The combined endocrine receptor in breast cancer, a novel approach to traditional hormone receptor interpretation and a better discriminator of outcome than ER and PR alone.

BACKGROUND: The functional role of progesterone receptor (PR) signalling was previously unclear and PR testing in breast cancer is controversial. Recent defining work has highlighted the functional crosstalk that exists between the oestrogen receptor (ER) and PR. The purpose of this retrospective cohort study was to compare the prognostic value of the combined ER and PR score with either ER or PR alone. METHODS: Tumour Allred ER and PR scores were reclassified as negative, low and high. The combined endocrine receptor (CER) was calculated as the average of the reclassified ER and PR scores, resulting in three groups: CER negative, impaired and high. Cox proportional hazards models were used to estimate disease-free survival (DFS) and breast cancer-specific survival (BCSS). RESULTS: The CER was a more powerful predictor of 5-year DFS and BCSS than either ER or PR alone. In multivariate analysis that included ER, PR and CER, only CER remained an independent prognostic variable for 5-year DFS (hazard ratio (HR) 0.393; CI: 0.283-0.548, P=0.00001) and BCSS (HR 0.553; CI: 0.423-0.722; P=2.506 × 10-8). In ER-positive (ER+) patients impaired CER was an independent marker of poor outcome for 5-year DFS (HR 2.469; CI: 1.049-5.810; P=0.038) and BCSS (HR 1.946; CI: 1.054-3.596; P=0.033) in multivariate analysis that included grade, lymph node, tumour size, HER2 status and PR status. The results were validated in a separate cohort of patients. CONCLUSIONS: Combined endocrine receptor is a more powerful discriminator of patient outcome than either ER or PR alone. Economical and simple, it can identify risk in ER+ early breast cancer and potentially be used for adjuvant cytotoxic chemotherapy decision-making.

Immunohistochemical detection improves the prognostic value of lymphatic and blood vessel invasion in primary ductal breast cancer.

BACKGROUND: Lymphovascular invasion (LBVI) including lymphatic (LVI) and blood (BVI) vessel invasion is a critical step in cancer metastasis. In breast cancer, the optimal detection method of LBVI remains unclear. This research aimed to compare the prognostic value of different assessments of the LVI and BVI in patients with early breast cancer. METHODS: The study cohort included 360 patients with a median follow-up of 168 months. LBVI on H&E sections (LBVIH&E) was reviewed centrally and blinded to the pathology report. Immunohistochemical staining for D2-40 and Factor VIII was performed to identify LVID2-40 and BVIFVIII. RESULTS: LBVIH&E, LVID2-40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node-negative patients (206), LBVIH&E, LVID2-40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple-negative patients (120), LBVIH&E, LVID2-40 and BVIFVIII were present in 35 (29%), 46 (38%) and 16 (13%) respectively. LBVIH&E was significantly associated with tumour recurrence in the whole cohort (P < 0.001), node-negative patients (P = 0.001) and triple-negative patients (P = 0.004). LVID2-40 and BVIFVIII were significantly associated with tumour recurrence in whole cohort, node-negative (all P < 0.001) and triple-negative patients (P = 0.002). In multivariate survival analysis, only LVID2-40 and BVIFVIII were independent predictors of cancer specific survival in the whole cohort (P = 0.023 and P < 0.001 respectively), node-negative patients (P = 0.004 and P = 0.001 respectively) and triple-negative patients (P = 0.014 and P = 0.001 respectively). CONCLUSION: Assessment of LVI and BVI by IHC using D2-40 and Factor VIII improves prediction of outcome in patients with node-negative and triple-negative breast cancer.

Expression of RUNX1 correlates with poor patient prognosis in triple negative breast cancer.

The RUNX1 transcription factor is widely recognised for its tumour suppressor effects in leukaemia. Recently a putative link to breast cancer has started to emerge, however the function of RUNX1 in breast cancer is still unknown. To investigate if RUNX1 expression was important to clinical outcome in primary breast tumours a tissue microarray (TMA) containing biopsies from 483 patients with primary operable invasive ductal breast cancer was stained by immunohistochemistry. RUNX1 was associated with progesterone receptor (PR)-positive tumours (P<0.05), more tumour CD4+(P<0.05) and CD8+(P<0.01) T-lymphocytic infiltrate, increased tumour CD138+plasma cell (P<0.01) and more CD68+macrophage infiltrate (P<0.001). RUNX1 expression did not influence outcome of oestrogen receptor (ER)-positive or HER2-positive disease, however on univariate analysis a high RUNX1 protein was significantly associated with poorer cancer-specific survival in patients with ER-negative (P<0.05) and with triple negative (TN) invasive breast cancer (P<0.05). Furthermore, multivariate Cox regression analysis of cancer-specific survival showed a trend towards significance in ER-negative patients (P<0.1) and was significant in triple negative patients (P<0.05). Of relevance, triple negative breast cancer currently lacks good biomarkers and patients with this subtype do not benefit from the option of targeted therapy unlike patients with ER-positive or HER2-positive disease. Using multivariate analysis RUNX1 was identified as an independent prognostic marker in the triple negative subgroup. Overall, our study identifies RUNX1 as a new prognostic indicator correlating with poor prognosis specifically in the triple negative subtype of human breast cancer.

