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.

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.

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.

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.

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.

Identification by differential display of annexin-VI, a gene differentially expressed during melanoma progression.

To identify genes involved in the melanocyte to malignant melanoma conversion, we have applied differential display to the comparison of syngeneic murine B16F10 (metastatic melanoma) and Melan-a-immortalized melanocyte cell lines. Approximately 7000 bands were analyzed, revealing approximately 80 to be differentially displayed. Reverse Northern blotting and subsequent Northern blotting confirmed the reproducible differential expression of four transcripts. Three B16F10-specific bands encode novel genes or partially sequenced cDNAs of unknown function. One Melan-a-specific band was found to be identical to the 3' end region of the mouse Annexin VI mRNA and shown to have a reduced message expression in B16F10 relative to Melan-a. Differential expression was confirmed at the protein level with Western blotting using a rabbit polyclonal antiserum. Immunohistochemistry of human melanoma specimens with this antiserum revealed a decrease or loss of Annexin VI expression as melanomas progressed from a benign to a more malignant phenotype. Our results provide further evidence for a potential role of Annexin VI in tumor suppression.

Analysis of foetal expression sites of human type II DNA topoisomerase alpha and beta mRNAs by in situ hybridisation.

We present the first analysis of the sites of expression of DNA topoisomerase II alpha and II beta mRNAs in human foetal tissues by in situ hybridisation, using 35S-radiolabelled probes. This revealed differential localisation of topoisomerase II alpha and II beta mRNAs in a range of foetal organs, including foetal kidney (developing structures within the neogenic zone), brain (cortical layers), small intestine (crypt epithelium and muscle), liver (hepatocytes), lung (smooth muscle, and epithelium in the lining of primitive lung buds) and placenta (trophoblastic epithelium). The intensity of expression of topoisomerase II alpha mRNA appeared higher than that of topoisomerase II beta, although topoisomerase II beta mRNA was expressed in a broader range of cell types. The distinct patterns of expression of topoisomerase II alpha and beta mRNAs indicate differential regulation of these genes, suggesting that the two isoforms may play important but different roles in foetal development, with topoisomerase II alpha being expressed most strongly in zones of proliferation.

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.

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.

An inter-observer and intra-observer variability study on the diagnosis of lymph node biopsy specimens.

One hundred lymph node biopsy specimens were examined on two separate occasions by seven pathologists differing in experience in lymphoreticular pathology. Neither history nor immunohistochemistry was provided and the study, therefore, focused on morphological interpretation alone. The participants evaluated each case using a constructed response form in which the confidence with which they entered each response was also entered. Agreement on various points, between pathologists, between the two rounds, and with the referring centre was assessed. Whilst there was a high level of agreement over a diagnosis of benign vs. malignant and non-Hodgkin lymphoma vs. Hodgkin's disease, there was considerably less agreement over both T vs. B cell phenotype and high vs. low grade. The lack of agreement over grade, an evaluation which is usually made independent of immunohistochemistry, is particularly important, because of the relevance to selection of treatment. Proliferation markers may be more appropriate determinants of treatment choice.

The androgen receptor exon 1 trinucleotide repeat does not act as a modifier of the age of presentation in breast cancer.

The CAG repeat in exon 1 of the androgen receptor (AR) genes has been postulated as both a susceptibility allele and phenotypic modifier in BRCA1-associated breast cancers. We have analysed this repeat in a set of 178 breast cancer cases who have been selected only for age of presentation at 65 years or less. No effect of repeat length on age of presentation was found and there was no association between repeat length and family history. In combination with the data from other workers, our findings suggest that the androgen receptor repeat does not act as a modifier gene or susceptibility locus outside the context of the hereditary breast/ovarian cancer syndrome.

Improving the detection of p53 mutations in breast cancer by use of the FASAY, a functional assay.

The aim of this investigation was to examine the ability of the yeast-based functional assay, the functional analysis for the separation of alleles in yeast (FASAY), to detect p53 mutations in breast cancers when compared with immunohistochemistry and automated sequencing of the whole p53 gene (exons 1-11). To achieve this, all three methods were carried out on a cohort of aggressive breast tumors. In those tumors, in which the FASAY analysis indicated the presence of a mutation, cDNA was extracted from red yeast colonies and was sequenced to identify the base change in the p53 gene. The FASAY detected all 24 mutations found in the series of 48 tumors, whereas initial automated sequencing of genomic DNA detected 18/24 mutations. A second round of automated sequencing carried out using an independent source of genomic DNA detected mutations in 3 of the 6 tumors that originally appeared to lack a mutation in genomic DNA. All but 1 of the mutations originally missed by sequencing of genomic DNA were point mutations. Five mutations in this series (21%) were outside the commonly investigated exons 5-8, reinforcing the need to extend sequencing beyond this region. Of 24 tumors, 14 had strong immunohistochemical staining, and all 14 had p53 mutations; the majority of mutations missed by immunohistochemistry produced a truncated protein. Strong staining was not seen in tumors lacking a p53 mutation. The FASAY proved to be a rapid, reliable, and effective method for identifying those breast tumors harboring p53 mutations.

Primary mucinous carcinomas of the skin express TFF1, TFF3, estrogen receptor, and progesterone receptors.

Mucinous carcinoma may present at various sites, including the breast and the gastrointestinal tract. Rarely, such tumors arise within the skin. Comparatively, breast lesions are relatively common and usually associated with a good prognosis. When pure, they are typically estrogen (ER) and progesterone receptor (PR) positive and responsive to tamoxifen. The authors studied 12 mucinous carcinomas of the skin and compared the morphology with that of typical mammary lesions. The authors also evaluated for expression of estrogen receptor, progesterone receptor, and the mucus-associated peptides of the trefoil factor family (TFF), TFF1 (formerly pS2) and TFF2 (formerly SP), using immunohistochemistry. The localization of mRNAs for TFF1, TFF2, and TFF3 (formally ITF) was also studied in a subset of three tumors, using in-situ hybridization with S35 labeled riboprobes. The Grimelius stain was used to look for evidence of neuroendocrine differentiation. Eight resembled type A mucinous carcinomas of the breast, two resembled type B, and one had composite features. The 12th was a papillary neoplasm. The two type B tumors exhibited argyrophilia. All showed strong nuclear staining with the estrogen receptor antibody but a more varied pattern with antibodies to progesterone receptor and TFF1. None labeled for TFF2. The detection of TFF1 in mammalian skin is a novel finding. Cutaneous mucinous carcinoma shows strong similarities to its mammary counterpart, including expression of estrogen receptor, TFF1, and TFF3 mRNA. These observations suggest that some mucinous carcinomas of the skin might respond to antiestrogenic therapies.

Tissue microarrays--big potential from small samples (review).

For well over a century, pathologists have used formalin-fixed paraffin-embedded tissues for microscopic examination and subsequent diagnosis of human disease. However, this type of approach is usually limited to the analysis and/or detection of one or two specific features on a single slide from an individual section. This area of histological study has been revolutionised by the development of tissue microarrays (TMAs). This review describes the development and use of TMAs and discusses their possible applications to clinical oncological practice.