This study was undertaken to determine the morphologic features and frequency of putative precursor lesions involved in the development of some pure forms of special types and low grade breast carcinoma. We reviewed 147 successive tumor cases, comprising tubular carcinoma (TC); pure type (n=56) and mixed type (n=20), invasive lobular carcinoma (ILC); classic type (n=57), and tubulolobular carcinoma (TLC; n=14). The presence of preinvasive lesions including columnar cell lesions (CCLs), usual epithelial hyperplasia, ductal carcinoma in situ (DCIS), and lobular neoplasia (LN) was determined. Estrogen receptor and E-cadherin immunohistochemistry was performed. Ninety-five percent (95%) of pure TCs had associated CCLs with the majority showing flat epithelial atypia. Atypical ductal hyperplasia (ADH)/DCIS was present in 89% patients. Colocalization of CCL, ADH/DCIS, and TC was seen in 85% patients, all displaying the same cytologic-nuclear morphology in most cases. LN was seen in 16%. In ILC, 91% cases showed LN. CCL and ADH/DCIS were seen in 60% and 42% cases, respectively. E-cadherin was positive in TLC but reduced in TC and completely absent in ILC. In conclusion, our findings support the hypothesis that CCLs are associated with pure and mixed forms of TC, and that LN is involved in ILC development. Our observations suggest that these lesions represent family members of low grade precursor, in situ and invasive neoplastic lesions of the breast. Molecular studies are being performed to substantiate the hypothesis that tubular and lobular carcinomas have direct evolutionary links to CCLs and flat epithelial atypia.

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n ¼ 1,297) and validated in an independent cohort of estrogen receptor (ER)-a-negative breast cancers (n ¼ 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia-mutated; ATM inhibitor) and NU7441 (DNAPKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P ¼ 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G 2 -M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5);

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ATR-CHEK1 signalling is critical for genomic stability. ATR-CHEK1 signalling may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. We investigated ATR, CHEK1 and phosphorylated CHEK1 (Ser345) protein (pCHEK1) levels in 1712 breast cancers. ATR and CHEK1 mRNA expression was evaluated in 1950 breast cancers. Pre-clinically, biological consequences of ATR gene knock down or ATR inhibition by the small molecule inhibitor (VE-821) were investigated in MCF7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). High ATR and high cytoplasmic pCHEK1 levels were significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analyses, high ATR and high cytoplasmic pCHEK1 levels were associated with poor breast cancer specific survival (BCSS). In multivariate analysis, high ATR level remains an independent predictor of adverse outcome. At the mRNA level, high CHEK1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple negative, aggressive molecular phenotypes and adverse BCSS. Pre-clinically, CHEK1 phosphorylation at serine(345) following replication stress was impaired in ATR knock down 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 was less toxic in non-tumorigenic breast epithelial cells (MCF10A). We provide evidence that ATR and CHEK1 are promising biomarkers and rational drug targets for personalized therapy in breast cancer.

Background: Interactions between the immune system and tumors are highly reciprocal in nature, leading to speculation that tumor recurrence or therapeutic resistance could be influenced or predicted by immune events that manifest locally, but can be detected systemically.Methods: Multi-parameter flow cytometry was used to examine the percentage and phenotype of natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), monocyte subsets and regulatory T (Treg) cells in the peripheral blood of of 85 patients with breast cancer (50 of whom were assessed before and after one cycle of anthracycline-based chemotherapy), and 23 controls. Transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) in 23 patients were generated using a NanoString gene profiling platform.Results: An increased percentage of immunosuppressive cells such as granulocytic MDSCs, intermediate CD14++CD16+ monocytes and CD127negCD25highFoxP3+ Treg cells was observed in patients with breast cancer, especially patients with stage 3 and 4 disease, regardless of ER status. Following neoadjuvant chemotherapy, B cell numbers decreased significantly, whereas monocyte numbers increased. Although chemotherapy had no effect on the percentage of Treg, MDSC and NK cells, the expression of inhibitory receptors CD85j, LIAR and NKG2A and activating receptors NKp30 and NKp44 on NK cells increased, concomitant with a decreased expression of NKp46 and DNAM-1 activating receptors. Transcriptomic profiling revealed a distinct group of 3 patients in the triple negative breast cancer (TNBC) cohort who expressed high levels of mRNA encoding genes predominantly involved in inflammation. The analysis of a large transcriptomic dataset derived from the tumors of patients with TNBC revealed that the expression of CD163, CXCR4, THBS1 predicted relapse-free survival.Conclusions: The peripheral blood immunome of patients with breast cancer is influenced by the presence and stage of cancer, but not by molecular subtypes. Furthermore, immune profiling coupled with transcriptomic analyses of peripheral blood cells may identify patients with TNBC that are at risk of relapse after chemotherapy.

IntroductionAtaxia telangiectasia mutated and Rad3 Related (ATR) protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response.MethodsIn the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed.ResultsATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells.ConclusionsOur data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.

We hypothesized that the functional status of p53 transcriptional pathways, rather than p53 protein expression alone, could accurately discriminate between low-and highrisk breast carcinoma (BC) and inform about individuals' tumour biological behaviour. To test this, we studied a well-characterized series of 990 BCs with long-term followup, immunohistochemically profiled for p53, its main regulators and downstream genes. Results were validated in an independent series of patients (n = 245) uniformly treated with adjuvant anthracycline-based chemotherapy. Eleven p53 transcriptional phenotypes were identified with just two main clinical outcomes. (a) Low risk/good prognosis group (active/partially inactive p53 pathways), defined as p53 + had the worst prognosis, particularly patients who received AT. Multivariate Cox regression models, including validated prognostic factors for both test and validation series, revealed that the functional status of p53 transcriptional pathways was an independent prognosticator for BC-specific survival (HR 2.64 and 4.5, p < 0.001, respectively) and disease-free survival (HR 1.93 and 2.5, p < 0.001, respectively). In conclusion, p53 functional status determined by assessment of p53 regulatory and downstream targets provides independent prognostic value and may help determine more adequate therapeutic regimens for specific subgroups of breast cancer patients.

RECQL4 helicase is a molecular motor that unwinds DNA, a process essential during DNA replication and DNA repair. Germ-line mutations in RECQL4 cause type II Rothmund-Thomson syndrome (RTS) characterised by a premature aging phenotype and cancer predisposition. RECQL4 is widely considered as a tumour suppressor, although its role in human breast cancer is largely unknown. As the RECQL4 gene is localized to chromosome 8q24, a site frequently amplified in sporadic breast cancers, we hypothesised that it may play an oncogenic role in breast tumorigenesis. To address this we analysed large cohorts for gene copy number changes (n=1977), mRNA expression (n=1977) and protein level (n=1902). Breast cancer incidence was also explored in 58 patients with type II RTS. DNA replication dynamics and chemo-sensitivity was evaluated in RECQL4-depleted breast cancer cells in vitro. Amplification or gain in gene copy number (30.6%), high level mRNA expression (51%) and high levels of protein (23%) significantly associated with aggressive tumour behaviour including lymph node positivity, larger tumour size, HER2 over expression, ER-negativity, triple negative phenotypes and poor survival. RECQL4 depletion impaired DNA replication rate and increased chemo-sensitivity in cultured breast cancer cells. Thus, although recognised as a “safe guardian of the genome”, our data provides compelling evidence that RECQL4 is tumour promoting in established breast cancers.