Grading of invasive breast carcinoma: the way forward.

Histologic grading has been a simple and inexpensive method to assess tumor behavior and prognosis of invasive breast cancer grading, thereby identifying patients at risk for adverse outcomes, who may be eligible for (neo)adjuvant therapies. Histologic grading needs to be performed accurately, on properly fixed specimens, and by adequately trained dedicated pathologists that take the time to diligently follow the protocol methodology. In this paper, we review the history of histologic grading, describe the basics of grading, review prognostic value and reproducibility issues, compare performance of grading to gene expression profiles, and discuss how to move forward to improve reproducibility of grading by training, feedback and artificial intelligence algorithms, and special stains to better recognize mitoses. We conclude that histologic grading, when adequately carried out, remains to be of important prognostic value in breast cancer patients.

Which lesions with a radiological or core biopsy diagnosis of fibroadenoma should be excised?

INTRODUCTION: A recent Association of Breast Surgery summary statement on fibroadenoma management recommends excision only for cellular fibroepithelial lesions and rapidly growing lesions with a core biopsy diagnosis of fibroadenoma; persistent pain is a relative indication for excision. METHODS: This retrospective study looked at the impact this approach would have on the diagnosis of phyllodes tumours. RESULTS: From 2014 to 2018, there were 1,058 core biopsy diagnoses of fibroadenoma; 112 lesions were excised, of which 98 were fibroadenomas, 4 were hamartomas and 10 were phyllodes tumours. In this group, an excision diagnosis of phyllodes tumour was associated with size more than 40 mm, age more than 40 years and radiological suspicion of phyllodes tumour or carcinoma. One hundred and sixty-six excised fibroepithelial lesions with no previous core biopsy included eight phyllodes tumours; in this group, rapid growth was associated with phyllodes tumour diagnosis. Twelve of the 26 fibroepithelial lesions classified as B3 (cellular fibroepithelial lesion or phyllodes tumour) were diagnosed as phyllodes tumours on excision. Using a combination of radiological, clinical and pathological features it was possible to create an excision policy that would recommend excision of 22 of the 31 phyllodes tumours in this period. Eight of the nine 'missed' phyllodes tumours were benign. CONCLUSION: The Association of Breast Surgery summary statement will reduce the number of fibroadenomas excised, but may also result in delayed diagnosis of some phyllodes tumours. Appropriate safety netting advice should be provided to identify rapidly growing lesions.

Patterns of biomarker expression in patients treated with primary endocrine therapy - a unique insight using core needle biopsy tissue microarray.

PURPOSE: Prediction of response to primary endocrine therapy (PET) in older women is based on measurement of oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor (HER)-2. This study uses a unique method for construction of core needle biopsy (CNB) tissue microarray (TMA), to correlate expression of a panel of 17 biomarkers with clinical outcome, in patients receiving PET. METHODS: Over 37 years (1973-2010), 1758 older (≥ 70 years) women with operable primary breast cancer were managed in a single institution. Of these, 693 had sufficient good-quality CNB to construct TMA, of which 334 had ER-positive tumours treated by PET with a minimum of 6-month follow-up. A panel of biomarkers was measured by immunohistochemistry (ER, PgR, HER2, Ki-67, p53, CK5/6, CK 7/8, EGFR, BCL-2, MUC1, VEGF, LKB1, BRCA1, HER3, HER4, PTEN and AIB1). Expression of each biomarker was dichotomised into 'low' or 'high' based on breast cancer-specific survival (BCSS). RESULTS: From the panel of biomarkers, multivariate analysis showed: High ER (p = 0.003) and PgR (p = 0.002) were associated with clinical benefit of PET at 6 months, as opposed to progressive disease. High ER (p = 0.0023), PgR (p < 0.001) and BCL-2 (p = 0.043) and low LKB1 (p = 0.022) were associated with longer time to progression. High PgR (p < 0.001) and low MUC1 (p = 0.021) were associated with better BCSS. Expression of other biomarkers did not show any significant correlation. CONCLUSIONS: In addition to ER and PgR; MUC1, BCL-2 and LKB1 are important in determining the outcome of PET in this cohort.