SIRT2: tumour suppressor or tumour promoter in operable breast cancer?

PURPOSE: Sirtuins comprise a family of genes involved in cellular stress, survival and damage responses. They have been implicated in a range of diseases including cancer, with most information pertaining to their function in tumourigenesis being derived from in vitro studies, or model organisms. Their putative roles as tumour suppressors or tumour promoters remain to be validated in vivo. Little is known about their role in breast tumourigenesis. We sought to evaluate the seven sirtuin family members (SIRT1-7) in a human breast cancer cohort, in relation to clinico-pathological features and outcome of the disease. MATERIALS AND METHODS: Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in 392 oestrogen receptor (ER+ve) and 153 ER-ve breast tumour samples. SIRT1-7 transcriptional levels were assessed in normal (n=25), non-malignant (n=73) and malignant (n=70) breast tissue using Relative Quantitative Real Time PCR. Statistical analyses determined if SIRT1-7 transcription or protein expression was associated with clinical parameters or outcome. RESULTS: In ER-ve tumours, high protein levels of nuclear SIRT2 were associated with reduced time to recurrence and disease-specific death. This association was only observed in Grade 3 tumours. In the ER+ve cohort, high SIRT2 nuclear levels were associated with shorter disease-free survival and time to recurrence whilst on Tamoxifen, in patients with Grade 3 tumours. Conversely, in Grade 2 tumours, high SIRT2 levels were associated with increased time to recurrence. CONCLUSIONS: Our data suggest that SIRT2 is the sirtuin predominantly involved in breast tumourigenesis and prognosis. It indicates that SIRT2 acts as a tumour suppressor or tumour promoter dependent upon breast tumour grade.

The role of lymphatic and blood vessel invasion in predicting survival and methods of detection in patients with primary operable breast cancer.

Lymphovascular invasion (LBVI) has long been recognized as an essential step of metastases in patients with cancer. However, the process of invasion into lymphatic and blood vessels is still not well defined in breast cancer. To examine the evidence for LBVI, lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) in predicting survival in patients with primary operable breast cancer, and to evaluate the detection methods of vessel invasion. A systematic review of data published from 1964 to 2012 was undertaken according to a pre-defined protocol. There is robust evidence that general LBVI and LVI are independent prognostic factors of poorer survival. The prognostic role of BVI remains unclear. Most studies detected LBVI using H&E stained sections. The overall weighted average of the LBVI rate using immunostaining was higher (35%) than H&E (24%). The LBVI rate using H&E was variable (9-50%) and less variable using immunostaining (32-41%). The overall weighted average of the LVI rate was similar using H&E and immunostaining (33% vs. 25%). The LVI rate using H&E was variable (10-49%) and less variable using immunostaining (21-42%). The overall weighted average of the BVI rate was similar using H&E and/or classical staining and immunostaining (16% vs. 10%). The BVI rates using H&E and/or classical staining approach (4-46%) and immunostaining (1-29%) were both variable. The LBVI and LVI are powerful prognostic factors in primary operable breast cancer. However, BVI was rarely specifically examined and its role in predicting survival is not clear. Further work is required using reliable specific staining to establish the routine use of LVI and BVI in the prediction of outcome in patients with primary operable breast cancer.

Comparison of visual and automated assessment of microvessel density and their impact on outcome in primary operable invasive ductal breast cancer.

Angiogenesis is essential for tumor growth and metastasis, and several studies have reported increased angiogenesis, as quantified by microvessel density, to be a powerful prognostic tool in breast cancer. Therefore, there is considerable interest in automated assessment of microvessel density with possible benefits in improved accuracy, increased precision, and handling workload. Visual and automated assessment of microvessel density (CD34(+)) and survival were examined in patients with primary operable invasive ductal breast cancer. Tissue microarrays (n = 356 patients) immunostained for microvessel density (CD34(+)) were scored visually and automatically with the Slidepath Tissue IA system (Dublin, Ireland). Visual and automated microvessel density (CD34(+)) were in agreement (interclass correlation coefficient = 0.69, P < .001). Visual but not the automated method for microvessel density (CD34(+)) was associated with locoregional treatment and metastasis. On univariate survival analysis, visual but not automated method for microvessel density (CD34(+)) was associated with recurrence-free and cancer-specific survival in patients with invasive ductal breast cancer (P < .01). Although automated assessment of microvessel density (CD34(+)) is in reasonable agreement, it poorly predicts outcome in patients with operable invasive ductal breast cancer.

