Assessment of algorithms for mitosis detection in breast cancer histopathology images

The proliferative activity of breast tumors, which is routinely estimated by counting of mitotic figures in hematoxylin and eosin stained histology sections, is considered to be one of the most important prognostic markers. However, mitosis counting is laborious, subjective and may suffer from low inter-observer agreement. With the wider acceptance of whole slide images in pathology labs, automatic image analysis has been proposed as a potential solution for these issues. In this paper, the results from the Assessment of Mitosis Detection Algorithms 2013 (AMIDA13) challenge are described. The challenge was based on a data set consisting of 12 training and 11 testing subjects, with more than one thousand annotated mitotic figures by multiple observers. Short descriptions and results from the evaluation of eleven methods are presented. The top performing method has an error rate that is comparable to the inter-observer agreement among pathologists

A multicentre validation of Metasin: a molecular assay for the intraoperative assessment of sentinel lymph nodes from breast cancer patients

Aims: Treatment strategies for breast cancer continue to evolve. No uniformity exists in the UK for the management of node‐positive breast cancer patients. Most centres continue to use conventional histopathology of sampled sentinel lymph nodes (SLNs), which requires delayed axillary clearance in up to 25% of patients. Some use touch imprint cytology or frozen section for intraoperative testing, although both have inherent sensitivity issues. An intraoperative molecular diagnostic approach helps to overcome some of these limitations. The aim of this study was to assess the clinical effectiveness of Metasin, a molecular method for the intraoperative evaluation of SLNs. Methods and results: RNA from 3296 lymph nodes from 1836 patients undergoing SLN assessment was analysed with Metasin. Alternate slices of tissue were examined in parallel by histology. Cases deemed to be discordant were analysed by protein gel electrophoresis. There was concordance between Metasin and histology in 94.1% of cases, with a sensitivity of 92% [95% confidence interval (CI) 88–94%] and a specificity of 97% (95% CI 95–97%). Positive and negative predictive values were 88% and 98%, respectively. Over half of the discordant cases (4.4%) were ascribed to tissue allocation bias (TAB). Conclusions: Clinical validation of the Metasin assay suggests that it is sufficiently sensitive and specific to make it fit for purpose in the intraoperative setting

Metasin — An Intra-Operative RT-qPCR Assay to Detect Metastatic Breast Cancer in Sentinel Lymph Nodes

Nodal status is one of the most important prognostic factors in breast cancer. Established tests such as touch imprint cytology and frozen sections currently used in the intra-operative setting show variations in sensitivity and specificity. This limitation has led to the development of molecular alternatives, such as GeneSearch, a commercial intra-operative real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows the surgeon to carry out axillary clearance as a one-step process. Since GeneSearch has been discontinued, we have developed the replacement Metasin assay, which targets the breast epithelial cell markers CK19 and mammaglobin mRNA and identifies metastatic disease in sentinel lymph nodes. The optimised assay can be completed within 32 min (6 min for RNA preparation and 26 min instrument run time), making its use feasible in the intraoperative setting. An analysis by Metasin of 154 archived lymph node homogenates previously analysed by both parallel histology and GeneSearch showed concordance for 148 cases. The sensitivity and specificity of Metasin compared with GeneSearch were 95% (CI 83%-99%) and 97% (CI 91%-99%) respectively; compared with histology they were 95% (CI 83%-99%) and 97% (CI 91%-99%), respectively. The sensitivity and specificity of GeneSearch compared with histology were 90% (CI 77%-96%) and 97% (CI 93%-99%) respectively. The positive predictive value of Metasin was 90% and negative predictive value was 98% for both histology and GeneSearch. The positive predictive value of GeneSearch was 92% and the negative predictive value was 97% compared to histology. The discordance rates of Metasin with both GeneSearch and histology were 3.89%. In comparison, the discordance rate of GeneSearch with histology was 4.5%. Metasin's robustness was independently evaluated on 193 samples previously analysed by GeneSearch from the Jules Bordet Institute, where Metasin yielded comparable results

Study of the expression of mismatch repair genes (MMR) in breast diseases: correlation with prognostic markers

