Molecular features of untreated breast cancer and initial metastatic event inform clinical decision-making and predict outcome: long-term results of ESOPE, a single-arm prospective multicenter study.

BACKGROUND: Prognosis evaluation of advanced breast cancer and therapeutic strategy are mostly based on clinical features of advanced disease and molecular profiling of the primary tumor. Very few studies have evaluated the impact of metastatic subtyping during the initial metastatic event in a prospective study. The genomic landscape of metastatic breast cancer has mostly been described in very advanced, pretreated disease, limiting the findings transferability to clinical use. METHODS: We developed a multicenter, single-arm, prospective clinical trial in order to address these issues. Between November 2010 and September 2013, 123 eligible patients were included. Patients at the first, untreated metastatic event were eligible. All matched primary tumors and metastatic samples were centrally reviewed for pathological typing. Targeted and whole-exome sequencing was applied to matched pairs of frozen tissue. A multivariate overall survival analysis was performed (median follow-up 64 months). RESULTS: Per central review in 84 patients (out of 130), we show that luminal A breast tumors are more prone to subtype switching. By combining targeted sequencing of a 91 gene panel (n = 67) and whole-exome sequencing (n = 30), a slight excess of mutations is observed in the metastases. Luminal A breast cancer has the most heterogeneous mutational profile and the highest number of mutational signatures, when comparing primary tumor and the matched metastatic tissue. Tumors with a subtype change have more mutations that are private. The metastasis-specific mutation load is significantly higher in late than in de novo metastases. The most frequently mutated genes were TP53 and PIK3CA. The most frequent metastasis-specific druggable genes were PIK3CA, PTEN, KDR, ALK, CDKN2A, NOTCH4, POLE, SETD2, SF3B1, and TSC2. Long-term outcome is driven by a combination of tumor load and metastasis biology. CONCLUSIONS: Profiling of the first, untreated, metastatic event of breast cancer reveals a profound heterogeneity mostly in luminal A tumors and in late metastases. Based on this profiling, we can derive information relevant to prognosis and therapeutic intervention, which support current guidelines recommending a biopsy at the first metastatic relapse. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov ( NCT01956552 ).

Biopathological Significance of PIWI-piRNA Pathway Deregulation in Invasive Breast Carcinomas.

The PIWI proteins emerging in the development of human cancers, edify PIWI-piRNA ribonucleoproteic complexes acting as pivotal regulators of genome integrity, differentiation and homeostasis. The aim of this study is to analyze the four PIWILs gene expression in invasive breast carcinomas (IBCs): at RNA level using quantitative RT-PCR (n = 526) and protein level using immunohistochemistry (n = 150). In normal breast tissue, PIWILs 2 and 4 were solely expressed, whereas an abnormal emergence of PIWIL1 and 3 was observed in respectively 30% and 6% of IBCs. Conversely, PIWIL2 was underexpressed in 48.3% and PIWIL4 downregulated in 43.3% of IBCs. Significant positive associations were observed between PIWIL4 underexpression, HR+ status and HR+ ERBB2+ molecular subtype and PIWIL2 underexpression, PR- status, ERBB2- status and molecular subtype. Similar patterns of PIWIL deregulation were observed in a multitumoral panel, suggesting a generic mechanism in most cancers. PIWIL2-4 underexpression was mainly regulated at epigenetic or post-transcriptional levels. PIWIL2 underexpression was significantly associated with DNA methylation and strong cytotoxic immune response. PIWIL2-4 were mainly associated with genes implicated in cell proliferation. As a result of this study, characterization of the PIWIL-piRNA pathway in IBCs opens interesting therapeutic perspectives using piRNAs, hypomethylating drugs, checkpoints immunotherapies and anti-PIWIL 1-3 antibodies.

Genomic Instability Signature of Palindromic Non-Coding Somatic Mutations in Bladder Cancer.

