Quantitative In Situ Measurement of Estrogen Receptor mRNA Predicts Response to Tamoxifen

Bordeaux, Jennifer; Cheng, Hao; Welsh, Allison W.; Haffty, Bruce G.; Lannin, Donald R.; Wu, Xinfeng; Su, Nan; Ma, Xiao‐Jun; Luo, Yuling; Rimm, David L.

doi:10.1371/journal.pone.0036559

Cited by 61 publications

(52 citation statements)

References 18 publications

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“…The difference might be explained, at least in part, by the distinct properties and independent information provided by PD-L1 protein and mRNA molecules, as reported (33). Methodologic differences might also account for the apparent inconsistency.…”

Section: Discussionmentioning

confidence: 87%

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Vassilakopoulou

Avgeris

Velcheti

et al. 2016

Clinical Cancer Research

198

151

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Purpose: Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). Experimental Design: A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. Results: PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Conclusions: Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors. Clin Cancer Res; 22(3); 704–13. ©2015 AACR.

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Section: Discussionmentioning

confidence: 87%

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Vassilakopoulou

Avgeris

Velcheti

et al. 2016

Clinical Cancer Research

198

151

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“…15 In one study, in situ RNA detection was not measurable in tissues prior to 1993. 16 DNA is thought to be most stable, but recent whole exome sequencing efforts in our group have noted increased specimen failure with age. Further understanding of age-dependent loss of biomarker information could also depend on storage conditions and other pre-analytic variables that can be optimized for signal preservation.…”

Section: Discussionmentioning

confidence: 90%

Loss of antigenicity with tissue age in breast cancer

Combs

Han

Mani

et al. 2016

Laboratory Investigation

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Archived tumor specimens, particularly those collected by large cooperative groups and trials, provide a wealth of material for post hoc clinical investigation. As these tissues are rigorously collected and preserved for many decades, subsequent use of the specimens to answer clinical questions must rely on the assumption that expression and detection of target biomarkers are not degraded with time. To test this assumption, we measured the expression of estrogen receptor (ER), human epidermal growth receptor 2 (HER2), and Ki67 in human breast carcinoma using quantitative immunofluorescence (QIF) in a series of formalin-fixed paraffin-embedded (FFPE) tissues from 1295 individual patients preserved for 7 to 53 years in four cohorts on tissue microarrays. Protein expression was measured using the automated quantitative analysis method for QIF. Change in quantitative protein expression over time was estimated in positive cases using both Pearson's correlation and a polynomial regression analysis with a random effects model. The average signal decreased with preservation time for all biomarkers measured. For ER and HER2, there was an average of 10% signal loss after 9.9 years and 8.5 years, respectively, compared with the most recent tissue. Detection of Ki67 expression was lost more rapidly, with 10% signal loss in just 4.5 years. Overall, these results demonstrate the need for adjustment of tissue age when studying FFPE biospecimens. The rate of antigenicity loss is biomarker specific and should be considered as an important variable for studies using archived tissues.

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“…Patient cohorts, tissue microarrays, and control preparations Two previously reported (33,34) retrospective stage I-III breast cancer collections from Yale University (New Haven, CT) represented in tissue microarray (TMA) format were used in this study, termed YTMA128 (N ¼ 238) and YTMA201 (N ¼ 398). Clinicopathologic information from patients in both cohorts was collected from clinical records and pathology reports.…”

Section: Methodsmentioning

confidence: 99%

“…In situ detection of PD-L1 transcripts in FFPE TMA samples was performed using the RNAscope assay with custom-designed in situ hybridization probes (Advanced Cell Diagnostics) coupled to automated quantitative fluorescence (QIF) detection as described (33)(34)(35). Briefly, 5 mm sections were deparaffinized, boiled with preamplification reagent for 15 minutes, and submitted to protease digestion followed by hybridization for 2 hours with target probes to human PD-L1 mRNA, Ubiquitin C (UbC) as a positive control, or the bacterial gene DapB mRNA as a negative control.…”

Section: In Situ Mrna Hybridizationmentioning

confidence: 99%

In Situ Tumor PD-L1 mRNA Expression Is Associated with Increased TILs and Better Outcome in Breast Carcinomas

Schalper

Velcheti

Carvajal

et al. 2014

Clinical Cancer Research

Self Cite

448

356

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Purpose: Blockade of the PD-1/PD-L1 axis emerged as a promising new therapeutic option for cancer that has resulted in lasting responses in metastatic renal, lung carcinomas, and melanomas. Tumor PD-L1 protein expression may predict response to drugs targeting this pathway. Measurement of PD-L1 protein is limited by the lack of standardized immunohistochemical methods and variable performance of antibodies. Our goal was to correlate PD-L1 mRNA expression with clinical variables in primary breast carcinomas.Experimental Design: The fluorescent RNAscope paired-primer assay was used to quantify in situ PD-L1 mRNA levels in 636 stage I-III breast carcinomas on two sets of tissue microarrays [YTMA128 (n ¼ 238) and YTMA201 (n ¼ 398)]. Tumor-infiltrating lymphocytes (TIL) were assessed by hematoxylin/eosin stain and quantitative fluorescence.Results: On YTMA128 and YTMA201, 55.7% and 59.5% of cases showed PD-L1 mRNA expression, respectively. Higher PD-L1 mRNA expression was significantly associated with increased TILs (P ¼ 0.04) but not with other clinical variables. Elevated TILs (scores 2 and 3þ) occurred in 16.5% on YTMA128 and 14.8% on YTMA201 and was associated with estrogen receptor-negative status (P ¼ 0.01 on YTMA128 and 0.0001 on YTMA201). PD-L1 mRNA expression was associated with longer recurrence-free survival (log-rank P ¼ 0.01), which remained significant in multivariate analysis including age, tumor size, histologic grade, nodal metastasis, hormone receptor, HER2 status, and the extent of TILs (HR, 0.268; CI, 0.099-0.721; P ¼ 0.009).Conclusions: PD-L1 mRNA expression is identified in nearly 60% of breast tumors and it is associated with increased TILs and improved recurrence-free survival. These observations support the evaluation of PD-

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Quantitative In Situ Measurement of Estrogen Receptor mRNA Predicts Response to Tamoxifen

Cited by 61 publications

References 18 publications

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Loss of antigenicity with tissue age in breast cancer

In Situ Tumor PD-L1 mRNA Expression Is Associated with Increased TILs and Better Outcome in Breast Carcinomas

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