Incidental findings in a series of 2500 gene panel tests for a genetic predisposition to cancer: Results and impact on patients.

With next generation sequencing, physicians are faced with more complex and uncertain data, particularly incidental findings (IF). Guidelines for the return of IF have been published by learned societies. However, little is known about how patients are affected by these results in a context of oncogenetic testing. Over 4 years, 2500 patients with an indication for genetic testing underwent a gene cancer panel. If an IF was detected, patients were contacted by a physician/genetic counsellor and invited to take part in a semi-structured interview to assess their understanding of the result, the change in medical care, the psychological impact, and the transmission of results to the family. Fourteen patients (0.56%) were delivered an IF in a cancer predisposition gene (RAD51C, PMS2, SDHC, RET, BRCA2, CHEK2, CDKN2A, CDH1, SUFU). Two patients did not collect the results and another two died before the return of results. Within the 10 patients recontacted, most of them reported surprise at the delivery of IF, but not anxiety. The majority felt they had chosen to obtain the result and enough information to understand it. They all initiated the recommended follow-up and did not regret the procedure. Information regarding the IF was transmitted to their offspring but siblings or second-degree relatives were not consistently informed. No major adverse psychological events were found in our experience. IF will be inherent to the development of sequencing, even for restricted gene panels, so it is important to increase our knowledge on the impact of such results in different contexts.

In-vivo and in-vitro impact of high-dose rate radiotherapy using flattening-filter-free beams on the anti-tumor immune response.

INTRODUCTION: Modern accelerators have the "flattening filter-free" (FFF) technique to deliver RT with a moderate high-dose rate, currently used in limited clinical indications. No scientifically established data are currently available on the possible effects of this high dose rate on the anti-tumor immune response. We therefore propose here to study these effects in a preclinical CT26 murine colorectal tumor model. MATERIAL AND METHODS: In-vitro, CT26 cells were irradiated on a Varian TrueBeam® linac at 3 different dose rates (4; 12 or 24 Gy/min) using the FFF mode. Activation of the anti-tumor immune response was evaluated by the analysis of induction of genes of the type I interferon pathway by RT-qPCR, and by the study of the induction of immunogenic death biomarkers. In-vivo, an efficacy study of RT delivering 16.5 Gy at 2 different dose rates was performed in immunocompetent Balb/c mice carrying CT26 syngeneic tumors, as well as an immunomonitoring analysed by flow cytometry and a transcriptomic analysis using RNA sequencing. Statistical analyzes were performed using non-parametric tests. RESULTS: In-vitro, no significant influence of an increase in FFF dose rate was shown for the induction of genes of the type I interferon pathway as well as for the studied immunogenic death markers (HMGB1 secretion). In-vivo, no difference in terms of tumor growth retardation between the 2 dose rates used was demonstrated, as well as for the composition of immune cell infiltrates within tumor microenvironment and the expression of immune checkpoints in immunomonitoring and RNAseq. CONCLUSION: In this study involving the CT26 model, no influence of a moderate high dose rate in FFF technique on the anti-tumor immune response was demonstrated, which would make studies of associations between RT and checkpoint inhibitors fit with this technique of RT. However, further explorations using other cellular models seem to be of interest.

A new ER-specific photosensitizer unravels (1)O2-driven protein oxidation and inhibition of deubiquitinases as a generic mechanism for cancer PDT.

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours.

BACKGROUND: Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy. METHODS: Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin. RESULTS: Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin. CONCLUSION: The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

FOXP3 expression in cancer cells and anthracyclines efficacy in patients with primary breast cancer treated with adjuvant chemotherapy in the phase III UNICANCER-PACS 01 trial.

BACKGROUND: Predictive markers of response to chemotherapy are lacking in breast cancer patients. Forkhead Box Protein 3 (FOXP3) is an anti-oncogene whose absence in cancer cells could confer resistance to DNA damaging agent. So we made the hypothesis that FOXP3 expression predicts the response to anthracyclines in breast cancer patients and that adjuvant chemotherapy adding taxanes to anthracyclines confers an overall survival (OS) benefit over anthracyclines alone, in patients with FOXP3-negative tumors. PATIENTS AND METHODS: Expression of FOXP3 in cancer cells was evaluated by immunohistochemistry in tumor samples from 1097 patients who participated in the PACS01 randomized trial that evaluated in adjuvant setting the adjunction of docetaxel (Taxotere) to anthracyclines in patients with localized breast cancer. Kaplan-Meier analysis and Cox regression model were used to assess OS according to the presence or absence of FOXP3 expression in tumor cells. RESULTS: Four hundred and five tumors were found to express FOXP3 (37%). FOXP3 expression in breast cancer cells was associated with better OS (P = 0.003). Uni- and multivariate survival analyses according to treatment arm revealed that FOXP3 expression in breast cancer cells is independently associated with improved OS in patients treated with anthracycline-based adjuvant chemotherapy, but not in patients treated with sequential anthracycline-taxane. Moreover, in vitro experiments showed that FOXP3 induction in breast cancer cell lines using histone deacetylase inhibitor enhances anthracyclines efficacy. CONCLUSION: FOXP3 expression in tumor cells may be an accurate predictive biomarker of anthracycline efficacy in breast cancer.

