Detection Rate and Spectrum of Pathogenic Variations in a Cohort of 83 Patients with Suspected Hereditary Risk of Kidney Cancer.

Hereditary predisposition to cancer affects about 3-5% of renal cancers. Testing criteria have been proposed in France for genetic testing of non-syndromic renal cancer. Our study explores the detection rates associated with our testing criteria. Using a comprehensive gene panel including 8 genes related to renal cancer and 50 genes related to hereditary predisposition to other cancers, we evaluated the detection rate of pathogenic variants in a cohort of 83 patients with suspected renal cancer predisposition. The detection rate was 7.2% for the renal cancer genes, which was 2.41-fold higher than the estimated 3% proportion of unselected kidney cases with inherited risk. Pathogenic variants in renal cancer genes were observed in 44.5% of syndromic cases, and in 2.7% of non-syndromic cases. Incidental findings were observed in CHEK2, MSH2, MUTYH and WRN. CHEK2 was associated with renal cancer (OR at 7.14; 95% CI 1.74-29.6; p < 0.003) in our study in comparison to the gnomAD control population. The detection rate in renal cancer genes was low in non-syndromic cases. Additional causal mechanisms are probably involved, and further research is required to find them. A study of the management of renal cancer risk for CHEK2 pathogenic variant carriers is needed.

Case Series of 11 CDH1 Families (47 Carriers) Including Incidental Findings, Signet Ring Cell Colon Cancer and Review of the Literature.

Germline pathogenic variants in E-cadherin (CDH1) confer high risk of developing lobular breast cancer and diffuse gastric cancer (DGC). The cumulative risk of DGC in CDH1 carriers has been recently reassessed (from 40-83% by age 80 to 25-42%) and varies according to the presence and number of gastric cancers in the family. As there is no accurate estimate of the risk of gastric cancer in families without DGC, the International Gastric Cancer Linkage Consortium recommendation is not straightforward: prophylactic gastrectomy or endoscopic surveillance should be proposed for these families. The inclusion of CDH1 in constitutional gene panels for hereditary breast and ovarian cancer and for gastrointestinal cancers, recommended by the French Genetic and Cancer Consortium in 2018 and 2020, leads to the identification of families with lobular cancer without DGC but also to incidental findings of pathogenic variants. Management of CDH1 carriers in case of incidental findings is complex and causes dilemmas for both patients and providers. We report eleven families (47 CDH1 carriers) from our oncogenetic department specialized in breast and ovarian cancer, including four incidental findings. We confirmed that six families did not have diffuse gastric cancer in their medical records. We discuss the management of the risk of diffuse gastric cancer in Hereditary Lobular Breast Cancer (HLBC) through a family of 11 CDH1 carriers where foci were identified in endoscopic surveillance. We also report a new colon signet ring cancer case in a CDH1 carrier, a rare aggressive cancer included in CDH1-related malignancies.

Diagnosis of PTEN mosaicism: the relevance of additional tumor DNA sequencing. A case report and review of the literature.

BACKGROUND: PTEN hamartoma syndrome (PHTS) is an autosomal dominant disorder characterized by pathogenic variants in the tumor suppressor gene phosphatase and tensin homolog (PTEN). It is associated with an increased risk of muco-cutaneous features, hamartomatous tumors, and cancers. Mosaicism has been found in a few cases of patients with de novo PHTS, identified from blood samples. We report a PHTS patient with no variant identified from blood sample. Constitutional PTEN mosaicism was detected through sequencing of DNA from different tumoral and non-tumoral samples. CASE PRESENTATION: Our patient presented clinical Cowden syndrome at 56 years of age, with three major criteria (macrocephaly, Lhermitte Duclos disease, oral papillomatosis), and two minor criteria (structural thyroid lesions, esophageal glycogenic acanthosis). Deep sequencing of PTEN of blood leukocytes did not reveal any pathogenic variants. Exploration of tumoral (colonic ganglioneuroma, esophageal papilloma, diapneusia fibroids) and non-tumoral stomach tissues found the same PTEN pathogenic variant (NM_000314.4 c.389G > A; p.(Arg130Gln)), with an allelic frequency of 12 to 59%, confirming genomic mosaicism for Cowden syndrome. CONCLUSIONS: This case report, and review of the literature, suggests that systematic tumor analysis is essential for patients presenting PTEN hamartoma syndrome in the absence of any causal variant identified in blood leukocytes, despite deep sequencing. In 65 to 70% of cases of clinical Cowden syndrome, no pathogenic variant in the PTEN is observed in blood samples: mosaicism may explain a significant number of these patients. Tumor analysis would improve our knowledge of the frequency of de novo variations in this syndrome. Finally, patients with mosaicism for PTEN may not have a mild phenotype; medical care identical to that of heterozygous carriers should be offered.

