Determination Of HER2 Status Research Articles

Molecular Testing for Gastrointestinal Cancer.

With recent advances in molecular diagnostic methods and targeted cancer therapies, several molecular tests have been recommended for gastric cancer (GC) and colorectal cancer (CRC). Microsatellite instability analysis of gastrointestinal cancers is performed to screen for Lynch syndrome, predict favorable prognosis, and screen patients for immunotherapy. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor has been approved in metastatic CRCs with wildtype RAS (KRAS and NRAS exon 2–4). A BRAF mutation is required for predicting poor prognosis. Additionally, amplification of human epidermal growth factor receptor 2 (HER2) and MET is also associated with resistance to EGFR inhibitor in metastatic CRC patients. The BRAF V600E mutation is found in sporadic microsatellite unstable CRCs, and thus is helpful for ruling out Lynch syndrome. In addition, the KRAS mutation is a prognostic biomarker and the PIK3CA mutation is a molecular biomarker predicting response to phosphoinositide 3-kinase/AKT/mammalian target of rapamycin inhibitors and response to aspirin therapy in CRC patients. Additionally, HER2 testing should be performed in all recurrent or metastatic GCs. If the results of HER2 immunohistochemistry are equivocal, HER2 silver or fluorescence in situ hybridization testing are essential for confirmative determination of HER2 status. Epstein-Barr virus–positive GCs have distinct characteristics, including heavy lymphoid stroma, hypermethylation phenotype, and high expression of immune modulators. Recent advances in next-generation sequencing technologies enable us to examine various genetic alterations using a single test. Pathologists play a crucial role in ensuring reliable molecular testing and they should also take an integral role between molecular laboratories and clinicians.

Abstract P5-03-10: Development of a novel HER2 testing strategy, using image-based cell-sorting to isolate pure cell populations from FFPE upstream of FISH

Abstract Fluorescent in Situ Hybridization (FISH) guidelines defined by American Society of Clinical Oncology (ASCO) and the College of American Pathologists for determining HER2 status are set to improve accuracy and usefulness as a diagnostic marker in breast cancer. Despite these guidelines, many factors can influence HER2 testing results such as sample preparation, assay-conditions and interpretation of test results due to heterogeneous breast cancer samples. In this multi-site study, sample preparation was carried out using the DEPArray™ to recover pure tumor cell populations from formalin-fixed, paraffin-embedded (FFPE) breast tumor samples. We then compared HER2/CEP17 ratios obtained from the DEPArray™ processed samples from each laboratory to routine FISH on tissue sections. Methods: Eight breast FFPE tumor tissue biopsies were obtained from commercial tissue banks. From the paraffin tissue blocks, four consecutive tissue curls (each 50 microns thick) were prepared. One curl from each of the 8 patient samples was distributed to four different laboratories for analysis following DEPArray™ based sample preparation. After an initial disassociation of each curl into a single-cell suspension, intact cells were sorted and then recovered based on cytokeratin/ vimentin/DAPI staining using the DEPArray™. Cytokeratin+/Vimentin-/DAPI+ tumor (~250) and Cytokeratin-/Vimentin+/DAPI+ stromal (~250) recovered cells were then deposited onto glass slides prior to standard dual-color HER2/CEP17 FISH analysis for comparison to conventional HER2 FISH result. Results: Serially sectioned breast tumors from 8 negative/positive cases: 7 infiltrating ductal carcinoma (IDC) and 1 metastatic carcinoma were studied. All four sites demonstrated 100% concordance between FISH results compared to the conventional HER2 FISH result. Overall, >60% of DEPArray™ isolated cells were recovered from FFPE samples that ranged from 1- 15 years of age and reported to contain 60% to 80% tumor content. The use of pure sorted cells permitted the accurate determination of HER2 amplification status in only the tumor cells while the stromal cells consistently yielded a more normalized ratio of HER2 to centromere 17. Conclusion: The preliminary results of this multi-site study demonstrate that use of DEPArray™ for sorted pure populations is reproducible as well as reliable method for subsequent analysis of HER2 by FISH on FFPE derived tumor cells. Given that traditional FFPE-based HER2 FISH results may be influenced by the tissue sectioning procedure, tissue heterogeneity and/or the scattering of few HER2 amplified tumor cells among normal stromal cells. The DEPArray™ allows analysis of immunofluorescence images and DNA content to isolate and recover pure and intact cell populations. This isolation of pure cell populations prior to FISH analysis is attractive for achieving precise determination of HER2 status on equivocal cases. A more formal analytical validation of this approach through CLIA is currently underway. Citation Format: Gerber A, Konig L, Millner L, Strotoman L, Khurana A, Kasimir-Bauer S, Moore MW, Cotter PD, Bischoff F. Development of a novel HER2 testing strategy, using image-based cell-sorting to isolate pure cell populations from FFPE upstream of FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-03-10.