Expression of Lamin A/C in early-stage breast cancer and its prognostic value.

PURPOSE: Lamins A/C, a major component of the nuclear lamina, play key roles in maintaining nuclear integrity, regulation of gene expression, cell proliferation and apoptosis. Reduced lamin A/C expression in cancer has been reported to be a sign of poor prognosis. However, its clinical significance in breast cancer remains to be defined. This study aimed to evaluate expression and prognostic significance of lamin A/C in early-stage breast cancer. METHODS: Using immunohistochemical staining of tissue microarrays, expression of lamin A/C was evaluated in a large well-characterised series of early-stage operable breast cancer (n = 938) obtained from Nottingham Primary Breast Carcinoma Series. Association of lamin A/C expression with clinicopathological parameters and outcome was evaluated. RESULTS: Positive expression rate of lamin A/C in breast cancer was 42.2% (n = 398). Reduced/loss of expression of lamin A/C was significantly associated with high histological grade (p < 0.001), larger tumour size (p = 0.004), poor Nottingham Prognostic Index score (p < 0.001), lymphovascular invasion (p = 0.014) and development of distant metastasis (p = 0.027). Survival analysis showed that reduced/loss of expression of lamin A/C was significantly associated with shorter breast cancer-specific survival (p = 0.008). CONCLUSION: This study suggests lamin A/C plays a role in breast cancer and loss of its expression is associated with variables of poor prognosis and shorter outcome.

Associations between genomic stratification of breast cancer and centrally reviewed tumour pathology in the METABRIC cohort.

The integration of genomic and transcriptomic profiles of 2000 breast tumours from the METABRIC [Molecular Taxonomy of Breast Cancer International Consortium] cohort revealed ten subtypes, termed integrative clusters (IntClust/s), characterised by distinct genomic drivers. Central histopathology (N = 1643) review was undertaken to explore the relationship between these ten molecular subtypes and traditional clinicopathological features. IntClust subtypes were significantly associated with histological type, tumour grade, receptor status, and lymphocytic infiltration (p < 0.0001). Lymph node status and Nottingham Prognostic Index [NPI] categories were also significantly associated with IntClust subtype. IntClust 3 was enriched for tubular and lobular carcinomas, the latter largely accounting for the association with CDH1 mutations in this cluster. Mucinous carcinomas were not present in IntClusts 5 or 10, but did not show an association with any of the remaining IntClusts. In contrast, medullary-like cancers were associated with IntClust 10 (15/26). Hormone receptor-positive tumours were scattered across all IntClusts. IntClust 5 was dominated by HER2 positivity (127/151), including both hormone receptor-positive (60/72) and hormone receptor-negative tumours (67/77). Triple-negative tumours comprised the majority of IntClust 10 (132/159) and around a quarter of IntClust 4 (52/217). Whilst the ten IntClust subtypes of breast cancer show characteristic patterns of association with traditional clinicopathological variables, no IntClust can be adequately identified by these variables alone. Hence, the addition of genomic stratification has the potential to enhance the biological relevance of the current clinical evaluation and facilitate genome-guided therapeutic strategies.

Current trials to reduce surgical intervention in ductal carcinoma in situ of the breast: Critical review.

The high proportion of ductal carcinoma in situ (DCIS) presented in mammographic screening and the relatively low risk of progression to invasive disease have raised questions related to overtreatment. Following a review of current DCIS management protocols a more conservative approach has been suggested. Clinical trials have been introduced to evaluate the option of avoiding surgical intervention in a proportion of patients with DCIS defined as "low-risk" using certain clinicopathological criteria. These trials can potentially provide evidence-based models of active surveillance (with or without endocrine therapy) as a future management approach. Despite the undisputable fact of our need to address the obvious overtreatment of screen-detected DCIS, some important questions need to be considered regarding these trials including the eligibility criteria and definition of risk, the proportion of patient eligible for inclusion, and the length of time required for proper analysis of the trials' outcome in view of the long-term natural history of DCIS progression particularly the low-risk group. These factors can potentially affect the practicality and future impact of such trials. This review provides critical analysis of current DCIS management trials and highlights critical issues related to their practicality and the expected outcome.

