Anja Høegh Brügmann

Digital image analysis of membrane connectivity is a robust measure of HER2 immunostains

The purpose of this study was to develop and validate a new software, HER2-CONNECTTM, for digital image analysis of the human epidermal growth factor receptor 2 (HER2) in breast cancer specimens. The software assesses immunohistochemical (IHC) staining reactions of HER2 based on an algorithm evaluating the cell membrane connectivity. The HER2-CONNECTTM algorithm was aligned to match digital image scorings of HER2 performed by 5 experienced assessors in a training set and confirmed in a separate validation set. The training set consisted of 167 breast carcinoma tissue core images in which the assessors individually and blinded outlined regions of interest and gave their HER2 score 0/1+/2+/3+ to the specific tumor region. The validation set consisted of 86 core images where the result of the automated image analysis software was correlated to the scores provided by the 5 assessors. HER2 fluorescence in situ hybridization (FISH) was performed on all cores and used as a reference standard. The overall agreement between the image analysis software and the digital scorings of the 5 assessors was 92.1% (Cohen’s Kappa: 0.859) in the training set and 92.3% (Cohen’s Kappa: 0.864) in the validation set. The image analysis sensitivity was 99.2% and specificity 100% when correlated to FISH. In conclusion, the Visiopharm HER2 IHC algorithm HER2-CONNECTTM can discriminate between amplified and non-amplified cases with high accuracy and diminish the equivocal category and thereby provides a promising supplementary diagnostic tool to increase consistency in HER2 assessment.

Testing HER2 in Breast Cancer: A Comparative Study on BRISH, FISH, and IHC

&NA;New brightfield in-situ hybridization (BRISH) methods based on the cohybridization of probes to the HER2 gene and chromosome 17 centromere have been developed and provide a promising alternative to fluorescence in-situ hybridization (FISH). The aim of this correlation study was to test HER2 status in primary breast carcinomas with 2 BRISH methods, FISH, and 2 immunohistochemical assays using tissue microarray technology. Materials and MethodsTissue cores (1.5 mm) were collected from 218 consecutive, archival formalin-fixed paraffin-embedded primary breast carcinomas into 4 duplicate tissue microarrays. Tumor tissue samples from 201 patients were successfully prepared in all 5 investigated methods comprising DuoCISH (Dako), Dual ISH (Ventana), HER-2 pharmDxFISH (Dako), HercepTest (Dako), and PATHWAY (4B5; Ventana). ResultsIn this study the overall agreement between Dual ISH and FISH was 98.5% with a specificity of 99% and a sensitivity of 96%. DuoCISH had an equivalent high-positive agreement with FISH (sensitivity of 96%), but a lower specificity of 93% and an overall agreement of 93% with FISH. The overall agreement between the 2 immunohistochemical methods and FISH was almost perfect (Dako HercepTest 97% and Ventana PATHWAY (4B5) 98%). With regard to specificity the 2 methods performed equally (99.4%). ConclusionsBRISH methods provide an alternative to FISH in evaluating HER2/CEN17 ratio in primary breast carcinomas. Dual ISH showed an almost perfect agreement with FISH and is a fast track method realistic to perform on all breast carcinomas. BRISH provide a permanent result that makes the method eligible for use in internal and external quality assurance.

Expression of PIK3CA, PTEN mRNA and PIK3CA mutations in primary breast cancer: association with lymph node metastases.

