Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1) deficiency is linked to aggressive breast cancer and predicts response to adjuvant therapy

Uracil in DNA is an important cause of mutagenesis. SMUG1 is a uracil DNA glycosylase that removes uracil through base excision repair. SMUG1 also processes radiation induced oxidative base damage as well as 5-fluorouracil incorporated into DNA during chemotherapy. We investigated SMUG1 mRNA expression in 249 primary breast cancers. SMUG1 protein expression was investigated in 1165 breast tumours randomised into two cohorts [training set (n=583) and test set (n=582)]. SMUG1 and chemotherapy response was also investigated in a series of 315 ER negative tumours (n=315). For mechanistic insights, SMUG1 was correlated to biomarkers of aggressive phenotype, DNA repair, cell cycle and apoptosis. Low SMUG1 mRNA expression was associated with adverse disease specific survival (p=0.008) and disease free survival (p=0.008). Low SMUG1 protein expression (25%) was associated with high histological grade (p<0.0001), high mitotic index (p<0.0001), pleomorphism (p<0.0001), glandular de-differentiation (p=0.0001), absence of hormonal receptors (ER-/PgR-/AR) (p<0.0001), presence of basal-like (p<0.0001) and triple negative phenotypes (p<0.0001). Low SMUG1 protein expression was associated with loss of BRCA1 (p<0.0001), ATM (p<0.0001) and XRCC1 (p<0.0001). Low p27 (p<0.0001), low p21 (p=0.023), mutant p53 (p=0.037), low MDM2 (p<0.0001), low MDM4 (p=0.004), low Bcl-2 (p=0.001), low Bax (p=0.003) and high MIB1 (p<0.0001) were likely in low SMUG1 tumours. Low SMUG1 protein expression was associated with poor prognosis in univariate (p<0.001) and multivariate analysis (p<0.01). In ER+ cohort that received adjuvant endocrine therapy, low SMUG1 protein expression remains associated with poor survival (p<0.01). In ER- cohort that received adjuvant chemotherapy, low SMUG1 protein expression is associated with improved survival (p=0.043). Our study suggests that low SMUG1 expression may correlate to adverse clinicopathological features and predict response to adjuvant therapy in breast cancer

DNA polymerase B deficiency is linked to aggressive breast cancer: a comprehensive analysis of gene copy number, mRNA and protein expression in multiple cohorts

Short arm of chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications in breast cancer. Although such genetic changes may have phenotypic consequences, the identity of candidate gene(s) remains to be clearly defined. Pol β gene is localized to chromosome 8p12 - p11 and encodes a key DNA base excision repair protein. Pol β may be a tumour suppressor and involved in breast cancer pathogenesis. We conducted the first and the largest study to comprehensively evaluate pol β in breast cancer. We investigated pol β gene copy number changes in two cohorts (n=128 & n=1952), pol β mRNA expression in two cohorts (n=249 & n=1952) and pol β protein expression in two cohorts (n=1406 & n=252). Artificial neural network analysis for pol β interacting genes was performed in 249 tumours. For mechanistic insights, pol β gene copy number changes, mRNA and protein levels were investigated together in 1 28 tumours and validated in 1952 tumours. Low pol β mRNA expression as well as low pol β protein expression was associated high grade, lymph node positivity, pleomorphism, triple negative, basal - like phenotypes and poor survival (ps<0.001). In oestrogen receptor (ER) positive sub - group that received tamoxifen, low pol β protein remains associated with aggressive phenotype and poor survival (ps<0.001). Artificial neural network analysis revealed ER as a top pol β interacting gene. Mechanistically, there was strong positive correlation between pol β gene copy number changes and pol β mRNA expression (p<0.0000001) and between pol β mRNA and pol β protein expression (p<0.0000001). This is the first study to provide evidence that pol β deficiency is linked to aggressive breast cancer and may have prognostic and predictive significance in patients

Breast cancer histologic grading using digital microscopy: concordance and outcome association