Numerous pan-genomic studies identified alterations in protein-coding genes and signaling pathways involved in bladder carcinogenesis, while non-coding somatic alterations remain weakly explored. The goal of this study was to identify clinical biomarkers in non-coding regions for bladder cancer patients. We have previously identified in bladder tumors two non-coding mutational hotspots occurring at high frequencies (≥30%). These mutations are located close to the GPR126 and PLEKHS1 genes, at the guanine or the cytosine of a TGAACA core motif flanked, on both sides, by a stretch of palindromic sequences. Here, we hypothesize that such a pattern of recurrent non-coding mutations could be a signature of somatic genomic instability specifically involved in bladder cancer. We analyzed 26 additional mutable non-coding sites with the same core motif in a cohort of 103 bladder cancers composed of 44 NMIBC cases and 59 MIBC cases using high-resolution melting (HRM) and Sanger sequencing. Five bladder cancers were additionally analyzed for protein-coding gene mutations using a targeted NGS panel composed of 571 genes. Expression levels of three members of the APOBEC3 family genes were assessed using real-time quantitative RT-PCR. Non-coding somatic mutations were observed for at least one TGAACA core motif locus in 62.1% (64/103) of bladder tumor samples. These non-coding mutations co-occurred in the bladder tumors but were absent in prostate tumor, HPV-positive Head and Neck Squamous Cell Carcinoma, and high microsatellite instability (MSI-H) colorectal tumor series. This signature of palindromic non-coding somatic mutations, specific to bladder tumors, was not associated with patients' outcome and was more frequent in females. Interestingly, this signature was associated with high tumor mutational burden (TMB) and high expression levels of APOBEC3B and interferon inducible genes. We identified a new type of somatic genomic instability targeting the TGAACA core motif loci flanked by palindromic sequences in bladder cancer. This mutational signature is a promising candidate clinical biomarker for the early detection of relapse and a major low-cost alternative to the TMB to monitor the response to immunotherapy for bladder cancer patients.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Immune gene expression in head and neck squamous cell carcinoma patients.

BACKGROUND: Nivolumab and pembrolizumab targeting programmed cell death protein 1 (PD-1) have recently been approved among patients with recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) who failed platinum therapy. We aimed to evaluate the prognostic value of selected immune gene expression in HNSCC. PATIENTS AND METHODS: We retrospectively assessed the expression of 46 immune-related genes and immune-cell subpopulation genes including immune checkpoints by real-time polymerase chain reaction among 96 patients with HNSCC who underwent primary surgery at Institut Curie between 1990 and 2006. Univariate and multivariate analyses were performed to assess the prognostic value of dysregulated genes. RESULTS: The Median age of the population was 56 years [range: 35-78]. Primary tumour location was oral cavity (45%), oropharynx (21%), larynx (18%) and hypopharynx (17%). Twelve patients (13%) had an oropharyngeal human papillomavirus-positive tumour. Most significantly overexpressed immune-related genes were TNFRSF9/4-1BB (77%), IDO1 (75%), TNFSF4/OX40L (74%) and TNFRSF18/GITR (74%), and immune-cell subpopulation gene was FOXP3 (62%). Eighty-five percent of tumours analysed overexpressed actionable immunity genes, including PD-1/PD-L1, TIGIT, OX40/OX40L and/or CTLA4. Among the immune-related genes, high OX40L mRNA level (p = 0.0009) and low PD-1 mRNA level (p = 0.004) were associated with the highest risk of recurrence. Among the immune-cell subpopulation genes, patients with high PDGFRB mRNA level (p < 0.0001) and low CD3E (p = 0.0009) or CD8A mRNA levels (p = 0.004) were also at the highest risk of recurrence. CONCLUSIONS: OX40L and PDGFRB overexpression was associated with poor outcomes, whereas PD-1 overexpression was associated with good prognosis in patients with HNSCC treated with primary surgery, suggesting their relevance as potential prognostic biomarkers and major therapeutic targets.

High Positive Correlations between ANRIL and p16-CDKN2A/p15-CDKN2B/p14-ARF Gene Cluster Overexpression in Multi-Tumor Types Suggest Deregulated Activation of an ANRIL-ARF Bidirectional Promoter.

