HER2 Assay Research Articles

Her2 expression in gastroesophageal cancer (GEC) FFPE tissue using mass spectrometry (MS) and correlation with HER2 gene amplification.

82 Background: HER2+ GEC derived benefit from trastuzumab. Her2 IHC is semi-quantitative, subjective, and sensitive to antigen instability; HER2 FISH is laborious, expensive, and subjective. False positivity/negativity have been described. Also, these are low throughput assays; there is known molecular heterogeneity, with several putative biomarkers, and only scarce tissue to assess for each. We sought to evaluate the association of Her2 MS expression with HER2 FISH, along with other markers within the ‘GEC-plex’. Methods: We utilized a previously described unique Her2 peptide and quantification method (Hembrough et al J Clin Oncol 32,2014(suppl 3;abstr17)). The assay was run on 27 cell lines, in parallel with HER2:CEP17 FISH. Her2 expression thresholds were established for HER2 amplification using ROC curves. We adjusted for Her3, Egfr, and Met MS expression levels and sample HER2:CEP17 ratio heterogeneity in a multiple linear regression model. The model/cut-offs were then validated prospectively on GEC tissues (n=142). Results: Her2 MS on cell lines revealed concordance with FISH (HER2:CEP17) ratio (R2=0.75), which improved by adjusting for Her3 MS, Egfr MS, Met MS and HER2:CEP17 heterogeneity (R2>0.98), with significant interactions. IHC correlation with HER2:CEP17 ratio was poor (R2=0.12). A wide range of expression was noted within IHC3+ patients (<150-24671amol/ug). Her2 MS expression (>150 amol/ug) was seen in 72.9% (102/142) of tissues (range <150-24671amol/ug); 9.9% (14/142) had Her2 > 750 amol/ug - all were HER2 FISH amplified. 6/142 (4%) cases <500 amol/ug were clinically ‘HER2amplified’. IHC/FISH results for cases with 550-750 amol/ug demonstrated an ‘equivocal’ zone, not unlike ‘IHC 2+’, but less frequent (8/142, 5.6% vs 46/142 32.4%). Conclusions: The MS HER2 assay can be multiplexed with other GEC biomarkers. A robust model was developed that showed strong correlation of Her2 MS with HER2:CEP17 ratio, particularly when adjusting for covariates Egfr MS, Met MS, Her3 MS, and HER2 FISH heterogeneity. Future analysis will assess the established model as a tool for better prediction of therapeutic benefit from anti-Her2 therapy compared to current methods.

Multiplex Ligation-dependent Probe Amplification Can Clarify HER2 Status in Gastric Cancers with "Polysomy 17".

Therapy with trastuzumab confers a survival benefit in HER2 positive advanced gastric and gastroesophageal adenocarcinoma. HER2 status is evaluated by immunohistochemistry (IHC) and in situ hybridization (ISH). An ISH ratio of HER2 to centromere 17 (CEP17) ≥2.0 is considered amplified. This assumes that CEP17 reflects chromosomal copy number. Cases where CEP17 exceeds 3 are classified as polysomic, but it's unknown if they represent true polysomy or centromeric amplification. This has implications on the validity of current ISH criteria. Multiplex ligation-dependent probe amplification (MLPA) allows simultaneous quantification of multiple loci and can distinguish between true polysomy and centromeric amplification. We selected 13 gastric cancers with CEP17 counts ≥3.0 (polyCEP17), and 8 non-polyCEP17 gastric cancer controls. Silver ISH for HER2 and CEP17 were performed and scored by manufacturer guidelines. We also performed an MLPA HER2 assay that evaluates 22 genes on chromosome 17. MLPA identified HER2 amplification in 7 polyCEP17 cases compared to 2 identified by ISH. Overall, 9 of 13 polyCEP17 cases had amplification of the peri-centromeric gene WSB1, compared to 1 of 8 non-polyCEP17 controls (p=0.02). This could account for ISH CEP17 counts ≥3.0. MLPA did not show any cases of complete chromosome 17 duplication and peri-centromeric amplification can explain most cases of ISH polyCEP17. Current ISH criteria may under-diagnose HER2 amplification in polyCEP17 cases due to flawed assumptions about polysomy. MLPA can detect HER2 amplification missed by IHC and ISH, and thus may be an effective ancillary technique in evaluating HER2 status.

Development of a robust RNA-based classifier to accurately determine ER, PR, and HER2 status in breast cancer clinical samples.

Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96 %, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10 % margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-014-3163-8) contains supplementary material, which is available to authorized users.

