The BIG 2.04 MRC/EORTC SUPREMO Trial: pathology quality assurance of a large phase 3 randomised international clinical trial of postmastectomy radiotherapy in intermediate-risk breast cancer.

INTRODUCTION: SUPREMO is a phase 3 randomised trial evaluating radiotherapy post-mastectomy for intermediate-risk breast cancer. 1688 patients were enrolled from 16 countries between 2006 and 2013. We report the results of central pathology review carried out for quality assurance. PATIENTS AND METHODS: A single recut haematoxylin and eosin (H&E) tumour section was assessed by one of two reviewing pathologists, blinded to the originally reported pathology and patient data. Tumour type, grade and lymphovascular invasion were reviewed to assess if they met the inclusion criteria. Slides from potentially ineligible patients on central review were scanned and reviewed online together by the two pathologists and a consensus reached. A subset of 25 of these cases was double-reported independently by the pathologists prior to the online assessment. RESULTS: The major contributors to the trial were the UK (75%) and the Netherlands (10%). There is a striking difference in lymphovascular invasion (LVi) rates (41.6 vs. 15.1% (UK); p = <0.0001) and proportions of grade 3 carcinomas (54.0 vs. 42.0% (UK); p = <0.0001) on comparing local reporting with central review. There was no difference in the locally reported frequency of LVi rates in node-positive (N+) and node-negative (N-) subgroups (40.3 vs. 38.0%; p = 0.40) but a significant difference in the reviewed frequency (16.9 vs. 9.9%; p = 0.004). Of the N- cases, 104 (25.1%) would have been ineligible by initial central review by virtue of grade and/or lymphovascular invasion status. Following online consensus review, this fell to 70 cases (16.3% of N- cases, 4.1% of all cases). CONCLUSIONS: These data have important implications for the design, powering and interpretation of outcomes from this and future clinical trials. If critical pathology criteria are determinants for trial entry, serious consideration should be given to up-front central pathology review.

The prognostic significance of tumour-stroma ratio in oestrogen receptor-positive breast cancer.

BACKGROUND: A high percentage of stroma predicts poor survival in triple-negative breast cancers but is diminished in studies of unselected cases. We determined the prognostic significance of tumour-stroma ratio (TSR) in oestrogen receptor (ER)-positive male and female breast carcinomas. METHODS: TSR was measured in haematoxylin and eosin-stained tissue sections (118 female and 62 male). Relationship of TSR (cutoff 49%) to overall survival (OS) and relapse-free survival (RFS) was analysed. RESULTS: Tumours with ≥49% stroma were associated with better survival in female (OS P=0.008, HR=0.2-0.7; RFS P=0.006, HR=0.1-0.6) and male breast cancer (OS P=0.005, HR=0.05-0.6; RFS P=0.01, HR=0.87-5.6), confirmed in multivariate analysis. CONCLUSIONS: High stromal content was related to better survival in ER-positive breast cancers across both genders, contrasting data in triple-negative breast cancer and highlighting the importance of considering ER status when interpreting the prognostic value of TSR.

Systematic review and meta-analysis of cytokeratin 19-based one-step nucleic acid amplification versus histopathology for sentinel lymph node assessment in breast cancer.

BACKGROUND: One-step nucleic acid amplification (OSNA) is a new rapid assay for detecting breast cancer metastases during surgery, saving a second procedure for patients requiring an axillary clearance. Many centres in the UK and abroad have adopted OSNA in place of routine histopathology, despite no published meta-analysis. The aim of this systematic review and meta-analysis was to determine whether intraoperative OSNA for lymph node assessment is comparable to routine histopathology in the detection of clinically relevant metastases. METHODS: PubMed, Embase, Web of Knowledge and regional databases were searched for relevant studies published before December 2012. Included studies compared OSNA and standard histology using fresh lymph nodes that were assessed in a clearly defined systematic manner in accordance with the index study. RESULTS: Twelve eligible studies were identified that included 5057 lymph nodes from 2192 patients. Although meta-analysis using a random-effects model showed a similar overall proportion of macrometastases detected (429 of 3234 versus 432 of 3234; odds ratio 0·99, 95 per cent confidence interval 0·86 to 1·15), analysis of concordance showed that the pooled positive predictive value for detecting macrometastases was 0·79. This suggests that up to 21 per cent of patients found to have macrometastases using OSNA would have an axillary clearance when histology would have classified the deposits as non-macrometastases. Furthermore, analysis of data from the index publication showed that the range of cytokeratin 19 titres for tumours of a given volume is too wide to predict tumour size. CONCLUSION: OSNA has an unacceptably low positive predictive value, leading to axillary clearances that would not be recommended if standard histology had been used to assess the sentinel node.