Comparison of visual and automated assessment of HER2 status and their impact on outcome in primary operable invasive ductal breast cancer.

AIMS: To compare visual and computerized image analysis of HER2 immunohistochemistry (IHC) with fluorescence in-situ hybridization (FISH) for HER2 status, and to examine the relationships with outcome in patients with primary operable invasive ductal breast cancer. METHODS AND RESULTS: Tissue microarrays for 431 breast cancer patients were used to compare different approaches to the assessment of HER2 status. The cores were scored visually and with the Slidepath Tissue IA system, using the NICE-approved scoring system for the HercepTest, as well as by FISH. The agreement between visual and image analysis of HER2 IHC was excellent [interclass correlation coefficient (ICCC) = 0.95, rs = 0.90, r = 0.91, k = 0.81, and P < 0.001]. The agreement of HER2 FISH with visual and image analysis of HER2 IHC was also excellent (ICCC = 0.95 and ICCC = 0.92, respectively). Univariate survival analysis showed equivalent associations of visual and image analysis of HER2 and HER2 FISH with both recurrence-free survival (all P < 0.01) and cancer-specific survival (all P < 0.05) in patients with invasive ductal breast cancer. CONCLUSION: Computerized image analysis of HER2 IHC gives results comparable to those obtained with visual assessment, with possible advantages in diagnostic pathology.

The role of the tumour inflammatory cell infiltrate in predicting recurrence and survival in patients with primary operable breast cancer.

Although the first studies highlighting the importance of the tumour inflammatory cell infiltrate were reported more than 80 years ago, the prognostic value of this response in breast cancer is still controversial. With the realisation of the importance of the inflammatory response in determining tumour progression there has been renewed interest in establishing the relationship between the type, density and location of inflammatory cell infiltrate and survival in patients with primary operable breast cancer. The aim was to undertake a systematic review of the literature examining the evidence for the role of the tumour inflammatory cell infiltrate in predicting recurrence and survival in patients with primary operable breast cancer. A systematic review of published papers up to September 2011 was undertaken according to a pre-defined protocol (Fig. 1). A total of 66 independent studies (34,086 patients) were identified. It can be concluded from the review that despite the large number of studies and considerable effort over an extended period, the relationship between different aspects of tumour inflammatory cell infiltrate and outcome in primary operable breast cancer remains unclear. This is in large part due to the absence of methodological validation, underpowered studies (small sample size and sample subtype heterogeneity, insufficient follow-up) and the absence of validation datasets. Therefore, although there are tantalising examples of the potential of the tumour inflammatory cell infiltrate to improve risk stratification patients with operable breast cancer (personalised care), this has not yet been realised. Future studies with standardised methodology, large and homogenous groups, sufficient follow-up and validation datasets should be undertaken to unlock the potential of the tumour inflammatory infiltrate to predict outcome in patients with primary operable breast cancer.

Breast cancer outcomes by steroid hormone receptor status assessed visually and by computer image analysis.

AIMS: To compare the assessment of steroid hormone receptor immunohistochemistry by eye and by computer-aided image analysis, and to examine their relationships with survival in breast cancer. METHODS AND RESULTS: Allred scores and weighted histoscores for oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry were determined by eye (visual histoscore) for 459 primary invasive ductal breast carcinomas in triplicate tissue microarrays. Histoscores were also determined by computerized image analysis (automated histoscore). ER and PR status determined by these different methods were compared with each other and in their ability to predict survival over at least 142 months of follow-up. Allred and visual histoscore were highly associated for ER and PR (both P < 0.001). By univariate analysis, Allred score and visual histoscore for ER and PR were highly associated with recurrence-free and cancer-specific survival (both P < 0.001) in patients with invasive ductal breast cancer overall, in those who received tamoxifen, and in those with recurrence on tamoxifen. Visual and automated histoscores were in excellent agreement for ER and PR (both P < 0.001), and were equally effective in predicting recurrence and survival for patients with invasive breast cancer who received tamoxifen. CONCLUSIONS: Automated histoscore appears to be a valid alternative to visual histoscore or Allred score for determining ER and PR status.