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

159. Comparison of hormone receptor and HER2 assay results between core needle biopsy and mastectomy specimen from the same patients

159. Comparison of hormone receptor and HER2 assay results between core needle biopsy and mastectomy specimen from the same patients

Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients

Abstract Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients. Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL. Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients. Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2014-4018

Abstract 4568: A new quantitative in-situ IHC method - validation of analytical performance

Abstract Background In routine pathology, in situ quantification of protein in formalin-fixed, paraffin-embedded (FFPE) tissue is limited to immunohistochemistry (IHC) which is semi-quantitative at best. Accurate and robust measurement of protein expression in FFPE samples, without loss of morphological information, is currently not achievable. Quantitative protein assessment of FFPE tissue can be destructive, and/or time consuming with risk of analyzing non-relevant tissue due to heterogeneity. We have developed a new reliable quantitative IHC (qIHC) method based on a novel amplification system that enables quantification of protein directly in FFPE tissue by counting dots using a standard bright field microscope. The qIHC method combines the ease, speed and morphological information gained from classical IHC staining together with quantitative assessment of protein expression in situ. The goal of this study was to assess the analytical performance of qIHC in a HER2 assay analyzing both FFPE cell pellets and breast cancer specimens. Methods Five different cell lines with HER2 expression ranging from undetectable with IHC to strongly positive (IHC 3+) were used as test samples. Flow cytometry, and ELISA were used as reference methods to confirm protein expression levels and stability of the cell lines. The qIHC assay was conducted using an automated Autostainer Link 48 stainer, and quantitative assessment was done by counting dots per cell using an image analysis algorithm. Repeatability, reproducibility, robustness, linearity, sensitivity, and quantification limits were evaluated. In addition, 44 breast cancer specimens were stained to compare standard HER2 IHC with qIHC. Results We validated the quantitative test through testing for accuracy, reproducibility, repeatability, robustness, and precision. Each of the different tests showed statistical significant results (p: &amp;lt;0.0001-0.005). The dynamic range and linearity of the assay were investigated by correlating the qIHC results to an in-house-developed flow cytometry assay as well as an FDA-approved ELISA kit for HER2 measurement. All tests demonstrated statistically significant correlation (p: 0.0002-0.0045). No overlap was seen between the different cell lines and a broad dynamic range could be demonstrated ranging from 1*103 receptors/cell up to 2.5*106 receptors/cell. Furthermore, HER2 was clearly detectable above background in the low expression cell line MDA-MB-468, thus showing the inherent high sensitivity of the qIHC assay. To demonstrate the capability of the qIHC assay in tumor tissue, we tested the qIHC concordance to HER2 IHC in 44 breast cancer specimens. We confirmed a strong correlation between IHC and qIHC (p: &amp;lt;0.0001). Conclusions The results show that qIHC provides a highly sensitive, accurate, quantitative, and reliable assay for protein expression in FFPE tissue, which could provide better and more precise diagnosis and thus improved personalized cancer treatment. Citation Format: Kristian Jensen, Rikke M. Jørgensen, Kenneth H. Petersen, Jesper Lohse, Helene Derand. A new quantitative in-situ IHC method - validation of analytical performance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2014-4568

Concomitant detection of HER2 protein and gene alterations by immunohistochemistry (IHC) and silver enhanced in situ hybridization (SISH) identifies HER2 positive breast cancer with and without gene amplification.

IntroductionHER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.MethodsTissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.ResultsOverall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.ConclusionsThe novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.

Quantification of HER2 from gastroesophageal cancer (GEC) FFPE tissue by mass spectrometry (MS).