The CDKN2B-AS1 gene, also called ANRIL, is located at the human CDKN2A/B locus at 9p21.3 and transcribed by RNA polymerase II into a long non-coding RNA of 3834 bp. The CDKN2B-AS1 gene overlaps a critical region of 125 kb covering the CDKN2B gene. The CDKN2A/B locus encompasses three major tumor suppressors juxtaposed and joined into a p16-CDKN2A/p15-CDKN2B/p14-ARF gene cluster. CDKN2A encodes splice variants p16-CDKN2A and p14-ARF, and CDKN2B encodes p15-CDKN2B. ANRIL shares a bidirectional promoter with the p14-ARF gene and is transcribed from the opposite strand to the cluster. We performed an analysis of the expression level of ANRIL and tumor suppressor p16-CDKN2A, p15-CDKN2B, and p14-ARF genes using quantitative RT-PCR in a multitumor panel. We observed the overexpression of the four genes ANRIL, p16-CDKN2A, p15-CDKN2B, and p14-ARF in the great majority of the 17 different cancer types. ANRIL was upregulated in 13/17 tumors compared to normal tissues, ranging from 5% (prostate cancer) to 91% (cervix cancer), with variable expression of p16-CDKN2A, p15-CDKN2B, and p14-ARF genes. A high positive correlation was identified between levels of expression of ANRIL and the three tumor suppressors. The strongest positive association was observed with p14-ARF (p < 0.001) in all but one (lung squamous cell carcinoma) of the examined tumor types. This correlation suggests coordinated deregulated mechanisms in all cancer types through aberrant activation of a bidirectional p14-ARF/ANRIL promoter. Furthermore, significant positive correlation was unexpectedly established in prostatic carcinomas, in contradiction with previous data.

Differential gene expression in growth factors, epithelial mesenchymal transition and chemotaxis in the diffuse type compared with the intestinal type of gastric cancer.

Gastric cancer (GC) is a highly heterogeneous disease and one of the major causes of cancer-related mortality worldwide. Diffuse-type gastric adenocarcinoma (or poorly cohesive- with independent cells) is characterized by aggressive behavior (rapid invasion, chemoresistance and peritoneal metastasis), as compared with intestinal-subtype adenocarcinoma. Diffuse subtype GC additionally has a substantially increasing incidence rate in Europe and the USA, and was often associated with younger age. Our objective was to analyze the expression and clinical significance of genes involved in several signaling pathways in diffuse-type GC. Tumors samples and non-malignant gastric tissues were obtained from patients with GC (diffuse-type and intestinal-subtype adenocarcinoma). The expression of 33 genes coding for proteins involved in four categories, growth factors and receptors, epithelial-mesenchymal transition, cell proliferation and migration, and angiogenesis was determined by reverse transcription-quantitative polymerase chain reaction. The expression of 22 genes was significantly upregulated in diffuse-type GC and two were downregulated (including CDH1) compared with normal tissues. Among these genes, acompared with intestinal-subtype adenocarcinoma, diffuse-type GC revealed elevated levels of IGF1 and IGF1R, FGF7 and FGFR1, ZEB2, CXCR4, CXCL12 and RHOA, and decreased levels of CDH1, MMP9 and MKI67. The expression of selected genes was compared with other genes and according to clinical parameters. Furthermore, TGF-ß expression was significantly increased in linitis, a sub-population of diffusely infiltrating type associated with extensive fibrosis and tumor invasion. Our study identified new target genes (IGF1, FGF7, CXCR4, TG-ß and ZEB2) whose expression is associated with aggressive phenotype of diffuse-type GC.

Inhibition of PI3K pathway increases immune infiltrate in muscle-invasive bladder cancer.