Concordance between vacuum assisted biopsy and postoperative histology: implications for the proposed Low Risk DCIS Trial (LORIS).

AIM: The recent Breast Cancer Screening Review has estimated that for one life saved three patients are overtreated. The dramatic increase in the diagnosis of Ductal carcinoma in-situ (DCIS) has not lead to the expected decrease in the incidence of invasive cancer. It is not clear if all DCIS progress to invasive cancer if untreated. The Low Risk DCIS Trial (LORIS) intends to compare the current treatment of low risk DCIS i.e. surgery, with active monitoring. For effective implementation, concordance between diagnostic biopsy using large volume vacuum assisted biopsy (VAB) and excision histology is vital. A two-centre UK audit was done to assess concordance in patients diagnosed with low grade DCIS diagnosed using VAB. METHODS: Data of DCIS diagnosed with VAB from year 2001-2010 in University Hospital Birmingham and Leeds Teaching Hospitals was retrospectively collected and concordance between diagnostic and excision histology was assessed. Low Grade DCIS diagnoses were further evaluated retrospectively with regard to their eligibility for LORIS. RESULTS: Of 225 DCIS diagnoses 128 (57%) were high grade, 66 (29%) intermediate grade and 31 (14%) low grade. Overall 18% were upgraded to invasive cancer. The upgrade rate to invasive cancer for high grade was 23% and for low grade DCIS was 10%. In the low grade group eligible for LORIS, there were no upgrades to invasive cancer. CONCLUSION: The upgrade rates to invasive cancer are comparable to series published in literature. The concordance for the low risk DCIS with zero upgrade to invasive cancer supports the stringent LORIS eligibility criteria for trial selection.

Lymphangiogenesis in abdominal aortic aneurysm.

BACKGROUND: Ongoing angiogenesis is implicated in the inflammatory environment that characterizes abdominal aortic aneurysm (AAA). Although lymphangiogenesis has been associated with chronic inflammatory conditions, it has yet to be demonstrated in AAA. The aim was to determine the presence of lymphangiogenesis and to delineate the relationship between inflammation and neovascularization in AAA tissue. METHODS: AAA samples and preoperative computed tomography images were obtained from patients undergoing elective AAA repair. Control samples were age-matched abdominal aortic tissue. Specific immunostains for blood vessels (CD31, CD105), lymphatic vessels (D2-40), vascular endothelial growth factor (VEGF) A and VEGF receptor (VEGFR) 3 allowed characterization and quantitation of vasculature. RESULTS: The AAA wall contained high levels of inflammatory infiltrate; microvascular densities of blood (P < 0·001) and lymphatic (P = 0·003) vessels were significantly increased in AAA samples compared with controls. Maximal AAA vascularity was observed in inflammatory areas, with vessels that stained positively for CD31 (ρ = 0·625, P = 0·017), CD105 (ρ = 0·692, P = 0·009) and D2-40 (ρ = 0·675, P = 0·008) correlating positively with the extent of inflammation. Increased VEGFR-3 and VEGF-A expression was also evident within inflammatory AAA areas. CONCLUSION: These findings demonstrated lymphatic vessel involvement in end-stage AAA disease, which was associated with the degree of inflammation, and confirmed the involvement of neovascularization.

Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity.

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

mRNA expression profiling of phyllodes tumours of the breast: identification of genes important in the development of borderline and malignant phyllodes tumours.

The aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over-expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation: PAX3, SIX1, TGFB2 and HMGA2. Over-expression was validated in a separate set of formalin-fixed, paraffin-embedded (FFPE) tumours, using either in situ hybridization or immunohistochemistry. This confirmed that expression of PAX3, SIX1, TGFB2 and HMGA2 in the stromal component of PTs was associated with the borderline/malignant phenotypes (p = 8.7 x 10(-5), p = 0.05, p = 0.009, p = 0.003, respectively; Fisher's exact test). The functional consequences of down-regulating these genes were studied using siRNA in short-term cultures and cell lines established from PTs. mRNA 'knock-down' of PAX3 resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA 'knock-down' of SIX1 and HMGA2 resulted in decreased cell proliferation only in the malignant PT cell line, and 'knock-down' of TGFB2 resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs. SIX1 over-expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT. PAX3, TGFB2 and HMGA2 were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype.