17 Background: Trastuzumab had a survival benefit in ‘HER2 positive’ GEC, determined by IHC/FISH. These companion diagnostics have limitations. IHC is semi-quantitative, subjective, and sensitive to antigen instability in FFPE; FISH is laborious, expensive, and subjective. Gene amplification may not correlate to protein expression. Moreover these are low throughput assays. There is increased recognition of profound interpatient molecular heterogeneity with several putative biomarkers, and only scarce tissue to assess for each one. We sought to evaluate our MS platform on GEC FFPE tissues for HER2 status compared to IHC/FISH. We also applied the “GEC-plex” of 11 other potentially predictive/prognostic markers for GEC. Methods: We utilized trypsin digestion mapping of rHER2 to identify unique peptides for SRM development. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in a complex eukaryotic matrix (PC3 cells) to determine LOD, LLOQ, accuracy, precision and linearity of the assays. The assay was run on 17 GEC cell lines, in parallel with FISH/IHC, and expression thresholds were established for HER2+/HER2-; the sensitivity/specificity of the established cutoffs were then tested prospectively in FFPE GEC tissues on 10uM FFPE LCM slides (n=121). HER2 stability from FFPE sections was assessed by assaying 33 freshly cut FFPE samples; the adjacent sections were processed one year later. Results: The HER2 peptide chosen (ELVSEFSR) had a LOD of 100 amol and CV&lt;20%. HER2 MS on GEC cell lines revealed concordance with FISH (HER2:CEP17) ratio (R2=0.96). The analysis suggested HER2 expression &gt; 750 amol/ug was indicative of HER2 amplification. The assay was stable in archival FFPE sections (R2=0.76). For GEC FFPE cases, ‘any’ HER2 expression was seen in 69.4% of cases; 8.2% showed HER2 &gt; 750 amol (10/121) - all were HER2 amplified. No cases &lt;550 amol/ug were HER2 amplified. IHC/FISH results for cases with 550-750 amol/ug demonstrated a heterogeneous ‘equivocal’ zone, not unlike ‘IHC 2+’, which may require FISH confirmatory testing. Conclusions: ‘GEC-plex’ has a quantitative, sensitive, and specific HER2 assay that can be multiplexed along with other GEC biomarkers.

Abstract P1-02-03: Clinical utility of combined brightfield HER2 gene-protein assay in IHC equivocal cases

Abstract The combined gene protein assay (GPA) for HER2 (Ventana Medical Systems Inc, Tucson AZ) is a newly developed technique where both HER2 gene and protein can be simultaneously assessed on a single slide using routine bright field microscopy. This is made possible by combining HER2 immunohistochemistry (using clone 4B5 from Ventana) with the HER2 Dual in-situ hybridization (DISH) DNA probe cocktail. The main goal of the study was to compare this new GPA methodology with standard fluorescence in situ hybridization (FISH) on the most challenging group of breast cancers, i.e. HER2 immunohistochemical (IHC) 2+ cases. This validation study included whole tissue sections of 36 invasive breast cancer cases with equivocal HER2 IHC results (IHC score 2+) on clinical testing. FISH and GPA were performed on the same tumor blocks. The GPA assay staining results were enumerated by counting at least 20 nuclei by a pathologist blinded to FISH results. Similar to FISH, areas with stronger IHC staining were counted in heterogeneous cases. For both FISH and GPA, HER2 amplification was defined as HER2/CEP17 ratio ≥ 2.0. Cases with HER2/CEP17 ratio &amp;lt;2.0 were considered as non-amplified. In addition, 25 known HER2 IHC 3+ invasive carcinoma cases (represented on a tissue microarray) and 22 known IHC negative (scores 0 and 1+ also represented on tissue microarray) were also evaluated by GPA. GPA was successful in all cases in the first attempt. All previously tested IHC 2+ cases were again scored as 2+ on GPA. The agreement for IHC 2+ cases between FISH and GPA with respect to HER2 gene status was 94.4% (see table). The 2 discordant cases were amplified by GPA and not amplified by FISH. The HER2 to CEP17 ratio on these 2 cases by GPA was 2.0 and 2.34 with average HER2 copies/cell of 5.7 and 5.5 respectively. For the 36 IHC 2+ cases, the mean HER2 copies per cell for FISH were 3.09 (std dev: 1.8; range: 1.9 to 11.07) versus 3.52 (std dev: 1.8; range: 1.8 to 9.95) for GPA. The mean CEP17 signals per cell were 2.24 (std dev: 0.6; range: 1.65 to 4.2) for FISH versus 2.3 (std dev: 0.6; range: 1.6 to 3.5) for GPA. The mean for HER2:CEP17 ratio was 1.34 (std dev: 0.6; range: 1.03 to 4.32) by FISH and 1.50 (std dev: 0.7; range: 1.02 to 4.72) by GPA. GPA versus FISH for IHC 2+ cases GPA amplifiedGPA not amplifiedTotalFISH amplified202FISH not amplified23234Total43236FISH: Fluorescence in-situ hybridization; GPA: Gene protien assay All 25 (100%) of IHC 3+ cases showed clusters of HER2 gene consistent with HER2 amplification by GPA. These 25 cases also showed strong membranous (“chicken wire”) reactivity consistent with 3+ IHC score. Similarly, the 22 known IHC negative cases on the TMA, were again IHC negative, and lacked HER2 gene amplification by GPA. The HER2 GPA shows excellent concordance with both HER2 IHC assay and FISH assay. It performs accurately not only for unequivocally positive and negative cases but also on the most challenging (IHC 2+) breast cancer cases. This new assay combines the advantages of both IHC and FISH, i.e. accuracy similar to FISH for IHC equivocal cases, faster turn around time (less than 24 hours) like IHC and evaluation under bright-field microscope where the tissue morphology is well preserved and heterogeneity is better appreciated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-03.

