Dataset Information

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

ABSTRACT: An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean?=?51.5%) and IDCA with DCIS (mean?=?53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P?>?0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P?

SUBMITTER: Gupta S

PROVIDER: S-EPMC6119113 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

Gupta Swati S Mani Navin R NR Carvajal-Hausdorf Daniel E DE Bossuyt Veerle V Ho Kenneth K Weidler Jodi J Wong Wendy W Rhees Brian B Bates Michael M Rimm David L DL

Laboratory investigation; a journal of technical methods and pathology 20180601 8

An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts ...[more]

PMID: 29858579

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Project description:BackgroundMany methodologies have been used in research to identify the "intrinsic" subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions.MethodsWe used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and "intrinsic" subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments.ResultsESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis.ConclusionsThe standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.

Project description:BACKGROUND:In melanoma, there is no companion diagnostic test to predict response to programmed cell death 1 (PD-1) axis immune checkpoint inhibitor (ICI) therapy. In the adjuvant setting, only one in five patients may benefit from ICI, so a biomarker is needed to select those that may or may not benefit. Here, we test a new 4-gene multiplex immunotherapy panel with research use only (RUO) prototype mRNA expression profile on the GeneXpert closed system using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) for association with clinical benefit after treatment with ICI therapy in metastatic melanoma patients. METHODS:Pretreatment formalin-fixed paraffin-embedded (FFPE) tissue sections from melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) between 2011 and 17 were selected from the Yale Pathology archives. FFPE sections were macrodissected to enrich for tumor for quantitative assessment of CD274 (PD-L1), PDCD1LG2 (PD-L2), CD8A, and IRF1 by RT-qPCR multiplex mRNA panel. Multiplex panel transcript levels were correlated with clinical benefit (complete response [CR], partial response [PR], stable disease [SD]); disease outcomes (progression-free survival [PFS] and overall survival [OS]); and protein levels assessed by quantitative immunofluorescence (QIF). RESULTS:Transcript levels were significantly higher in responders (CR/PR/SD) than in nonresponders (PD) for CD8A (p = 0.0001) and IRF1 (p = 0.0019). PFS was strongly associated with high CD274 (p = 0.0046), PDCD1LG2 (p = 0.0039), CD8A (p = 0.0002), and IRF1 (p = 0.0030) mRNA expression. Similar associations were observed for OS with high CD274 (p = 0.0004), CD8A (p = 0.0030), and IRF1 (p = 0.0096) mRNA expression. Multivariate analyses revealed significant PFS and OS associations with immunotherapy panel markers independent of baseline variables. Exploratory analyses revealed a novel significant association of high combined CD274 & PDCD1LG2 (L1/L2) transcript expression with PFS (p < 0.0001) and OS (p = 0.0011), which remained significant at a multivariate level for both PFS (HR = 0.31) and OS (HR = 0.39). CONCLUSIONS:Individual immunotherapy panel markers CD274, PDCD1LG2, CD8A, IRF1 and a combined L1/L2 mRNA levels show promising associations with melanoma immunotherapy outcome. The turnaround time of the test (2 h) and easy standardization of the platform makes this an attractive approach for further study in the search for predictive biomarkers for ICI.

Dataset Information

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

Publications

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

Similar Datasets

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