Although immune checkpoint inhibitors have shown improvement in survival in comparison to chemotherapy in urothelial bladder cancer, many patients still fail to respond to these treatments and actual efforts are made to identify predictive factors of response to immunotherapy. Understanding the tumor-intrinsic molecular basis, like oncogenic pathways conditioning the presence or absence of tumor-infiltrating T cells (TILs), should provide a new rationale for improved anti-tumor immune therapies. In this study, we found that urothelial bladder cancer from human samples bearing PIK3CA gene mutations was significantly associated with lower expression of a defined immune gene signature, compared to unmutated ones. We identified a reduced 10-gene immune gene signature that discriminates muscle-invasive bladder cancer (MIBC) samples according to immune infiltration and PIK3CA mutation. Using a humanized mouse model, we observed that BKM120, a pan-PI3K inhibitor, significantly inhibited the growth of a human bladder cancer cell line bearing a PIK3CA mutation, associated to increased immune cell infiltration (hCD45+). Using qRT-PCR, we also found an increase in the expression of chemokines and immune genes in PIK3CA-mutated tumors from mice treated with BKM120, reflecting an active immune infiltrate in comparison to untreated ones. Moreover, the addition of BKM120 rendered PIK3CA-mutated tumors sensitive to PD-1 blockade. Our results provide a relevant rationale for combination strategies of PI3K inhibitors with immune checkpoint inhibitors to overcome resistance to immune checkpoint inhibitors.

Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility.

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.

High Prevalence of a Hotspot of Noncoding Somatic Mutations in Intron 6 of GPR126 in Bladder Cancer.

Numerous pangenomic studies identified protein-coding genes and signaling pathways involved in bladder carcinogenesis. However, noncoding somatic alterations remain unexplored. A recent study revealed a mutational hotspot in intron 6 of GPR126 gene in 2.7% of a large breast cancer series. As GPR126 is highly expressed in bladder tissues, we investigated here the prevalence and the prognostic significance of these mutations in bladder cancer. We analyzed a cohort of 103 bladder cancers including 44 nonmuscle-invasive bladder cancers (NMIBC) and 59 muscle-invasive bladder cancers (MIBC). GPR126 mutations were analyzed by high-resolution melting and Sanger sequencing, and GPR126 expression levels were assessed using real-time quantitative RT-PCR. In NMIBC, somatic GPR126 noncoding mutations occurred in 47.7% of samples and were negatively associated with GPR126 mRNA levels. GPR126 mutations had higher frequencies in nonsmoker patients and were associated with a prior history of NMIBC. GPR126 overexpression was detected in 70.5% of samples. GPR126 mutation and overexpression status were not associated with outcome. In MIBC, somatic GPR126 mutations occurred in 44.1% of samples. Mutations were more frequent in females. GPR126 overexpression was detected in 27.1% of the sample. A trend toward significance was observed between GPR126 overexpression and better outcome. We identified the second most frequent mutational hotspot after TERT promoter (∼70%) in bladder cancer, with a mutation rate of approximately 50%. IMPLICATIONS: The GPR126 intronic mutational hotspot could be a promising clinical biomarker candidate to monitor tumor burden using circulating tumor DNA in bladder cancer.

Molecular profiling of hormone receptor-positive, HER2-negative breast cancers from patients treated with neoadjuvant endocrine therapy in the CARMINA 02 trial (UCBG-0609).

BACKGROUND: Postmenopausal women with large, hormone receptor (HR)-positive/HER2-negative and low-proliferative breast cancer derived a benefit from neoadjuvant endocrine therapy (NET) in the CARMINA02 trial. This study was designed to correlate gene expression and mutation profiles with both response to NET and prognosis. METHODS: Gene expression profiling using RNA sequencing was performed in 86 pre-NET and post-NET tumor samples. Targeted next-generation sequencing of 91 candidate breast cancer-associated genes was performed on DNA samples from 89 patients. Molecular data were correlated with radiological response and relapse-free survival. RESULTS: The transcriptional profile of tumors to NET in responders involved immune-associated genes enriched in activated Th1 pathway, which remained unchanged in non-responders. Immune response was confirmed by analysis of tumor-infiltrating lymphocytes (TILs). The percentage of TILs was significantly increased post-NET compared to pre-NET samples in responders (p = 0.0071), but not in non-responders (p = 0.0938). Gene expression revealed that lipid metabolism was the main molecular function related to prognosis, while PPARγ is the most important upstream regulator gene. The most frequently mutated genes were PIK3CA (48.3%), CDH1 (20.2%), PTEN (15.7%), TP53 (10.1%), LAMA2 (10.1%), BRCA2 (9.0%), MAP3K1 (7.9%), ALK (6.7%), INPP4B (6.7%), NCOR1 (6.7%), and NF1 (5.6%). Cell cycle and apoptosis pathway and PIK3CA/AKT/mTOR pathway were altered significantly more frequently in non-responders than in responders (p = 0.0017 and p = 0.0094, respectively). The average number of mutations per sample was significantly higher in endocrine-resistant tumors (2.88 vs. 1.64, p = 0.03), but no difference was observed in terms of prognosis. ESR1 hotspot mutations were detected in 3.4% of treatment-naive tumors. CONCLUSIONS: The Th1-related immune system and lipid metabolism appear to play key roles in the response to endocrine therapy and prognosis in HR-positive/HER2-negative breast cancer. Deleterious somatic mutations in the cell cycle and apoptosis pathway and PIK3CA/AKT/mTOR pathway may be relevant for clinical management. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov ( NCT00629616 ) on March 6, 2008, retrospectively registered.