Rare duplication of the CDC73 gene and atypical hyperparathyroidism-jaw tumor syndrome: A case report and review of the literature.

BACKGROUND: Hyperparathyroidism jaw-tumor syndrome (HPT-JT) is the rarest familial cause of primary hyperparathyroidism, with an incidence <1/1000000, caused by a pathogenic variant in the CDC73 (or HRPT2) gene that encodes parafibromin, a protein involved in many cellular mechanisms. Patients with HPT-JT have a 15-20% of risk of developing parathyroid carcinoma, whereas it accounts for only 1% of all cases of primary hyperparathyroidism. Patients also develop jaw tumors in 30% of cases, kidney abnormalities in 15% of cases, and uterine tumors in 50% of patients. CASE REPORT: Here are report two atypical cases of HPT-JT with variable expressivity in the same family. In front of an isolated primary hyperparathyroidism at 28 years of age of incidental discovery following a weight gain, the propositus benefited a first-line panel by Next-Generation Sequencing of the genes involved in familial hyperparathyroidism: CaSR, CDC73, MEN1, and RET. Genetic testing revealed the presence of a pathogenic germline variation CDC73: c.687_688dup; p.Val230Glufs*28, found only in nine families in the literature and allowing the diagnosis of HPT-JT. Given a history of primary hyperparathyroidism at 52 years and adenomyosis, the patient's mother also underwent a genetic analysis that found her daughter's variation and established her inherited trait. CONCLUSION: In view of the clinical and genotypic heterogeneity, we confirm the interest of using an extended gene panel for the diagnosis of familial primary hyperparathyroidism. CDC73 variations could be more frequent than described in the literature. The association of primary hyperparathyroidism with uterine involvement could be a new indication for analysis.

RNA sequencing reveals the differential expression profiles of RNA in metastatic triple negative breast cancer and identifies SHISA3 as an efficient tumor suppressor gene.

Breast cancer metastasis is the second leading cause of female mortality worldwide. Because of the heterogeneity within the group, metastatic biomarkers for triple-negative breast cancer (TNBC) providing predictive and prognosis values are urgently needed. Using RNA-Seq, we analyzed the transcriptome profiles of two groups of TNBCs tumors with or without distant metastasis. Whole transcriptome sequencing identified a set of genes implicated in TNBC metastasis with major roles in cell-cell adhesion, immune-modulation, and Wnt/ß-catenin pathways. We further selected the SHISA3 gene and studied its biological significance through a series of in vitro and in vivo experiments. SHISA3 is a tumor suppressor gene, involved in several types of cancer. However, little is known concerning the role of SHISA3 in TNBC. Our in vitro and in vivo studies demonstrate that overexpression of SHISA3 inhibits TNBCs cell proliferation, metastasis and colony formation, and TNBC growth in xenografts. Mechanistically, SHISA3 inhibits TNBCs development and growth via downregulation of the epithelial-mesenchymal transition. Taken together, these results identified SHISA3 as a novel tumor suppressor gene in TNBC and suggest that SHISA3 could serve as a therapeutic target for TNBC patients.

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach.

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

Recurrence biomarkers of triple negative breast cancer treated with neoadjuvant chemotherapy and anti-EGFR antibodies.