Tumour-promoting activity of altered WWP1 expression in breast cancer and its utility as a prognostic indicator.

WWP1 is a ubiquitin ligase, associated with the post-translational regulation of several tumour-promoting and tumour suppressor proteins. Here we show that WWP1 expression is up-regulated in a subset of breast tumour cell lines and primary breast tumours. We overexpressed WWP1 in MCF10A breast epithelial cells and demonstrated increased cell growth and anchorage-independent colony formation. RNAi knockdown of WWP1 expression in T47D and MCF7 breast tumour cell lines reduced anchorage-independent colony formation. We used WWP1 protein expression levels, in combination with its sub-cellular localization, to classify breast tumours into four categories. Surprisingly, a category with low/absent WWP1 expression displayed a consistently worse prognosis compared with WWP1-expressing tumours. Importantly, the association with disease-free survival was independent of the status of other commonly used prognostic indicators. Thus, WWP1 is a prognostic marker and may be a potential therapeutic target for a subset of breast tumours.

Small is beautiful: microRNAs and breast cancer-where are we now?

MicroRNAs are a recently discovered class of small regulatory RNAs that influence the stability and translational efficiency of target mRNAs. They have been implicated in an increasing number of biological processes, including neoplasia. Recent studies have shown an involvement for these regulatory molecules in breast cancer. For example, miRNA profiling studies have identified microRNAs that are deregulated in breast cancer. Furthermore, functional studies have uncovered their roles in breast cancer as both tumour suppressor genes (eg miR-335) and oncogenes (eg miR-21). miRNAs deregulated in breast cancer influence the translational regulation of well-established regulatory molecules, such as oestrogen receptor-alpha, which is regulated by miR-206, and novel cancer-related molecules whose functions are not yet fully understood.. Here we present an overview of our current understanding of miRNA in breast cancer.

HER2 testing in the UK: further update to recommendations.

These guidelines update the previous UK HER2 testing guidelines and have been formulated to give advice on methodology, interpretation and quality assurance to ensure that HER2 testing results are accurate, reliable and timely with the expansion of testing to all patients with breast cancer at the time of primary diagnosis. The recommendations for testing are the use of immunohistochemistry but with analysis of equivocal cases by in situ hybridisation to clarify their HER2 status or the use of frontline fluorescence in situ hybridisation (FISH) testing for those laboratories wishing to do so; the inclusion of a chromosome 17 probe is strongly recommended. Laboratories using chromogenic or silver in situ hybridisation should perform an initial validation against FISH. For immunohistochemistry and in situ hybridisation there must be participation in the appropriate National External Quality Assurance scheme.

A comprehensive genetic profile of phyllodes tumours of the breast detects important mutations, intra-tumoral genetic heterogeneity and new genetic changes on recurrence.

The aims of this study were to identify genetic changes associated with malignant progression of the fibroepithelial neoplasms, phyllodes tumours of the breast (PTs), and to ascertain whether genetic progression occurs when PTs recur locally. A further aim was to assess whether the genetic data support the classification of these tumours into three subtypes, benign, borderline and malignant. 126 PTs (37 benign, 41 borderline, 48 malignant) were analysed by either array-CGH or the Illumina Goldengate assay. The large-scale genetic changes associated with malignant/borderline phenotypes were +1q, +5p, +7, +8, -6, -9p, -10p and -13. Cluster analysis of the array-CGH data supported the division of malignant and borderline PTs into two separate groups, one comprising almost all malignant lesions and the other, benign and borderline tumours. Interstitial deletions of 9p21 that involved the p16INK4a locus were present in many malignant/borderline PTs, and some of these appeared to cause homozygous loss. Loss of expression of p16INK4a was found frequently and this was associated with 9p deletion; we also identified one p16INK4a mutation and evidence of methylation of p16INK4a in malignant PTs. Our evidence shows that inactivation of this gene is important in the development of malignant PTs. In selected PTs, multiple areas of stroma were isolated and analysed separately by array-CGH. We found considerable intra-tumoral genetic heterogeneity. Analysis of paired primary and recurrent tumours showed that recurrent tumours often acquired new genetic changes; in particular, benign tumours tended to acquire changes characteristic of the malignant/borderline phenotype. We believe it likely that unfavourable sub-clones not easily identified by histology account for the unpredictable clinical behaviour of these tumours.

In situ and invasive lobular neoplasia of the breast.

