Correction: The clinical significance of oestrogen receptor expression in breast ductal carcinoma in situ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The clinical significance of oestrogen receptor expression in breast ductal carcinoma in situ.

BACKGROUND: Oestrogen receptor (ER) in invasive breast cancer (BC) predicts response to endocrine therapy (ET) and provides prognostic value. In this study, we investigated the value of ER expression in ductal carcinoma in situ (DCIS) in terms of outcome and the impact on ET decision. METHODS: In total, 643 pure DCIS, diagnosed at Nottingham University Hospitals, were assessed for ER. Clinicopathological data were correlated against ER status, together with assessment of recurrence rate. RESULTS: ER positivity was observed in 74% (475/643) of cases. ER positivity was associated with clinicopathological variables of good prognosis; however, outcome analysis revealed that ER status was not associated with local recurrence. In the intermediate- and high-grade ER-positive DCIS, 58% (11/19) and 63% (15/24) of the recurrences were invasive, respectively, comprising 7% and 6% of all ER-positive DCIS, respectively. Invasive recurrence in low-grade DCIS was infrequent (2%), and none of these patients died of BC. The ER status of the recurrent invasive tumours matched the primary DCIS ER status (94% in ipsilateral and 90% of contralateral recurrence). CONCLUSION: The strong correlation between DCIS and invasive recurrence ER status and the clinical impact of ET justify discussion of the use of ET in ER-positive DCIS treated by breast-conserving surgery. The excellent outcome of low-grade DCIS, which was almost always ER-positive, does not, in the opinion of authors, justify the use of risk-reducing ET. Therefore, the decision on ET for DCIS should be personalised and consider grade, ER status and other characteristics.

Intra-operative spectroscopic assessment of surgical margins during breast conserving surgery.

BACKGROUND: In over 20% of breast conserving operations, postoperative pathological assessment of the excised tissue reveals positive margins, requiring additional surgery. Current techniques for intra-operative assessment of tumor margins are insufficient in accuracy or resolution to reliably detect small tumors. There is a distinct need for a fast technique to accurately identify tumors smaller than 1 mm2 in large tissue surfaces within 30 min. METHODS: Multi-modal spectral histopathology (MSH), a multimodal imaging technique combining tissue auto-fluorescence and Raman spectroscopy was used to detect microscopic residual tumor at the surface of the excised breast tissue. New algorithms were developed to optimally utilize auto-fluorescence images to guide Raman measurements and achieve the required detection accuracy over large tissue surfaces (up to 4 × 6.5 cm2). Algorithms were trained on 91 breast tissue samples from 65 patients. RESULTS: Independent tests on 121 samples from 107 patients - including 51 fresh, whole excision specimens - detected breast carcinoma on the tissue surface with 95% sensitivity and 82% specificity. One surface of each uncut excision specimen was measured in 12-24 min. The combination of high spatial-resolution auto-fluorescence with specific diagnosis by Raman spectroscopy allows reliable detection even for invasive carcinoma or ductal carcinoma in situ smaller than 1 mm2. CONCLUSIONS: This study provides evidence that this multimodal approach could provide an objective tool for intra-operative assessment of breast conserving surgery margins, reducing the risk for unnecessary second operations.

MYC functions are specific in biological subtypes of breast cancer and confers resistance to endocrine therapy in luminal tumours.