To find metastatic recurrence biomarkers of triple-negative breast cancer (TNBC) treated by neoadjuvant chemotherapy and anti-EGFR antibodies (NAT), we evaluated tumor genomic, transcriptomic, and immune features, using MSK-IMPACT assay, gene arrays, Nanostring technology, and TIL assessment on H&E. Six patients experienced a rapid fatal recurrence (RR) and other 6 had later non-fatal recurrences (LR). Before NAT, RR had low expression of 6 MHC class I and 13 MHC class II genes but were enriched in upregulated genes involved in the cell cycle-related pathways. Their TIL number before NAT in RR was very low (<5%) and did not increase after treatment. In post-NAT residual tumors, RR cases showed high expression of SOX2 and CXCR4. Our results indicate that high expression of cell cycle genes, combined with cold immunological phenotype, may predict strong TNBC resistance to NAT and rapid progression after it. This biomarker combination is worth validation in larger studies.

New insight into the role of ANXA1 in melanoma progression: involvement of stromal expression in dissemination.

ANXA1, first described in the context of inflammation, appears to be deregulated in many cancers and increased in melanomas compared with melanocytes. To date, few studies have investigated the role of ANXA1 in melanoma progression. Furthermore, this protein is expressed by various cell types, including immune and endothelial cells. We therefore analyzed the specific roles of ANXA1 using melanoma and stromal cells in two human cell lines (A375-MA2 and SK-MEL-28) in vitro and in Anxa1 null C57Bl6/J mice bearing B16Bl6 tumors. We report decreased proliferation in both ANXA1 siRNA A375-MA2 and SK-MEL-28, but cell-dependent effects of ANXA1 in migration in vitro. However, we also observed a significant decrease of B16Bl6 tumor growth associated with a reduction of Ki-67 positive cells in Anxa1 null mice compared with wild-type mice. Interestingly, we also found a significant reduction of spontaneous metastases, which can be attributed to decreased angiogenesis concomitantly with greater immune cell presence in the Anxa1 null stromal context. This study highlights the pejorative role of ANXA1 in both tumor and stromal cells in melanoma, due to its involvement in proliferation and angiogenesis.

Analysis of 11 candidate genes in 849 adult patients with suspected hereditary cancer predisposition.

Hereditary predisposition to cancer concerns between 5% and 10% of cancers. The main genes involved in the most frequent syndromes (hereditary breast and ovarian cancer syndrome, hereditary nonpolyposis colorectal cancer syndrome) were identified in the 1990s. Exploration of their functional pathways then identified novel genes for hereditary predisposition to cancer, and candidate genes whose involvement remains unclear. To determine the contribution of truncating variants in 11 candidate genes (BARD1, FAM175A, FANCM, MLH3, MRE11A, PMS1, RAD50, RAD51, RAD51B, RINT1, and XRCC2) to cancer predisposition in a population of interest, panel sequencing was performed in 849 patients with a suspected hereditary predisposition to cancer for whom a diagnostic panel of 38 genes identified no causal mutation. Sixteen truncating variants were found in FANCM (n = 7), RINT1 (n = 4), RAD50 (n = 2), BARD1, PMS1, and RAD51B. FANCM (adjusted P-value: .03) and RINT1 (adjusted P-value: .04) were significantly associated with hereditary breast and ovarian cancer. However, further studies are required to determinate the risk of cancer, including the segregation of the variants in the families of our cases. No mutation was identified in RAD51, MRE11A, FAM175A, XRCC2, or MLH3. The involvement of these genes in the hereditary predisposition to cancer cannot be ruled out, although if it exists it is rare or does not seem to involve truncating variants.

Feedback of extended panel sequencing in 1530 patients referred for suspicion of hereditary predisposition to adult cancers.

High-throughput sequencing analysis represented both a medical diagnosis and technological revolution. Gene panel analysis is now routinely performed in the exploration of hereditary predisposition to cancer, which is becoming increasingly heterogeneous, both clinically and molecularly. We present 1530 patients with suspicion of hereditary predisposition to cancer, for which two types of analyses were performed: a) oriented according to the clinical presentation (n = 417), or b) extended to genes involved in hereditary predisposition to adult cancer (n = 1113). Extended panel analysis had a higher detection rate compared to oriented analysis in hereditary predisposition to breast / ovarian cancer (P < .001) and in digestive cancers (P < .094) (respectively 15% vs 5% and 19.3%, vs 12.5%). This higher detection is explained by the inclusion of moderate penetrance genes, as well as the identification of incident mutations and double mutations. Our study underscores the utility of proposing extended gene panel analysis to patients with suspicion of hereditary predisposition to adult cancer.

