Recent results of clinical trials in antibody drug conjugate (ADC) therapies have significantly broadened treatment options for the HER2 low and ultra-low breast cancer patients. However, sensitive, accurate and quantitative evaluation of HER2 expression based on current immunohistochemistry (IHC) assays remains challenging, especially in low and ultra-low HER2 expression ranges. We developed a novel methodology for quantifying HER2 protein expression, targeting breast cancer cases in the HER2 IHC 0 and 1+ categories. We measured HER2 expression using quantitative IHC (qIHC) that enables precise and tunable HER2 detection across different expression levels as demonstrated in formalin-fixed paraffin-embedded cell lines. Additionally, we developed an AI-based interpretation of HercepTest™ mAb pharmDx (Dako Omnis) (HercepTest™ mAb) using qIHC measurements as the ground truth. Both methodologies allowed spatial resolution and visualization of low and ultra-low levels of HER2 expression across entire tissue sections to demonstrate and enable quantification of heterogeneity of HER2 expression. Serial sections of 82 formalin-fixed paraffin-embedded tissue blocks of invasive breast carcinoma with HER2 IHC scores 0 or 1+ were stained with H&E, HercepTest™ (mAb), qIHC and p63, then scanned and digitally aligned. Tumor areas were manually selected and reviewed by expert pathologists. HER2 expression was quantitatively evaluated based on the qIHC assay in each 128x128μm2 area within tumor regions. We observed statistically significant differences in HER2 expression between IHC 0, 0 < IHC < 1+, and IHC 1+ groups, and a high degree of spatial heterogeneity of the HER2 expression levels within the same tissue, up to five-fold in some cases. We demonstrated high slide-level tumor region agreement of estimates of HER2 expression between the AI-based interpretation of HercepTest™ mAb and the qIHC ground truth with a Pearson correlation of 0.94, and R2 of 0.87. The developed methodologies can be used to stratify HER2 low-expression patient groups, potentially improving the interpretation of IHC assays and maximizing therapeutic benefits. This method can be implemented in histology labs without requiring a specialized workflow.

e13603 Background: Recent advancements in antibody drug conjugate therapies have significantly broadened treatment options for a substantial subgroup of breast cancer patients. However, sensitive, accurate and quantitative evaluation of HER2 expression based on current immunohistochemistry (IHC) assays remains challenging, especially in low and ultra-low HER2 expression ranges. Advanced computational approaches can improve the interpretation of such IHC assays and could be of high benefit for identifying the best treatment options for current and future HER2 targeted therapies. More importantly, it may provide a basis for objectively assessing the lower level of HER2 protein expression that maximizes therapeutic benefits, while minimizing drug exposure risks for patients. Methods: We developed a novel methodology for quantifying HER2 protein expression, targeting breast cancer cases in the HER2 IHC 0 and 1+ categories. We measured HER2 expression using a quantitative IHC (qIHC) assay (Jensen et al, Mod Path 2017) that enables precise and tunable HER2 detection across different expression levels as demonstrated in formalin-fixed paraffin-embedded (FFPE) cell lines . Additionally, we developed an AI-based interpretation of HercepTest™ mAb pharmDx (Dako Omnis) (HercepTest™ mAb) using qIHC measurements as the ground truth. Both methodologies allowed spatial resolution and visualization of low and ultra-low levels of HER2 expression across entire tissue sections to demonstrate and enable quantification of heterogeneity of HER2 expression. Serial sections of 82 FFPE patient tissue blocks of invasive breast carcinoma with HER2 IHC scores 0 or 1+ were stained with H&E, HercepTest™ mAb, qIHC and p63, then scanned and digitally aligned. Tumor areas were manually selected and reviewed by expert pathologists. HER2 expression was quantitatively evaluated based on the qIHC assay in each 128x128µm 2 area within tumor regions. Results: We observed statistically significant differences in HER2 expression between IHC 0, 0 < IHC < 1+, and IHC 1+ groups, and a high level of spatial heterogeneity of the HER2 expression levels within the same tissue, up to five-fold in some cases. We demonstrated high slide-level tumor region agreement of estimates of HER2 expression between the AI-based interpretation of HercepTest™ mAb and the qIHC ground truth with a Pearson correlation of 0.94, and R 2 of 0.87. Conclusions: The developed methodologies can be used to stratify HER2 low-expression patient groups, potentially improving the interpretation of IHC assays and maximizing therapeutic benefits. This method can be implemented in histology labs without requiring a specialized workflow. Disclaimer: This study is for proof of concept only. This study does not imply any clinical functionality, nor off-label use for any products mentioned.

This Special Issue features contributions from leading international researchers in the field of MET (hepatocyte growth factor (HGF) receptor) biology and therapeutics [...].

