Expression of proteins involved in DNA damage response in familial and sporadic breast cancer patients.

Understanding the expression of proteins involved in DNA damage response could improve knowledge of the pathways that contribute to familial and sporadic breast cancer (BC). We aimed to assess the different roles of BRCA1, poly(ADP-ribose) polymerase-1 (PARP1), BRCT-repeat inhibitor of hTERT expression (BRIT1) and novel SWItch 5 (SWI5) expression in 130 sporadic and 73 familial BC samples, by immunohistochemistry. In the sporadic group, negative nuclear BRCA1 (nBRCA1) expression was associated with positive PgR (p = 0.037). Negative association was found between nBRCA1 expression and HER2 (p = 0.001). In the familial group, nBRCA1 expression was associated with ER (p = 0.002). Reduced nBRCA1 expression was associated with higher histological grade and positive Ki67 both in sporadic (p = 0.0010, p = 0.047) and familial groups (p < 0.001, p = 0.001). Nuclear PARP1 (nPARP1) expression was associated with histological grade (p = 0.035) and positive PgR (p = 0.047) in sporadic cases. High cytoplasmic and low nuclear BRIT1 (cBRIT1 and nBRIT1) expression were associated with high histological grade in the familial group (p = 0.013, p = 0.025). Various statistical associations between the protein expressions were observed in the sporadic group, while in familial group only few associations were found. Univariate analyses showed that nPARP1 expression is able to discriminate between sporadic and familial tumors (OR 2.80, p = 0.002). Multivariate analyses proved that its overexpression is an independent factor associated with a high risk of sporadic tumor (OR 2.96, p = 0.017). Our findings indicate that nPARP1 expression is an independent factor for sporadic BCs and PARP1 inhibitors could be a promising therapy for different phenotypes.

Nuclear PARP1 expression and its prognostic significance in breast cancer patients.

Poly(adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) plays important roles in DNA damage response pathways and is often overexpressed in various human tumors. Currently, the use of PARP inhibitors for breast cancer (BC) therapy is the subject of debate, and there is an urgent need to understand much the expression and prognostic role of the PARP1 protein. The aim was to investigate the clinicopathological and prognostic significance of PARP1 in BC patients. The PARP1 and breast cancer susceptibility gene 1 (BRCA1) protein expressions were evaluated in 114 BCs by immunohistochemistry. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan-Meier method. Our results showed that nuclear PARP1 expression was significantly associated with peritumoral vascular invasion (P = 0.046), chemotherapeutic treatment (P = 0.026), oestrogen receptor (ER; P = 0.013), human epidermal growth factor receptor 2 (HER2; P = 0.003) and BRCA1 (P < 0.001) expression. Survival analyses showed a significant association with clinical outcome in the subgroup of ER-negative patients (P = 0.017 for DFS and P = 0.048 for OS) and in the subgroup of patients treated with chemotherapeutic agents (P = 0.042 for DFS and P = 0.046 for OS). A significant correlation was also found for DFS in patients characterized by tumors without peritumoral vascular invasion (P = 0.022). More importantly, multivariate analyses revealed that high nuclear PARP1 expression was associated with decreased DFS (P = 0.012) and OS (P = 0.026). In conclusion, PARP1 expression may be used as an independent prognostic factor in BC patients. In addition, this study demonstrated that high PARP1 expression may represent a marker of poorer prognosis both for patients with worse clinical outcome and in less aggressive clinical conditions.

CES2, ABCG2, TS and Topo-I primary and synchronous metastasis expression and clinical outcome in metastatic colorectal cancer patients treated with first-line FOLFIRI regimen.

Enzymatic activation of irinotecan (CPT-11) is due to carboxylesterase (CES), and its pharmacological behavior is influenced by drug resistance-related proteins. We previously reported that the clinical response and prognosis of metastatic colorectal cancer (mCRC) patients did not differ in tumors with different thymidylate synthase (TS) or topoisomerase-I (Topo-I) expression. Using immunohistochemistry (IHC), we evaluated the biological role of CES2 and the expression of breast cancer resistance protein (BCRP/ABCG2) in 58 consecutive mCRC patients, who had undergone a first-line CPT-11/5-FU/leucovirin (FOLFIRI) regimen. The expression of these proteins was also examined in a group of synchronous lymph nodes and liver metastases. Furthermore, all samples were revaluated for TS and Topo-I expression. High expression of CES2, ABCG2, TS and Topo-I was observed in 55%, 56%, 38% and 49% of patients, respectively. There was a significant association between high TS and high ABCG2 expression (p = 0.049). Univariate analysis showed that only TS expression significantly impacted on time to progression (p = 0.005). Moreover, Cox' multivariate analysis revealed that TS expression was significantly associated with overall survival (p = 0.01). No significant correlation was found between investigated markers expression and clinical response. Topo-I expression resulted in being significantly higher in liver metastases with respect to the corresponding primary tumors (p < 0.0001), emphasizing the role of Topo-I expression in metastatic cancer biology. In primary tumor tissues, CES2 expression tended to be higher than that observed in liver metastasis tissues (p = 0.05). These preliminary data may suggest CES2 over-expression as a potential marker of malignant phenotype. In light of these findings, we suggest that Topo-I expression together with TS expression could be associated with metastatic progression of CRC. Further studies are warranted with the aim of evaluating the potential predictive and prognostic role of CES2 and ABCG2 in larger series of patients.

