Phil Quinlan

A Pin1/Mutant p53 Axis Promotes Aggressiveness in Breast Cancer

TP53 missense mutations dramatically influence tumor progression, however, their mechanism of action is still poorly understood. Here we demonstrate the fundamental role of the prolyl isomerase Pin1 in mutant p53 oncogenic functions. Pin1 enhances tumorigenesis in a Li-Fraumeni mouse model and cooperates with mutant p53 in Ras-dependent transformation. In breast cancer cells, Pin1 promotes mutant p53 dependent inhibition of the antimetastatic factor p63 and induction of a mutant p53 transcriptional program to increase aggressiveness. Furthermore, we identified a transcriptional signature associated with poor prognosis in breast cancer and, in a cohort of patients, Pin1 overexpression influenced the prognostic value of p53 mutation. These results define a Pin1/mutant p53 axis that conveys oncogenic signals to promote aggressiveness in human cancers.

Tissue confirmation of disease recurrence in breast cancer patients: pooled analysis of multi-centre, multi-disciplinary prospective studies.

BACKGROUND Treatment decisions in recurrent breast cancer are usually based on the estrogen (ER), progesterone (PgR) and HER2 receptor status of the primary tumour. Retrospective studies suggest that discordance between receptor expression of primary and recurrent breast cancer exists. METHODS A pooled analysis of individual patient data from two large prospective studies comprising biopsy of recurrent lesions obtained from consenting patients was undertaken. Tissue was analyzed for ER, PgR by immunohistochemistry and HER2 by FISH. Receptor status of recurrent disease was compared with that of the primary tumour. Recruiting clinicians assessed whether or not receptor discordance affected subsequent systemic treatment. RESULTS Two hundred and eighty-nine patients underwent biopsy. Recurrent biopsy specimens were obtained from locoregional recurrence in 48.1% and from distant metastases in 51.9%. Distant sites included skin/soft tissue (25.0%), bone/bone marrow (19.2%) and liver (15.8%). Benign disease or second primary cancer was observed in 7.6% of biopsies. Discordance in ER, PgR or HER2 between confirmed primary and recurrent breast cancer was 12.6%, 31.2% and 5.5%, respectively (all p<0.001). Biopsy results altered management in 14.2% of patients undergoing biopsy (95% confidence intervals 10.4-18.8%, p≤0.0001). The duration between primary and recurrent disease, the site of recurrence and the receptor profile of the primary tumour did not affect discordance rates. CONCLUSIONS There is substantial discordance in receptor status between primary and recurrent breast cancer. The number needed to biopsy in order to alter treatment was 7.1. Patients with recurrent breast cancer should have tissue confirmation of receptor status of recurrent disease.

Progesterone receptor expression is an independent prognostic variable in early breast cancer: a population-based study

Background:Progesterone receptor (PR) expression assessment in early invasive breast cancer remains controversial. This study sought to re-evaluate PR expression as a potential therapeutic guide in early breast cancer; particularly in oestrogen receptor (ER)-positive, lymph node (LN)-negative disease.Methods:A population cohort of 1074 patients presenting to a single Cancer Centre over 4 years (2000–2004) underwent surgery for primary invasive breast cancer with curative intent. Prospective data collection included patient demographics, pathology, ER and PR expression, HER2 status, adjuvant chemotherapy and endocrine therapy. Progesterone receptor expression was compared with (all causes) overall survival (OS), breast cancer-specific survival (BCSS) and disease-free survival (DFS).Results:Overall survival was 71.0% and BCSS was 83.0% at median follow-up of 8.34 years. Absent PR expression was significantly associated with poorer prognosis for OS, BCSS and DFS (P<0.0001, log-rank), even within the ER-positive, LN-negative group (hazard ratio for BCSS 3.17, 95% CI 1.43–7.01) and was not influenced by endocrine therapy. Cox’s regression analysis demonstrated that PR expression was an independent prognostic variable.Conclusion:Absence of PR expression is a powerful, independent prognostic variable in operable, primary breast cancer even in ER-positive, LN-negative patients receiving endocrine therapy. Absence of PR expression should be re-evaluated as a biomarker for poor prognosis in ER-positive breast cancer and such patients considered for additional systemic therapy.

ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR

IntroductionEpithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.MethodsBasal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.ResultsHere we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.ConclusionsTaken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

High CCND1 amplification identifies a group of poor prognosis women with estrogen receptor positive breast cancer

CCND1 encodes for the cyclin D1 protein involved in G1/S cell cycle transition. In breast cancer the mechanism of CCND1 amplification, relationship between cyclin D1 protein expression and the key clinical markers estrogen receptor (ER) and HER2 requires elucidation. Tissue microarrays of primary invasive breast cancer from 93 women were evaluated for CCND1 amplification by fluorescent in‐situ hybridization and cyclin D1 protein overexpression by immunohistochemistry. CCND1 amplification was identified in 27/93 (30%) cancers and 59/93 (63%) cancers had overexpression of cyclin D1. CCND1 amplification was significantly associated with cyclin D1 protein overexpression (p < 0.001; Fishers exact test) and both CCND1 amplification and cyclin D1 protein expression with oestrogen receptor (ER) expression (p = 0.003 and p < 0.001; Fishers exact test). Neither CCND1 amplification nor cyclinD1 expression was associated with tumor size, pathological node status or HER2 amplification, but high CCND1 amplification (Copy Number Gain (CNG) ≥ 8) was associated with high tumor grade (p = 0.005; chi square 7.915, 2 df) and worse prognosis by Nottingham Prognostic Index (p = 0.001; 2 sample t‐test). High CCND1 amplification (CNG ≥ 8) may identify a subset of patients with poor prognosis ER‐positive breast cancers who should be considered for additional therapy.