Dysregulation of the MET tyrosine kinase receptor is a known oncogenic driver, and multiple genetic alterations can lead to a clinically relevant oncogenesis. Currently, a number of drugs targeting MET are under development as potential therapeutics for different cancer indications, including non-small cell lung cancer (NSCLC). However, relatively few of these drugs have shown sufficient clinical activity and obtained regulatory approval. One of the reasons for this could be the lack of effective predictive biomarkers to select the right patient populations for treatment. So far, capmatinib is the only MET-targeted drug approved with a companion diagnostic (CDx) assay, which is indicated for the treatment of metastatic NSCLC in patients having a mutation resulting in MET exon 14 skipping. An alternative predictive biomarker for MET therapy is MET amplification, which has been identified as a resistance mechanism in patients with EGFR-mutated NSCLC. Results obtained from different clinical trials seem to indicate that the MET/CEP7 ratio detected by FISH possesses the best predictive properties, likely because this method excludes MET amplification caused by polysomy. In this article, the concept of CDx assays will be discussed, with a focus on the currently FDA-approved MET targeted therapies for the treatment of NSCLC.

Background MET gene aberrations are found in several human cancers including gastric, ovarian and lung. In a large multinational cohort of patients with gastric/gastroesophageal junction/esophageal (G/GEJ/E) adenocarcinoma we assessed the MET status with respect to amplification and deletion and correlate the results with the phenotypical gene signal distribution pattern. Methods Tissue specimens from 1,580 patients were analyzed using a novel fluorescence in situ hybridization (FISH) assay employing a MET/CEN-7 IQFISH Probe Mix. MET amplification and deletions were defined as a MET/CEN-7 ratio ≥2.0 and a MET/CEN-7 ratio <0.8, respectively. Furthermore, the link between the MET gene status and the phenotypical signal distribution was investigated. Results The prevalence of MET amplification and deletions was found to be 7.2% and 8.7%, respectively. Significant differences were observed with regard to geographic regions and sex. The Asian population had the highest percentage of MET amplification (9.4%) and the lowest percentage of deletions (3.2%). MET deletions was found more frequently among males (10.1%) compared to females (5.3%) and in esophagus (17.6%) compared to the stomach (5.7%). More than 50% of the patients who harbored MET gene amplification had a heterogeneous distribution of the FISH signals. Patients with a focal signal distribution were solely to be found among the MET amplified population. MET deletion were mainly observed in the group of patients with a homogenous signal distribution. Conclusions The screening data from this cross-sectional study showed that MET deletion and amplification are frequent events in G/GEJ/E cancer, which are linked to different phenotypical signal distribution patterns. The role of MET deletion in relation to tumor development is not fully understood but it is likely to play a role in the oncogenic transformation of the cells.

The development of trastuzumab (Herceptin ® ) was one of the most significant cancer drug development projects of the 20th century. Not only was it a scientific and medical achievement but it also paved the way for the drug-diagnostic codevelopment model, where a predictive biomarker assay is developed in parallel to the drug. One of the challenges in the development of trastuzumab was to select the right patient population likely to respond and here, it was critical to have access to an accurate, robust and reliable assay for detection of HER2 overexpression in tumors. In the clinical development of trastuzumab, a clinical trial assay (CTA), developed by Genentech, was used for selection of HER2 positive patients. However, during the phase III trial with trastuzumab, a new optimized IHC assay, HercepTest™ was designed and developed by Dako. In the final stage of its development, a comparative study with the CTA was conducted in order to show concordance between the two assays. In September 1998, the Food and Drug Administration (FDA) simultaneously granted approval to trastuzumab and HercepTest™. The assay has been used for patient selection in a number of significant breast cancer clinical trials such as the HERA, CLEOPATRA, EMILIA and more. In these trials, HercepTest™ demonstrated its clinical utility in the neoadjuvant, adjuvant, and metastatic setting as well as in relation to different types of HER2 targeted therapies. Likewise, the assay was used for selection of HER2 positive gastric cancer patients in the important ToGA trail. HercepTest™ was the first companion diagnostic ever approved by the FDA, and more than 20 years of use has documented its clinical impact.

