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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.

Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy.We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55).We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

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

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.

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 development of BRAF and MEK inhibitors (BRAFi/MEKi) has led to major advances in melanoma treatment. However, the emergence of resistance mechanisms limits the benefit duration and a complete response occurs in less than 20% of patients receiving BRAFi ± MEKi. In this study, we evaluated the impact of an intermittent versus continuous dosing schedule of BRAF/MEK inhibition in a melanoma model mildly sensitive to a BRAF inhibitor. The combination of a BRAFi with three different MEKi was studied with a continuous or intermittent dosing schedule in vivo, in a xenografted melanoma model and ex vivo using histoculture drug response assays (HDRAs) of patient-derived xenografts (PDX). To further understand the underlying molecular mechanisms of therapeutic efficacy, a biomarker pharmacodynamic readout was evaluated. An equal impact on tumor growth was observed in monotherapy or bitherapy regimens whether we used continuous and intermittent dosing schedules, with no significant differences in biomarkers expression between the treatments. The antitumoral effect was mostly due to modulations of expression of cell cycle and apoptotic mediators. Moreover, ex vivo studies did not show significant differences between the dosing schedules. In this context, our preclinical and pharmacodynamic results converged to show the similarity between intermittent and continuous treatments with either BRAFi or MEKi alone or with the combination of both.

International audience

6520 Background: Real-world progression-free survival (rwPFS) and time to next line of therapy (TTNT) are two endpoints of clinical interest in patients with metastatic breast cancer (MBC). Their validation as intermediate endpoints for overall survival (OS), in a real-world setting, remains not fully established. Methods: We conducted a retrospective cohort study using the nationwide US Electronic Health Record-derived de-identified Flatiron Health database. The study population included pts diagnosed with MBC from Jan 1, 2011 to Feb 30, 2021. rwPFS was defined as the time from start of first-line systemic therapy for MBC to disease progression or death. TTNT was defined as the time from start of first-line systemic therapy until start of next line of therapy. The nonparametric Kendall tau correlation between each surrogate endpoint (rwPFS and TTNT) and OS was evaluated using the Hougaard copula model in the Weibull margin distribution. Kendall’s tau (τ) with its 95%CI was calculated across the entire dataset, and within each disease subgroup defined by receptor (ER and/or PR status defined as hormone receptor [HR], and HER2) status. This work was conducted on behalf of the imCORE network and the Dana-Farber Cancer Institute. Results: Overall, 9,770 patients with MBC were included. Median age was 63 years (IQR: 54-72 years). HR+/HER2- disease represented the most frequent MBC subtype (n=6,287; 64.4%), followed by HER2+ (n=2,096; 21.5%) and triple negative (n=1,387; 14.2%) disease. Median f/u was 41.5 months (95%CI: 40.4 to 42.8). Median OS in the overall population was 32.4 months (95%CI: 31.2 to 33.3). Median rwPFS was 11.5 months (95%CI: 11.1 to 11.9), and median TTNT was 11.1 months (95%CI: 10.7 to 11.5). Across the entire population, the correlation of rwPFS with OS was 0.54 (95%CI: 0.53-0.56), while the correlation of TTNT with OS was 0.47 (95%CI: 0.46-0.48) (Table). Conclusions: rwPFS and TTNT may represent meaningful intermediate endpoints for OS in patients with MBC overall, and within the different disease subgroups. [Table: see text]