Factors influencing local control in patients undergoing breast conservation surgery for ductal carcinoma in situ.

BACKGROUND: The aim of our study was to assess various predictors for local recurrence (LR) in patients undergoing breast conservation surgery (BCS) for ductal carcinoma in situ (DCIS). MATERIALS AND METHODS: An audit was performed of 582 consecutive patients with DCIS between Jan 1975 to June 2008. In patients undergoing BCS, local guidelines reported a margin of ≥10 mm during the above period. Guideline with regard to margin of excision changes soon after this period. We retrospectively analysed clinical and pathological risk factors for local recurrence in patients undergoing BCS. Statistical analysis was carried out using SPSS version 19, and a cox regression model for multivariate analysis of local recurrence was used. RESULTS: Overall 239 women had BCS for DCIS during the above period. The actuarial 5-year recurrence rate was 9.6%. The overall LR rate was 17% (40/239. LR was more common in patients ≤50 years: (10/31 patients, 32%) compared to patients > 50 years (30/208, 14%, P = 0.02). Forty three per cent of patients (6/14) with <5 mm margin developed LR which was significantly higher compared to patients with 5-9 mm margin (12%, 3/25) and with ≥10 mm margin (14%, 27/188, P = 0.01). On multivariate analysis age ≤50 years, <5 mm pathological margin were independent prognostic factors for local recurrence. CONCLUSION: Our study shows that younger age (≤50 years) and a margin < 5 mm are poor prognostic factors for LR in patients undergoing breast conservation surgery for DCIS.

Tumour cell membrane laminin expression is associated with basal-like phenotype and poor survival in Nigerian breast cancer.

INTRODUCTION: Laminin is a glycoprotein with diverse functions in carcinogenesis including cell proliferation, invasion, metastases and epithelial-mesenchymal transition (EMT). In breast cancer (BC) laminin expression is speculated to be associated with unfavourable clinicopathological and molecular characteristics. We hypothesize that laminin expression would contributed to the aggressive nature of basal like and triple negative BC phenotype observed in Black women. METHODS: The expression of laminin was determined in a well-characterised Nigerian cohort of 255 BC using tissue microarray and immunohistochemistry. Laminin expression was compared with clinical, pathological and survival characteristics. RESULTS: Laminin was expressed in 146 (57.3%) cases and significantly correlated with younger age at diagnosis (p=0.005), premenopausal status (p=0.003), expression of EGFR (p=0.002), ID4 and MTA1, basal cytokeratin 5/6, p53, and triple negative tumours (all p<0.001). In addition, there was an inverse association of laminin expression with E-cadherin (p=0.03), ER and PgR (all p<0.001) and a trend with BRCA1 (p=0.05). Univariate survival analysis showed tumours positive for laminin had significantly poorer breast cancer specific survival (BCSS, p=0.009) and disease free interval (p=0.03), but not associated in Cox multivariate analysis. CONCLUSION: This study demonstrates that laminin expression may have important roles in the aggressive nature observed in the basal-like and triple negative molecular subtype of Nigerian BC women.

Impact of intratumoural heterogeneity on the assessment of Ki67 expression in breast cancer.

