BACKGROUND: Presently, therapy for treatment of breast cancer is based on the evaluation of formalin fixed, paraffin embedded (FFPE) pathological specimens using a combination of immunohistochemistry (IHC) and gene copy assessment by in-situ hybridization (-ISH) techniques, following current testing guidelines from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). Patients with specimens found to have marked overexpression and amplification of the human epidermal receptor 2 (HER2) are approved for treatment with trastuzumab. While IHC and –ISH assays represent the current clinical standard, these assays are subject to pre-analytical variation, which could lead to false negative results. There are novel laboratory technologies which may improve the accuracy of HER2 measurements and lead to improved patient selection for therapy. AIM: In this comparative study, we assessed the utility of testing HER2 expression by the fluorescence IHC using AQUA® - a novel computer assisted platform to enable quantitative assessment of protein expression in FFPE tissue specimens – against current clinical assays (IHC and –ISH). METHODS: Local cases from 2008-2010 clinically evaluated for HER2 were identified for further pathological review. FFPE tissue specimens were retrieved from a total of 207 cases with sufficient tumor present, and placed into a tissue microarray (TMA). TMA sections underwent assessment for IHC HER2 (Clone 4B5, Ventana), HER2/Chromosome 17 gene copy number (Inform HER2 dual-ISH DNA Probe Cocktail Assay, Ventana), and fluorescence IHC (Clone SP3, Thermo Fisher). RESULTS: HER2 results were available for 142 patients for IHC, and 134 patients for dual–ISH and fluorescence IHC. A comparison of the current clinical methods revealed 11 discordant cases. The average median fluorescence IHC cytoplasmic HER2 expression (cAQUA) was found to be 225.65, (70.65-419.95). HER2 cAQUA was strongly correlated with dual-ISH, and cases with low level amplification had low cAQUA expression. There were cases having high cAQUA expression that did not show amplification by dual-ISH. Only a few amplfied cases demonstrated low cAQUA expression. Dichotomizing cAQUA at the 256 showed an improvement of the receiver operating characteristic compared to the clinical HER2 IHC assay (cAQUA=0.903, p<0.001; IHC=0.833, p=0.006). CONCLUSIONS: Measurement of HER2 expression using human interpretation can be imprecise as there is still some discordance between HER2 IHC and dual-ISH assays. Evaluation of HER2 protein expression using the novel AQUA assay showed correlation with IHC and dual-ISH, and AQUA may present a more precise way to quantify HER2 protein expression. HER2 cAQUA used a different antibody clone than the clinical IHC assay; however, previous studies have shown strong correlation between these two antibodies. The AQUA assay may identify a previously unrecognized group of breast cancers with elevated HER2 expression. The significance of this finding requires further investigation, particularly in regards with cAQUA HER2 serving as a marker for response to anti-HER2 therapy. Citation Format: Kornaga EN, Feng X, Klimowicz AC, Dean ML, Guggisberg N, Morris DG, Magliocco AM. Fluorescence quantitative image analysis of HER2 evaluation against current clinical HER2 assays in breast cancer testing. [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 P5-07-07.
