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Aim: Our goal was to describe a precision medicine program in a regional academic hospital, characterize features of included patients and present early data on clinical impact. Materials and methods: We prospectively included 163 eligible patients with late-stage cancer of any diagnosis from June 2020 to May 2022 in the Proseq Cancer trial. Molecular profiling of new or fresh frozen tumor biopsies was done by WES and RNAseq with parallel sequencing of non-tumoral DNA as individual reference. Cases were presented at a National Molecular Tumor Board (NMTB) for discussion of targeted treatment. Subsequently, patients were followed for at least 7 months. Results: 80% (N = 131) of patients had a successful analysis done, disclosing at least one pathogenic or likely pathogenic variant in 96%. A strongly or potentially druggable variant was found in 19% and 73% of patients, respectively. A germline variant was identified in 2.5%. Median time from trial inclusion to NMTB decision was one month. One third (N = 44) of patients who underwent molecularly profiling were matched with a targeted treatment, however, only 16% were either treated (N = 16) or are waiting for treatment (N = 5), deteriorating performance status being the primary cause of failure. A history of cancer among 1st degree relatives, and a diagnosis of lung or prostate cancer correlated with greater chance of targeted treatment being available. The response rate of targeted treatments was 40%, the clinical benefit rate 53%, and the median time on treatment was 3.8 months. 23% of patients presented at NMTB were recommended clinical trial participation, not dependent on biomarkers. Conclusions: Precision medicine in end-stage cancer patients is feasible in a regional academic hospital but should continue within the frame of clinical protocols as few patients benefit. Close collaboration with comprehensive cancer centers ensures expert evaluations and equality in access to early clinical trials and modern treatment.

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.

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 (κ=1). Using H-score, a very high agreement between VDS and visual ER assessment was seen (R=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.

Background The three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1β, and HPγ, are involved in chromatin packing and epigenetic gene regulation. HP1α is encoded from the CBX5 gene and is a suppressor of metastasis. CBX5 is down-regulated at the transcriptional and protein level in metastatic compared to non-metastatic breast cancer. CBX5 shares a bi-directional promoter structure with the hnRNPA1 gene. But whereas CBX5 expression is down-regulated in metastatic cells, hnRNAP1 expression is constant. Here, we address the regulation of CBX5 in human breast cancer. Methods Transient transfection and transposon mediated integration of dual-reporter mini-genes containing the bi-directional hnRNPA1 and CBX5 promoter was performed to investigate transcriptional regulation in breast cancer cell lines. Bioinformatics and functional analysis were performed to characterize transcriptional events specifically regulating CBX5 expression. TSA treatment and Chromatin Immunoprecipitation (ChIP) were performed to investigate the chromatin structure along CBX5 in breast cancer cells. Finally, expression of hnRNPA1 and CBX5 mRNA isoforms were measured by quantitative reverse transcriptase PCR (qRT-PCR) in breast cancer tissue samples. Results We demonstrate that an hnRNPA1 and CBX5 bi-directional core promoter fragment does not comprise intrinsic capacity for specific CBX5 down-regulation in metastatic cells. Characterization of transcriptional events in the 20 kb CBX5 intron 1 revealed existence of several novel CBX5 transcripts. Two of these encode consensus HP1α protein but used autonomous promoters in intron 1 by which HP1α expression could be de-coupled from the bi-directional promoter. In addition, another CBX5 transcriptional isoform, STET, was discovered. This transcript includes CBX5 exon 1 and part of intron 1 sequences but lacks inclusion of HP1α encoding exons. Inverse correlation between STET and HP1α coding CBX5 mRNA expression was observed in breast cancer cell lines and tissue samples from breast cancer patients. Conclusion We find that HP1α is down-regulated in a mechanism involving CBX5 promoter downstream sequences and that regulation through alternative polyadenylation and splicing generates a transcript, STET, with potential importance in carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2059-x) contains supplementary material, which is available to authorized users.

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.

The purpose of this study was to develop and validate a new software, HER2-CONNECT(TM), 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-CONNECT(TM) 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-CONNECT(TM) 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.

Introduction: The Human Epidermal Growth Factor Receptor 4 (HER4) of the EGF receptor family has been characterized in both normal and malignant human breast tissue and HER4 overexpression has been shown to predict prolonged survival compared to HER4 receptor negative disease. In our study we investigated the HER4 expression in normal breast tissue, primary breast carcinoma and in ipsilateral metastatic axillary lymphnodes at the time of primary breast cancer surgery. Material and methods: Paired tissue samples from normal breast tissue and primary breast carcinomas were obtained from 169 patients. Out of these a third sample was obtained from 66 patients with metastatic lymphnodes. The primary tumour specimens were sampled uniformly at random. The mRNA expression of HER4 was quantified with real time RT-PCR and expressed relative to the householdgene (HMBS) in arbitrary units (arb.u.). Results: The mRNA expression of HER4 was significantly higher in breast carcinoma with a mean of 2.26 arb.u. [95% c.i.:1.87 to 2.65 arb.u.] than in the paired sample of normal breast tissue with a mean of 0.82 arb.u. [95% c.i.: 0.47 to 1.16 arb.u.] (p=0.0001). The mRNA expression of HER4 was also significantly higher in breast carcinoma than in the corresponding lymphnode mean 0.75 arb. u. [95% c.i.: 0.43 to 1.07 arb.u.] (p=0.015). There was no significant difference observed in mRNA expression of HER4 between the metastatic lymphnode and the corresponding normal breast tissue (P>0.05). When HER4 expression was compared between the primary tumours that had metastasised and those that had not, there was no significant difference (P>0.05). Conclusion: The HER4 expression was high in the primary tumour as compared to normal breast tissue and the corresponding lymphnode. In view of previously published relations between a low expression of HER4 and a poor prognosis our results warrant further studies in order to evaluate whether suppression of HER4 in tumour cells could be involved in lymphogenic metastatic spread. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-01-09.