The transcription factor GATA-1 is overexpressed in breast carcinomas and contributes to survivin upregulation via a promoter polymorphism.

Expression of survivin, a member of the inhibitor of apoptosis protein family, is elevated in human cancers and considered as a new therapeutic target. Mechanism upregulating survivin expression in tumour cells is poorly understood. In this study, we show that breast cancer patients harbouring a polymorphism G235A in the survivin promoter present a higher level of survivin expression. This polymorphism creates a binding site for the transcription factor GATA-1 inducing a second GATA-1-binding site in survivin promoter. At the mRNA level, GATA-1 was present in breast carcinomas and adjacent normal tissues, whereas the protein was only detected in carcinomas by western blot and immunohistochemistry. Transfection of wild-type and different constitutively active GATA-1 mutants (serine 26, 178 or 310) showed that only phospho-serine 26 GATA-1 was able to increase survivin expression. This increase was higher in G235A than in G235G cell lines. Phospho-serine 26 GATA-1 bound directly survivin promoter, with a stronger interaction in G235A than in G235G polymorphism indicating that both GATA-1-binding sites are functional. These data identify GATA-1 as a key feature in tumour aggressiveness by enhancing survivin expression and delineate its targeting as a possible new therapeutic strategy in breast carcinomas.

Gene expression profile and response to trastuzumab-docetaxel-based treatment in breast carcinoma.

BACKGROUND: Resistance to trastuzumab is often observed in women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer and has been shown to involve multiple potential mechanisms. We examined the ability of microarray analyses to determine the potential markers of pathological complete response (pCR). METHODS: We conducted an analysis of tumours from 38 patients with locally advanced HER2-positive breast cancer who had received trastuzumab combined with docetaxel. Quantitative reverse transcriptase (RT)-PCR was used to assess the expression of 30 key genes; microarray analyses were carried out on 25 tumours to identify a prognostic gene expression profile, with 13 blinded samples used to validate the identified profile. RESULTS: No gene was found to correlate with response by RT-PCR. The microarray analysis identified a gene expression profile of 28 genes, with 12 upregulated in the pCR group and 16 upregulated in non-pCR. The leave-one-out cross-validation test exhibited 72% accuracy, 86% specificity, and 55% sensitivity. The 28-gene expression profile classified the 13 validation samples with 92% accuracy, 89% specificity, and 100% sensitivity. CONCLUSION: Our results suggest that genes not involved in classical cancer pathways such as apoptosis or DNA repair could be involved in responses to a trastuzumab-docetaxel-based regimen. They also describe for the first time a gene expression signature that predicts trastuzumab response.

Association of p53 gene alterations with the expression of antiapoptotic survivin splice variants in breast cancer.

Survivin, a member of the inhibitory apoptosis protein family, gives rise, by an alternative splicing, to four variants with different functions. Many experimental studies indicate that p53 can regulate the expression of survivin and some of its splice variants. Although both the expression of survivin splice variants and the p53 gene were frequently altered in human cancers, nothing is known about their interactions in in vivo tumour samples. Here, we report that, in 162 breast carcinomas, p53 mutations are significantly associated with an increased expression of survivin and, in particular, its antiapoptotic splice variants (survivin-DeltaEx3 and survivin-3B). The upregulation of these variant expressions is particularly related to p53 mutations occurring in the residues belonging to the tetramerization domain. The loss of heterozygosity in the p53 gene is also associated with an increased expression of the survivin-DeltaEx3 variant. The expression of the proapoptotic variants (survivin-2B and survivin-2alpha) is not affected by any of these alterations. Our results provide for the first time in vivo evidence that, in human breast cancer, the survivin expression as well as its splicing depends on the p53 status. The results also suggest that the upregulation of antiapoptotic survivin variant expression by the mutant p53 may increase breast cancer cells survival and resistance to therapy.