HER2 Gene Protein Assay Is Useful to Determine HER2 Status and Evaluate HER2 Heterogeneity in HER2 Equivocal Breast Cancer

Approximately 15% of breast cancers show equivocal human epidermal growth factor receptor 2 (HER2) results on HER2 immunohistochemistry (IHC) and are reflexed for fluorescence in situ hybridization (FISH). However, some cases remain equivocal. In this study, we evaluated these double-equivocal cases by using a novel gene protein assay (GPA), which can simultaneously assess HER2 gene copy number and protein on a single slide using bright-field microscopy. GPA was performed on 42 HER2 IHC and FISH double-equivocal cases. GPA was negative for amplification in 28 cases, equivocal in three cases, and positive in 11 cases. The GPA results showed excellent concordance with either repeat FISH using a chromosome 17 centromere probe or FISH using an alternative probe. Furthermore, HER2 heterogeneity was identified in three of 11 GPA-positive cases. HER2 GPA performs accurately and is very useful to determine HER2 status in HER2 IHC and FISH double-equivocal breast cancer cases and identify HER2 heterogeneity.

Human epidermal growth factor receptor 2 amplification detection by droplet digital polymerase chain reaction in formalin-fixed paraffin-embedded breast and gastric cancer samples.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for the precise individualized treatment including trastuzumab of HER2-positive breast and gastric cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the routine analyses for formalin-fixed paraffin-embedded (FFPE) samples. However, IHC is variable and depends on the evaluator, and FISH is a labor intensive and expensive method. We evaluated the feasibility of droplet digital polymerase chain reaction (ddPCR) as a precise and quantitative method for HER2 amplification test. We used ddPCR to confirm HER2 amplification status in 24 breast cancer and 29 gastric cancer samples to validate the HER2 cutoff value in ddPCR. After setting cutoff value, all the above-mentioned samples were tested by IHC. Afterward, another 51 equivocal IHC 2+ gastric cancer samples were further determined by FISH and ddPCR, respectively, and the concordance between ddPCR and FISH was calculated. We set the HER2 cutoff value at 1.8. The concordance rate of HER2 status between ddPCR and IHC was 94.4% (17 out of 18) in 24 breast cancer samples. In 29 gastric cancer specimens, the concordance rate of HER2 amplification between ddPCR and IHC was 100% (22 out of 22). At last, compared with FISH determined HER2 status, ddPCR HER2 scores correctly classified 44 of 51 cases with 86.3% concordance in 51 equivocal IHC 2+ gastric cancer samples. ddPCR was able to identify HER2 amplification status in breast and gastric cancers with precise correlation with IHC and FISH results. This method might become a standard method for testing FFPE samples. However, the technology requires further research.

Dual-color chromogen In Situ hybridization (CISH) for the determination of Her2 status in breast cancer tissue sections

Background: A factor associated with breast cancer development is the mutation of the Her2/neu gene located on chromosome 17. Chromogen in situ hybridization (CISH) in which the color is generated with an enzymatic reaction is a promising alternative detection method to fluorescence in situ hybridization (FISH) which is nowadays used in clinical practice. The resulting color is stable and therefore usable for permanent storage, the use of a normal brightfield microscope is possible and the morphology of the sample can be evaluated more easily than with a fluorescence microscope. For the visualization of Her2/neu it is however important to also detect chromosome 17 and use it as a reference chromosome. The aim of the study is: to select the best substrates considering color production, background staining, reactivity and localization. Secondly to develop a dualcolor procedure visualizing Her2/neu and chromosome 17 to examine the possibility of using CISH as a standardized method for the detection of Her2 positive tumors and finally to compare these results with FISH. Methods: Single target CISH with eight substrates for the enzyme horseradish peroxidase (PO) and six substrates for alkaline phosphatase (AP) was performed on cervical cancer cell lines with centromere 1 (C1) and 1p36 as targets. The best substrates were subjectively selected with the use of a brightfield microscope. The procedure was expanded to a dual-color detection on cells and formalin-fixed, paraffin embedded (FFPE) breast cancer tissue sections with Her2/neu and centromere 17 (C17) as targets. In this procedure the best conjugate detection systems were worked out. Results: In the single target detection procedure the substrates DAB, Vina Green and Seramun Grun for PO and Ferangi Blue and Vulcan Fast Red for AP were found to give the best color reactions. The dual-color detection on tissue sections showed strong and good distinguishable color reactions when combining the substrates Vina Green for C17 and Vulcan Fast Red for Her2. Results of CISH and FISH were comparable when using immersion oil for the detection of the fluorescent signals. Discussion and Conclusion: Vina Green for C17 and Vulcan Fast Red for Her2/neu are the best substrates considering background staining, color distinguishability and localization and give comparable results to FISH. The study gives a further step in the direction of introducing CISH as a standardized method for the detection of Her2/neu amplification.