In breast cancer (BC), the prognostic value of Ki67 expression is well-documented. Intratumoural heterogeneity (ITH) of Ki67 expression is amongst the several technical issues behind the lag of its inclusion into BC prognostic work-up. The immunohistochemical (IHC) expression of anti-Ki67 antibody (MIB1 clone) was assessed in four full-face (FF) sections from different primary tumour blocks and their matched axillary nodal (LN) metastases in a series of 55 BC. Assessment was made using the highest expression hot spots (HS), lowest expression (LS), and overall/average expression scores (AS) in each section. Heterogeneity score (Hes), co-efficient of variation, and correlation co-efficient were used to assess the levels of Ki67 ITH. Ki67 HS, LS, and AS scores were highly variable within the same section and between different sections of the primary tumour, with maximal variation observed in the LS (P < 0.001). The least variability between the different slides was observed with HS scoring. Although the associations between Ki67 and clinicopathological and molecular variables were similar when using HS or AS, the best correlation between AS and HS was observed in tumours with high Ki67 expression only. Ki67 expression in LN deposits was less heterogeneous than in the primary tumours and was perfectly correlated with the HS Ki67 expression in the primary tumour sections (r = 0.98, P < 0.001). In conclusion, assessment of Ki67 expression using HS scoring method on a full-face BC tissue section can represent the primary tumour growth fraction that is likely to metastasise. The association between Ki67 expression pattern in the LN metastasis and the HS in the primary tumour may reflect the temporal heterogeneity through clonal expansion.

Nottingham prognostic index plus (NPI+) predicts risk of distant metastases in primary breast cancer.

The Nottingham prognostic index plus (NPI+) is based on the assessment of biological class combined with established clinicopathologic prognostic variables providing improved patient outcome stratification for breast cancer superior to the traditional NPI. This study aimed to determine prognostic capability of the NPI+ in predicting risk of development of distant disease. A well-characterised series of 1073 primary early-stage BC cases treated in Nottingham and 251 cases from Budapest were immunohistochemically assessed for cytokeratin (Ck)5/6, Ck18, EGFR, oestrogen receptor (ER), progesterone receptor, HER2, HER3, HER4, Mucin 1 and p53 expression. NPI+ biological class and prognostic scores were assigned using individual algorithms for each biological class incorporating clinicopathologic parameters and investigated in terms of prediction of distant metastases-free survival (MFS). The NPI+ identified distinct prognostic groups (PG) within each molecular class which were predictive of MFS providing improved patient outcome stratification superior to the traditional NPI. NPI+ PGs, between series, were comparable in predicting patient outcome between series in luminal A, basal p53 altered and HER2+/ER+ (p > 0.01) tumours. The low-risk groups were similarly validated in luminal B, luminal N, basal p53 normal tumours (p > 0.01). Due to small patient numbers the remaining PGs could not be validated. NPI+ was additionally able to predict a higher risk of metastases at certain distant sites. This study may indicate the NPI+ as a useful tool in predicting the risk of metastases. The NPI+ provides accurate risk stratification allowing improved individualised clinical decision making for breast cancer.

Tumor Infiltrating T Lymphocytes and Apoptosis in Colorectal Cancer.

Dysfunction of the immune system in colorectal cancer (CRC) can be due to a number of reasons including apoptosis of tumour infiltrating lymphocytes (TILs). The aims of this study was to investigate TILs in colorectal cancer and characterize apoptosis of TILs using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for detecting DNA fragments. We used monoclonal antibodies (mAbs) to T lymphocytes to detect TILs and double immunohistochemistry to assess apoptosis. T lymphocytes were detected in the immune infiltrate in CRC. TUNEL staining disclosed a high level of cell death among TILs. Apoptosis of T lymphocytes showed significant correlation with Dukes' stage (P = 0.02), lymphatic metastasis (P = 0.03), vascular metastasis (P = 0.01), lymph node metastasis (P = 0.02) and age of patient (P = 0.01). In conclusion, CRC may elude immunological surveillance by inducing apoptosis of TILs.

Markers of progression in early-stage invasive breast cancer: a predictive immunohistochemical panel algorithm for distant recurrence risk stratification.