The pathology of the breast consists a great chapter in pathology of the women. Both benign and malignant lesions are thought to develop due to genetic and/or epigenetic defects. One of the mechanisms involved is genetic instability and the DNA repair system. The purpose of the present study was to investigate the expression of three of proteins encoded by the DNA mismatch repair genes, namely hMLH1, hMSH2 and hMSH6 and also the expression of BRCA1 gene product that is also involved in the DNA repair system in benign breast lesions, in situ carcinomas and invasive carcinomas of the breast, familial and sporadic. The expression of these proteins was monitored immunohistochemically in 190 benign breast lesions (adenomas, fibroadenomas, radial scars, papillomas, fibrocystic disease with or without epithelial hyperplasia etc.), 60 in situ carcinomas, 30 familial invasive carcinomas and 124 invasive sporadic carcinomas of various types and grades. We did not detect any loss of hMLH1, hMSH2 and hMSH6 expression in the material that included the benign lesions and the in situ carcinomas. Concerning invasive carcinomas, we detected loss of the mismatch repair proteins in 43,3% (MLH1), 16,7% (MSH2) and 46,7% (MSH6) in familial cases and 40,3% (MLH1), 26% (MSH2) 51,6% (MSH6) in sporadic cases examined. The reduced or absent expression of these proteins was correlated with higher histological grade, negative estrogen receptor status, overexpression of the c-erbB-2 oncogene and higher lymphatic density, peritumoral and intratumoral. Especially, loss of MSH2 protein was strongly statistically correlated with increased frequency of lymph node metastases and loss of BRCA1 protein. BRCA1 reduction in breast carcinomas usually characterizes highly malignant tumors. In addition, loss or reduction of hMLH1 and BRCA1 protein was associated with worse overall survival in the group of sporadic carcinomas, but it had no effect on the survival curves in the familial cases examined. The published data concerning the mismatch repair protein expression and its relationship with breast pathology are few and the reports are variable and controversial. Recent studies have shown that there is a high correlation between MMR-deficiency and loss of expression of the related proteins. Additionally, imuunohistochemistry is a rapid and simple technique and it can be used in everyday practice in order to detect the patients that carry these genetic/epigenetic disorder. Our results suggest that the DNA mismatch repair system is involved in breast carcinogenesis, in both familial and sporadic cases, but it is not an early event. Defects in the DNA mismatch repair system seem to occur during progression from in situ to invasive carcinoma. The MMR-deficiency affects prognosis and therefore the management and therapeutic procedures of the patients.