Melanoma, the most serious form of skin cancer, frequently involves the dysregulation of key signaling pathways. Treatment strategies presently target the MAPK/ERK pathway, which is overactive in melanomas due in part to BRAF and NRAS mutations, and involve inhibitors against mutated BRAF (vemurafenib or dabrafenib) or MEK kinases (cobimetinib or trametinib), or a combination of the two. Using an established biochip technology, we assessed base excision repair (BER) and nucleotide excision repair (NER) activities in a collection of BRAF mutated (A-375, Colo 829, HT-144, Malme-3M, SK-mel5, SK-mel24 and SK-mel28) and NRAS mutated (M18, MZ2 and SK-mel2) melanoma cell lines, as well as wild-type controls (A7, CHL-1). We evaluated both basal activities (i.e., without treatment) and repair capacities after treatment with vemurafenib or cobimetinib alone, or in combination. Our results indicate that globally the DNA repair capacity of the cell lines was determined by the mutation status of the BRAF and NRAS genes, indicating that the MAPK pathway participates in the regulation of both BER and NER. Treatment of BRAF mutated melanoma cells with vemurafenib alone or the vemurafenib/cobimetinib combination, but not cobimetinib alone, led to reduced DNA repair capacity in about 60% of the BRAF mutated samples, indicating that signaling pathway inhibition can alter DNA repair activity. Upregulation of some DNA repair activities was also observed in several of the treated samples, suggesting activation of compensatory signaling pathways upon treatment. The data collectively indicate that mutations in the BRAF and NRAS genes exert distinct regulatory effects on the excision/synthesis steps of the BER and NER pathways and that targeted pharmacological inactivation of the signaling mechanism can translate into specific consequences in DNA repair capacity. The heterogeneity of the responses reported herein could help define subtypes of melanoma that are associated with resistance to targeted therapies.

Nanoparticles have been used for decades in breast cancer. More recently, anti-human epidermal receptor 2 (Her2) immunoliposomes are of rising interest. However, recent studies have questioned the actual relevance of using anti-Her2 antibodies to improve liposome distribution and efficacy. Using standard thin-film method and maleimide linker, we have synthesized a 140-nm docetaxel-trastuzumab immunoliposome. This nanoparticle was then tested on a canonical Her2-overexpressing breast cancer model (i.e., SKBR3), using 3D spheroids and xenografted mice. Its efficacy was compared with free docetaxel + trastuzumab, liposomal docetaxel + free trastuzumab and to reference antibody-drug conjugate trastuzumab-emtansine (T-DM1). Immunoliposomes resulted in better efficacy as compared with all other treatments, both in vitro and in vivo. To explain such an improvement, immunoliposome biodistribution was investigated using live imaging in xenografted mice. Surprisingly, no difference in tumor uptake was found between anti-Her2 immunoliposomes and standard docetaxel liposomes (i.e., 1.9 ± 1.2 vs. 1.7 ± 0.5% at the end of treatment and 1.4 ± 0.6 vs. 1.6 ± 0.4% at the end of the study, respectively, P > 0.05). We hypothesized that passive targeting (i.e., enhanced permeation and retention effect) contributed more to tumor distribution than active targeting and that the observed differences in efficacy could come from a better internalization of immunoliposomes into Her2+ cells as compared with standard liposomes, and not from a higher specificity towards tumor tissue.

548 Background: Real-world data (RWD) linking clinical outcomes with comprehensive genomic profiling (CGP) may enable identification of biomarkers to guide treatment selection and stratification in future trials. The primary objective was to characterize patients with metastatic urothelial carcinoma (mUC) included in a clinic-genomic database (CGDB), comprised of the electronic health record-derived Flatiron Health database with linked FoundationOne CGP results. As secondary objective, a novel Bladder Immune Prognostic Index (BIPI) was developed. Methods: A retrospective exploratory analysis was performed of de-identified RWD, retrieved from the CGDB. Data from mUC patients starting first-line single-agent immune checkpoint inhibitors (ICIs) and an unmatched group treated with front-line platinum-based chemotherapy (CHT) between Jan 1, 2011, and Sept 30, 2019, were analyzed and correlated with overall survival (OS). Known driver alterations, tumor mutational burden (TMB), and PD-L1 expression were described. A BIPI predicting outcome with ICIs was developed using a Cox-LASSO model and validated externally in a phase II trial (NCT02951767). Results: Of the 1021 patients with mUC identified in CGDB, 118 ICI-treated and 268 CHT-treated patients were included. Median follow-up duration was 9.4 and 14.5 months, respectively. Median OS was 5.4 months (95%CI, 3.3–9.2) with ICIs and 8.2 months (95%CI, 6.8–10.0) with CHT. In ICI-treated patients, low albumin and metastatic disease at initial presentation were associated with worse OS [HR (95%CI) 2.15 (1.18–3.90), p =.012; 2.58 (1.30–5.10), p =.007, respectively] whereas surgery for organ-confined disease and high TMB (≥10 mut/Mb) were associated with improved OS (HR (95%CI) 0.56 (0.36–0.88), p =.012; 0.58 [0.35–0.95]; p=.03), respectively. In CHT-treated patients, those with high APOBEC had worse OS (HR 1.43 [95% CI, 1.06–1.94]; p=.02). Neither PD-L1 (HR 0.96 [0.37-2.46]; p =.93), FGFR3 mutations (HR 0.98 [0.65-1.47]; p =.92) nor DNA damage-repair pathway alterations (HR 1.06 [0.73-1.52]; p =.77) were associated with OS. A novel BIPI for ICI-treated patients combining clinical and genomic variables (non-metastatic at initial diagnosis, normal albumin level, previous surgery for organ-confined disease, high TMB) was developed. Patients were categorized in 3 groups (low, intermediate, high risk) which correlated with OS. Median OS (95%CI) for low, intermediate and high risk was 11.7 (8.9−17.7), 4.1 (2.5–NE) and 2.4 months (1.0–4.0), (p