A high expression ratio of RhoA/RhoB is associated with the migratory and invasive properties of basal-like Breast Tumors.

Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate. qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins. We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity. These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.

Methyl-donor supplementation prevents intestinal colonization by Adherent-Invasive E. coli in a mouse model of Crohn's disease.

Deficiencies in methyl-donor molecules (folate, B12 vitamin), DNA methylation alteration and high prevalence of Adherent-Invasive Escherichia coli (AIEC) are frequently observed in Crohn's disease (CD) patients. AIEC bacteria adhere to the enterocytes through abnormally expressed carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) glycoprotein on host cells. This work aims at studying the relationship between methyl-donor molecules and AIEC-induced intestinal inflammatory response. CEABAC10 mice, a mouse model of CD, were fed a control or Methyl-donor Supplemented diet (MS diet). CEACAM6 promoter was hypermethylated in intestinal epithelial cells from mice fed an MS diet, which was associated with a significant decrease in CEACAM6 expression. Transcriptomic analysis revealed increased expression of anti-microbial peptides, increase in HSP70 gene family expression and a decreased expression of inflammatory marker Calprotectin upon MS diet, associated to a lower ability of AIEC bacteria to colonize gut mucosa. We observed in a cohort of CD patients that serum folate concentration was inversely correlated to Crohn's disease endoscopic index of severity and to fecal inflammatory markers. This study demonstrates that methyl-donor supplementation through the diet induces a specific intestinal micro-environment limiting pathobiont colonization of the gut. Clinicians may wish to consider methyl-donor supplementation for methyl-donor deficient CD patients.

Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12.

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

Is BRCA2 involved in early onset colorectal cancer risk?

A, Closed symbols indicate patients affected with cancer. Open symbols indicate healthy individuals. The type of cancer and age at presentation are given in brackets. Blue circle represents c.4471_4474del variant and red circle represents the c.9648 + 1G > A. B, RNA was extracted from blood of patient III-3 and his sisters III-1 and III-4. RT-PCR analysis was performed with primers mapping to exons 25 and 27, and PCR products were separated by Bioanalyzer electrophoresis. The sizes of the DNA marker (M) are indicated to the left. LM, lower marker; UM, upper marker. C, Each RT-PCR product from patient III-3 was gel-purified and analyzed by Sanger sequencing. The 297-bp band corresponds to the reference BRCA2 transcript and the 150-bp band corresponds to a BRCA2 transcript lacking exon 26.

Early Onset Multiple Primary Tumors in Atypical Presentation of Cowden Syndrome Identified by Whole-Exome-Sequencing.

A family with an aggregation of rare early onset multiple primary tumors has been managed in our oncogenetics department: the proband developed four early onset carcinomas between ages 31 and 33 years, including acral melanoma, bilateral clear cell renal carcinoma (RC), and follicular variant of papillary thyroid carcinoma. The proband's parent developed orbital lymphoma and small intestine mucosa-associated lymphoid tissue (MALT) lymphoma between 40 and 50 years old. Whole-exome-sequencing (WES) of the nuclear family (proband, parents, and sibling) identified in the proband a de novo deleterious heterozygous mutation c.1003C > T (p.Arg335∗) in the phosphatase and tensin homolog (PTEN) gene. Furthermore, WES allowed analysis of the nuclear family's genetic background, and identified deleterious variants in two candidate modifier genes: CEACAM1 and MIB2. CEACAM1, a tumor suppressor gene, presents loss of expression in clear cell RC and is involved in proliferation of B cells. It could explain in part the phenotype of proband's parent and the occurrence of clear cell RC in the proband. Deleterious mutations in the MIB2 gene are associated with melanoma invasion, and could explain the occurrence of melanoma in the proband. Cowden syndrome is a hereditary autosomal dominant disorder associated with increased risk of muco-cutaneous features, hamartomatous tumors, and cancer. This atypical presentation, including absence of muco-cutaneous lesions, four primary early onset tumors and bilateral clear cell RC, has not been described before. This encourages including the PTEN gene in panel testing in the context of early onset RC, whatever the histological subtype. Further studies are required to determine the implication of CEACAM1 and MIB2 in the severity of Cowden syndrome in our proband and occurrence of early onset MALT lymphoma in a parent.

Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines.

Basal-like breast cancers are among the most aggressive cancers and effective targeted therapies are still missing. In order to identify new therapeutic targets, we performed Methyl-Seq and RNA-Seq of 10 breast cancer cell lines with different phenotypes. We confirmed that breast cancer subtypes cluster the RNA-Seq data but not the Methyl-Seq data. Basal-like tumor hypermethylated phenotype was not confirmed in our study but RNA-Seq analysis allowed to identify 77 genes significantly overexpressed in basal-like breast cancer cell lines. Among them, 48 were overexpressed in triple negative breast cancers of TCGA data. Some molecular functions were overrepresented in this candidate gene list. Genes involved in antioxydation, such as SOD1, MGST3 and PRDX or cadherin-binding genes, such as PFN1, ITGB1 and ANXA1, could thus be considered as basal like breast cancer biomarkers. We then sought if these genes were linked to BRCA1, since this gene is often inactivated in basal-like breast cancers. Nine genes were identified overexpressed in both basal-like breast cancer cells and BRCA1 mutated cells. Amongst them, at least 3 genes code for proteins implicated in epithelial cell migration and epithelial to mesenchymal transition (VIM, ITGB1 and RhoA). Our study provided several potential therapeutic targets for triple negative and BRCA1 mutated breast cancers. It seems that migration and mesenchymal properties acquisition of basal-like breast cancer cells is a key functional pathway in these tumors with a high metastatic potential.

TERT promoter status and gene copy number gains: effect on TERT expression and association with prognosis in breast cancer.

Upregulation of the telomerase reverse transcriptase (TERT) gene in human cancers leads to telomerase activation, which contributes to the growth advantage and survival of tumor cells. Molecular mechanisms of TERT upregulation are complex, tumor-specific and can be clinically relevant. To investigate these mechanisms in breast cancer, we sequenced the TERT promoter, evaluated TERT copy number changes and assessed the expression of the MYC oncogene, a known transcriptional TERT regulator, in two breast cancer cohorts comprising a total of 122 patients. No activating TERT promoter mutations were found, suggesting that this mutational mechanism is not likely to be involved in TERT upregulation in breast cancer. The T349C promoter polymorphism found in up to 50% of cases was not correlated with TERT expression, but T349C carriers had significantly shorter disease-free survival. TERT gains (15-25% of cases) were strongly correlated with increased TERT mRNA expression and worse patient prognosis in terms of disease-free and overall survival. Particularly aggressive breast cancers were characterized by an association of TERT gains with MYC overexpression. These results evidence a significant effect of gene copy number gain on the level of TERT expression and provide a new insight into the clinical significance of TERT and MYC upregulation in breast cancer.

Quantification of hypoxia-related gene expression as a potential approach for clinical outcome prediction in breast cancer.

Breast cancers are solid tumors frequently characterized by regions with low oxygen concentrations. Cellular adaptations to hypoxia are mainly determined by "hypoxia inducible factors" that mediate transcriptional modifications involved in drug resistance and tumor progression leading to metastasis and relapse occurrence. In this study, we investigated the prognostic value of hypoxia-related gene expression in breast cancer. A systematic review was conducted to select a set of 45 genes involved in hypoxia signaling pathways and breast tumor progression. Gene expression was quantified by RT-qPCR in a retrospective series of 32 patients with invasive ductal carcinoma. Data were analyzed in relation to classical clinicopathological criteria and relapse occurrence. Coordinated overexpression of selected genes was observed in high-grade and HER2+ tumors. Hierarchical cluster analysis of gene expression significantly segregated relapsed patients (p = 0.008, Chi2 test). All genes (except one) were up-regulated and six markers were significantly expressed in tumors from recurrent patients. The expression of this 6-gene set was used to develop a basic algorithm for identifying recurrent patients according to a risk score of relapse. Analysis of Kaplan-Meier relapse-free survival curves allowed the definition of a threshold score of 2 (p = 0.021, Mantel-Haenszel test). The risk of recurrence was increased by 40% in patients with a high score. In addition to classical prognostic factors, we showed that hypoxic markers have potential prognostic value for outcome and late recurrence prediction, leading to improved treatment decision-making for patients with early-stage invasive breast cancer. It will be necessary to validate the clinical relevance of this prognostic approach through independent studies including larger prospective patient cohorts.