PurposeHigh activity of the intracellular phosphatidylinositol-3 kinase (PI3K) pathway is common in breast cancer. Here, we explore differences in expression of important PI3K pathway regulators: the activator, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA), and the tumour suppressor, phosphatase and tensin homolog (PTEN), in breast carcinoma tissue and normal breast tissue. Furthermore, we examine whether expression of PIK3CA and PTEN mRNA and occurrence of PIK3CA mutations are associated with lymph node metastases in patients with primary breast cancer.MethodsPaired tissue samples of breast carcinoma and normal breast tissue were obtained from 175 breast cancer patients at the time of primary surgery, of these 105 patients were lymph node positive. Expression of PIK3CA and PTEN mRNA was quantified with Quantitative Real Time PCR. Somatic mutations in exon 9 and exon 20 of the PIK3CA gene were identified by genotyping.ResultsBoth PIK3CA and PTEN mRNA expression was significantly increased in breast carcinoma tissue compared to normal breast tissue (p = 2 × 10-11) and (p < 0.001), respectively. PIK3CA mutations were present in 68 out of 175 patients (39%), but were not associated with PIK3CA expression (p = 0.59). Expression of PIK3CA and PTEN mRNA, and PIK3CA mutations in breast carcinomas were not associated with presence of lymph node metastases.ConclusionsThe expression of PTEN and PIK3CA mRNA is increased in breast carcinoma tissue compared to normal breast tissue, and PIK3CA mutations are frequent in primary breast carcinoma, however these factors were not associated with lymph node metastases.

Identifying responders to trastuzumab therapy in breast cancer

In breast cancer, HER2-targeted therapy with trastuzumab has gained significant attention, owing to the dramatic response observed in a subset of HER2-positive patients. The mechanisms of action are complex and not fully understood, and much effort has been spent in order to identify responders. Good patient management, side effects of the humanized monoclonal antibody and socioeconomics all demand that the drug should be administered only to the patients who will benefit from it. This has been a difficult task and contributions to solve it have been proposed from a variety of research. In this article we describe some of these contributions based on the literature and provide our viewpoint as to which identifiers will emerge in the following decade.

An inter-observer Ki67 reproducibility study applying two different assessment methods : on behalf of the Danish Scientific Committee of Pathology, Danish breast cancer cooperative group (DBCG)

Abstract Introduction: In 2011, the St. Gallen Consensus Conference introduced the use of pathology to define the intrinsic breast cancer subtypes by application of immunohistochemical (IHC) surrogate markers ER, PR, HER2 and Ki67 with a specified Ki67 cutoff (>14%) for luminal B-like definition. Reports concerning impaired reproducibility of Ki67 estimation and threshold inconsistency led to the initiation of this quality assurance study (2013–2015). The aim of the study was to investigate inter-observer variation for Ki67 estimation in malignant breast tumors by two different quantification methods (assessment method and count method) including measure of agreement between methods. Material and methods: Fourteen experienced breast pathologists from 12 pathology departments evaluated 118 slides from a consecutive series of malignant breast tumors. The staining interpretation was performed according to both the Danish and Swedish guidelines. Reproducibility was quantified by intra-class correlation coefficient (ICC) and Lights Kappa with dichotomization of observations at the larger than (>) 20% threshold. The agreement between observations by the two quantification methods was evaluated by Bland–Altman plot. Results: For the fourteen raters the median ranged from 20% to 40% by the assessment method and from 22.5% to 36.5% by the count method. Light’s Kappa was 0.664 for observation by the assessment method and 0.649 by the count method. The ICC was 0.82 (95% CI: 0.77–0.86) by the assessment method vs. 0.84 (95% CI: 0.80–0.87) by the count method. Conclusion: Although the study in general showed a moderate to good inter-observer agreement according to both ICC and Lights Kappa, still major discrepancies were identified in especially the mid-range of observations. Consequently, for now Ki67 estimation is not implemented in the DBCG treatment algorithm.

Virtual Double Staining: A Digital Approach to Immunohistochemical Quantification of Estrogen Receptor Protein in Breast Carcinoma Specimens