Anne Rodallec,1 Guillaume Sicard,1 Sarah Giacometti,1 Manon Carré,1 Bertrand Pourroy,2 Fanny Bouquet,3 Ariel Savina,3 Bruno Lacarelle,1 Joseph Ciccolini,1 Raphaelle Fanciullino1 1SMARTc Unit, Laboratory of Pharmacokinetics and Toxicology UFR Pharmacy, Center for Research on Cancer of Marseille, Inserm UMR1068, CNRS UMR7258, Aix Marseille University, Marseille, France; 2Pharmacy Department, APHM La Conception, Marseille, France; 3Roche Institute, Boulogne Billancourt, France Purpose: Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo. Materials and methods: Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging. Results: In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and invivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments. Conclusion: We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies. Keywords: docetaxel, trastuzumab, breast cancer, immunoliposome, spheroids, distribution, tumor xenograftErratumfor this paper has been published

Background Trastuzumab plus docetaxel is a mainstay to treat HER2-positive breast cancers. However, developing nanoparticles could help to improve the efficacy/toxicity balance of this doublet by improving drug trafficking and delivery to tumors. This project aimed to develop an immunoliposome in breast cancer, combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab, and comparing its performances on human breast cancer cell lines with standard combination of docetaxel plus trastuzumab. Methods Several strategies to engraft trastuzumab to pegylated liposomes were tested. Immunoliposomes made of natural (antibody nanoconjugate-1 [ANC-1]) and synthetic lipids (ANC-2) were synthesized using standard thin film method and compared in size, morphology, docetaxel encapsulation, trastuzumab engraftment rates and stability. Antiproliferative activity was tested on human breast cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to overexpressing (SKBR3) HER2. Finally, cell uptake of ANC-1 was studied by electronic microscopy. Results ANC-1 showed a greater docetaxel encapsulation rate (73%±6% vs 53%±4%) and longer stability (up to 1 week) as compared with ANC-2. Both ANC presented particle size ≤150 nm and showed similar or higher in vitro antiproliferative activities than standard treatment, ANC-1 performing better than ANC-2. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7 and 6±2 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. The IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were 1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. Cellular uptake appeared to depend on HER2 expression, the higher the expression, the higher the uptake. Conclusion In vitro results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models using nanoparticles., Video abstract

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.

Purpose Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo. Materials and methods Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging. Results In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and in vivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments. Conclusion We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies.

