Background: Immunohistochemistry (IHC) staining for ER and progesterone (PR) receptors in breast cancer tissue is the current standard for testing for eligibility for hormone targeted therapies. However, these markers are imperfect predictors of response, and ER/PR expression can be heterogeneous, especially in the metastatic setting. Within an adjuvant tamoxifen-treated ER positive patient group functional ER pathway activity was associated with improved patient outcome (Verhaegh et al, Cancer Research 2014). This study investigated how ER/PR staining correlates with ER pathway activity in a cohort of metastatic breast cancers. Methods: Cases of metastatic breast cancer with variable reported ER expression by IHC were selected from the Stanford Pathology Database. Cases were scored by a breast pathologist for percent cellularity of sample, percent and intensity of ER and PR staining, and a qualitative rating of heterogeneity of staining. Digital slides were annotated for areas of cellularity to perform ER pathway activity analysis. Functional ER pathway activitywas measured in a quantitative manner using a biologically validated method, based on Bayesian computational model inference of functional pathway activity from RT-qPCR measurements of mRNA levels of target genes of the pathway transcription factor, providing an ER Pathway Activity Score (PAS)(Verhaegh et al, Cancer Research 2014). Results: A total of 64 samples were tested for ER/PR expression as well as for ER pathway analysis. Annotated tumor areas of 57 samples were used for measurement of ER PAS. 61.4% were ER expression high (defined as >50% ER IHC positive, mean 94.0%) and 38.6 % were ER low (defined as <50% ER IHC positive, mean 23.0%). ER high cases had higher mean ER PAS when compared to ER low cases (ER high, mean PAS 47.6, SD 19.2; versus ER low, mean PAS 19.4, SD 11.7; unpaired one-tailed 2-sample t-test p<0.001). Importantly, there was wide variation in ER PAS even in cases with high ER expression levels. PR levels did not correlate with ER PAS. Three cases had clustered areas with different ER expression levels within the tissue slide (heterogeneous cases), resulting in 7 separately analyzed areas; ER pathway activity was separately analyzed in these areas. ER PAS was remarkably similar across areas with variable ER staining, e.g. within one tissue slide, areas with 20% and 90% ER expression had nearly the same ER PAS. Discussion: Grouping cases into high vs low ER IHC staining reveals expected differences in ER PAS. However, nuclear ER expression (IHC) levels may be an imperfect predictor of actual ER pathway activity on an individual case basis. Preliminary results on cases with regional heterogeneity for levels of ER IHC expression suggest that ER PAS is more homogenous than IHC levels. Clinical studies examining value of ER PAS to predict response to hormonal therapies are ongoing. Citation Format: Yang S-R, van de Stolpe A, van Brussel A, van Ooijen H, Galimzianova A, Cohn DM, Beca F, Rubin DL, Allison KH. Does hormone expression by IHC predict ER pathway activity? An analysis in a metastatic breast cancer patient cohort [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 P5-11-06.
Background: Previous studies have found that particular pathologic features are more common in breast cancers arising in BRCA mutation carriers. However, the biologic and molecular bases for the morphologic associations are not clear. This study is conducted to analyze pathologic and molecular features in tumors stratified by BRCA1 or BRCA2 mutation status using the breast cancer samples that have comprehensive molecular portraits characterized by the Cancer Genome Atlas (TCGA). Methods: The digital slides of breast cancer samples submitted for comprehensive molecular profiling to the TCGA were reviewed by expert breast pathologists, who were unaware of the BRCA status or other molecular signatures. Each tumor was evaluated and scored for histologic type, nuclear pleomorphism, tubule formation, mitosis, stromal inflammation, and necrosis. 562 cases had both pathology and tumor exome sequencing data available and constituted the current study population. We determined the association of somatic BRCA1 and BRCA2 mutation status with pathologic features and molecular characteristics (mutation of PIK3CA and TP53, and molecular subtypes defined by PAM50 mRNA data) using the Fisher exact test for categorical variables and the Wilcoxon test for ordinal variables. Results: Of the 562 tumors, 514 had no BRCA1 or BRCA2 mutation, while 48 (8.5%) of tumors were found to harbor a BRCA1 mutation (n = 16, 3%), BRCA2 mutation (n = 30, 5%), or mutation in both (n = 2, 0.3%). BRCA1 and BRCA2 mutational status showed no significant association with lobular features, tubule formation, nuclear pleomorphism, or stromal inflammation (all p > 0.05), although there was a trend for increased nuclear pleomorphism in BRCA2 mutant cases (p = 0.07). The lack of significant association of BRCA1/2 mutational status with these features may be due to our study's relatively small number of BRCA1/2 mutant cases. Both BRCA1 and BRCA2 mutations were associated with a higher mitotic count (p = 0.03 and 0.04, respectively). BRCA2 mutation showed no association with necrosis (p = 1), while BRCA1 mutation status was associated with increased necrosis (OR = 2.7, p = 0.04). BRCA2 mutation status showed no significant association with PAM50 subtype (p = 0.37), while BRCA1 mutation status was significantly associated with PAM50 molecular subtype (p = 0.005), with the greatest enrichment among Basal-like (7/70 Basal-like with BRCA1 mutation, 10%) and depletion among Luminal-B (0/79 Luminal-B with BRCA1 mutation, 0%). Neither BRCA1 nor BRCA2 mutations were significantly association with PIK3CA mutations (p = 0.39, 0.08, respectively). BRCA2 mutation status was not associated with TP53 mutations (p = 0.65), while BRCA1 mutation status was associated with increased TP53 mutations (OR = 4.0, p = 0.005). Conclusion: Tumors with BRCA1 and BRCA2 alterations are associated with specific pathologic and molecular features. However, there is molecular and morphologic heterogeneity within these cancers. These factors need to be considered when designing algorithms for BRCA testing and targeted therapy in BRCA-related cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-15.