Background: HER2 serves as an important therapeutic target in gastroesophageal cancer. Differences in HER2 gene signal distribution patterns can be observed at the tissue level, but how it influences the HER2 amplification status has not been studied so far. Here, we investigated the link between HER2 amplification and the different types of gene signal distribution. Methods: Tumor samples from 140 patients with gastroesophageal adenocarcinoma where analyzed using the HER2 IQFISH pharmDx™ assay. Specimens covered non-amplified and amplified cases with a preselected high proportion of HER2 amplified cases. Based on the HER2/CEN-17 ratio, specimens were categorized into amplified or non-amplified. The signal distribution patterns were divided into homogeneous, heterogeneous focal or heterogeneous mosaic. The study was conducted based on anonymized specimens with limited access to clinicopathological data. Results: Among the 140 analyzed specimens 83 had a heterogeneous HER2 signal distribution, with 62 being focal and 21 of the mosaic type. The remaining 57 specimens had a homogeneous signal distribution. HER2 amplification was observed in 63 of the 140 specimens, and nearly all (93.7%) were found among specimens with a heterogeneous focal signal distribution (p<0.0001). The mean HER2/CEN-17 ratio for the focal heterogeneous group was 8.75 (CI95%: 6.87 - 10.63), compared to 1.53 (CI95%: 1.45 - 1.61) and 1.70 (CI95%: 1.22 - 2.18) for the heterogeneous mosaic and homogeneous groups, respectively, (p<0.0001). Conclusions: A clear relationship between HER2 amplification and the focal heterogeneous signal distribution was demonstrated in tumor specimens from patients with gastroesophageal cancer. Furthermore, we raise the hypothesis that the signal distribution patterns observed with FISH might be related to different subpopulations of HER2 positive tumor cells.

BACKGROUND:The gene mesenchymal epithelial transition factor (MET) is a proto-oncogene that encodes a transmembrane receptor with intrinsic tyrosine kinase activity known as Met or cMet. MET is found to be amplified in several human cancers including gastroesophageal cancer.METHODS:Here we report the MET amplification prevalence data from 159 consecutive tumor specimens from patients with gastric (G), gastroesophageal junction (GEJ) and esophageal (E) adenocarcinoma, using a novel fluorescence in situ hybridization (FISH) assay, MET/CEN-7 IQFISH Probe Mix [an investigational use only (IUO) assay]. MET amplification was defined as a MET/CEN-7 ratio ≥2.0. Furthermore, the link between the MET signal distribution and amplification status was investigated.RESULTS:The prevalence of MET amplification was found to be 6.9%. The FISH assay demonstrated a high inter-observer reproducibility. The inter-observer results showed a 100% overall agreement with respect to the MET status (amplified/non-amplified). The inter-observer CV was estimated to 11.8% (95% CI: 10.2-13.4). For the signal distribution, the inter-observer agreement was reported to be 98.7%. We also report an association of MET amplification and a unique signal distribution pattern in the G/GEJ/E tumor specimens. We found that the prevalence of MET amplification was markedly higher in tumors specimens with a heterogeneous (66.7%) versus homogeneous (2.0%) signal distribution. Furthermore, specimens with a heterogeneous signal distribution had a statically significantly higher median MET/CEN-7 ratio (2.35 versus 1.04; P<0.0001).CONCLUSIONS:The novel FISH assay showed a high inter-observer reproducibility both with respect to amplification status and signal distribution. Based on the finding in the study it is suggested that MET amplification mainly is associated with tumor cells that is represented by a heterogonous growth pattern.

BACKGROUND:Chromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques.METHODS:Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory.RESULTS:The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit.CONCLUSIONS:The concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.

Abstract Introduction: HER2 assessment for selection of patients that may benefit from HER2 targeting treatment can be performed by either immunohistochemistry (IHC), fluorescence or chromogen in situ hybridization (FISH or CISH). FISH is a robust and reliable technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells, but FISH protocols are time-consuming and involve toxic reagents. By introducing a new non-toxic ethylene carbonate based hybridization buffer that perform with very short hybridization times, the total FISH assay time on breast cancer tissue sections can be reduced from the traditional 16–20 hours to 3½–4½ hours. Material and methods: The new Dako HER2 IQFISH pharmDx™ was compared with Dako HER2 FISH pharmDx™ in a comparative study on 120 breast cancer specimens, and reproducibility of the HER2 IQFISH pharmDx™ assay was investigated in a study comprising 3 different sites and a total of 6 different observers. Samples for the comparative study was evaluated by Dako HercepTest™ to include all IHC scoring groups (0, 1+, 2+, 3+). Slides were stained according to manufacturer's instructions using microwave oven for heat pretreatment and RTU pepsin for 3–5 minutes at 37 °C. Hybridization was performed for 2 hours when using HER2 IQFISH pharmDx™ and for 17–20 hours when using HER2 FISH pharmDx™ Kit. All slides were blinded before evaluation. HER2 status was classified as “Non-amplified” when the HER2/CEN17 ratio < 2.0 and “Amplified” when the HER2/CEN17 ratio ≥ 2.0. Results: The new non-toxic hybridization buffer introduces a major safety improvement since formamide is no longer needed. Significantly shorter hybridization times are required to generate the same signal intensity (1–2 hour hybridization versus overnight). HER2 IQFISH pharmDx™ was compared with the traditional HER2 FISH pharmDx™ in a comparative study on 120 breast tissue specimens of human breast carcinoma. The preliminary data on HER2 status for 78 patients obtained by the two assays gave an overall agreement of 98.7% with lower and upper 95% confidence limits at 94.2% and 99.9%. The Kappa value was 0.96 (95% CL: 0.89–1.00). The p-value for McNemars test was 1.00 indicating absence of bias between the two assays. Disagreement between the two assays was observed for one specimen - a heterogeneous tissue with a small amplified area. Data from the reproducibility study that included site-to-site variation, day-to-day variation and inter-observer variation showed that the assay has a high degree of reproducibility. Conclusion: The validation studies of the new HER2 IQFISH pharmDx™ showed a very high concordance to the traditional HER2 FISH pharmDx™ and also that the assay is robust and reproducible. Reduction of the overall assay time from a two-day to a half-day procedure for HER2 FISH, offers more flexible laboratory routines and same day reporting for all working days of the week, which could be used for fast and simultaneous FISH and IHC answers and improved patient care. Taken together, the study demonstrates the potential of a new revolutionary platform that enables optimization and acceleration of FISH analysis to the benefit of cancer patients and laboratory personnel. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-02.