International audience; PURPOSE:The primary objective of this study was to compare the ability of PET and MRI biomarkers to predict treatment efficacy in a preclinical model of recurrent glioblastoma multiforme.METHODS:MRI (anatomical, diffusion, vasculature and oxygenation) and PET ([(18)F]FDG and [(18)F]FLT) parameters were obtained 3 days after the end of treatment and compared with late tumour growth and survival.RESULTS:Early after tumour recurrence, no effect of treatment with temozolomide combined with bevacizumab was observed on tumour volume as assessed by T2-W MRI. At later times, the treatment decreased tumour volume and increased survival. Interestingly, at the earlier time, temozolomide + bevacizumab decreased [(18)F]FLT uptake, cerebral blood volume and oedema. [(18)F]FLT uptake, oedema and cerebral blood volume were correlated with overall survival but [(18)F]FLT uptake had the highest specificity and sensitivity for the early prediction of treatment efficacy.CONCLUSION:The present investigation in a preclinical model of glioblastoma recurrence underscores the importance of multimodal imaging in the assessment of oedema, tumour vascular status and cell proliferation. Finally, [(18)F]FLT holds the greatest promise for the early assessment of treatment efficacy. These findings may translate clinically in that individualized treatment for recurrent glioma could be prescribed for patients selected after PET/MRI examinations.

Over 60% of human melanoma tumors bear a mutation in the

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.

Ataxia Telangiectasia (AT) is an autosomal recessive disorder characterized by a wide variety of progressive clinical symptoms. This includes neuronal degeneration, oculocutaneous telangiectasias, diabetes mellitus, immunodeficiency, increased risk of cancer and sensitivity to ionizing radiation. The gene mutated in this disease, ATM (Ataxia Telangiectasia Mutated), encodes a protein kinase involved in DNA double strand breaks signalling and repair. ATM deficient cells also display an increase in oxidative stress, by poorly characterized mechanism(s), which clearly contributes to the neurodegenerative aspect of the disease. Despite these advances, the occurrence of the vascular abnormalities, glucose intolerance and insulin resistance remains poorly understood. In different cellular models where ATM expression was disrupted, we demonstrated that the absence of ATM leads to an increased expression of both subunits of the transcription factor Hypoxia Inducible Factor 1 (HIF-1). We also observed enhanced trans-activating functions of HIF-1. HIF-1 is the central regulator of responses to hypoxia which induces the transcription of genes involved in angiogenesis (e.g., VEGF-Vascular Endothelial Growth Factor) and cellular metabolism (e.g., GLUT-1). Interestingly, we demonstrated that ATM disruption positively regulates both expression and function of the basal glucose transporter GLUT-1 as well as the proangiogenic factor, VEGF. In addition, our results suggest that the absence of ATM increases HIF-1 proteins biosynthesis, and this effect is dependant on the oxidative stress existing in ATM deficient cells. Our compelling results highlight a new link between ATM deficiency and the clinical features of the disease and provide a molecular link between ATM downregulation and the increase in tumor angiogenesis observed in human breast cancers.

e21559Background: Vismodegib , a selective Hh pathway inhibitor that targets Smoothened (SMO) is a major therapeutic breakthrough for locally advanced (la) or metastatic BCC. During treatment some ...

The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to high toxicity and genetic instability. Various approaches have been undertaken to quantify the number of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation of the variant histone H2AX (named gammaH2AX), quantified by specific immunodetection approaches, has provided a valuable and highly sensitive method to monitor DSBs formation. Although it is admitted that the number of gammaH2AX foci reflected that of DSBs, contradictory reports leave open the question of a link between the disappearance of gammaH2AX signal and DSBs repair. We determined gammaH2AX expression (i) in cells either proficient or not in DSBs repair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin gamma1, a radiomimetic compound, (iii) and by three different immunodetection methods, foci numbering, flow cytometry or Western blotting. We showed here that gammaH2AX loss correlates with DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaks per genome are produced, independently of the method used. In addition, in DNA repair proficient cells, the early decrease in the number and intensity of gammaH2AX foci observed after a 2 Gy exposure was not associated with a significant change in the global gammaH2AX level as determined by Western blotting or flow cytometry. These results suggest that the dephosphorylation step of gammaH2AX may be limiting and that the loss of foci is mediated not only by gammaH2AX dephosphorylation but also through its redistribution towards the chromatin.

Purpose: To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. Experimental Design: TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Results: Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. Conclusion: These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. Clin Cancer Res; 17(21); 6754–65. ©2011 AACR.

DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1–1% O2) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4–DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways.

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