BACKGROUND: MYC is amplified in approximately 15% of breast cancers (BCs) and is associated with poor outcome. c-MYC protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in BCs. We hypothesised that the functional role of c-MYC differs between molecular subtypes of BCs. METHODS: We therefore investigated the correlation between c-MYC protein expression and other proteins involved in different cellular functions together with clinicopathological parameters, patients' outcome and treatments in a large early-stage molecularly characterised series of primary invasive BCs (n=1106) using immunohistochemistry. The METABRIC BC cohort (n=1980) was evaluated for MYC mRNA expression and a systems biology approach utilised to identify genes associated with MYC in the different BC molecular subtypes. RESULTS: High MYC and c-MYC expression was significantly associated with poor prognostic factors, including grade and basal-like BCs. In luminal A tumours, c-MYC was associated with ATM (P=0.005), Cyclin B1 (P=0.002), PIK3CA (P=0.009) and Ki67 (P<0.001). In contrast, in basal-like tumours, c-MYC showed positive association with Cyclin E (P=0.003) and p16 (P=0.042) expression only. c-MYC was an independent predictor of a shorter distant metastases-free survival in luminal A LN+ tumours treated with endocrine therapy (ET; P=0.013). In luminal tumours treated with ET, MYC mRNA expression was associated with BC-specific survival (P=0.001). In ER-positive tumours, MYC was associated with expression of translational genes while in ER-negative tumours it was associated with upregulation of glucose metabolism genes. CONCLUSIONS: c-MYC function is associated with specific molecular subtypes of BCs and its overexpression confers resistance to ET. The diverse mechanisms of c-MYC function in the different molecular classes of BCs warrants further investigation particularly as potential therapeutic targets.

Biological and clinical significance of PARP1 protein expression in breast cancer.

Poly(ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair. PARP inhibitors have gained recent attention as promising therapeutic agents for the treatment of solid tumours including breast cancer (BC). However, the biological and clinical significance of PARP1 expression in BC and its role in DNA-damage response (DDR) remain to be defined. We investigated the expression of PARP1 expression, cleaved (PARP1c) and non-cleaved (PAR1nc) forms, in a large and well-characterised cohort of clinically annotated stage I-III operable BCs (n = 1,269) and 43 BRCA1-mutated BCs using immunohistochemistry. PARP1 expression was correlated to clinicopathological variables, outcome and expression of other key DNA repair proteins (BRCA1, RAD51, Ku70/80, PIASγ and CHK1). Expression of PARP1 was exclusively nuclear. 49 and 85 % of sporadic BC showed expression PARP1nc and PARP1c, respectively. In BRCA1-mutated tumours, PARP1nc/PARP1c was highly expressed (95 and 79 %, respectively). PARP1nc expression was positively associated with premenopausal younger age patients, larger size and higher tumour grade. PARP1 was positively associated with DDR-proteins; RAD51, BRCA1, CHK1 and PIASγ (p < 0.001). Negative association was found between PARP1nc and Ki67. PARP1c was associated with ER (p < 0.001). Different associations between PARP1 and DDR-proteins were observed when stratified based on ER/BRCA1 status. PARP1 was not an independent predictor of outcome in sporadic or BRCA1-mutated BC. Our results demonstrate a potential biological role for PARP1c and PARP1nc in DNA repair in BC based on the significant association with other key DNA damage repair proteins. These associations were not restricted to ER-negative or triple-negative subgroup.

DNA damage response markers are differentially expressed in BRCA-mutated breast cancers.

Cells have stringent DNA repair pathways that are specific for each different set of DNA lesions which is accomplished through the integration of complex array of proteins. However, BRCA-mutated breast cancer (BC) has defective DNA repair mechanisms. This study aims to investigate differential expression of a large panel of DNA repair markers to characterise DNA repair mechanisms in BRCA-associated tumours compared to sporadic tumours in an attempt to characterise these tumours in routine practice. Immunohistochemistry and tissue microarray technology were applied to a cohort of clinically annotated series of sporadic (n = 1849), BRCA1-mutated (n = 48), and BRCA2-mutated (n = 27) BC. The following DNA damage response (DDR) markers are used; BRCA1, BRCA2, RAD51, Ku70/Ku80, BARD, PARP1 (cleaved), PARP1 (non-cleaved), and P53 in addition to basal cytokeratins, ER, PR, and HER2. A significant proportion of BRCA1 tumours were positive for PARP1 (non-cleaved), and negative for BARD1 and RAD51 compared with sporadic BC. BRCA2 tumours were significantly positive for PARP1 (non-cleaved) compared with sporadic tumours. RAD51 was significantly higher in BRCA1 compared with BRCA2 tumours (p = 0.005). When BRCA1/2 BCs were compared to triple-negative (TN) sporadic tumours of the studied DDR proteins, BARD1 (p < 0.001), PARP1 (non-cleaved) (p < 0.001), and P53 (p = 0.002) remained significantly different in BRCA1/2 tumours compared with TN BC. DNA repair markers showed differential expression in BRCA-mutated tumours, with a substantial degree of disruption of DNA repair pathways in sporadic BC especially TN BC. DNA double-strand break (DSB) repair is assisted by PARP1 expression in BRCA-mutated tumours, whereas the loss of DSB repair via RAD51 is predominant in BRCA1 rather than BRCA2 BC.