Background: Ki67, an indicator of proliferation, has been shown to be a useful prognostic and predictive marker for breast cancer. Ki67 can be used to identify two distinct estrogen-receptor positive subtypes: luminal A and luminal B. Luminal A breast cancers have been identified as having a lower proliferation and better outcome compared to luminal B. Furthermore, early clinical trials suggest that Ki67 may be useful in identifying a subset of patients that are sensitive to adjuvant docetaxel treatment. Currently, only estrogen-receptor (ER), progesterone-receptor (PR), and human epidermal growth-factor (HER2) are routinely performed. We have optimized and validated an immunohistochemical (IHC) Ki67 assay and automated computerized image analysis platform for routine clinical testing. Materials and Methods: Immunohistochemical staining was quantitatively assessed using the ACIS® III platform on a cohort (N=761) of tamoxifen treated patients who were diagnosed with breast cancer in Calgary between 1990 and 2001. Tissue microarrays were constructed using three 0.6 mm cores. Ki67 results were available for 510 patients, 461 of which are ER/PR positive and HER2 negative. Staining was performed using the DAKO FLEX ready-to-use system. The percent nuclear area positive was calculated using ACIS III and the maximum value was used in statistical analysis Results: X-tile statistical software was used to identify an optimal Ki67 cut point to distinguish differential overall survival in node negative ER positive cancers of 18.75%. This cut point was then used to categorize the 461 ER/PR positive and HER2 negative breast cancers into luminal A (407; 88.3%) or luminal B (54; 11.7%) subtypes. The 8-year breast cancer specific survival was 85.2% (95% CI = 81.3% - 89.1%) for luminal A and 53.6% (95% CI = 39.3% - 67.9%) for luminal B (P<0.0001). Cox regression showed a hazard ratio of 1.63 (95% CI = 0.99 — 2.67, p=0.055), adjusting for age, tumor size, grade and lymph node status. Discussion: Quantification of Ki67 expression using automated image analysis can be used clinically to distinguish luminal A from luminal B in ER/PR positive and HER2 negative breast cancers. The purpose of this project was to develop a reliable Ki67 assay that can be easily adopted by other testing centers. Using a ready-to-use IHC system — such as DAKO FLEX — allows for consistent results between other clinical laboratories. Additionally, using an automated, quantitative imaging system — such as the ACIS® III — reduces inter-observer variation that can occur by human visual assessment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-35.
Background: Access to cancer screening, diagnosis and treatment in the United States (US) is affected by insurance status; whereas, access within a publicly-funded health care system is similar across the whole population. The aim of this study was to compare overall survival (OS) of de novo stage IV (metastatic) breast cancer (BC) pts in a Canadian province and in the US according to insurance status. Methods: All female pts 18-64 yrs of age diagnosed with de novo stage IV BC from Jan 1, 2010 through Dec 31, 2014 with available biomarker information were included. Pts diagnosed by death certificate or autopsy and pts ≥ 65 yrs were excluded due to unreliable insurance status classification in the US SEER database. The Alberta cohort (AB) was obtained from the Alberta Health Services CancerControl Breast Data Mart (BDM), a repository of information on all pts diagnosed with their first BC diagnosis from Jan 1, 2004 onwards in the province of Alberta, Canada. The U.S. cohort was obtained from the US Surveillance, Epidemiology, and End Results (SEER) program cancer database. A total of 9,604 pts from the SEER database and 294 pts from the BDM were analyzed. OS was evaluated over a 2 yr period and median and 2 yr OS were estimated. Unadjusted associations were compared using the log-rank test, and hazard ratios (HR) were estimated using the Cox proportional hazards model with US insured set as reference group. Results: Comparison of AB and US cohorts showed no differences based on age group (18-49 vs 50-64), yr of diagnosis or receipt of primary surgery. The AB cohort had a higher incidence of hormone receptor positive (HR+), similar frequency of HER2+, and a lower incidence of triple negative (TN) BC relative to the US cohort: HR+ 60.5% vs 56.4%; HER2+ 30.6% vs 28.8%; and TN 8.8% vs 14.8%, respectively [p=0.017]. The distribution of HR+, HER2+ and TN BC was consistent between the SEER insured, Medicaid and uninsured groups. AB cohort estimated 2 yr OS was 70.1%, similar to the insured group of 66.0% and significantly better than the Medicaid or uninsured pts [53.2% and 50.9%; p<0.0001]. Subgroup analysis based on biomarker status, surgery