PMD6 - Data Mining Used To Characterize Discordance In Gastric Cancers HER2 Status Determination To Help For A Better Treatment Decision

PMD6 - Data Mining Used To Characterize Discordance In Gastric Cancers HER2 Status Determination To Help For A Better Treatment Decision

Use of a multiplexed immunoassay (PRO Onc assay) to detect HER2 abnormalities in circulating tumor cells of women with HER2-negative metastatic breast cancer: lack of response to HER2-targeted therapy

Determination of HER2 status by testing circulating tumor cells (CTCs), compared to sampling tumor biopsies, may improve patient management by allowing ongoing assessment of HER2 status during the disease course. The PRO Onc assay (Prometheus Laboratories; San Diego, CA) is a multiplexed immunoassay that measures the expression and activation of HER2 in CTCs. In this study, we screened patients with metastatic HER2-negative breast cancer with the PRO Onc assay; patients with HER2 overexpression or activation received a trial of HER2-targeted therapy. In Part 1 of the trial, patients with HER2-negative breast cancer were screened with the PRO Onc assay to confirm the presence of a cohort that tested HER2-positive. After this finding was confirmed, patients in Part 2 of the study with HER2 abnormalities received a trial of treatment with trastuzumab/pertuzumab. In Part 1, 31 of 57 specimens contained CTCs; of these, 12 (38%) showed HER2 abnormalities by PRO Onc assay. In Part 2, 129 of 226 patients (57%) had CTCs; 24 of these patients (19%) had HER2 abnormalities detected. Fourteen patients were treated with HER2-targeted therapy. Twelve of 14 patients progressed within 6weeks, one patient had a brief (12weeks) partial response, and one patient was stable for 12weeks. HER2 overexpression or activation was detected by the PRO Onc assay in 22% of HER2-negative patients with CTCs. However, HER2-targeted therapy was not effective in such patients. FISH and IHC staining remain the standards for HER2 determination.

Copy Number Variation Analysis of Circulating Tumor Cells at a Single Cell Level Based on Hydrogel Encapsulation

