Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels Using Quantitative Dot Blot (QDB) Method

Hao, Junmei; Lyu, Yan; Zou, Jiarui; Zhang, Yunyun; Xie, Sheng Quan; Jing, Lili; Tang, Fangrong; Lyu, Jiahong; Zhang, Wenfeng; Zhang, Jianbo; Wang, Xunting; Chen, Kuisheng; Zhang, Jiandi

doi:10.3389/fonc.2021.737781

Cited by 7 publications

(15 citation statements)

References 25 publications

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“…An optimized cyclinD1 cutoff value was determined for OS analysis using the “surv_cutpoint” function of the “surviminer” R package at 0.44 μmol/g, and this value was used to separate these Luminal-like specimens into cyclinD1-high (C h ) and cyclinD1-low (C l ) groups. Meanwhile, these specimens were also separated into Ki67-high (K h ) and Ki67-low (K l ) groups using the 2.31 nmol/g defined in the previous study ( 8 ). In the accompanying manuscript III (submitted), the proposed 0.44 μmol/g cutoff was validated using an independent cohort from another hospital.…”

Section: Resultsmentioning

confidence: 99%

“…The IHC analyses for both Ki67 and cyclinD1 were performed using a procedure described elsewhere ( 8 ), using SA38-08 antibody for cyclinD1 and SP6 for Ki67 from LBP ( www.gzlbp.com ) at 1:100 dilution, respectively.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…In a previous study, we measured the Ki67 protein levels absolutely and quantitatively in 155 Luminal-like formalin-fixed paraffin-embedded (FFPE) specimens using our independently developed Quantitative Dot Blot (QDB) method (8). When replacing IHC-based Ki67 scores with absolutely quantitated Ki67 levels, we found that the prognosis of surrogate assay was improved significantly for the overall survival (OS) of Luminallike tumors, with 10-year survival probability (10y SP) for Luminal A-like patients at 91 vs. 63% for Luminal B-like patients (8). In comparison, the 10y SP for Luminal A and Blike group was 88% and 68%, respectively, when using a Ki67 score of 14% as cutoff based on the 2013 St. Gallen consensus (2), and 84% and 70%, respectively, when 20% was used as cutoff based on the 2015 St. Gallen consensus (5).…”

Section: Introductionmentioning

confidence: 99%

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Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients

Hao

Zhang²,

Lyu³

et al. 2021

Front. Oncol.

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BackgroundKi67 is a biomarker of proliferation to be used in immunohistochemistry (IHC)-based surrogate assay to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients. cyclinD1 is another frequently used biomarker of proliferation. A retrospective study was performed here to investigate if these two biomarkers may be combined to improve the prognosis of Luminal-like patients.MethodsBoth Ki67 and cyclinD1 protein levels were measured absolutely and quantitatively using Quantitative Dot Blot method in 143 Luminal-like specimens. Optimized cutoffs for these two biomarkers were developed to evaluate their prognostic roles using Kaplan–Meier overall survival (OS) analysis.ResultscyclinD1 was found as an independent prognostic factor from Ki67 in univariate and multivariate OS analyses. At optimized cutoffs (cyclinD1 at 0.44 μmol/g and Ki67 at 2.31 nmol/g), the subgroup with both biomarkers below the cutoffs (n = 65) had 10-year survival probability at 90% in comparison to those with both biomarkers above the cutoffs (n = 18) with 8-year survival probability at 26% (log-rank test, p <0.0001). This finding was used to modify the surrogate assay using IHC-based cyclinD1 scores, with p-value decreased from 0.031 to 0.00061 or from 0.1 to 0.02, when the Ki67 score of 14 or 20% was used as cutoff, respectively, in the surrogate assay.ConclusionThe current study supports the prospective investigation of cyclinD1 relevance in the clinic.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients

Hao

Zhang²,

Lyu³

et al. 2021

Front. Oncol.