Explantation with Lateral Pedicle Mastopexy.

BACKGROUND: While breast explantation combined with mastopexy is an increasingly common procedure, it does present certain technical difficulties. We present a technique of explantation mastopexy with the use of an extended lateral pedicle for auto-augmentation. METHODS: A consecutive series of 40 cases was retrospectively reviewed, with patient reported outcome questionnaire and photography at 3 and 12 months. RESULTS: The mean age was 57 years (range 40 - 70 years), and mean duration of implantation was 20.4 years (range 7 - 42 years). 12 women had undergone previous mastopexy (30%). Minor wound complications requiring simple dressings were seen in 7 cases (17.5%). Major infected wound problems occurred in 1 case, who was a smoker and had other co-morbidities. All except 1 case reported being satisfied or very satisfied with the outcome, with a mean patient reported satisfaction score of 4.9/5. When the photographs were independently assessed by a cosmetic practitioner, all cases were rated as average, good or very good, with a mean score of 4.1/5. CONCLUSIONS: The procedure is associated with low risk of post-operative complications, good cosmetic outcomes, and a high degree of patient satisfaction. We feel this technique provides a logical, reproducible method for combined explantation and mastopexy.

The microRNA maturation regulator Drosha is an independent predictor of outcome in breast cancer patients.

Drosha is a protein that plays a key role in the biogenesis of microRNAs which are well known to be deranged in human breast cancer (BC). The purpose of the current study was to assess the biological and prognostic value of Drosha protein expression in BC. Drosha protein expression was assessed immunohistochemically in two sets of BC: (1) full-face sections of selected BC series with distinct stages of tumour progression (Normal parenchymal cells, ductal carcinoma in situ (DCIS), primary invasive BC and nodal metastases) to evaluate its differential expression, (2) tissue microarray comprising a large and well-characterised series of unselected clinically annotated invasive BC to investigate its correlation with clinicopathological features and patient outcome. A gradual loss of Drosha cytoplasmic expression was observed along tumour progression from DCIS, to invasive and to metastatic cancer cells. In invasive BC, loss of Drosha cytoplasmic expression was associated with BRCA1 and ER expression and with shorter BC specific survival (BCSS), disease free interval (DFI) and distant metastasis free interval (DMFI). This correlation was maintained in ER negative, HER2 negative, triple negative and LN negative cases. Moreover, loss of cytoplasmic Drosha was predictive of better response to chemotherapy and endocrine therapy. This study provides evidence that Drosha protein potentially plays an important role in BC progression and assessment of its expression provides an independent predictor of patient outcome. These observations provide further evidence that alterations in miRNA regulation influence tumour behaviour.

Pleomorphic lobular carcinoma of the breast: is it a prognostically significant pathological subtype independent of histological grade?