Circulating tumor cells (CTCs) are defined as tumor cells circulating in peripheral blood of patients with metastatic cancer. CTCs have potential as a biomarker for cancer diagnosis or drug discovery. Recently, much attention has been paid to genomic analyses such as copy number variation (CNV) analysis of CTCs, because it will provide important information related to treatment for personalized therapy. These analyses should be performed at a single cell level because of the heterogeneity of cancer cells. However, CTCs are extremely rare cell in blood (1 in 109blood cells), so highly efficient technique for CTC recovery and single-cell isolation is required. Our group has developed “microcavity array system” on which rare CTCs can be efficiently captured from whole blood on the basis of differences in the size and deformability between tumor and blood cells. Furthermore, a novel technique for a single cell manipulation using a photo-polymerized hydrogel, “hydrogel-based cell encapsulation” has been demonstrated. In this study, to evaluate the performance of “microcavity array system” and “hydrogel-based cell encapsulation”, whole genome amplification (WGA) was performed using single-CTCs isolated by our proposed method. Furthermore, the utility of the proposed method in CNV analysis were evaluated. The microcavity array (φ= 8 μm, 4×103 cavities) was created by electroforming technique as microfabricated filter. The microcavity array was fabricated with PDMS structure equipped with a vacuum microchannel to apply negative pressure for cell entrapment. A549 cells (lung cancer cell line), SKBR-3 cells (breast cancer cell line) or MCF-7 cells (breast cancer cell line) were used as model CTCs. These cells were stained with fluorescent dyes, and suspended in PBS or healthy whole blood. These cell suspensions were introduced into the microcavity array at a flow rate of 150 µl/min. After cell recovery process, the number of cancer cells captured on the microcavity array was counted under a fluorescence microscope. As a results, more than 95% of cancer cells was successfully recovered on the microcavity array from PBS and whole blood. Then, poly (ethylene glycol) diacrylate (PEGDA) hydrogel was introduced onto the MCA. An excitation light (λ=365 nm) was irradiated to the targeted cancer cells for 30 seconds, leading to cross-linking reaction of PEGDA hydrogel and encapsulation of a single-cell. Solidified PEGDA hydrogel can be easily recovered from microcavity array by tweezers. Fluorescent microscopic analysis revealed that targeted single-cells were successfully encapsulated with PEGDA hydrogel. The hydrogel-based cell encapsulation process for 20 single-cells was completed within 10 min. To evaluate the effect of hydrogel on genome amplification, hydrogels with single-cells were subjected to the WGA, and the yields of WGA products were measured by PicoGreen assay. Genome amplification was also successfully carried out in all single-cells encapsulated on the hydrogel, and the yields of WGA products were the same with those obtained by using the micromanipulation as a conventional method. The amplification bias of WGA was evaluated based on the success rate of PCR amplification using 9 different primer sets (multiple loci). The PCR amplification of 9 fragments revealed that no significant bias was observed between WGA samples from single-cells isolated by hydrogel encapsulation and micromanipulation. Based on these results, we concluded that our single cell isolation method could be utilized for WGA at single cell level. Furthermore, quantitative PCR was performed to evaluate CNV analysis using SKBR-3 and MCF-7 cells, which have different HER2 status. As a result, the level of HER2 copy number in WGA products obtained from single-cells was similar with that from bulk analysis (103cells).Determination and monitoring of HER2 status in genome is well known to serve as a useful biomarker for diagnosis of breast cancer. Therefore, our proposed method for CNV analysis of CTCs is expected to be widely utilized towards real-time cancer diagnosis without repeating surgical biopsy.

Determining HER2 (ERBB2) amplification status in women with breast cancer: final results from the Australian in situ hybridisation program

Appropriate and accurate determination of HER2 status in women with breast cancer is critical for stratifying anti-HER2 therapies, and for access to subsidised treatment in the Australian setting. We conducted a regulated, nationwide program providing HER2 in situ hybridisation (ISH) testing for patients with newly diagnosed breast cancer. Cases with equivocal or non-diagnostic ISH test results at the local laboratory were sent to a high volume central testing laboratory for analysis using fluorescence ISH (FISH). We tested 78,408 early breast cancers and 3469 metastatic cancers using ISH. Of these, 12,405 early breast cancers (15.8%) and 798 metastatic cancers (23.0%) were HER2 positive. During the testing period, the proportion of core biopsy samples increased, the number of repeat tests remained stable and testing turnaround time declined. Discordant 3+ IHC, ISH negative results dropped from 20% to 13% in early breast cancers and from 35% to 8% among metastatic breast cancers. Following central laboratory FISH testing only 87 samples remained non-diagnostic (1.9% of FISH-tested samples, 0.1% of the whole cohort), most being decalcified specimens. This is a successful story of a cohesive service determining HER2 status in women with breast cancer in a 'real-world' setting.

Concordance between HER-2 status determined by qPCR in Fine Needle Aspiration Cytology (FNAC) samples compared with IHC and FISH in Core Needle Biopsy (CNB) or surgical specimens in breast cancer patients

Determining the status of HER2-neu amplification and overexpression in breast cancer is crucial for prognosis but mostly for treatment purposes. Standard techniques include the determination of IHC in combination with in situ hybridization techniques to confirm a HER2-neu amplification in case of IHC2+ using either a core-needle biopsy or a surgical specimen. qPCR has been also demonstrated to be able to determine HER2 status, mostly in core biopsies or in surgical specimens. Fine-needle aspiration is a reliable, quicker and less invasive technique that is widely used for diagnosis of invasive breast cancer. In this study, we assessed the performance of qPCR in invasive breast carcinomas to determine HER2-neu status by using fine-needle aspiration samples and comparing to standard IHC and FISH. From a total of 154 samples from patients who had nodular breast lesions and attended the 1-day-stop clinic at the Gustave Roussy from March 2013 to October 2014, qPCR was able to determine the HER2 status in a mean of 3.7 days (SD 3.1). The overall concordance with standard HER2-testing was very high: 97% (95% CI 0.94 to 0.99); sensitivity was 96% (0.87–1), specificity 98% (0.95–1) and positive and negative predictive values 88% (0.75–1) and 99% (0.98–1), respectively.In conclusion, our study demonstrates that qPCR performed using fine-needle aspiration samples from a primary tumour is a reliable and fast method to determine HER2/neu status in patients with early breast cancer.