Ο μαστός αποτελεί ένα όργανο στο οποίο αναπτύσσεται μεγάλος αριθμός παθήσεων, καλοήθων και κακοήθων. Πολλές από τις παθήσεις αυτές εμφανίζονται λόγω γενετικών ή/και επιγενετικών αλλαγών. Σημαντικούς μηχανισμούς αποτελούν η γενετική αστάθεια και τα συστήματα επιδιόρθωσης του DNA. Σκοπός της παρούσας διατριβής είναι η μελέτη της έκφρασης των πρωτεϊνών επιδιόρθωσης και διατήρησης του DNA, hMLH1, hMSH2 και hMSH6, καθώς και της πρωτεΐνης BRCA1, η οποία εμπλέκεται επίσης στον ίδιο μηχανισμό, σε καλοήθεις και προκαρκινικές αλλοιώσεις, καθώς και σε δείγματα οικογενή και σποραδικού καρκίνου του μαστού με ανοσοϊστοχημικές τεχνικές. Ιδιαίτερη έμφαση δόθηκε στην έκφραση των πρωτεϊνών από τα φυσιολογικά και τα καρκινικά κύτταρα και οι μεταβολές που παρατηρούνται, καθώς και στη διερεύνηση της προγνωστικής αξίας των δεικτών αυτών και τη συσχέτισή τους με κλινικοπαθολογοανατομικές παραμέτρους. Εξετάσθηκαν 190 περιστατικά καλοήθων παθήσεων, 60 μη διηθητικού καρκινώματος, 124 σποραδικού καρκίνου και 30 οικογενή καρκίνου. Δεν παρατηρήθηκε μείωση ή/και απώλεια των πρωτεϊνών επιδιόρθωσης και διατήρησης του DNA hMLH1, hMSH2 και hMSH6 στις καλοήθεις παθήσεις του μαστού και τα μη διηθητικά καρκινώματα (in situ) που εξετάσθηκαν και στα οποία συμπεριλήφθησαν σχεδόν όλοι οι ιστολογικοί τύποι. Στα περιστατικά οικογενούς καρκίνου του μαστού παρατηρήθηκε απώλεια της έκφρασης των hMLH1, hMSH2 και hMSH6 σε ποσοστό 43,3%, 16,7% και 46,7% αντίστοιχα. Η απώλεια αυτή σχετίζεται με υψηλότερο βαθμό κακοηθείας, απουσία έκφρασης οιστρογονικών υποδοχέων, υπερέκφραση του ογκογονιδίου c-erbB-2 και αύξηση της λεμφαγγειακής πυκνότητας. Επιπλέον, η απώλεια της hMSH2 συνδέεται με αυξημένη συχνότητα λεμφαδενικών μεταστάσεων και μείωση της πρωτεΐνης BRCA1, η οποία χαρακτηρίζει επιθετικότερους κλινικά όγκους. Η απώλεια των πρωτεϊνών hMLH1, hMSH2, hMSH6 και BRCA1, αν και σχετίζεται με αρκετές κλινικοπαθολογοανατομικές παραμέτρους που θεωρούνται ότι επηρεάζουν αρνητικά την πρόγνωση, δε φαίνεται να επηρεάζει τη συνολική επιβίωση των ασθενών με οικογενή καρκίνο του μαστού. Στα περιστατικά σποραδικού καρκίνου του μαστού παρατηρήθηκε απώλεια της έκφρασης των hMLH1, hMSH2 και hMSH6 σε ποσοστό 40,3%, 26% και 51,6% αντίστοιχα. Η απώλεια των πρωτεϊνών επιδιόρθωσης και διατήρησης του DNA στο σποραδικό καρκίνο του μαστού σχετίζεται με υψηλότερο βαθμό κακοηθείας, αρνητικούς οιστρογονικούς υποδοχείς, υπερέκφραση του c-erbB-2, αύξηση της εμφάνισης μεταστάσεων στους μασχαλιαίους λεμφαδένες, αύξηση των νεοπλασματικών εμβόλων και απώλεια της BRCA1 πρωτεΐνης. Επιπλέον, η μείωση των πρωτεϊνών hMLH1 και BRCA1 στο σποραδικό καρκίνο του μαστού φαίνεται ότι επηρεάζει αρνητικά την επιβίωση των ασθενών. Οι υπάρχουσες μελέτες που ασχολούνται με το MMR σύστημα και την επίδρασή του στην παθολογία του μαστού είναι ελάχιστες και τα αποτελέσματα ποικίλουν και είναι επιπλέον και αντικρουόμενα. Πρόσφατες μελέτες εξαίρουν την αξία της ανοσοϊστοχημικής ανίχνευσης των βλαβών του συστήματος MMR θεωρώντας τη ως μία γρήγορη και αξιόπιστη μέθοδο για την εντόπιση των ασθενών στους οποίους εμφανίζεται αυτή η γενετική ή/και επιγενετική διαταραχή. Τα αποτελέσματά μας υποδεικνύουν ότι τα γονίδια επιδιόρθωσης και διατήρησης του DNA εμπλέκονται στους παθογενετικούς μηχανισμούς της καρκινογένεσης του μαστού, αλλά όχι σε πρώιμο στάδιο. Η διαταραχή του συστήματος αυτού εμφανίζεται κατά την εξέλιξη του in situ σε διηθητικό καρκίνωμα, τόσο στην οικογενή μορφή όσο και στη σποραδική. Η ανεπάρκεια του συστήματος MMR επηρεάζει την πρόγνωση, και επακόλουθα τον τρόπο αντιμετώπισης και θεραπείας των ασθενών

Variations in sentinel node isolated tumour cells/micrometastasis and non-sentinel node involvement rates according to different interpretations of the TNM definitions

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images. (c) 2008 Elsevier Ltd. All rights reserved