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“…Recently, the quantitative dot blot method (QDB) has been used to measure Her2 and Ki67 protein levels in breast cancer specimens. This is an enzyme-linked immunosorbent assay (ELISA)-like method that can be adopted in routine clinical practice [ 26 27 28 29 ]. In the analytical process, the signal is quantitated by a microplate reader rather than visual scoring, thus eliminating subjectivity.…”

Section: Introductionmentioning

confidence: 99%

Objective Quantitation of EGFR Protein Levels using Quantitative Dot Blot Method for the Prognosis of Gastric Cancer Patients

Xin

Tang²,

Song

et al. 2021

J Gastric Cancer

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Purpose An underlying factor for the failure of several clinical trials of anti-epidermal growth factor receptor (EGFR) therapies is the lack of an effective method to identify patients who overexpress EGFR protein. The quantitative dot blot method (QDB) was used to measure EGFR protein levels objectively, absolutely, and quantitatively. Its feasibility was evaluated for the prognosis of overall survival (OS) of patients with gastric cancer. Materials and Methods Slices of 2×5 μm from formalin-fixed paraffin-embedded gastric cancer specimens were used to extract total tissue lysates for QDB measurement. Absolutely quantitated EGFR protein levels were used for the Kaplan-Meier OS analysis. Results EGFR protein levels ranged from 0 to 772.6 pmol/g (n=246) for all gastric cancer patients. A poor correlation was observed between quantitated EGFR levels and immunohistochemistry scores with ρ=0.024 and P=0.717 in Spearman's correlation analysis. EGFR was identified as an independent negative prognostic biomarker for gastric cancer patients only through absolute quantitation, with a hazard ratio of 1.92 (95% confidence interval, 1.05–3.53; P=0.034) in multivariate Cox regression OS analysis. A cutoff of 208 pmol/g was proposed to stratify patients with a 3-year survival probability of 44% for patients with EGFR levels above the cutoff versus 68% for those below the cutoff based on Kaplan-Meier OS analysis (log rank test, P=0.002). Conclusions A QDB-based assay was developed for gastric cancer specimens to measure EGFR protein levels absolutely, quantitatively, and objectively. This assay should facilitate clinical trials aimed at evaluation of anti-EGFR therapies retrospectively and prospectively for gastric cancer.

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Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal‐like breast cancer patients

Lyu²,

et al. 2022

Clinical Laboratory Analysis

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Aims To translate a clinical research finding into daily clinical practice requires well‐controlled clinical trials. We have demonstrated the usage of absolute quantitation of Ki67 and cyclinD1 protein levels to improve prognosis of Luminal‐like patients based on overall survival (OS) analysis of a cohort of 155 breast cancer specimens (cohort 1). However, this finding is considered the D level of evidence (LOE) to require subsequent validation before it may be used in daily clinical practice. To set the stage for future clinical trials, our findings were validated through OS analysis of an independent cohort (cohort 2) of 173 Luminal‐like patients. Methods Both Ki67 and cyclinD1 levels were measured absolutely and quantitatively using the Quantitative Dot Blot (QDB) method in cohort 2. The proposed cutoffs for both biomarkers from cohort 1 were re‐evaluated in cohort 2 and in the merged cohort of 1 and 2, respectively, through univariate, multivariate and Kaplan–Meier survival analysis. Results The proposed cutoffs of 2.31 nmol/g for Ki67 and 0.44 μmol/g for cyclinD1 were validated as effective cutoffs in cohort 2 and the merged cohort through OS analysis. The combined use of both biomarkers allowed us to identify patients with both biomarker levels below the cutoffs (59.3%) with10‐year survival probability (SP) of 89%, in comparison to those above the cutoffs (8.3%) with 8 year SP of 28% through OS analysis in the merged cohort. Conclusions This study validated our findings that absolute quantitation of Ki67 and cyclinD1 allows effective subtyping of luminal‐like patients. It sets the stage for prospective or prospective‐retrospective clinical studies.

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Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels Using Quantitative Dot Blot (QDB) Method

Cited by 7 publications

References 25 publications

Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients

Combined Use of cyclinD1 and Ki67 for Prognosis of Luminal-Like Breast Cancer Patients

Objective Quantitation of EGFR Protein Levels using Quantitative Dot Blot Method for the Prognosis of Gastric Cancer Patients

Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal‐like breast cancer patients

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