ERK expression of melanoma cell lines under Vemurafenib treatment.

Vemurafenib and Batimastat modulate the expression of NK cell ligands by melanoma cells.

Effect of Vemurafenib on BRAF non mutated melanoma cell lines

List of antibodies used for flow cytometry.

Supplementary figures and table described in the manuscript

Mutational profile of the BRAF non mutated melanoma cell lines.

Effect of Vemurafenib treatment on melanoma cell lysis.

Supplementary Figure from Clinico-Genomic Characterization of Patients with Metastatic Urothelial Carcinoma in Real-World Practice Identifies a Novel Bladder Immune Performance Index (BIPI)

Supplementary Data from Clinico-Genomic Characterization of Patients with Metastatic Urothelial Carcinoma in Real-World Practice Identifies a Novel Bladder Immune Performance Index (BIPI)

Purpose:This retrospective analysis of the largest available clinico-genomic database used de-identified patient-level electronic health record–derived real-world data (RWD) combined with FoundationOne comprehensive genomic profiling (CGP) to characterize patients with metastatic urothelial carcinoma (mUC) treated in the real-world setting, detect potential biomarkers, and develop a bladder immune performance index (BIPI).Experimental Design:Patients with mUC who started front-line single-agent immune checkpoint inhibitors (ICI) and an unmatched group treated with front-line platinum-based chemotherapy between January 1, 2011, and September 30, 2019, were selected. Clinical and genomic data were correlated with overall survival (OS). A novel BIPI predicting outcome with ICIs was developed using machine learning methods and validated using data from a phase II trial (NCT02951767).Results:In ICI-treated patients (n = 118), high tumor mutational burden (≥10 mutations/megabase) was associated with improved OS (HR, 0.58; 95% CI, 0.35–0.95; P = 0.03). In chemotherapy-treated patients (n = 268), those with high APOBEC mutational signature had worse OS (HR, 1.43; 95% CI, 1.06–1.94; P = 0.02). Neither FGFR3 mutations nor DNA damage–repair pathway alterations were associated with OS. A novel BIPI combining clinical and genomic variables (nonmetastatic at initial diagnosis, normal or above normal albumin level at baseline, prior surgery for organ-confined disease, high tumor mutational burden) identified ICI-treated patients with longest OS and was validated in an independent dataset.Conclusions:Contemporary RWD including FoundationOne CGP can be used to characterize outcomes in real-world patients according to biomarkers beyond PD-L1. A validated, novel clinico-genomic BIPI demonstrated satisfactory prognostic performance for OS in patients with mUC receiving front-line ICI therapy.

Supplementary Figure from Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma

Purpose:Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms.Experimental Design:Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC).Results:We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate.Conclusions:IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

Purpose: Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms. Experimental Design: Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC). Results: We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate. Conclusions: IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

The benefit of EGFR–TKI in non–small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non–small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). We establish here that cisplatin pretreatment specifically sensitizes wild-type EGFR-expressing cells to erlotinib, contrary to what happens in mutant EGFR cells and with a blocking EGFR antibody, both in vitro and in vivo. The sensitization entails the activation of the kinase Src upstream of EGFR, thereafter transactivating EGFR through a ligand-independent activation. We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. These markers might be useful for the selection of patients who would benefit from erlotinib as a maintenance therapy. Mol Cancer Ther; 16(8); 1634–44. ©2017 AACR.

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF-mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell–mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582–93. ©2017 AACR.