Accurate distant metastasis (DM) prediction is critical for risk stratification and effective treatment decisions in breast cancer (BC). Many prognostic markers/models based on tissue marker studies are continually emerging using conventional statistical approaches analysing complex/dimensional data association with DM/poor prognosis. However, few of them have fulfilled satisfactory evidences for clinical application. This study aimed at building DM risk assessment algorithm for BC patients. A well-characterised series of early invasive primary operable BC (n = 1902), with immunohistochemical expression of a panel of biomarkers (n = 31) formed the material of this study. Decision tree algorithm was computed using WEKA software, utilising quantitative biomarkers' expression and the absence/presence of distant metastases. Fifteen biomarkers were significantly associated with DM, with six temporal subgroups characterised based on time to development of DM ranging from <1 to >15 years of follow-up. Of these 15 biomarkers, 10 had a significant expression pattern where Ki67LI, HER2, p53, N-cadherin, P-cadherin, PIK3CA and TOMM34 showed significantly higher expressions with earlier development of DM. In contrast, higher expressions of ER, PR and BCL2 were associated with delayed occurrence of DM. DM prediction algorithm was built utilising cases informative for the 15 significant markers. Four risk groups of patients were characterised. Three markers p53, HER2 and BCL2 predicted the probability of DM, based on software-generated cut-offs, with a precision rate of 81.1 % for positive predictive value and 77.3 %, for the negative predictive value. This algorithm reiterates the reported prognostic values of these three markers and underscores their central biological role in BC progression. Further independent validation of this pruned panel of biomarkers is therefore warranted.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

BACKGROUND: It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. METHODS: A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. RESULTS: KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. CONCLUSIONS: This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Checkpoint kinase1 (CHK1) is an important biomarker in breast cancer having a role in chemotherapy response.

BACKGROUND: Checkpoint kinase1 (CHK1), which is a key component of DNA-damage-activated checkpoint signalling response, may have a role in breast cancer (BC) pathogenesis and influence response to chemotherapy. This study investigated the clinicopathological significance of phosphorylated CHK1 (pCHK1) protein in BC. METHOD: pCHK1 protein expression was assessed using immunohistochemistry in a large, well-characterized annotated series of early-stage primary operable invasive BC prepared as tissue microarray (n=1200). RESULT: pCHK1 showed nuclear and/or cytoplasmic expression. Tumours with nuclear expression showed positive associations with favourable prognostic features such as lower grade, lower mitotic activity, expression of hormone receptor and lack of expression of KI67 and PI3K (P<0.001). On the other hand, cytoplasmic expression was associated with features of poor prognosis such as higher grade, triple-negative phenotype and expression of KI67, p53, AKT and PI3K. pCHK1 expression showed an association with DNA damage response (ATM, RAD51, BRCA1, KU70/KU80, DNA-PKCα and BARD1) and sumoylation (UBC9 and PIASγ) biomarkers. Subcellular localisation of pCHK1 was associated with the expression of the nuclear transport protein KPNA2. Positive nuclear expression predicted better survival outcome in patients who did not receive chemotherapy in the whole series and in ER-positive tumours. In ER-negative and triple-negative subgroups, nuclear pCHK1 predicted shorter survival in patients who received cyclophosphamide, methotrexate and 5-florouracil chemotherapy. CONCLUSIONS: Our data suggest that pCHK1 may have prognostic and predictive significance in BC. Subcellular localisation of pCHK1 protein is related to its function.

Prognostic factors in metaplastic carcinoma of the breast: a multi-institutional study.