PTU-157 Her2 Positivity in upper Gastrointestinal Tumours in a Large Multi-Centre UK Cohort Identifies Similar Positivity Rates but different Trends than Previously Reported and Suggests new Potential Avenues for her2 Therapies

IntroductionEvaluation of HER2 status is standard practise for people with inoperable locally advanced or recurrent and/or metastatic adenocarcinoma of the stomach or gastroesophageal junction (GOJ). HER2 positivity is defined as...

Mo1134 HER2 Positivity in Metastatic Upper Gastrointestinal Tumours in a Large Multi-Centre UK Cohort Identifies Similar Positivity Rates but Different Trends Than Previously Reported and Suggests New Potential Avenues for HER2 Therapies

Introduction Evaluation of HER2 status is standard practise for people with inoperable locally advanced or recurrent and/or metastatic adenocarcinoma of the stomach or gastroesophageal junction (GOJ). HER2 positivity is defined as a histology score of 3+, or 2+ with HER2 gene amplification. This study analysed gastroesophageal HER2 testing in UCL-AD over the past 2.5 years. Methods All referrals for HER2 testing (n = 844) were reviewed. Data was captured for age (n = 367), sex (n = 320), tumour origin (n = 104), referring lab (n = 768), sample adequacy, HER2 grading (0–3+) and tumour type. Sections were immunostained with the Roche Tissue Diagnostics Pathway HER2 assay. For cases scoring 2+, F/DDISH testing was performed and the turn-around-time (TAT) from receipt to result analysed. Results Cases were received from 61 labs, mean age 63yo (26–93yo, SD = 13) and the majority were men (71.5%). Specimens originated from oesophageal (OE) (33.7%), GOJ (13.5%), stomach (48%) and UGI metastases (4.8%). The majority (98.7%, n = 833) were HER2-graded. The tumour types were intestinal (INT) (49.6%, n = 257), diffuse (D) (29%, n = 150), mixed D/INT (14.3%, n = 74), carcinoma-in-situ (HGD/IMC) (6.4%, n = 33), squamous (Sq) and adenosquamous (ASq) (0.4%, n = 2 each). HER2 scores were 0 (44%, n = 366), 1+ (14.5%, n = 121), 2+ (18.8%, n = 156) and 3+ (22.7%, n = 189).Gene amplification of 2+ cases identified 124 (79.5%) negative, 23 (14.7%) positive and 9 (1.1%) unrecordable results. HER2 positivity (3+/2+&F/DDISH+) was noted in 10% (15/150) of D, 30.4% (78/257) of INT, 13.5% (10/74) of mixed D/INT, 54.5% (18/33) of HGD/IMC and in 50% (1/2) of Sq tumours. A TAT of Conclusion Overall HER2-positivity rate was 25.8% in a cohort twice the size of the UK cohort reported in the benchmark ToGA trial. When compared with ToGA, subgroup analysis showed comparable but higher rates in D cancers (10% vs 6.1%), lower in both INT/mixed cancers (30.4/13.5% vs 32.2/20.4%), showed opposite proportions of GOJ/stomach cancers (28.6/31.3% vs 33.1/20.9%), and grouping proximal (OE+GOJ) cancers together, a higher ratio compared to stomach cancers was seen (0.51 vs 0.33). Positivity in Sq cancer is of uncertain significance and warrants further research into new potential applications for HER2 targeting. Disclosure of Interest None Declared

Abstract 44: Tissue image analysis tool to quantify HER2 RNA and protein expression by colorimetric in situ hybridization and immunohistochemistry for breast cancer diagnosis.