Background: Improving drug delivery in oncology by developing antibodies targeted nanoparticles loaded with cytotoxics is a rising strategy in oncology. Determination of the optimal density of antibodies on nanoparticles surface remains an open question, mainly due to the difficulty in measuring accurately the actual level of coating. We have developed a stealth immunoliposome encapsulating docetaxel and harboring anti-Her2 trastuzumab. To better prototyping the nanoparticles, we have developed a method for the absolute quantitation of trastuzumab, so as to compare the performance of the immunoliposomes in terms of efficacy in breast cancer depending on the number of antibodies. Methods: Using flow cytometry we have developed a quick and quantitative assay to measure the amount of trastuzumab coated per docetaxel immunoliposome. Three batches of immunoliposomes exhibiting different densities of trastuzumab were synthesized using the standard thin-film method and a maleimide linker. Quality control was operated regarding size, docetaxel encapsulation and trastuzumab coating levels, including stability studies. In vitro and in vivo efficacy studies were performed on MDA-MB-453 used as a canonical model of Her-2+ human breast cancer cells. Results: The three batches of Immunoliposomes exhibited 330 ± 30 (batch1), 480 ± 110 (batch2) and 690 ± 80 (batch3) trastuzumab IgG molecules per liposome, respectively (p Citation Format: Anne Rodallec, Corentin Franco, Stéphane Robert, Guillaume Sicard, Sarah Giacometti, Ariel Savina, Fanny Bouquet, Bruno Lacarelle, Joseph Ciccolini, Philippe Poncelet, Raphaelle Fanciullino. Impact of trastuzumab coating when prototyping immunoliposomes in breast cancer models: The more the merrier [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 3000.

In HER2-positive breast cancer, the Trastuzumab + Docetaxel doublet has a significant efficacy but is hampered by frequent toxicities that could be addressed with nanoparticles. We have developed an Antibody-Nano Conjugate (ANC), combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab. This entity is expected to improve the efficacy/toxicity balance of this doublet, by improving trafficking and delivery to the tumors. ANCs were synthesized using thin-film method and trastuzumab was grafted via sulfydryl groups on maleimide polyethylene glycol. Two compositions (ANC-1 and ANC-2) were tested and compared (i.e., size, morphology, encapsulation rate, engrafting rate, and stability). Antiproliferative activity was tested on a panel of human breast cancer models ranging fromHER2-negative (MDA-MB-231) to HER2-positive (MDA-MB-453) or -overexpressing (SKBR3), both on 2D and spheroid models. Biodistribution, efficacy and survival were tested in MDAMB-231 and MDA-MB-453 bearing nude mice. ANC-1 presented a greater size (140 ± 3 nm), docetaxel encapsulation rate (73 ± 6 %) and stability as compared with ANC-2. In vitro, both ANCs were found to be more efficient than standard docetaxel + trastuzumab, with a better performance for ANC-1. In vivo, ANC-1 accumulated better in tumors, resulting in a higher reduction in tumor growth (i.e. >+36%) and prolonged survival (i.e. >+33%) as compared with standard treatment. Both in vitro and in vivo results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models. Of note, although modest, even Her2- model (i.e., MDA231) was found to benefit from the nanoparticle. Citation Format: Anne Rodallec, Jean Michel Brunel, Stephane Robert, Sarah Giacometti, Ariel Savina, Fanny Bouquet, Bruno Lacarelle, Joseph Ciccolini, Raphaelle Fanciullino. Characterization, in vitro and in vivo efficacy studies of docetaxel trastuzumab stealth immunoliposome in human breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3709.