Abstract 4236: SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides

Abstract In recent years, patients with Her2-positive breast cancer have benefitted greatly from targeted therapies such as Trastuzumab and Pertuzumab. However, there are currently no FDA-approved Her2-targeted imaging agents to diagnose and monitor Her2-positive breast cancer leaving physicians to rely on biopsies to determine Her2 status. Whole-body visualization of Her2 would allow the noninvasive identification of all Her2 primary and metastatic lesions as well as serve as a powerful tool to monitor the effectiveness of Her2-targeted therapeutics. We recently described the generation of Her2-targeted SUPR (Scanning Unnatural Protease Resistant) peptides that selectively bind the Her2 receptor in vitro with low nanomolar affinity. Cy5-labeled SUPR peptides showed rapid and Her2-specific tumor uptake and minimal retention in non-tumor tissues after 24 hours of washout. In this study, we describe 18F radiolabeling of Her2-targeted SUPR peptides and their evaluation as PET radiotracers to visualize Her2 expression in vivo. The lead compound, SUPR4, was labeled with 18F-fluoroethylazide in high radiochemical yield and specific activity on an automated radiochemical synthesis platform. The resulting radiotracer (SUPR-18F) showed rapid and Her2-selective tumor uptake between 30-60 minutes post-injection with minimal liver uptake. The majority of the tracer was cleared by the kidneys at 2 hours post-injection although some activity was observed in the GI tract suggesting hepatobiliary excretion. No significant uptake was observed in the brain. 90 minute dynamic PET scans were performed to estimate the rate of tumor uptake and clearance in major organ systems and the biodistribution quantified by autoradiography post-mortem. Pre-injection of unlabeled SUPR peptide, Trastuzumab, and Pertuzumab followed by PET imaging with SUPR-18F was used to confirm tumor uptake as a function of specific binding to the Her2 receptor. Having established the utility of SUPR-18F in pre-clinical mouse models, we anticipate that this class of PET tracers could be employed in same-day imaging procedures throughout the administration of Her2-targeted therapy. Citation Format: Lindsay E. Kelderhouse, Amanda Hardy, Federica Pisaneschi, Joshua P. Gray, Seth Gammon, Richard W. Roberts, Terry T. Takahashi, Steve Fiacco, Steven W. Millward. SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4236.

Novel method for rapid in-situ hybridization of HER2 using non-contact alternating-current electric-field mixing.

Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating HER2-positive breast cancer patients. However, the lack of survival benefit in HER2-negative patients as well as the toxic effects and high cost of the drugs highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel rapid dual in-situ hybridization (RISH) method developed to facilitate hybridization. The method takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. One hundred sixty-three specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+). The specimens were all tested using conventional dual in-situ hybridization (DISH), DISH with an automated slide stainer, and RISH. With RISH the HER2 test was completed within 6 h, as compared to 20–22 h needed for the standard protocol. Although RISH produced results more promptly using smaller amounts of labeled antibody, the staining and accuracy of HER2 status evaluation with RISH was equal to or greater than with DISH. These results suggest RISH could be used as a clinical tool to promptly determine HER2 status.

Clinical evaluation of HER2 molecular diagnostic assay with internal quality controls in breast cancer.

e23130Background: Determination of HER2 status before taking therapeutic regimens plays an essential role in breast cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) ...

Use of next generation sequencing and quantitative mass spectrometry to determine HER2 status.

e23237Background: Traditional methods for HER2 assessment such as IHC and FISH are semi-quantitative and often limited by reproducibility issues. This may be a problem in tumors with borderline HER...

Fusion of FISH image analysis methods of HER2 status determination in breast cancer

The paper presents an application of several methods of markers localization in fluorescent in situ hybridization images in order to determine HER2 status of the breast cancer samples. The applied methods implement different mathematical foundations of image processing and perform the analysis in an independent way. The paper proposes their fusion to obtain better results of spot recognition. The integration process of many methods takes into account different points of view on the classification problem and takes the best from each of them in developing the final solution. Thanks to this the improvement of the performance of the system is achieved. The efficiency of the developed system in the form of sensitivity and specificity has been verified on the examples of analysis of many FISH images. They confirm the advantage of using several methods of image processing combined in the form of an ensemble.