Abstract Early cancer diagnosis and risk assessment are powerful drivers in discovery of oncology biomarkers for diagnostic tests and simultaneous improvement of already identified biomarkers. Molecular tools such as RNA Colorimetric in situ hybridization (CISH) and immunohistochemistry (IHC) combined with conventional histopathology promise to increase the sensitivity and specificity of cancer staging and overall survival prediction. Changes in oncogene and proto-oncogene expression are a hallmark of cancer. For this reason, simultaneous quantification of mRNA and protein expression using RNA CISH and IHC combined with image-analysis is of interest. One such proto-oncogene is HER2, which is encoded by ERBB2 gene located at the long arm of human chromosome 17 (17q12). Here, we combine RNA CISH and IHC with image-analysis to develop a reliable quantitative HER2 assay that enables quantification of HER2 mRNA and protein expression in the context of human breast cancer. Since HER2 is only over-expressed in a subset of human breast cancers, we initially focused our analysis by screening tissue microarrays (TMAs) using RNA CISH and IHC. TMAs containing 20 normal and 20 breast tumor tissues were tested and whole-slide imaging was used to obtain CISH and IHC signals. HER2 mRNA and protein expression were analyzed, quantified, and compared both at tissue- and individual cell-levels using proprietary image-analysis based tools. Moreover, we compared heterogeneity of RNA and protein expression and further extended our studies to several other cancer-related genes. Finally, since oncogene expression plays a role in cancer pathologies, successful quantification of oncogene in situ expression in human cancer tissues may prove to be beneficial to further understanding of cancer pathology, for drug screening and development of biomarkers and ultimately diagnostic tests. Citation Format: Mirza Peljto, Joseph Krueger, Erik Hagendorn, David Young, Steve Potts, Quan Nguyen, Botoul Maqsodi, Takuro Yaoi, Gary McMaster. Tissue image analysis tool to quantify HER2 RNA and protein expression by colorimetric in situ hybridization and immunohistochemistry for breast cancer diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 44. doi:10.1158/1538-7445.AM2013-44

Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

IntroductionComparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.MethodsWe compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.ResultsIn early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.ConclusionsAll CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.

Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence in Situ Hybridization

The accuracy of diagnostic assays for HER-2/neu in breast cancer is extremely important as HER-2/neu status is essential in tailoring adjuvant and/or neoadjuvant treatment in every patient. FNAC is widely practiced in Egypt in preoperative diagnosis of breast cancer for its low cost and high diagnostic accuracy. Since the determination of HER-2/neu protein expression on cytological preparations was previously found to be unreliable for clinical use, we opted for the assessment of HER-2/neu status in fine needle aspirates using FISH. The main objective of this study was to evaluate the reliability of HER-2/neu status assessment by FISH on fine needle aspirates of breast cancers by comparing the results with IHC and FISH on FFPE tissue sections obtained from corresponding surgically resected specimens. Fine needle aspirates from 40 breast cancer patients with pathologically confirmed breast cancer were included in the study. They were submitted for HER-2/neu evaluation by FISH. After surgery, the corresponding FFPE sections were evaluated for HER-2/neu by FISH and by IHC. FNAs from 11 cases proved to be amplified by FISH, while 29 cases were not amplified. Apart from two cases that showed lack of signals, all specimens evaluated by FISH on the corresponding FFPE sections showed matched results. The Measurement of Agreement between FISH on FNAs and FISH on FFPE sections was 86.7%, while that between FISH on FNAs and IHC was 72.5%. The high concordance rate in the present study between FISH evaluation of HER-2/neu gene amplification on FNAC samples and their corresponding FFPE samples indicate that FISH may be a reliable technique for HER-2/neu assessment on FNAs. Furthermore, FISH on FNAs gave us better hybridization signals than their corresponding FFPE tissue sections. Finally, we also conclude that all score (2+) cases by IHC should be reevaluated by FISH which is crucial for the patient management.

50P Silver-Enhanced in Situ Hybridization as an Alternative to Fluorescence in Situ Hybridization for Assaying Her2 Amplification in Clinical Breast Cancer

ABSTRACT Purpose Valid determination of HER2 status is a prerequisite to establish adequate treatment strategy for breast cancer patient regardless of the disease stage. The goal of this study was to examine the feasibility of the newly developed silver-enhanced in situ hybridization technique as an alternative to fluorescence in situ hybridization for HER2 assay in primary invasive breast cancer. Methods Fluorescence in situ hybridization (FISH) and silver-enhanced in situ hybridization (SISH) for HER2 amplification were performed using tissue microarray. Both methods were used in 257 consecutive primary breast cancers. Results HER2 amplification was observed in 62 (23.1%) of 257 breast cancers based on silver-enhanced in situ hybridization. Of the 257 breast cancers measured using both methods, the results of the two methods were consistent in 248 (concordance: 96.5%; kappa = 0.903). When we compared HER2 amplification in the primary tumor with the metastatic lymph nodes of the same patients, HER2 amplification was observed in nine cases (14.0%) out of 64 cases in which HER2 was not amplified in the primary tumors. In contrast, HER2 status was completely preserved in metastatic lymph nodes showing HER2 amplification in the primary tumor. Conclusions These results indicate that silver-enhanced in situ hybridization can be a feasible alternative to FISH in the clinical setting. In node-positive breast cancer, confirmation of the HER2 status of the metastatic lymph nodes appears to be mandatory, regardless of the HER2 status of the primary tumors. Disclosure All authors have declared no conflicts of interest.