Refinement of Bos taurus sequence assembly based on BAC-FISH experiments.

BACKGROUND: The sequencing of the cow genome was recently published (Btau_4.0 assembly). A second, alternate cow genome assembly (UMD2), based on the same raw sequence data, was also published. The two assemblies have been subsequently updated to Btau_4.2 and UMD3.1, respectively. RESULTS: We compared the Btau_4.2 and UMD3.1 alternate assemblies. Inconsistencies were grouped into three main categories: (i) DNA segments showing almost coincidental chromosomal mapping but discordant orientation (inversions); (ii) DNA segments showing a discordant map position along the same chromosome; and (iii) sequences present in one chromosomal assembly but absent in the corresponding chromosome of the other assembly. The latter category mainly consisted of large amounts of scaffolds that were unassigned in Btau_4.2 but successfully mapped in UMD3.1. We sampled 70 inconsistencies and identified appropriate cow BACs for each of them. These clones were then utilized in FISH experiments on cow metaphase or interphase nuclei in order to disambiguate the discrepancies. In almost all instances the FISH results agreed with the UMD3.1 assembly. Occasionally, however, the mapping data of both assemblies were discordant with the FISH results. CONCLUSIONS: Our work demonstrates how FISH, which is assembly independent, can be efficiently used to solve assembly problems frequently encountered using the shotgun approach.

NHERF1 together with PARP1 and BRCA1 expression as a new potential biomarker to stratify breast cancer patients.

It has been recognized that Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) in breast cancer (BC) acts as a tumor suppressor or as an oncogenic protein, depending on its subcellular localization. This study aims to correlate NHERF1 expression to BRCA1 and PARP1 proteins, to investigate their relationship, and their biological and clinical significance. Using immunohistochemistry on tissue microarrays, we evaluated subcellular NHERF1, BRCA1 and PARP1 expression in 308 BCs including a subgroup (n=80) of triple negative BCs (TNBCs). Herein, we show that nuclear NHERF1 (nNHERF1) expression was significantly associated with nuclear BRCA1 (nBRCA1) expression (p=0.0008), and an association was also found between nuclear PARP1 (nPARP1) and nBRCA1 (p<0.0001). Cytoplasmic NHERF1 (cNHERF1) was correlated to nPARP1 (p<0.0001). Survival analyses showed that the patients with positive nPARP1 and nNHERF1 tended toward a shorter 5-year overall survival (OS) (p=0.057). In TNBCs, the association between nBRCA1 and nPARP1 was maintained (p<0.0001), and an association between nNHERF1 and nPARP1 was observed (p=0.010). Univariate analysis revealed that TNBCs with positive cNHERF1 and nPARP1 had a shorter 5-year OS (p=0.048). Our data suggest that NHERF1 could be a new potential biomarker in combination with PARP1 and BRCA1 expression to stratify BC patients. In particular, in TNBCs, cNHERF1 associated with nPARP1 expression identified a patient subgroup with a shorter survival, for whom it may be useful to develop novel therapeutic strategies.

Fine Needle Aspiration Cytology: A Tool to Study NHERF1 Expression as a Potential Marker of Aggressiveness in Lung Cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide and cytology is often the only diagnostic approach. Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a protein implicated in various cancers. However, the function of NHERF1 in lung cancer has not been described. The aim of this study was to evaluate the biological role of NHERF1 in lung tumors using fine needle aspirates cytology (FNAC) and to verify the utility and reliability of FNACs. We assessed NHERF1 expression by immunohistochemistry on 26 advanced NSCLC FNAC and on 18 surgical NSCLC samples. The relationship of its expression with clinicopathological features including stage and histotype was analyzed. In FNACs, cytoplasmic and nuclear NHERF1 expression was significantly higher than membranous expression. In surgical samples, cytoplasmic NHERF1 was significantly higher than both membranous and nuclear NHERF1 expression. In these tumors, a higher cytoplasmic and lower nuclear NHERF1 expression was observed compared to advanced NSCLCs (p < 0.0001, p < 0.05, respectively). In all 44 NSCLCs, a significant correlation was found between cytoplasmic NHERF1 expression and stage (p = 0.001), and between nuclear NHERF1 expression and histotype (p = 0.015). To our knowledge, this is the first study analyzing the immunohistochemical expression of NHERF1 in lung cancer using FNAC samples. We conclude that FNACs provide useful material for detection of NHERF1 localization and expression, and that high nuclear NHERF1 expression may be a potential marker of aggressiveness in NSCLC.