Visual assessment of immunohistochemically detected estrogen receptor protein is prone to interobserver and intraobserver variation due to its subjective evaluation. The aim of this study was to validate a new image analysis system based on virtual double staining (VDS) by comparing visual and automated scorings of ER in tissue microarrays of breast carcinomas. Tissue microarrays were constructed of 112 consecutive resection specimens of breast carcinomas. Immunohistochemistry assays for ER and pancytokeratin was applied on separate serial sections. ER scoring was visually performed by 5 observers using the histoscore (H-score) method. The Visiopharm ER image analysis protocol (APP) software application using VDS technique was applied separating stromal cells from carcinoma and other epithelial cells based on the pancytokeratin reaction. Using color deconvolution, polynomial filters, and nuclear segmentation the APP determined the percentage of positive cells and their intensity, and calculated the resulting H-score. On the basis of 1% cutoff VDS was perfectly correlated with visual assessment (&kgr;=1). Using H-score, a very high agreement between VDS and visual ER assessment was seen (R2=0.950). Image analysis has the attributes to eliminate the shortcomings of visual ER evaluation by generating automated, reproducible, and objective results of ER assessment.

The HER2 CISH pharmDx™ Kit in the assessment of breast cancer patients for anti-HER2 treatment

Testing for amplification of the human EGF receptor 2 (HER2) gene by in situ hybridization is a central principle for the identification of breast cancer patients likely to respond to treatments directed toward HER2. However, its application in clinical routine has been somewhat restricted by the typical use of a visualization system based on fluorescence (FISH), which requires skilled, work-intensive, high-magnification quantitative microscopy. The US FDA has recently approved the HER2 CISH pharmDx™ Kit, which is characterized by employing a chromogenic visualization system that allows quantification of the HER2 gene and centromere 17 reference signals by relatively low-magnification brightfield microscopy. It is indicated as an aid in the assessment of patients for whom Herceptin® (trastuzumab) treatment is being considered.

Histological definition of a lymph node - based on a survey among Danish pathologists

Introduction: Harvesting a specific number of lymph nodes for pathological examination of surgical cancer specimens is recommended in national and international guidelines including the TNM classification. The number of lymph nodes with metastasis is also a parameter for the different N-stages in some types of cancer. In contrast the exact minimal histological criteria for a lymph node are not clearly defined in the literature. We have conducted an investigation on which definition is used by Danish pathologists and residents in pathology. Material and methods: A questionnaire in Survey Monkey was sent by email to members of the Danish Society of Pathology on December 11th 2013. The responses were collected on January 6th 2014. The questionnaire offered four different options for the histological criteria for a lymph node, three options given as a yes/no answer and one option as free text. A final question of the position of the responder was also given. Results: 261 members of the Danish Pathology Society received the questionnaire and 138 (53%) answered. 99 (72%) were specialists in pathology, 34 (25%) were resident doctors in pathology, 3 (2%) designated their position as ‘other’ and 2 (1%) did not answer the question of their position. 47 (34%) had as the minimal requirement for a lymph node a nodular collection of lymphatic tissue with both a capsule and a sinus. Of these 35 (74%) were specialists, 10 (21%) residents, 1 (2%) ‘other’ and 1 (2%) had not given their position. 29 (21%) required a nodular collection of lymphatic tissue with either a capsule or a sinus. Of these 21 (72%) were specialists, 6 (21%) residents and 2 (7%) ‘other’. 38 (28%) required a nodular collection of lymphatic tissue with a capsule. Of these 25 (66%) were specialists, 12 (32%) residents and 1 (3%) had not given their position. 21 (15%) required a nodular collection of lymphatic tissue with a sinus. Of these 16 (76%) were specialists and 5 (24%) residents. 3 (2%) had a personal definition (a bigger collection of lymphocytes (1), a lymphocytic collection with a capsule and lymph node architecture (2)). All these were specialists. Discussion and conclusion: It seems clear from our questionnaire that the pathologists in Denmark do not have a consistent definition of what a lymph node is. This missing uniformity may invalidate the comparison between different pathology departments e.g. in the number of identified lymph nodes. A consensus upon a common definition is advisable. O1-2 Processing times for gastrointestinal biopsies with suspected malignancy can be reduced by a simple intervention Tina Prangsgaard, Ulla Engel Hvidovre University Hospital, Department of Pathology, Hvidovre, Denmark; Department of Pathology, Herlev Hospital, Denmark