Background: HER2 gene amplification status may be tested by FISH using either HER2 signals per cell or HER2:CEP17 ratio. Established HER2 testing CAP/ASCO reporting guidelines allow for interpretation and reporting using either method. Both methods classify cases as amplified, not amplified or equivocal yet the two methods may yield different interpretations for the same case. We analyzed a large dataset to quantify concordance between these two testing and reporting methodologies. Materials and Methods: We collected individual cell HER2 and CEP17 counts in 32,116 tumor cells from 1329 consecutive breast cancer cases. Data from Excel counting sheets were imported to a SQL Server database for analysis and statistics; histograms and analyses were also performed in Excel. We specifically computed concordances between methods for HER2 signals per cell and HER2:CEP17 ratio reporting. Results: Concordance between HER2 signals per cell and HER2:CEP17 ratio case averages was 89.8%. Most discordances involved cases classified as equivocal by either method. 7.8% of cases reported by HER2 signals per cell and 4.0% of cases reported by HER2:CEP17 ratio were classified as equivocal. 1% of cases were amplified by one method and not amplified by the other. Discussion: This very large dataset shows that HER2 FISH reporting by established CAP/ASCO guidelines is generally concordant between the two testing methods. However, a substantial number of cases are discordant and patient treatment could vary depending on reporting criteria used. These results indicate the need for reporting by both methods to prevent missing patients who could potentially benefit from HER2 targeted therapy. Further study is necessary to determine the most clinically relevant reporting method and criteria. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-34.
Background: Current recommendations for reporting Her2 amplification by FISH have several problems, including imperfect concordance between different reporting methods, presence of an ‘equivocal’ category that is clinically problematic, and lack of accommodation for the common presence of intratumoral heterogeneity (ITH). Using a large dataset, we sought to better define ITH and develop a FISH reporting system that is more useful for patient management. Materials and Methods: We analyzed HER2 and CEP17 counts in 32,116 individual tumor cells from 1329 consecutive breast cancer cases. Based on the individual tumor cell ratios (ICRs) and the overall average case ratios, cases were divided into three groups to explore the significance of ITH. These groups were as follows: 1) Uniformly non-amplified cases defined as < 5% of individual cells with a HER2:CEP17 ratio > 2.2 and an overall average case ratio < 1.8 (n=763). 2) Uniformly amplified cases defined as > 90% of individual cells with a HER2:CEP17 ratio > 2.2 and the overall average case ratio > 2.2 (n=137). 3) Non-uniformly amplified defined as between 5-90% of cells with ratio > 2.2. (n=429). The Her2:CEP17 ratios for individual cells were then compared between groups to identify the ICRs characteristic of uniformly amplified cancers. Results: ITH for HER2 gene amplification is very common and is present in non-amplified, equivocal and amplified cases by traditional reporting criteria. Cells with Her2:CEP17 ratios >= 4 are characteristic of uniformly amplified cases, are essentially absent in uniformly non-amplified cases and represent only 5.3% of cells in non-uniformly amplified cases (as defined above). Lower ratios are not characteristic of uniformly amplified cancers. Using an individual cell ratio >= 4 to define significantly amplified (“High- ICR”) cells, the following diagnostic categories are proposed: uniformly amplified (defined as having >90% High-ICR cells), uniformly non-amplified (<10% High-ICR cells and heterogeneous with X% amplified cells (10-90% of High-ICR cells). The distribution of cases using this proposed reporting system versus the classic CAP/ASCO categories is shown in the below table. Proposed ITH reporting system vs classic CAP/ASCO categories Discussion: Intratumoral heterogeneity for HER2 gene amplification is common in amplified, non-amplified and equivocal cases by current reporting guidelines but not all heterogeneity is likely to be clinically significant. The High-ICR scoring system categorizes cases based on the number of cells with significant amplification, eliminates the problematic equivocal category. and directly reports the fraction of cells that are presumed to be most amenable to treatment. We propose that the High-ICR reporting system be further validated and adopted for clinical use. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-04.
Background: The College of American Pathologists (CAP) recently published recommendations for reporting intratumoral heterogeneity for HER2 gene amplification in breast cancers. These guidelines recommend reporting cases with between 5-50% of cells with HER2:CEP17 ratios > 2.2 (or > 6 HER2 signals/cell) as “heterogeneous for HER2 gene amplification.” Because these recommendations lack biological and clinical foundation, we applied them to a large number of historical breast cancers and compared the ITH classification to routine FISH Her2 results. Materials and Methods: We collected Her2 and CEP17 counts in 32,116 tumor cells from 1329 consecutive breast cancer cases analyzed by FISH. Data from Excel counting sheets were imported to a SQL Server database for analysis and statistics; histograms and analyses were also performed in Excel. We specifically computed concordances between guidelines for traditional and intra-tumoral heterogeneity (ITH) reporting. Results: 309 of 1,329 cases (23%) met the criteria for heterogeneous HER2 gene amplification by HER2:CEP17 ratio, but > 79% of these were non-amplified by standard FISH reporting criteria. Using Her2 signals per cell, ITH was found in 87 (6.5%) of cases and most of these were not amplified or equivocal by standard criteria. Discussion: A significant proportion of breast cancers contain ITC heterogeneity based on CAP/ASCO reporting recommendations. The majority of these cases are non-amplified without consideration of ITH. Because it is not known whether these patients (who would not currently be treated) would benefit from HER2 targeted therapies, it may be premature to use the ITH reporting recommendations for clinical decision-making. More validation of ITH criteria and biological significance are needed. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-03.
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