and age group showed similar results. Adjusting for these variables, AB OS remained similar to the insured group [HR=0.92 (0.74-1.15) p=0.474] with worse OS noted in the Medicaid and uninsured populations [HR=1.44 (1.32-1.56) and HR=1.53 (1.33-1.77) p<0.001, respectively]. HR+HER2+TNNo SurgerySurgery18-4950-64InsuredReference Group: HR=1.00AB0.92 (0.68-1.24) p=0.5841.15 (0.76-1.72) p=0.5090.84 (0.49-1.42) p=0.5070.91 (0.71-1.17) p=0.4660.71 (0.45-1.11) p=0.1330.84 (0.56-1.27) p=0.4080.87 (0.67-1.12) p=0.284Medicaid1.43 (1.27-1.61) p<0.0011.57 (1.32-1.87) p<0.0011.37 (1.18-1.60) p<0.0011.46 (1.33-1.59) p<0.0011.55 (1.33-1.81) p<0.0011.75 (1.52-2.00) p<0.0011.47 (1.34-1.61) p<0.001Uninsured1.76 (1.44-2.15) p<0.0011.64 (1.20-2.22) p<0.0011.56 (1.20-2.05) p=0.0011.56 (1.34-1.80) p<0.0011.59 (1.16-2.18) p=0.0041.76 (1.35-2.29) p<0.0011.67 (1.43-1.94) p<0.001 Conclusion: OS in women with de novo stage IV BC in AB was similar to US insured. AB and US insured experienced superior OS compared with US Medicaid and uninsured. Citation Format: Kornaga EN, Matutino AR, Pereira AA, Verma S, Lupichuk S. Survival in women with de novo metastatic breast cancer: Comparison of real-world evidence from publicly-funded Canadian province and the United States by insurance status [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-13.
BACKGROUND: Approximately 15% of newly diagnosed breast cancers are classified as triple negative (TNBC). TNBCs are considered more aggressive and have a worse prognosis as no targeted therapies are currently available. These tumors are routinely treated with chemotherapy agents with only modest proven efficacy. Temozolomide (TMZ) is an oral chemotherapy agent commonly used for the treatment of brain tumors and melanoma. TMZ is an alkylating agent, and its therapeutic benefit depends on its ability to alkylate/methylate DNA, most commonly at the N-7 or O-6 positions of guanine residues. This process leads to DNA damage and subsequently triggers cell death. Cells that express the enzyme O6-Methylguanine-DNA Methyltransferase (MGMT) are able to repair damage caused by TMZ. Tumors that lack expression of MGMT, owing to methylation of the gene promoter, demonstrate a better response to TMZ treatment as a result of synthetic lethality. It was first reported in 2012 that TNBCs were more likely to be MGMT methylated, which was confirmed by another group that reported up to 64% of wild-type BRCA1 TNBC exhibited MGMT gene methylation. In 2013 it was found that basal-like breast cancers were more likely to be MGMT methylated and linked to larger tumor size. Together these findings suggest that a sub-population of TNBCs lack MGMT expression, due to promoter methylation. Currently, TMZ is not a treatment option for breast cancers given the modest efficacy of TMZ noted in breast cancer clinical trials; however, most of these trials have focused on using this agent to either treat or prevent brain metastases, due to TMZs ability to cross the blood-brain barrier. Importantly, none of these trials investigated MGMT expression or specifically TNBC populations. We hypothesize that TMZ may be a viable and efficacious treatment option for TNBCs that lack MGMT expression, due to promoter methylation. METHODS: We analyzed 12 archival specimens and 4 TNBC cell lines (HTB132, HTB26, HTB126 and HCC1806) for MGMT expression using a qRT-PCR clinical assay available from Calgary Laboratory Services. Additionally, we also looked at MGMT protein expression in the cell lines using Western Blot analysis to confirm the qRT-PCR results. Finally, we performed an in vitro assay with TNBC cell lines to determine cytotoxicity of TMZ. RESULTS: Analysis of the archival specimens found that 33% of samples analyzed had MGMT promoter methylation by qRT-PCR. Additionally, we found that HTB26 and HTB126 cell lines showed MGMT promoter methylation by qRT-pCR analysis. Western Blot analysis confirmed lack of MGMT expression in these two cell lines, and also identified another cell line (HCC1806) lacking MGMT protein that was classified as unmethylated by the qRT-PCR clinical assay. Moreover, our in vitro assay found that two cell lines (HTB26 and HCC1806) showed a noticeable response to treatment with TMZ. Interestingly, HTB126 did not show response to TMZ, suggesting that there may be another putative resistance pathway. CONCLUSIONS: Preliminary findings suggest that TMZ may be a viable targeted treatment option for TNBCs. Currently, we are investigating drug response using in vivo mouse models, as well as investigating synergistic combination therapy options. Citation Format: Kornaga EN, Gratton K, Shi Q, Yang A, Nixon NA, Roldan Urgoiti G, Morris DG. Temozolomide as a targeted therapy strategy for triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-08.