Pleomorphic lobular carcinoma is regarded as a biologically aggressive variant of invasive lobular carcinoma of the breast. However, there is no consensus on the definition and whether this subtype adds useful information to histological grade. Two-hundred and two grade 2 or grade 3 invasive lobular carcinomas were studied. Tumours were categorised according to the components of histological grade: tubules, pleomorphism and mitoses. Pleomorphic lobular carcinoma was defined as a carcinoma with a lobular growth pattern and marked nuclear pleomorphism (pleomorphism 3). Breast cancer-specific survival was used in analysis of prognosis. Grade 3 pleomorphic lobular carcinomas (tubules 3, pleomorphism 3, mitoses 2 and tubules 3, pleomorphism 3, mitoses 3) had a worse prognosis than grade 2 (tubules 3, pleomorphism 2, mitoses 1) carcinomas. Grade 2 lobular carcinomas with marked nuclear pleomorphism (tubules 3, pleomorphism 3, mitoses 1) had a similar prognosis to grade 2 carcinomas with moderate pleomorphism (tubules 3, pleomorphism 2, mitoses 1). Survival was associated with mitotic score, but not with nuclear pleomorphism on both univariate and multivariate analysis. A non-classical growth pattern was seen more frequently in all subgroups with marked nuclear pleomorphism and was associated with worse survival. Histological grade and nodal status were independent of prognostic factors. This study shows that histological grade (in particular the mitotic component) in invasive lobular carcinomas is of prognostic importance, but pleomorphic type does not provide useful additional prognostic information.

EpCAM expression is an indicator of recurrence in basal-like breast cancer.

Advances in the understanding of the molecular basis of breast cancer have necessitated a definition of more sensitive and specific indicators of prognosis that are central to the underlying cancer biology and that reflect the complicated and heterogeneous nature of the disease. This study investigates the expression of epithelial cell adhesion molecule (EpCAM) in breast cancer particularly basal-like phenotype group which remains unclear. EpCAM expression was assessed using immunohistochemistry in a large well-characterised series of breast carcinomas prepared as tissue microarrays. Relationships between EpCAM expression with molecular subtypes, clinicopathological variables and patients' outcome were examined. EpCAM expression was associated with higher tumour grade (P < 0.001), larger tumour size (P < 0.001) and basal phenotype (P = 0.03). Importantly, within the basal-like tumours those positive for EpCAM showed a significantly shorter DFI (LR = 7.97, P = 0.005) and MFS (LR 4.01, P = 0.045). EpCAM may play a role in breast cancer progression and its expression is associated with poor patient outcome in basal-like breast cancer, independent of other prognostic factors.

Objective assessment of lymphatic and blood vascular invasion in lymph node-negative breast carcinoma: findings from a large case series with long-term follow-up.

In a previous study on a small series of breast cancers, we developed objective methods for the assessment of vascular invasion (VI), using immunohistochemical staining. We found that VI was predominantly lymphovascular invasion (LVI), with minimal contribution of blood vascular invasion (BVI). The aims of the current study were: (a) to assess the frequency, extent and prognostic role of LVI and BVI in a large, well-characterized series of LN-negative breast cancers; and (b) to assess the ability of VI to stratify early breast cancer into different prognostic groups. Paraffin-embedded sections from 1005 lymph-node (LN)-negative primary invasive breast cancers were stained for CD34, CD31 and podoplanin/D240 to detect BVI and LVI. VI lesions were assessed and the results were correlated with clinicopathological criteria and survival. VI was detected in 218 (22%); 211/218 (97%) were LVI, while BVI was detected in 7/218 (3%). The frequency of LVIs/section ranged from 1 to 79, with no significant difference between the frequency of LVI and outcome. The presence of LVI was significantly associated with adverse disease-free interval (DFI) and poor overall survival (OS) in both univariate and multivariate analyses. The results from the study indicated that VI in early stage breast cancer is predominantly LVI and that its objective assessment is a powerful independent prognostic factor. Efforts to detect early metastatic activity, such as diligent pathological examination of sentinel LN biopsies would be complimented by the objective evaluation of VI status of the primary tumour. VI status should be included routinely in breast cancer staging systems.

MIB1/Ki-67 labelling index can classify grade 2 breast cancer into two clinically distinct subgroups.

