Laura G. Fulford

Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype.

Purpose: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast

Aims : Cytokeratin (CK) 14, a myoepithelial marker, is also expressed in a proportion of breast carcinomas. There is evidence that these tumours show a differing metastatic pattern and clinical outcome from other invasive ductal carcinomas (IDCs) and may need different management. Currently, they are not identified in routine practice and no morphological guidelines exist to aid their identification. The aim of this study was to analyse the histological features of CK14+ IDC.

Expression Profiling of Purified Normal Human Luminal and Myoepithelial Breast Cells Identification of Novel Prognostic Markers for Breast Cancer

The normal duct-lobular system of the breast is lined by two epithelial cell types, inner luminal secretory cells and outer contractile myoepithelial cells. We have generated comprehensive expression profiles of the two normal cell types, using immunomagnetic cell separation and gene expression microarray analysis. The cell-type specificity was confirmed at the protein level by immunohistochemistry in normal breast tissue. New prognostic markers for survival were identified when the luminal- and myoepithelial-specific molecules were evaluated on breast tumor tissue microarrays. Nuclear expression of luminal epithelial marker galectin 3 correlated with a shorter overall survival in these patients, and the expression of SPARC (osteonectin), a myoepithelial marker, was an independent marker of poor prognosis in breast cancers as a whole. These data provide a framework for the interpretation of breast cancer molecular profiling experiments, the identification of potential new diagnostic markers, and development of novel indicators of prognosis.

Oestrogen receptor status, pathological complete response and prognosis in patients receiving neoadjuvant chemotherapy for early breast cancer

The aim of this study was to ascertain if oestrogen receptor (ER) status predicts for pathological complete response (pCR) to neoadjuvant chemotherapy in operable breast cancer, and the effects of pCR on survival. Using a single-institution database, 435 patients were identified, who received neoadjuvant chemotherapy for operable breast cancer and were eligible for the analysis. Patients whose tumours were ER negative were more likely to achieve a pCR than patients who were ER positive (21.6 vs 8.1%, P<0.001). Owing to a strong correlation between ER status and grade, these variables were not shown to be independent predictors of pCR. Overall survival (OS) was better in those patients who achieved a pCR compared to those who did not (5-year OS 91 vs 73%; P=0.02). This was still the case when only patients with ER-negative tumours were examined (5-year OS 90 vs 52%, P=0.005), but not in the subset of patients with ER-positive tumours (5-year OS 93 vs 79%; P=0.3). Therefore, patients with ER-negative tumours were found to be more likely to achieve a pCR to neoadjuvant chemotherapy than those with ER-positive tumours, and pathological response did not have prognostic significance in patients with ER-positive tumours.

Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. Results: We show that basal-like and HER-2 tumors are characterized by “sawtooth” and “firestorm” genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Molecular Cytogenetic Identification of Subgroups of Grade III Invasive Ductal Breast Carcinomas with Different Clinical Outcomes

Tumor grade is an established indicator of breast cancer outcome, although considerable heterogeneity exists even within-grade. Around 25% of grade III invasive ductal breast carcinomas are associated with a “basal” phenotype, and these tumors are reported to be a distinct subgroup. We have investigated whether this group of breast cancers has a distinguishing pattern of genetic alterations and which of these may relate to the different clinical outcome of these patients. We performed comparative genomic hybridization (CGH) analysis on 43 grade III invasive ductal breast carcinomas positive for basal cytokeratin 14, as well as 43 grade- and age-matched CK14-negative controls, all with up to 25 years (median, 7 years) of clinical follow-up. Significant differences in CGH alterations were seen between the two groups in terms of mean number of changes (CK14+ve − 6.5, CK14−ve − 10.3; P = 0.0012) and types of alterations at chromosomes 4q, 7q, 8q, 9p, 13q, 16p, 17p, 17q, 19p, 19q, 20p, 20q and Xp. Supervised and unsupervised algorithms separated the two groups on CGH data alone with 76% and 74% accuracy, respectively. Hierarchical clustering revealed distinct subgroups, one of which contained 18 (42%) of the CK14+ve tumors. This subgroup had significantly shorter overall survival (P = 0.0414) than other grade III tumors, regardless of CK14 status, and was an independent prognostic marker (P = 0.031). These data provide evidence that the “basal” phenotype on its own does not convey a poor prognosis. Basal tumors are also heterogeneous with only a subset, identifiable by pattern of genetic alterations, exhibiting a shorter overall survival. Robust characterization of this basal group is necessary if it is to have a major impact on management of patients with breast cancer.