HER2-positive gastric cancer identified by serum HER2: A case report.

Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the current standards methods for the determination of tissue human epidermal growth factor receptor 2 (HER2) status in gastric cancer, as for breast cancer. However, HER2-positive gastric cancer occasionally exhibits heterogeneous tissue HER2 overexpression, raising concern regarding false-negative results in unresectable cases diagnosed by biopsy samples. Serum HER2, the concentration of the extracellular domain of HER2 protein shed into the bloodstream, has the potential to supplement the use of IHC or FISH to determine HER2 status. However, the clinical significance of serum HER2 has not been well studied in gastric cancer. The present study describes an illustrative case of metastatic gastric cancer initially diagnosed as HER2-negative (IHC score 1+). The patient exhibited an elevated serum HER2 level, which prompted a reevaluation of the tissue by IHC, using an alternative antibody, and FISH; re-biopsy analyses confirmed the case as HER2-positive, and trastuzumab was subsequently added to the combination chemotherapy with capecitabine and cisplatin. Serum HER2 may aid in avoiding false-negative diagnoses of HER2 gastric cancer.

Abstract P3-07-06: Objective measurement of HER2 (ERBB2) intracellular and extracellular domain spatial co-localization stratifies benefit from adjuvant trastuzumab

Abstract Background: The ASCO/CAP guidelines consider chromogen-based immunohistochemistry (IHC) as the primary assay to determine HER2 status in breast cancer. U. S. Food and Drugs Administration (FDA) approved HER2 antibody assays target the protein's intracellular domain (ICD). Studies suggest that quantitative, domain-specific measurement of HER2 might predict benefit from trastuzumab therapy, further classifying traditional HER2-positive breast cancer. Here we define a method of simultaneous, objective measurement of HER2 ICD and extracellular (ECD) domains, and determine its effect on trastuzumab benefit in the adjuvant setting. Methods: We measured co-expression of HER2 ICD and ECD using a proximity ligation assay (PLA) and quantitative immunofluorescence (QIF) in a HER2 standardization tissue microarray (TMA) with CLIA-lab defined HER2 status. Previously validated, standardized HER2 antibodies were used to detect ICD and ECD (CB11 and SP3, respectively). We determined the relationship between HER2 PLA scores, HER2 clinical status and domain-specific scores. Finally, we measured HER2 ICD/ECD PLA in 180 patients from a clinical trial of adjuvant chemotherapy followed by trastuzumab (HeCOG 10/05). Median cut-point was used to stratify patients according to HER2 PLA scores. Cut-points for HER2 ICD and ECD were obtained using Joinpoint software. All statistical tests were two-sided. Results: In the standardization TMA, HER2 PLA levels were associated to HER2 CLIA status (P<0.0001). There was a good correlation between HER2 PLA scores and HER2 ICD and ECD (R2=0.57 and R2=0.54, respectively). In trastuzumab-treated patients from HeCOG 10/05, a similarly good correlation was observed between HER2 PLA scores and HER2 ICD and ECD (R2=0.41 and R2=0.3, respectively). In univariate analysis, HER2 PLA-low status was associated with ER-positive status (P=0.005). There was no association with age, histological grade, tumor size, lymph node status and TNM stage. Although all tumors were HER2-positive, HER2 PLA-high status was significantly associated with longer 5-year disease-free survival (DFS) (log-rank P=0.036, HR=0.32, 95% CI: 0.132-0.935). HER2 PLA status was superior to ICD status (log-rank P=0.67) and numerically comparable to ECD status (log-rank P=0.049, HR=0.31, 95% CI: 0.144-0.997) to predict benefit from adjuvant trastuzumab, as previously published by our group. HER2 PLA-high status was independent predictor of better outcome in a Cox proportional hazards model including age, histological grade, ER status, tumor size, lymph node status and TNM stage. Discussion: Using an objective, quantitative HER2 assay for synchronous, domain-specific measurement, we stratified benefit from adjuvant trastuzumab treatment in patients from a prospective cohort. Our results further support the concept that benefit from HER2 ECD-targeted therapies might be modulated by the presence of truncated HER2 protein variants and that tyrosine kinase inhibitors (ICD-directed) may be advantageous for a subset of HER2-positive patients. Furthermore, this technique that uses two antibodies has the potential to increase both sensitivity and specificity of the IHC assay to predict response to HER2 pathway inhibitors. Citation Format: Carvajal-Hausdorf DE, Toki M, Schalper KA, Pusztai L, Psyrri A, Kalogeras KT, Kotoula V, Fountzilas G, Rimm DL. Objective measurement of HER2 (ERBB2) intracellular and extracellular domain spatial co-localization stratifies benefit from adjuvant trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-06.