About 50% of melanomas carry activating mutation in BRAF or NRAS genes. BRAF/MEK inhibitors elicit a transient effective response but resistance rapidly develops through various MAPK/PI3K/AKT pathway activating mechanisms. DNA Repair mechanisms are regulated by these signaling pathways. We hypothesized that effective inhibition of the MAPK pathway should translate into decrease of DNA Repair capacities. To gain insights into this hypothesis we measured the DNA Repair capacities of 12 melanoma cell lines treated or not by BRAF and MEK inhibitors (respectively Vemurafenib V and Cobimetinib C, Roche laboratories), alone and in combination. We evaluated various excision synthesis repair mechanisms using a multiplexed functional DNA Repair assay using cell extracts. We thus obtained a comprehensive overview of the cell lines DNA Repair capacities. Significant qualitative and quantitative differences were observed between the DNA Repair profiles of the 3 mutation groups in non-treated cells. Globally DNA Repair capacities of BRAFm cells significantly decreased following treatment by V and V+C confirming that excision synthesis repair mechanisms are under the control of the MAPK pathway. When the cell lines were examined individually, interesting features were observed. 3/7 showed drastic decrease of DNA repair with V and V+C treatment. On the contrary, C alone activated repair in 3/7, possibly reflecting a paradoxical activation of some pathways. In addition, among the 3 NRASm cell lines, only 1 exhibited a marked decreased of DNA Repair after C treatment. Surprisingly V and V+C activated some repair activities in one WT cell line, possibly reflecting some paradoxical activation of the complex kinase network. Our results suggest that mutations in signaling kinase pathways impact the so-called DNA Damage Response and translate into specific DNA Repair Enzyme Signatures. Crosstalk of tyrosine kinases with the DNA damage signaling pathway is well known. It is possible to take advantage of their intricate regulation through characterization of DNA Repair Enzymatic Signature to gain information on the inhibition efficacy of targeted drugs. Interestingly this approach could be conducted on clinical samples and we propose to use the DNA Repair Enzyme Signature as a new tool to investigate effectiveness of targeted therapies in metastatic melanoma. This study was sponsored by Institut Roche. Citation Format: Sylvie Sauvaigo, Manel Benkhiat, Florian Braisaz, Florence de Fraipont, Caroline Aspord, Stéphane Mouret, Joël Plumas, Fanny Bouquet, Marie-Thérèse Leccia. DNA Repair Enzyme Signature as a biomarker of MAPK pathway inhibition by targeted therapies in melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2888.

Lung cancer remains one of the most prevalent and deadliest malignancies worldwide. Radiotherapy (RT) is a major therapeutic modality for inoperable non-small cell lung cancer (NSCLC). There is substantial evidence that ionizing radiation triggers activation of TGFβ especially in lung, breast and liver tumor. TGFβ inhibition prior to IR inhibits the DNA damage response in epithelial cells via blockade of ATM kinase activity (Cancer Res 62:5627, 2002; Cancer Res 66:10861, 2006) and sensitizes radiation responses in breast cancer cell lines and tumors (Clin Cancer Res 2011;17:6754-6765.). The current studies test whether TGFβ blockade prior to irradiation modulates radiation response of NSCLC cell lines, NCI-H1299, NCI-H292, H460 and Lewis Lung Cancer (LLC), assessed in vitro and in vivo. All NSCLC cell lines responded to TGFβ by phosphorylation of Smad2, which was blocked by LY364947, a small molecule TGFβ type 1 receptor inhibitor. NCI-H1299 and LLC were insensitive to TGFβ growth inhibition in vitro. LY364947 increased radiosensitivity in NCI-H1299 and H460 in clonogenic assays. Consistent with increased clonogenic cell death, TGFβ inhibition also compromised radiation-induced phosphorylation of H2AX and ATM in NCI-H1299. Treatment of cultured LLC cells with either small molecule TGFβ type 1 receptor inhibitor or TGFβ neutralizing antibody, 1D11, significantly increased LLC radiosensitivity as measured by clonogenic assay. Furthermore, C57BL mice bearing subcutaneous LLC tumors treated before and after RT with 1D11 or 13C4 isotype control antibody (20 mg/kg i.p.) resulted in greater tumor growth delay compared to RT and control antibody. Together, these data indicate that TGFβ inhibition prior to radiation attenuates DNA damage responses, enhances clonogenic cell killing, and promotes tumor growth delay. Given the recognized role of radiation-induced TGFβ in lung fibrosis, the therapeutic index for RT could be significantly increased if circumstances are defined in which TGFβ inhibition during RT both enhances lung cancer radiosensitivity and protects normal lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5715. doi:1538-7445.AM2012-5715