Aims: Virtual microscopy utilising digital whole slide imaging (WSI) is increasingly used in breast pathology. Histologic grade is one of the strongest prognostic factors in breast cancer (BC). This study aims at investigating the agreement between BC grading using traditional light microscopy (LM) and digital whole slide imaging (WSI) with consideration of reproducibility and impact on outcome prediction. Methods: A large (n=1675) well-characterised cohort of BC originally graded by LM was re-graded using WSI. Two separate virtual-based grading sessions (V1 and V2) were performed with a three months washout period. Outcome was assessed using breast cancer specific and distant metastasis free survival. Results: The concordance between LM grading and WSI was strong (LM/SWI Cramer’s V: V1=0.576, and V2=0.579). The agreement regarding grade components was as follows: Tubule formation=0.538, Pleomorphism=0.422 and Mitosis=0.514. Greatest discordance was observed between adjacent grades whereas high/low grade discordance was uncommon (1.5%). The intra-observer agreement for the two WSI sessions was substantial for grade (V1/V2 Cramer’s V=0.676; kappa=0.648) and grade components (Cramer’s V T=0.628, P=0.573 and M=0.580). Grading using both platforms showed strong association with outcome (All p-value <0.001). Although mitotic scores assessed using both platforms were strongly associated with outcome, WSI tends to underestimate mitotic counts. Conclusions: Virtual microscopy is a reliable and reproducible method for assessing BC histologic grade. Regardless of the observer or assessment platform, histologic grade is a significant predictor of outcome. Continuing advances in imaging technology could potentially provide improved performance of WSI BC grading and in particular mitotic count assessment

Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy

BRCA1, a key factor in homologous recombination repair may also regulate base excision repair (BER). Targeting BRCA1-BER deficient cells by blockade of ATM and DNA-PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression in two cohorts (n=1602 sporadic and n=50 germ-line BRCA1 mutated) and mRNA expression in two cohorts (n=1952 and n=249). Artificial neural network analysis for BRCA1-DNA repair interacting genes was conducted in 249 tumours. Pre-clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA-PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol β at mRNA and protein levels (p<0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol β expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol β expressing BRCA1 negative tumours (ps<0.05). Pre-clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol β. BRCA1-BER deficient cells were sensitive to ATM and DNA-PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol β expression status in BRCA1 negative tumours may have prognostic significance. BRCA1-BER deficient cells could be targeted by ATM or DNA-PKcs inhibitors for personalized therapy

Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer

Background: ATR-Chk1 signalling network is critical for genomic stability. ATR-Chk1 may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. Patients and methods: We investigated ATR and phosphorylated CHK1Ser345 protein (pChk1) expression in 1712 breast cancers (Nottingham Tenovus series). ATR and Chk1 mRNA were evaluated in 1950 breast cancers (METABRIC cohort). Pre-clinically, biological consequences of ATR gene knockdown or ATR inhibition by small molecule inhibitor (VE-821) were investigated in MCF-7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). Results: High ATR and high cytoplasmic pChk1 expression was significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analysis, high ATR and high cytoplasmic pChk1 protein expression was associated with shorter breast cancer specific survival (BCSS). In multivariate analysis, high ATR remains an independent predictor of adverse outcome. At the mRNA level, high Chk1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple-negative phenotype and molecular classes associated with aggressive behaviour and shorter survival.. Pre-clinically, Chk1 phosphorylation at serine 345 following replication stress (induced by gemcitabine or hydroxyurea treatment) was impaired in ATR knockdown and in VE-821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re-expressed. Similarly, VE-821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA-MB-231) but had no effect on non-tumorigenic breast epithelial cells (MCF10A). Conclusions: We provides evidence that ATR and Chk1 are promising biomarkers and rational drug target for personalized therapy in breast cancer

Things change: Women’s and men’s marital disruption dynamics in Italy during a time of social transformations, 1970-2003

We study women’s and men’s marital disruption in Italy between 1970 and 2003. By applying an event-history analysis to the 2003 Italian variant of the Generations and Gender Survey we found that the spread of marital disruption started among middle-highly educated women. Then in recent years it appears that less educated women have also been able to dissolve their unhappy unions. Overall we can see the beginning of a reversed educational gradient from positive to negative. In contrast the trend in men’s marital disruption risk appears as a change over time common to all educational groups, although with persisting educational differentials.determinants, educational differences, event history analysis, gender difference, Italy, marital disruption

Expression of CDK7, cyclin H and MAT1 is elevated in breast cancer and is prognostic in estrogen receptor-positive breast cancer

PURPOSE: CDK-activation kinase (CAK) is required for the regulation of the cell-cycle and is a trimeric complex consisting of Cyclin Dependent Kinase 7 (CDK7), Cyclin H and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-alpha(ERalpha).). Deregulation of cell cycle and transcriptional control is aare general featurefeatures of cancertumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics in cancer. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential co-factors cyclinH and MAT1, were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathological features and patient outcome. RESULTS: We show that expression of CDK7, cyclinH and MAT1 are all closely linked at the mRNA and protein level and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumour grade and size and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ERalpha expression and in particular with phosphorylation of ERalpha at serine 118, a site important for ERalpha transcriptional activity. CONCLUSIONS: Expression of components of the CAK complex, CDK7, MAT1 and Cyclin H are elevated in breast cancer and correlates with ERalpha.. Like ERalpha, CDK7 expression is inversely proportional to poor prognostic factors and survival