Estrogen-Receptor, Progesterone-Receptor and HER2 Status Determination in Invasive Breast Cancer. Concordance between Immuno-Histochemistry and MapQuant™ Microarray Based Assay.

BackgroundHormone receptor status and HER2 status are of critical interest in determining the prognosis of breast cancer patients. Their status is routinely assessed by immunohistochemistry (IHC). However, it is subject to intra-laboratory and inter-laboratory variability. The aim of our study was to compare the estrogen receptor, progesterone receptor and HER2 status as determined by the MapQuant™ test to the routine immuno-histochemical tests in early stage invasive breast cancer in a large comprehensive cancer center.Patients and MethodsWe retrospectively studied 163 invasive early-stage breast carcinoma with standard IHC status. The genomic status was determined using the MapQuant™ test providing the genomic grade index.ResultsWe found only 4 tumours out of 161 (2.5%) with discrepant IHC and genomic results concerning ER status. The concordance rate between the two methods was 97.5% and the Cohen’s Kappa coefficient was 0.89.Comparison between the MapQuant™ PR status and the PR IHC status gave more discrepancies. The concordance rate between the two methods was 91.4% and the Cohen’s Kappa coefficient was 0.74.The HER2 MapQuant™ test was classified as « undetermined » in 2 out of 163 cases (1.2%). One HER2 IHC-negative tumour was found positive with a high HER2 MapQuant™ genomic score. The concordance rate between the two methods was 99.3% and the Cohen’s Kappa coefficient was 0.86.ConclusionOur results show that the MapQuant™ assay, based on mRNA expression assay, provides an objective and quantitative assessment of Estrogen receptor, Progesterone receptor and HER2 status in invasive breast cancer.

Genetic variants in the HER2 gene: Influence on HER2 overexpression and loss of heterozygosity in breast cancer

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is an indicator of poor prognosis and is the pre-requisite for treatment with the agents targeting this member of the epidermal growth factor receptor family. In order to determine the influence of these common single-nucleotide polymorphisms (SNPs) in the HER2 gene, genomic DNA was obtained from 361 patients with breast cancer, aged between 29 and 82 years. Samples of tumour tissue were obtained from 241 (66%) patients and material for extraction of DNA is isolated from surrounding normal tissue by laser capture microdissection. Genotyping was performed using the Taqman fluorogenic 5′ nuclease assay. Of the 360 patients with definitive determination of HER2 status, 49% were positive. The Ile655Val SNP had no influence on the frequency of HER2 expression. However, the proline allele of the Ala1170Pro SNP was associated with a higher frequency of HER2 overexpression (56% versus 43%, p = 0.015). Where the germline genotype was homozygous, the tumour genotype was identical in every case and for both SNPs. In HER2-positive tumours, heterozygosity was maintained in only 15% and 18% of the Ile655Val and Ala1170Pro SNPs, respectively. This was lower than in the HER2-negative tumours (46% and 43%, respectively). Normal breast tissue (n = 23) retained the germline genotype in all but one case. The underlying link between the Ala1170Pro SNP and HER2 positivity is not known, nor is the significance of HER2 overexpression and loss of heterozygosity in breast cancer. However, these results illustrate the complexity of HER2 genotype and overexpression in this disease.

HER2 testing in gastric cancer: An update.

Human epidermal growth factor receptor 2 (HER2) overexpression is increasingly recognized as a frequent molecular abnormality in gastric and gastroesophageal cancer. With the recent introduction of HER2 molecular targeted therapy for patients with advanced gastric cancer, determination of HER2 status is crucial in order to select patients who may benefit from this treatment. This paper provides an update on our knowledge of HER2 in gastric and gastroesophageal cancer, including the prognostic relevance of HER2, the key differences between HER2 protein expression interpretation in breast and gastric cancer, the detection methods and the immunohistochemistry scoring system.