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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.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.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.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.

Purpose: In breast cancer, the EGF receptors host an increasing number of therapeutic targets and the interactive mechanisms of actions of the receptors and their ligands justify investigation of the EGF family as an entity. Experimental design: Paired tissue samples of normal breast tissue and primary breast carcinomas were examined in a prospective study of 163 patients. A third sample was obtained from the paired ipsilateral metastatic lymph node from 58 of these patients. The mRNA expression of four EGF receptors (HER1 - HER4) and 11 activating ligands was quantified with real-time RT-PCR. Results: Expression of HER2, HER3, and HER4 mRNA was upregulated in primary carcinomas compared to normal breast tissue while HER1 was downregulated. The mRNA expression of HER3 and HER4 differed between primary breast carcinomas and lymph node metastases whereas there was no difference in the expression of HER1 and HER2. The combination of low HER3 and low HER4 expression in the primary carcinoma was significantly more frequent in lymph node-negative patients as compared to lymph node positive patients. Distinct correlation patterns of the receptors and their corresponding activating ligands appeared in both normal breast tissue and in carcinomas, notably for the HER3 and HER4 receptors and their 3 specific ligands: HB-EGF, NRG2, and NRG4. Conclusion: HER2, HER3, and HER4 showed increased mRNA expression in carcinomas and were positively correlated to each other and to specific activating ligands. Furthermore, low HER3 and HER4 expression in the carcinomas correlated to the absence of lymph node metastases.

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.

Purpose High 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. Methods Paired 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. Results Both PIK3CA and PTEN mRNA expression was significantly increased in breast carcinoma tissue compared to normal breast tissue (p = 2 × 10-11) and (p 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. Conclusions The 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.

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.

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.Tissue 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).In 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%).BRISH 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.