Prognostic Impact of Residual HPV ctDNA Detection after Chemoradiotherapy for Anal Squamous Cell Carcinoma.

Purpose: Chemoradiotherapy (CRT) is the current standard of care for patients diagnosed with locally advanced anal squamous cell carcinoma (ASCC), but some patients develop local and/or distant relapse during follow-up. This study was designed to monitor human papillomavirus (HPV) circulating tumor DNA (ctDNA) levels during CRT in patients with ASCC.Experimental Design: We analyzed samples from patients with HPV16- or HPV18-positive locally advanced ASCC. Blood samples were collected before and after CRT. HPV16 or HPV18 ctDNA detection was performed by droplet digital-PCR.Results: HPV ctDNA was detected before CRT in 29 of 33 patients with stages II-III ASCC [sensitivity: 88%; 95% confidence interval (CI), 72-95]; ctDNA positivity rate was associated with tumor stage (64% and 100% in stages II and III, respectively; P = 0.008). Among ctDNA-positive patients at baseline, ctDNA levels were higher in N+ than in N- tumors (median 85 copies/mL, range = 8-9,333 vs. 32 copies/mL, range = 3-1,350; P = 0.03). ctDNA detection at baseline had no significant prognostic impact. After CRT, three of 18 (17%) patients displayed residual detectable HPV ctDNA; ctDNA detection after CRT was strongly associated with shorter disease-free survival (P < 0.0001).Conclusions: This is the first proof-of-concept study assessing the prognostic value of ctDNA after CRT in locally advanced ASCC. In most patients, HPV ctDNA can be detected before CRT and becomes undetectable during CRT. In this study, we show that residual ctDNA levels after CRT are associated with very poor outcome. Clin Cancer Res; 24(22); 5767-71. ©2018 AACR.

Morphology and genomic hallmarks of breast tumours developed by ATM deleterious variant carriers.

BACKGROUND: The ataxia telangiectasia mutated (ATM) gene is a moderate-risk breast cancer susceptibility gene; germline loss-of-function variants are found in up to 3% of hereditary breast and ovarian cancer (HBOC) families who undergo genetic testing. So far, no clear histopathological and molecular features of breast tumours occurring in ATM deleterious variant carriers have been described, but identification of an ATM-associated tumour signature may help in patient management. METHODS: To characterise hallmarks of ATM-associated tumours, we performed systematic pathology review of tumours from 21 participants from ataxia-telangiectasia families and 18 participants from HBOC families, as well as copy number profiling on a subset of 23 tumours. Morphology of ATM-associated tumours was compared with that of 599 patients with no BRCA1 and BRCA2 mutations from a hospital-based series, as well as with data from The Cancer Genome Atlas. Absolute copy number and loss of heterozygosity (LOH) profiles were obtained from the OncoScan SNP array. In addition, we performed whole-genome sequencing on four tumours from ATM loss-of-function variant carriers with available frozen material. RESULTS: We found that ATM-associated tumours belong mostly to the luminal B subtype, are tetraploid and show LOH at the ATM locus at 11q22-23. Unlike tumours in which BRCA1 or BRCA2 is inactivated, tumours arising in ATM deleterious variant carriers are not associated with increased large-scale genomic instability as measured by the large-scale state transitions signature. Losses at 13q14.11-q14.3, 17p13.2-p12, 21p11.2-p11.1 and 22q11.23 were observed. Somatic alterations at these loci may therefore represent biomarkers for ATM testing and harbour driver mutations in potentially 'druggable' genes that would allow patients to be directed towards tailored therapeutic strategies. CONCLUSIONS: Although ATM is involved in the DNA damage response, ATM-associated tumours are distinct from BRCA1-associated tumours in terms of morphological characteristics and genomic alterations, and they are also distinguishable from sporadic breast tumours, thus opening up the possibility to identify ATM variant carriers outside the ataxia-telangiectasia disorder and direct them towards effective cancer risk management and therapeutic strategies.