BACKGROUND: Metaplastic breast carcinoma (MBC) is a rare type of breast cancer that has basal-like characteristics and is perceived to have poorer prognosis when compared with conventional no specific type/ductal carcinomas (ductal/NST). However, current data on MBC are largely derived from small case series or population-based reports. This study aimed to assess the clinicopathological features and outcome of MBC identified through an international multicentre collaboration. METHODS: A large international multicentre series of MBC (no=405) with histological confirmation and follow-up information has been included in this study. The prognostic value of different variables and outcome has been assessed and compared with grade, nodal status and ER/HER2 receptor-matched ductal/NST breast carcinoma. RESULTS: The outcome of MBC diagnosed in Asian countries was more favourable than those in Western countries. The outcome of MBC is not different from matched ductal/NST carcinoma but the performance of the established prognostic variables in MBC is different. Lymph node stage, lymphovascular invasion and histologic subtype are associated with outcome but tumour size and grade are not. Chemotherapy was associated with longer survival, although this effect was limited to early-stage disease. In this study no association between radiotherapy and outcome was identified. Multivariate analysis of MBC shows that histologic subtype is an independent prognostic feature. CONCLUSIONS: This study suggests that MBC is a heterogeneous disease. Although the outcome of MBC is not different to matched conventional ductal/NST breast carcinoma, its behaviour is dependent on the particular subtype with spindle cell carcinoma in particular has an aggressive biological behaviour. Management of patients with MBC should be based on validated prognostic variables.

KSR1 regulates BRCA1 degradation and inhibits breast cancer growth.

Kinase suppressor of Ras-1 (KSR1) facilitates signal transduction in Ras-dependent cancers, including pancreatic and lung carcinomas but its role in breast cancer has not been well studied. Here, we demonstrate for the first time it functions as a tumor suppressor in breast cancer in contrast to data in other tumors. Breast cancer patients (n>1000) with high KSR1 showed better disease-free and overall survival, results also supported by Oncomine analyses, microarray data (n=2878) and genomic data from paired tumor and cell-free DNA samples revealing loss of heterozygosity. KSR1 expression is associated with high breast cancer 1, early onset (BRCA1), high BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels. Phospho-profiling of major components of the canonical Ras-RAF-mitogen-activated protein kinases pathway showed no significant changes after KSR1 overexpression or silencing. Moreover, KSR1 stably transfected cells formed fewer and smaller size colonies compared to the parental ones, while in vivo mouse model also demonstrated that the growth of xenograft tumors overexpressing KSR1 was inhibited. The tumor suppressive action of KSR1 is BRCA1 dependent shown by 3D-matrigel and soft agar assays. KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance. These data link these proteins in a continuum with clinical relevance and position KSR1 in the major oncoprotein pathways in breast tumorigenesis.

A study of ductal versus non-ductal invasive breast carcinomas in older women: long-term clinical outcome and comparison with their younger counterparts.

Ductal carcinoma is the commonest histological type found in invasive breast carcinomas and may be associated with worse prognosis, when compared to non-ductal carcinoma. Older patients tend to display more favourable tumour biology than younger patients. This study aimed to investigate the significance of histological type and its relationship with clinical outcome in the older group. A total of 808 older (≥70 years) women with early operable primary breast cancer underwent surgery as their primary treatment, followed by optimal adjuvant therapies, in the Nottingham Breast Unit between 1973 and 2009. The histological types of the surgical specimens were reviewed and compared with those in a previously characterised younger (<70 years) series (N = 1,733), in terms of distribution and correlation with clinical outcome. Ductal type was associated with a significantly worse clinical outcome when compared to non-ductal type in the older group in terms of 10-year rates of metastasis-free survival (75 vs 79 %, p = 0.028) and overall survival (44 vs 52 %; p = 0.015). Similar worse clinical outcome was found with the ductal type in the younger group in terms of 10-year rates of metastasis-free survival (65 vs 79 %; p = 0.001) and overall survival (60 vs 78 %; p = 0.001). For all patients with ductal type carcinomas, the older series showed significantly better 10-year metastasis-free survival (75 vs 65 %, p < 0.001) and breast cancer-specific survival (75 vs 69 %, p = 0.025) when compared to the younger series. In both old and the young, ductal cancers were associated with poor survival outcome when compared to non-ductal cancers. When compared to their younger counterparts, older patients with ductal type carcinomas had better metastasis-free and breast cancer-specific survival rates (their lower overall survival was likely to be due to death from other causes), despite having a lower likelihood of receiving adjuvant systemic therapy.