CGH analysis of ductal carcinoma of the breast with basaloid/myoepithelial cell differentiation

2–18% of ductal carcinoma-No Special Type (NST) are reported to express basal cell keratin 14 and such tumours may have a different metastatic pattern and prognosis. We performed immunohistochemistry for cytokeratins 19 (luminal) and 14 (basal) on 92 ductal carcinoma-NST. Those tumours showing CK14 expression were further characterized by immunohistochemistry for myoepithelial cell phenotype and analysed by comparative genomic hybridization. The 7 cases of ductal carcinoma-NST exhibiting a basal cell phenotype were all grade III tumours and showed a molecular cytogenetic profile similar to more conventional myoepithelial cell carcinomas. Therefore it appears that grade III invasive ductal carcinomas contain a subset of tumours with specific morphological and cytogenetic characteristics, and probably prognosis for the patient.

The diagnosis and management of pre-invasive breast disease: Pathology of atypical lobular hyperplasia and lobular carcinoma in situ

The term lobular neoplasia refers to a spectrum of lesions featuring atypical lobular hyperplasia and lobular carcinoma in situ (LCIS). The histopathological characteristics of these lesions are well documented. What is less well understood is the management implications of a patient diagnosed with LCIS; treatment regimes vary and are somewhat controversial. LCIS is now considered a risk factor and a non-obligate precursor for the subsequent development of invasive cancer.

Expression of ATM, p53, and the MRE11-Rad50-NBS1 complex in myoepithelial cells from benign and malignant proliferations of the breast

Aims: To analyse the expression of proteins involved in DNA double strand break detection and repair in the luminal and myoepithelial compartments of benign breast lesions and malignant breast tumours with myoepithelial differentiation. Methods: Expression of the ataxia telangiectasia (ATM) and p53 proteins was immunohistochemically evaluated in 18 benign and malignant myoepithelial tumours of the breast. Fifteen benign breast lesions with prominent myoepithelial compartment were also evaluated for these proteins, in addition to those in the MRE11–Rad50–NBS1 (MRN) complex, and the expression profiles were compared with those seen in eight independent non-cancer (normal breast) samples and in the surrounding normal tissues of the benign and malignant tumours examined. Results: ATM expression was higher in the myoepithelial compartment of three of 15 benign breast lesions and lower in the luminal compartment of eight of these lesions compared with that found in the corresponding normal tissue compartments. Malignant myoepithelial tumours overexpressed ATM in one of 18 cases. p53 was consistently negative in benign lesions and was overexpressed in eight of 18 malignant tumours. In benign breast lesions, expression of the MRN complex was significantly more reduced in myoepithelial cells (up to 73%) than in luminal cells (up to 40%) (p  =  0.0005). Conclusions: Malignant myoepithelial tumours rarely overexpress ATM but are frequently positive for p53. In benign breast lesions, expression of the MRN complex was more frequently reduced in the myoepithelial than in the luminal epithelial compartment, suggesting different DNA repair capabilities in these two cell types.

p63-driven nuclear accumulation of beta-catenin is not a frequent event in human neoplasms

deltaN-p63 isoforms may act as oncogenes owing to their ability to bind to p53-reporter genes without inciting their transcription, thus blocking the p53-driven cell cycle arrest and apoptosis. A novel mechanism linking p63 and Wnt pathways has recently been proposed. Briefly, in vitro studies using squamous cell carcinoma cell lines have suggested that deltaN-p63 may block the phosphorylation of beta-catenin, leading to its nuclear accumulation and triggering beta-catenin-responsive transcription of genes related to proliferation and oncogenic biological behavior. To test this new mechanism, the coexpression of deltaN-p63 and beta-catenin was evaluated in a large cohort of human neoplasms. Two serial sections of TARP-4 multi-tumor tissue microarray, composed of 51 normal tissue cores and 400 human neoplasms [breast (n = 75), colon (n = 75), lung (n = 75), prostate (n = 75) and ovary (n = 50) neoplasms, melanoma (n = 25), and glioblastoma (n = 25)] were subjected to immunohistochemistry with deltaN-p63 and beta-catenin monoclonal antibodies. p63 nuclear expression and beta-catenin membranous, cytoplasmic, membranous + cytoplasmic, and nuclear localization were evaluated. deltaN-p63 expression and beta-catenin nuclear localization were found in 92.6% and 0% of squamous cell carcinomas, 8.9% and 0% of breast carcinomas, 13.8% and 0% of lung adenocarcinomas, 1.4% and 23.2% of colon adenocarcinomas, 0% and 4.8% of prostate adenocarcinomas, 11.1% and 5% of ovary carcinomas, 9.0% and 9.1% of malignant melanomas, and 12.5% and 40.0% of glioblastomas, respectively. No statistically significant association between deltaN-p63 and nuclear beta-catenin expression was found for all tumors. At variance with squamous cell carcinoma cell lines, p63-driven nuclear accumulation of beta-catenin is an unusual phenomenon in human neoplasms. Caution should be exercised when translating the results of studies performed on cell lines to human neoplasms.