miR-10b, miR-26a, miR-146a And miR-153 Expression in Triple Negative Vs Non Triple Negative Breast Cancer: Potential Biomarkers.

MicroRNAs (miRNAs) are small non-coding RNAs composed of 18-25 nucleotides that can post-transcriptionally regulate gene expression and have key regulatory roles in cancer, acting as both oncogenes and tumor suppressors. About 1000 genes in humans encode miRNAs, which account for approximately 3% of the human genome, and up to 30% of human protein coding genes may be regulated by miRNAs. The objective of this article is to evaluate the expression profile of four miRNAs previously implicated in triple negative breast cancer: miR-10b, miR-26a, miR-146a and miR-153, and to determine their possible interaction in triple negative and non triple negative breast cancer based on clinical outcome and the expression of BRCA1. 24 triple-negative and 13 non triple negative breast cancer cases, were studied by q-RT-PCR and immunohistochemistry to determine the expression of the four studied miRNAs and the BRCA1 protein, respectively. We observed that the BRCA1 protein was absent in 62.5% of the triple negative cases. Besides, the miR-146a and miR-26a were over expressed in triple negative breast cancer. These two miRNAs, miR-10b and miR-153 were significantly associated to lymph node metastases occurrence in triple negative breast carcinoma. All the analyzed microRNAs were not associated with the expression of BRCA1 in our conditions. Our work provides evidence that miR-146a, miR-26a, miR-10b and miR-153 could be defined as biomarkers in triple negative breast cancer to predict lymph node metastases (LNM).

ERCC1 and telomere status in breast tumours treated with neoadjuvant chemotherapy and their association with patient prognosis.

Dysfunctional telomeres and DNA damage repair (DDR) play important roles in cancer progression. Studies have reported correlations between these factors and tumour aggressiveness and clinical outcome in breast cancer. We studied the characteristics of telomeres and expression of ERCC1, a protein involved in a number of DNA repair pathways and in telomere homeostasis, to assess their prognostic value, alone or in combination, in 90 residual breast tumours after treatment with neoadjuvant chemotherapy (NCT). ERCC1 status was investigated at different molecular levels (protein and gene expression and gene copy-number variations) by immunohistochemistry, qRT-PCR and quantitative multiplex fluorescent-PCR (QMF-PCR). A comprehensive analysis of telomere characteristics was performed using qPCR for telomere length and qRT-PCR for telomerase (hTERT), tankyrase 1 (TNKS) and shelterin complex (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) gene expression. Short telomeres, high hTERT and TNKS expression and low ERCC1 protein expression were independently associated with worse survival outcome. Interestingly, ERCC1 gains and losses correlated with worse disease-free (p = 0.026) and overall (p = 0.043) survival as compared to survival of patients with normal gene copy-numbers. Unsupervised hierarchical clustering of all ERCC1 and telomere parameters identified four subgroups with distinct prognosis. In particular, a cluster combining low ERCC1, ERCC1 gene alterations, dysfunctional telomeres and high hTERT and a cluster with high TNKS and shelterin expression correlated with poor disease-free (HR= 5.41, p= 0.0044) and overall survival (HR= 6.01, p= 0.0023) irrespective of tumour stage and grade. This comprehensive study demonstrates that telomere dysfunction and DDR can contribute synergistically to tumour progression and chemoresistance. These parameters are predictors of clinical outcome in breast cancer patients treated with NCT and could be useful clinically as prognostic biomarkers to tailor adjuvant chemotherapy post-NCT.