CONTEXT New guidelines for HER2 testing have been introduced. OBJECTIVES To evaluate the difference in HER2 assessment after introduction of new cutoff levels for both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and to compare interobserver agreement and time to score between image analysis and conventional microscopy. DESIGN Samples from 150 patients with breast cancer were scored by 7 pathologists using conventional microscopy, with a cutoff of both 10% and 30% IHC-stained cells, and using automated microscopy with image analysis. The IHC results were compared individually and to HER2 status as determined by FISH, using both the approved cutoff of 2.0 and the recently introduced cutoff of 2.2. RESULTS High concordance was found in IHC scoring among the 7 pathologists. The 30% cutoff led to slightly fewer positive IHC observations. Introduction of a FISH equivocal zone affected 4% of the FISH scores. If cutoff for FISH is kept at 2.0, no difference in patient selection is found between the 10% and the 30% IHC cutoff. Among the 150 breast cancer samples, the new 30% IHC and 2.2 FISH cutoff levels resulted in one case without a firm diagnosis because both IHC and FISH were equivocal. Automated microscopy and image analysis-assisted IHC led to significantly better interobserver agreement among the 7 pathologists, with an increase in mean scoring time of only about 30 seconds per slide. CONCLUSIONS The change in cutoff levels led to a higher concordance between IHC and FISH, but fewer samples were classified as HER2 positive.

CONTEXT:The need for higher efficiency, maximum quality, and faster turnaround time is a continuous focus for anatomic pathology laboratories and drives changes in work scheduling, instrumentation, and management control systems.OBJECTIVE:To determine the costs of generating routine, special, and immunohistochemical microscopic slides in a large, academic anatomic pathology laboratory using a top-down approach.DESIGN:The Pathology Economic Model Tool was used to analyze workflow processes at The Nebraska Medical Center's anatomic pathology laboratory. Data from the analysis were used to generate complete cost estimates, which included not only materials, consumables, and instrumentation but also specific labor and overhead components for each of the laboratory's subareas. The cost data generated by the Pathology Economic Model Tool were compared with the cost estimates generated using relative value units.RESULTS:Despite the use of automated systems for different processes, the workflow in the laboratory was found to be relatively labor intensive. The effect of labor and overhead on per-slide costs was significantly underestimated by traditional relative-value unit calculations when compared with the Pathology Economic Model Tool. Specific workflow defects with significant contributions to the cost per slide were identified.CONCLUSIONS:The cost of providing routine, special, and immunohistochemical slides may be significantly underestimated by traditional methods that rely on relative value units. Furthermore, a comprehensive analysis may identify specific workflow processes requiring improvement.

Aims:Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results.Methods and results:We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (rho) of 0.96.Conclusions:We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.

The apoptosis linked gene-2 (ALG-2), discovered as a proapoptotic calcium binding protein, has recently been found upregulated in lung cancer tissue indicating that this protein may play a role in the pathology of cancer cells and/or may be a tumor marker. Using immunohistochemistry on tissue microarrays we analysed the expression of ALG-2 in 7371 tumor tissue samples of various origin as well as in 749 normal tissue samples. Most notably, ALG-2 was upregulated in mesenchymal tumors. No correlation was found between ALG-2 staining intensity and survival of patients with lung, breast or colon cancer. siRNA mediated ALG-2 downregulation led to a significant reduction in viability of HeLa cells indicating that ALG-2 may contribute to tumor development and expansion.