Involvement of the FOXO6 transcriptional factor in breast carcinogenesis.

In mammals, FOXO transcriptional factors form a family of four members (FOXO1, 3, 4, and 6) involved in the modulation proliferation, apoptosis, and carcinogenesis. The role of the FOXO family in breast cancer remains poorly elucidated. According to the cellular context and the stage of the disease, FOXOs can have opposite effects on carcinogenesis. To study the role of FOXOs in breast carcinogenesis in more detail, we examined their expression in normal tissues, breast cell lines, and a large series of breast tumours of human origin. We found a very low physiological level of FOXO6 expression in normal adult tissues and high levels of expression in foetal brain. FOXO gene expressions fluctuate specifically in breast cancer cells compared to normal cells, suggesting that these genes may have different roles in breast carcinogenesis. For the first time, we have shown that, among the various FOXO genes, only FOXO6 was frequently highly overexpressed in breast cell lines and tumours. We also found that inhibition of the endogenous expression of FOXO6 by a specific siRNA inhibited the growth of the human breast cell lines MDA-MB-468 and HCC-38. FACS and Western blot analysis showed that inhibition of endogenous expression of FOXO6 induced accumulation of cells in G0/G1 phase of the cell cycle, but not apoptosis. These results tend to demonstrate that the overexpression of the human FOXO6 gene that we highlighted in the breast tumors stimulates breast carcinogenesis by activating breast cancer cell proliferation.

High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism.

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed.

Embryonic signature distinguishes pediatric and adult rhabdoid tumors from other SMARCB1-deficient cancers.

Extra-cranial rhabdoid tumors (RT) are highly aggressive malignancies of infancy, characterized by undifferentiated histological features and loss of SMARCB1 expression. The diagnosis is all the more challenging that other poorly differentiated cancers lose SMARCB1 expression, such as epithelioid sarcomas (ES), renal medullary carcinomas (RMC) or undifferentiated chordomas (UC). Moreover, late cases occurring in adults are now increasingly reported, raising the question of differential diagnoses and emphasizing nosological issues. To address this issue, we have analyzed the expression profiles of a training set of 32 SMARCB1-deficient tumors (SDT), with ascertained diagnosis of RT (n = 16, all < 5 years of age), ES (n = 8, all > 10 years of age), UC (n = 3) and RMC (n = 5). As compared with other SDT, RT are characterized by an embryonic signature, and up-regulation of key-actors of de novo DNA methylation processes. Using this signature, we then analysed the expression profiling of 37 SDT to infer the appropriate diagnosis. Thirteen adult onset tumors showed strong similarity with pediatric RT, in spite of older age; by exome sequencing, these tumors also showed genomic features indistinguishable from pediatric RT. In contrary, 8 tumors were reclassified within carcinoma, ES or UC categories, while the remaining could not be related to any of those entities. Our results demonstrate that embryonic signature is shared by all RT, whatever the age at diagnosis; they also illustrate that many adult-onset SDT of ambiguous histological diagnosis are clearly different from RT. Finally, our study paves the way for the routine use of expression-based signatures to give accurate diagnosis of SDT.

MED12 mutations in breast phyllodes tumors: evidence of temporal tumoral heterogeneity and identification of associated critical signaling pathways.