Introduction: Adjuvant tamoxifen is the standard therapy for early stage hormone receptor+ breast cancers (BC). Estrogen receptor (ER) and progesterone receptor (PR) analysis is routinely performed by immunohistochemical (IHC) testing on BC specimens to assist in determining hormone receptor status and patient treatment. ER/PR IHC methodologies and guidelines have recently come under review. Many clinical laboratories have opted to use platform specific, ready-to-use (RTU) ER/PR assays provided by three companies: Dako, Leica and Ventana. While each of these companies are using antibodies that were validated on BC clinical outcome series, these platform specific RTU assays have never been directly compared using the same clinical outcome series. We present a systematic comparison of the three platform specific RTU ER/PR assays, using a retrospective BC cohort, to evaluate the concordance and reproducibility of the RTU ER/PR assays, and to assess the ability of the RTU ER assays to predict tamoxifen response. Methods: The Calgary Tamoxifen Cohort is a retrospective database containing demographic, clinical and pathological data for 820 BC patients diagnosed between 1985–2000 at the Tom Baker Cancer Centre (Calgary, Canada). Formalin-fixed paraffin-embedded tissue blocks were available for 511 patients, and replicate 0.6mm cores were taken and built into tissue microarrays (TMAs). The TMAs were stained using the platform specific assays on the DakoLink Plus, Ventana BenchMark Ultra, or Bond-III Leica autostainers. Slides were manually scored by the Allred method. Results: Ventana and Dako had the best concordance for ER (κ=0.90). Substantial agreement was seen for ER staining between Leica and Ventana (κ=0.79), and Dako and Leica (κ=0.66). Agreement was more consistent between the three platforms for PR staining (κ=0.78–0.82). Inter-observer reproducibility was evaluated for all three platforms between three observers: Dako ER (κ=0.80–0.92) and PR (κ=0.69–0.90); Leica ER (κ=0.67–0.83) and PR (κ=0.70–0.89); Ventana ER (κ=0.88–1.00) and PR (κ=0.78–0.94). TMAs were rescored and intra-observer agreement was calculated: Dako ER (κ=1.00) and PR (κ=0.98); Leica ER (κ=0.91) and PR (κ=0.94); Ventana ER (κ=1.00) and PR (κ=0.94). ER Allred scores were dichotomized using current standards and univariate analysis for 5-year disease free survival was performed. All platforms achieved significance with the logrank test and hazard ratio (HR) estimates (p < 0.0001). Cox models were also run to adjust for lymph node status, grade, size and HER2 status. ER status determined by Dako [HR=0.37(0.19–0.74), p = 0.005] and Ventana [HR=0.40(0.18–0.87), p = 0.021] maintained significance, while ER status determined by Leica [HR=0.61(0.31–1.20), p = 0.154] did not. Conclusions: Concordance between RTU assays demonstrated more variation for ER than PR. All assays showed substantial agreement for inter- and intra- observer reproducibility. Although ER RTU assays from all vendors performed as expected in univariate analysis, multivariate models demonstrated differences. Dako and Ventana appeared equivalent in the multivariate analysis, each providing prognostic information, whereas Leica did not achieve independence in this analysis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-10.
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