Histological grade is recognized as one of the strongest prognostic factors in operable breast cancer (BC). Although grade 1 and grade 3 tumours are biologically and clinically distinct, grade 2 tumours bear considerable difficulty in outcome prediction and planning therapies. Several attempts such as genomic grade index have been performed to subclassify grade 2 into two subgroups with clinical relevance. Here, we present evidence that the routinely evaluable immunohistochemical MIB1/Ki67 labelling index (MIB-LI) can classify grade 2 tumours into two clinically distinct subgroups. In this study, growth fractions of 1,550 primary operable invasive breast carcinomas were immunohistochemically assayed on full-face tissue sections using the MIB1 clone of Ki-67. Growth fractions were assessed as number of MIB1 positive nuclei in 1,000 tumour nuclei at high-power magnification and expressed as MIB1-LI. Using a 10% cut-point of MIB1-LI, grade 2 BCs were classified into low (49.8%) and high (50.2%) proliferative subgroups. Univariate and multivariate survival analysis revealed statistically significant differences between these subgroups regarding patients' BC specific survival (P < 0.001), and metastasis free survival (P < 0.001) which was independent of the well-established prognostic factors (HR = 2.944, 95% CI = 1.634-5.303, P < 0.001). In conclusion, our results further demonstrate that grade 2 BCs may represent at least two biological or behaviourally different entities. Assay of growth fraction in BC using MIB1/Ki67 immunohistochemistry is a robust cost-effective diagnostic tool that subdivides grade 2 tumours into low and high risk populations providing additional prognostic information in planning therapies and outcome prediction.

An evaluation of the clinical significance of FOXP3+ infiltrating cells in human breast cancer.

Studies in mice have shown that thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3(+) lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and the patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs. Tissue microarrays were used, and the density of FOXP3(+) cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up. FOXP3(+) cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3(+) cells significantly correlated with higher tumour grade (r (s) = 0.37, P < 0.001) and ER negativity (Mann-Whitney U test, P < 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3(+) cells were associated with a worse prognosis (P = 0.012, log rank = 6.36). This association was found for intratumoural FOXP3(+) and for tumour-adjacent stromal FOXP3(+)-cells (tumour-cell associated FOXP3, P = 0.001 and log rank 10.35). However, the number of FOXP3(+) cells was not found to be an independent prognostic factor in multivariate analysis. We therefore conclude that FOXP3(+) infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.

Growth fraction as a predictor of response to chemotherapy in node-negative breast cancer.

Accurate predictive markers of chemotherapeutic response in early breast cancer are still lacking. The role of tumour growth fraction as a predictor of response to chemotherapy was assessed in early breast cancer. In this study, immunohistochemical expression of MIB1 was studied in a well-characterised series of early (Stages I and II) node-negative breast carcinoma cases (n = 100) with long-term follow-up that have received adjuvant chemotherapy (cyclophosphamide/methotrexate/5-fluorouracil regimen). In addition, 728 cases who did not receive adjuvant chemotherapy were used as a control group. Increased tumour growth fraction was associated with a better response to adjuvant chemotherapy in terms of longer breast cancer specific survival and disease-free interval [hazard ratio (HR) = 0.354, 95% CI = 0.177-0.688, p = 0.003 and HR = 0.396, 95% CI = 0.205-0.768, p = 0.006, respectively]. In contrast to the control group, patients with high growth fraction tumour (>70%) showed an excellent outcome with infrequently reported events during the period of follow-up. Importantly, patients with a low growth fraction (< or =10%) showed frequent recurrences and shorter survival time with outcome comparable to those of high growth fraction who did not receive chemotherapy. Therefore, tumour growth fraction can be used to assign patients into distinct groups showing differential response to adjuvant chemotherapy. Patients with a high growth fraction appear to be ideal candidates for adjuvant chemotherapy while those with low growth fraction are less likely to benefit and are prone to the potential serious side effects of adjuvant chemotherapy.

The prognostic significance of PELP1 expression in invasive breast cancer with emphasis on the ER-positive luminal-like subtype.