Exome sequencing has recently identified highly recurrent MED12 somatic mutations in fibroadenomas (FAs) and phyllodes tumors (PTs). In the present study, based on a large series, we confirmed the presence of MED12 exon 1 and 2 mutations in 49% (41/83) of PTs, 70% (7/10) of FAs and 9.1% (1/11) of fibromatoses. We show that MED12 mutations are associated with benign behavior of phyllodes tumors, as they are detected less frequently in malignant PTs (27.6%) compared to benign (58.3%) and borderline (63.3%) PTs, respectively (p = 0.0036). Phyllodes tumors presented marked temporal heterogeneity of MED12 mutation status, as 50% (3/6) of primary and recurrent phyllodes tumor pairs with MED12 mutation presented different MED12 mutations between the primary and recurrent tumors. There was no correlation between MED12 status and genomic profiles obtained by array-CGH. MED12 mutations are associated with altered expressions of the genes involved in the WNT (PAX3, WNT3A, AXIN2), TGFB (TAGLN, TGFBR2, CTGF) and THRA (RXRA, THRA) signaling pathways.In conclusion, this study confirmed that MED12 plays a central oncogenic role in breast fibroepithelial tumorigenesis and identified a limited number of altered signaling pathways that maybe associated with MED12 mutations. MED12 exon 1 and 2 mutation status and some of the altered genes identified in this study could constitute useful diagnostic or prognostic markers, and form the basis for novel therapeutic strategies for PTs.

ERBB2 mutations associated with solid variant of high-grade invasive lobular breast carcinomas.

ERBB2 and ERBB3 somatic gain-of-function mutations, which may be targeted by anti-ERBB2 therapies, were reported by high-throughput sequencing studies in 1% and 2% of invasive breast cancers respectively. Our study aims to determine ERBB2 and ERBB3 mutations frequencies in grade 3 and/or ERBB2-positive invasive lobular breast carcinomas (ILC). All the 529 ILC surgically-excised registered at Institut Curie in the years 2005 to 2008 were reviewed. Thirty-nine grade 3 ERBB2-negative ILC and 16 ERBB2-positive ILC were retrieved and subjected to Sanger sequencing of the ERBB2 and ERBB3 activation mutation hotspots (ERBB2: exons 8, 17, 19, 20, 21; ERBB3: exons 3, 6, 7, 8). Among the 39 grade 3 ERBB2-negative ILC, six tumors were found to have at least one detectable ERBB2 activating mutation (incidence rate: 15%, 95%CI [4%-27%]). No ERBB2 mutation was found among the 16 ERBB2-positive ILC. No ERBB3 mutation was found in any of the 55 ILC. ERBB2 mutations were statistically associated with solid ILC features (p=0.01). Survival analyses showed no significant prognostic impact of ERBB2 mutations. Our study demonstrates that high grade ERBB2-negative ILC display a high frequency of ERBB2 mutations, and should be subjected to systematic genetic screening.

Identification of new candidate therapeutic target genes in head and neck squamous cell carcinomas.

BACKGROUND: We aimed at identifying druggable molecular alterations at the RNA level from untreated HNSCC patients, and assessing their prognostic significance. METHODS: We retrieved 96 HNSCC patients who underwent primary surgery. Real-time quantitative RT-PCR was used to analyze a panel of 42 genes coding for major druggable proteins. Univariate and multivariate analyses were performed to assess the prognostic significance of overexpressed genes. RESULTS: Median age was 56 years [35-78]. Most of patients were men (80%) with a history of alcohol (70.4%) and/or tobacco consumption (72.5%). Twelve patients (12%) were HPV-positive. Most significantly overexpressed genes involved cell cycle regulation (CCND1 [27%], CDK6 [21%]), tyrosine kinase receptors (MET [18%], EGFR [14%]), angiogenesis (PGF [301%], VEGFA [14%]), and immune system (PDL1/CD274 [28%]). PIK3CA expression was an independent prognostic marker, associated with shorter disease-free survival. CONCLUSIONS: We identified druggable overexpressed genes associated with a poor outcome that might be of interest for personalizing treatment of HNSCC patients.