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Oncotype DX

Researchers find discordance between standard human epidermal growth factor receptor 2 (HER2) testing and HER2 status reported on Onco<i>type</i> DX

Multiplication of Chromosome 17 Centromere Is Associated with Prognosis in Patients with Invasive Breast Cancers Exhibiting NormalHER2andTOP2AStatus

PurposeThis study aimed to investigate the clinical significance of chromosome 17 centromere (CEP17) multiplication (increased copy number of CEP17) related to human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) status in patients with invasive breast cancer.MethodsWe constructed tissue microarrays using 594 invasive breast cancer samples and performed single-color silver-enhanced in situ hybridization (SISH) assay for HER2, TOP2A, and CEP17 to assess for copy number aberrations. The association of CEP17 multiplication with patient survival was analyzed according to HER2 and TOP2A status.ResultsAmong 567 informative cases, HER2 amplification was noted in 22.8%, TOP2A amplification in 8.3% and TOP2A deletion in 11.1%. CEP17 multiplication was identified in 33.2% and was significantly associated with worse overall survival (OS) (p=0.02) and disease-free survival (DFS) (p=0.02). CEP17 multiplication correlated with patient survival in patients with normal TOP2A or non-amplified HER2 status, but the prognostic significance was lost in those with altered TOP2A or amplified HER2. On multivariate analyses, CEP17 multiplication was an independent prognostic factor for poorer OS (p=0.02) and DFS (p=0.01) in patients with normal TOP2A and non-amplified HER2.ConclusionCEP17 multiplication was identified as a promising prognostic marker in patients with invasive breast cancer exhibiting either non-amplified HER2 or normal TOP2A status.

Correlation Between Quantitative HER-2 Protein Expression and Risk for Brain Metastases in HER-2+ Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy

Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.

P2-12-05: Correlation between Quantitative HER2 Protein Expression and Risk of Brain Metastases in HER2−Positive Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy.

Abstract Background. Patients with HER2−positive breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Within HER2−positive breast cancer tumors, it is possible to resolve a ∼1.5-log range of HER2 protein expression using a novel quantitative HER2 assay (HERmark®). We investigated the correlation between quantitative HER2 protein expression in primary breast cancers and the time to brain metastases (TTBM) in HER2−positive advanced breast cancer patients treated with trastuzumab. Methods. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER2−positive metastatic breast cancer, defined as 3+ categorical staining by immunohistochemistry (IHC). HER-2/neu gene copy number was subsequently quantified as HER2/CEP17 ratio by central laboratory fluorescence in situ hybridization (FISH). HER2 protein was quantified as total HER2 protein expression (H2T) by the HERmark assay in formalin-fixed, paraffin-embedded primary tumor samples. HER2 variables were correlated with clinical features and TTBM measured from the initiation of trastuzumab-containing therapy. Results. H2T level (continuous variable) was correlated with shorter TTBM (HR=2.3; p=0.013), whereas HER2 gene amplification by FISH (p=0.28) and continuous HER2/CEP17 ratio (p=0.25) had no significant prognostic impact. The correlation between continuous H2T level and TTBM was confirmed in a multivariate analysis (HR=3.2; p=0.021). Controlling for the competing risk of death from causes other than brain metastases, continuous H2T remained a strong correlate of TTBM (HR=2.7; p=0.0009). In the subset of patients that was centrally-determined HER2 positive by FISH (117 patients), above-median H2T level was significantly associated with shorter TTBM (HR=2.4; p=0.005), whereas this was not true for median FISH/CEP17 ratio (p=0.4). In a multivariate analysis of this subset, continuous H2T (p=0.021) and a time dependent covariate capturing time to non-brain metastases (p=0.0044) were prognostic for TTBM, whereas FISH/CEP17, ER, PgR and grade were not. Conclusions. These data reveal a strong relationship between quantitative HER2 protein expression levels and the risk of brain relapse in HER2−positive advanced breast cancer patients. Consequently, quantitative assessment of HER2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-05.