The transcription functions of oestrogen receptors (ER) are influenced by several coregulators such as PELP1 (proline, glutamate and leucine rich protein 1). The aim of the present study, which uses tissue microarrays and immunohistochemistry, is to explore the clinical and biological relevance of PELP1 protein expression in a large series of consecutive patients (1,162 patients) with invasive breast cancers with particular emphasis on its role in the ER-positive/luminal-like class of tumours. Our results showed that increased PELP1 expression is associated with tumours of larger size, higher histological grade, higher mitotic count, and with positive expression of basal cytokeratins (CK) (CK14; P = 0.018 and CK5/6; P = 0.029), P-cadherin (P = 0.002), p53 and MIB1 (P = 0.018). There was an inverse association between PELP1 expression and ER (P = 0.002), progesterone (PgR) (P = 0.004), androgen (AR) receptor (P < 0.001), and luminal CK (CK18; P = 0.027) expression. A significant association between PELP1 expression and shorter breast cancer specific survival (BCSS) (P = 0.002) and disease-free survival (DFI) (P = 0.006) was found. Multivariate Cox hazard analysis showed that PELP1 expression was an independent predictor of shorter BCSS (Hazard ratio (HR) = 1.349, P = 0.006) and shorter DFI (HR = 1.255, P = 0.011). In the ER-positive/luminal-like group (n = 768), PELP1 expression showed similar association with other clinicopathological variables and was an independent predictor of shorter DFI (HR = 1.256, P = 0.036). In conclusion, PELP1 protein expression is an independent prognostic predictor of shorter BCSS and DFI in breast cancer and its elevated expression is positively associated with markers of poor outcome. PELP1 appears to have a potential application in assessing the clinical outcome of patients with ER-positive breast cancer.

PIK3CA expression in invasive breast cancer: a biomarker of poor prognosis.

The implications of Phosphatidylinositol 3-kinase (PIK3CA) mutations and its aberrant protein expression in breast cancer (BC) different molecular subtypes and patients' outcome remain controversial. The aims of this study were to assess the prevalence and clinical significance of PIK3CA protein expression in BC and to determine its association with its different molecular classes. PIK3CA protein expression was assessed in a well-characterized series of early stage BC (n = 1,394) with long-term follow-up, using tissue microarrays and immunohistochemistry. Associations between PIK3CA expression and clinicopathological variables, molecular classes, and patients' outcome were investigated. Positive PIK3CA expression was associated with poor prognostic variables including higher grade, larger size, nodal involvement, vascular invasion, and higher proliferative fraction (P < 0.001). Increased PIK3CA expression was associated with the basal-like breast cancer (BLBC) and HER2-positive classes as well as triple negative non-basal (TNnon-B) tumors (P < 0.001). The luminal class showed reduced PIK3CA expression relative to other classes. Patients with PIK3CA positive tumors had shorter BC specific and disease free survival, independent of other prognostic factors except grade. Similar associations with outcome were found when the analysis was restricted to the large luminal class of tumors. PIK3CA is an oncogenic biomarker associated with poor prognosis in BC. Although, PIK3CA over-expression was more prevalent in BLBC and HER2-positive tumors it appeared to be a marker of poor differentiation rather than of a particular subtype. Thus, targeting of PIK3CA using specific inhibitors could potentially be beneficial, particularly for patients with more aggressive poorly differentiated tumors, irrespective of their molecular subtype.

Comparison of margin assessment by radial and shave sections in wide local excision specimens for invasive carcinoma of the breast.

AIMS: Standard margin assessment of breast carcinoma surgical specimens uses radial sections perpendicular to the margin. Shave sections assess a larger surface area of margin than radial sections. The aim was to assess the value of additional shave sections of the margin. METHODS AND RESULTS: Both types of section were used to assess 471 wide local excision specimens for invasive carcinoma. One hundred and seventy-nine specimens had positive margins: only radial margins were involved (tumour within 5 mm of margin) in 76, only shave margins in 45, and both shave and radial margins in 58. Residual carcinoma was found in re-excision specimens (immediate or later) in 43% when the closest distance to the radial margin was 0-1 mm, 25% for 2-4 mm, 18% for 5-9 mm and 13% for >9 mm. Residual carcinoma was found in 44% of specimens if any shave section was positive and in 9% if all shaves were negative. Residual carcinoma was found in 32% if either radial or shave sections were positive and in 4% if neither was positive. CONCLUSIONS: The combination of radial and shave sections appears to be good at separating patients into two groups with high and low risk of residual carcinoma.