Lobular neoplasia of the breast represents a group of related malignancies with clinical implications ranging from risk lesions [atypical lobular hyperplasia and lobular carcinoma in situ (LCIS)] through to aggressive invasive lesions, notably invasive pleomorphic lobular carcinoma. The diversity in lobular carcinoma is evident at the morphological level, at the molecular marker level and in cytogenetic profiles. Research in these areas is already changing the face of the disease group, for example suggesting that some lobular and ductal carcinomas are closely related and even that one of the lobular group, the tubulo-lobular carcinomas, should, in fact, be regarded as a ductal cancer. More research is required to understand the long-term pathogenic implications of a diagnosis of in situ lobular neoplasia, particularly pleomorphic LCIS, and to understand the genetics behind the well-recognized high risk of bilateral disease. For invasive carcinoma, molecular studies will allow refinement of therapy and the possibility of novel targeted therapies, for example directed against fibroblast growth factor receptor 1.

Correlation of energy dispersive diffraction signatures and microCT of small breast tissue samples with pathological analysis.

Identification of specific tissue types in conventional mammographic examinations is extremely limited. However, the use of x-ray diffraction effects during imaging has the potential to characterize the tissue types present due to the fact that each tissue type produces its own unique diffraction signature. Nevertheless, the analysis and categorization of these diffraction signatures by tissue type can be hampered by the inhomogeneous nature of breast tissue, leading to categorization errors where several types are present. This work aims to reduce sample categorization errors by combining spectral diffraction signature collection with sample imaging, giving more detailed data on the composition of each sample. Diffraction microCT was carried out on 19 unfixed breast tissue samples using an energy resolving translate-rotate CT system. High-resolution transmission microCT images were also recorded for comparison and sample composition analysis. Following imaging, the samples were subjected to histopathological analysis. Reconstructing on various momentum transfer regions allows different tissue types to be identified in the diffraction images. Results show a correlation between measured x-ray diffraction images and stained histopathological tissue sections. X-ray diffraction signatures generated from the measured data were categorized and analysed, with a t-test indicating that they have the potential for use in tissue type identification.

HER2 testing in the UK: consensus from a national consultation.

OBJECTIVE: To gain an understanding of current attitudes among oncologists and pathologists to prospective HER2 testing in breast cancer and to gauge whether a national consensus exists regarding extent and quality of testing. DESIGN: Qualitative study, with semi-quantitative components, using emailed questionnaires and open-ended discussion documents. PARTICIPANTS: 186 relevant specialists, including 76 breast oncologists and 99 pathologists, representing all but three of the UK cancer networks. RESULTS: A strong consensus was seen in favour of universal, non-selective testing for HER2 at the point of breast cancer diagnosis. Similarly, an overwhelming majority of participants agreed that, to optimise the quality of test results, all laboratories undertaking HER2 testing should be CPA-accredited, participate in the recognised national external quality assessment scheme (UK NEQAS), and carry out a formal annual audit of its testing service. A further recommendation that testing be restricted to laboratories undertaking a minimum 250 tests per annum for immunohistochemistry and 100 tests per annum for in situ hybridisation techniques met with majority support. However, this was not a clear consensus; a significant minority of participants favoured continued use of local services falling short of these criteria. CONCLUSION: This study was successful in gauging national specialist opinion regarding the extent and quality assurance of HER2 testing in the UK.

The pathology of breast cancer and the role of the histopathology laboratory.

Histopathology plays an important part in determining the treatment strategy for women with breast cancer, with the evaluation of breast specimens determining the surgical and the oncological therapeutic options used. The correct approach to specimens requires integration of clinical and imaging findings. This work is not trivial. It is time-consuming and skilled, and requires (and has in place) safeguards and checks in the form of national audit and quality-control schemes. The pathobiology of breast cancer is diverse, and the current taxonomy, rooted in morphological interpretation, has been underscored by molecular observations, such as the relationship of E-cadherin mutations to lobular carcinomas. Investigation of ductal carcinoma of no special type (NST) reveals covert tumour types, such as those with basal or myoepithelial features, whose distinctive features are only now being widely recognised. With the rise of modern molecular techniques, the demise of diagnostic histopathology has been predicted, but, for now and the intermediate future, the histopathologist remains a key element of the integrated breast-care team.

Angiogenesis in breast cancer: how should we measure this? (Review).

Despite the established importance of angiogenesis in the pathogenesis of solid tumours, there is still no consensus on how this is best measured or which method is the most appropriate in the determination of prognosis. Here we review the pros and cons of current methods of assessing angiogenesis, both clinical and in the laboratory, and discuss with respect to breast cancer.