The prolyl 3-hydroxylases P3H2 and P3H3 are novel targets for epigenetic silencing in breast cancer

Expression of P3H2 (Leprel1) and P3H3 (Leprel2) but not P3H1 (Leprecan) is down-regulated in breast cancer by aberrant CpG methylation in the 5′ regulatory sequences of each gene. Methylation of P3H2 appears specific to breast cancer as no methylation was detected in a range of cell lines from other epithelial cancers or from primary brain tumours or malignant melanoma. Methylation in P3H2, but not P3H3, was strongly associated with oestrogen-receptor-positive breast cancers, whereas methylation in P3H3 was associated with higher tumour grade and Nottingham Prognostic Index. Ectopic expression of P3H2 and P3H3 in cell lines with silencing of the endogenous gene results in suppression of colony growth. This is the first demonstration of epigenetic inactivation of prolyl hydroxylases in human cancer, implying that this gene family represents a novel class of tumour suppressors. The restriction of silencing in P3H2 to breast carcinomas, and its association with oestrogen-receptor-positive cases, suggests that P3H2 may be a breast-cancer-specific tumour suppressor.

Increased mortality in HER2 positive, oestrogen receptor positive invasive breast cancer: a population-based study

Background:This study assessed the impact of human epidermal growth factor receptor 2 (HER2) status on the outcomes in an unselected population of breast cancer patients who did not receive HER2-targeted therapy.Methods:HER2 status by immunohistochemistry and fluorescence in situ hybridisation was compared with clinicopathological data, overall survival (OS) and disease-free survival (DFS) for all patients presenting with breast cancer over 3 years.Results:In 865 patients (median follow up 6.02 years), HER2 positivity was identified in 13.3% of all cancers and was associated with higher tumour grade (P<10−8), lymphovascular invasion (P<0.001) and axillary nodal metastasis (P=0.003). There was a negative association with oestrogen-receptor (ER) and progesterone-receptor expression (P<10−8), but the majority (57%) of HER2+tumours were ER+HER2 positivity was associated with poorer OS (P=0.0046) and DFS (P=0.0001) confined to the lymph node-positive (LN+) and ER+ subgroups.Conclusion:HER2-positive cancers were less common in this population-based cohort than most selected series. The association of HER2 positivity with poor prognosis was confined to the ER+ and LN+ subgroups. The survival deficit for the 7.5% of patients with ER+/HER2+ cancer compared with ER+/HER2– patients points to a significant subgroup of women who may not (currently) be considered for HER2-directed therapy.

CYP2D6 genotype affects outcome in postmenopausal breast cancer patients treated with tamoxifen monotherapy

Tamoxifen efficacy may be influenced by a number of factors, including CYP2D6 genotype, co-administration of drugs that inhibit CYP2D6, and adherence to tamoxifen therapy. CYP2D6 plays a major role in catalyzing the conversion of tamoxifen to its active metabolite endoxifen. Studies of the relevance of CYP2D6 genotyping have had conflicting results due to various limitations: differences in CYP2D6 allele coverage, phenotype classification and other confounding variables.

Multiparameter DNA content analysis identifies distinct groups in primary breast cancer.

Background:Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.Methods:The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox’s Regression (CR) analysis.Results:Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76–1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18–1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI⩾1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and ‘multiploid tumours’ (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (⩾10%) independently associated with poor overall survival (P=0.027, CR).Conclusion:Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

An integrated informatics platform to facilitate transforming tissue into knowledge

Tissue banks provide a core facility that enable translational research to be undertaken on quality-assured patient-donated samples. These samples require clinical and pathological annotations to allow comparisons to be drawn between the research-generated data and the conventional pathological and clinical outcome parameters. The challenge is to develop a flexible database system to capture these parameters and to provide a statistical framework for automated analysis. Various database solutions have been developed to capture the data at the numerous stages of research. A tissue banking platform allows complete tracking of samples as they are frozen, extracted and released, combined with an independent pathology database to capture the pathological and clinical outcome data for all breast cancer patients. This web-based interface allows the appointed research nurse to submit data as they are approved at the Multi Disciplinary Team Meetings. The patient records are also routinely checked to capture any data relating to disease recurrence, treatments and surgical procedures. A digital pathology database is also used to capture and store the tissue microarray results. All of these databases are then combined using another locally designed system, INSPIRE, which brings all the data together to perform statistical analyses and present the results via a user-friendly, web-based interface. The various systems developed allow tissue-banked samples to be tracked throughout the research process and to be annotated with high-quality pathology and research-derived data. INSPIRE then completes the cycle by aggregating these data to perform statistical analyses and present these in a user-friendly, web-based interface. This allows the researcher to focus purely on generating high-quality research and enhancing our knowledge in breast cancer.