Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers

Zhang, Wenfeng; Yu, Guohua; Zhang, Yunyun; Tang, Fangrong; Lv, Jiahong; Tian, Geng; Zhang, Yuan; Liu, Jing; Mi, Jia; Zhang, Jiandi

doi:10.1016/j.ab.2019.04.003

Cited by 17 publications

(20 citation statements)

References 8 publications

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“…The QDB process was described previously with minor modifications [8][9][10][11]. In brief, FFPE tissue lysates (about 0.35 g/unit) and MCF-7 cell lysate (about 0.08 g/unit) were applied onto the QDB plate at 2 l/unit in triplicate.…”

Section: Qdb Analysismentioning

confidence: 99%

Combined use of absolutely quantitated cyclin D1 and Ki67 protein levels to improve prognosis of Luminal-like breast cancer

Hao

Zhang²,

Lv³

et al. 2020

Preprint

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Purpose: Both Ki67 and cyclin D1 are routinely used protein biomarkers of cell proliferation for breast cancer patients. Ki67 is used to differentiate Luminal A-like from Luminal-B like subtype in surrogate assay. These two proliferative factors are investigated in this retrospective study to evaluate their prognostic role on the overall survival (OS) of Luminal-like breast cancer patients. Method: The cyclin D1 protein level was measured absolutely and quantitatively using Quantitative Dot Blot (QDB) method in 143 Luminal-like FFPE breast cancer specimens. An optimized cutoff at 0.71 μmole/g was identified and used to separate these specimens into cyclin D1 high and low groups alone, or in combination with Ki67, for overall survival (OS) analyses of these patients. Results: Cyclin D1 was found to be an independent prognostic factor from Ki67 in univariate and multivariate analysis. When both biomarkers were used to separate these Luminal-like specimens, the group with low expression of both biomarkers (n=52) had significantly improved 10 year survival probability at 94%, while the one with high expression of both markers (n=34) were at 41% based on Kaplan-Meier survival analysis of OS (Log rank test p<0.0001). Conclusion: We demonstrated cyclin D1 as an independent prognostic protein biomarker from Ki67 for Luminal-like breast cancers. The combined usage of cyclin D1 and Ki67 significantly improved the prognosis over current prevailing surrogate assay. We propose to incorporate cyclin D1 in surrogate assay to improve prognosis for Luminal-like breast cancer patients in future clinical practice.

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Section: Qdb Analysismentioning

confidence: 99%

Combined use of absolutely quantitated cyclin D1 and Ki67 protein levels to improve prognosis of Luminal-like breast cancer

Hao

Zhang²,

Lv³

et al. 2020

Preprint

Self Cite

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“…We hypothesized the inherent inaccuracy associated with Ki67 IHC scores may negatively affect the performance of surrogate assay for Luminal-like patients. Therefore, we attempted to measure Ki67 levels absolutely and objectively using a recently developed Quantitative Dot Blot (QDB) method in Formalin Fixed Para n Embedded (FFPE) specimens [10][11][12][13]. The specimens were separated into luminal A (LumA q ) and luminal B subtypes (LumB q ) based on the absolute quantitated Ki67 levels in these specimens in a retrospective study (Table 1).…”

Section: Introductionmentioning

confidence: 99%

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

Hao

Liu²,

Zou

et al. 2021

Preprint

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Background: Immunohistochemistry (IHC) based-surrogate assay is the prevailing method in daily clinical practice to determine the necessity of cytotoxic therapy for Luminal-like breast cancer patients worldwide. It relies on Ki67 scores to separates Luminal A-like from Luminal B-like breast cancer subtypes. Yet, IHC method is plagued with subjectivity and inconsistency. We attempted to circumvent these issues by measuring Ki67 levels absolutely, quantitatively and objectively.Methods: The Ki67 protein levels in a cohort of 253 specimens were measured with IHC and Quantitative Dot Blot (QDB) methods respectively, and used to assign these specimens into Luminal A-like and Luminal B-like subtypes accordingly. Their performances were compared with the Kaplan-Meier, univariate and multivariate survival analyses of the overall survival (OS) of Luminal-like patients.Results: The surrogate assay based on absolutely quantitated Ki67 levels (cutoff at 2.31 nmole/g) subtyped the Luminal-like patients more effectively than those based on Ki67 scores (cutoff at 14%) (Log rank test, p=0.00052 vs p=0.031). It is also correlated better with OS in multivariate survival analysis [Hazard Ratio (HR) at 6.89 (95%CI: 2.66-17.84, p=0.0001) vs 2.14 (95%CI: 0.89-5.11, p=0.087)].Conclusions: Our study showed the performance of surrogate assay may be improved significantly by measuring Ki67 levels absolutely, quantitatively and objectively.

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“…Nonetheless, a validated antibody used in other immunoblot processes, including IHC, flow cytometry, and Western blot analysis with single band at detection, can be used directly in a QDB-based high throughput assay . In a proof of concept (POC) study, we have measured HER2 levels quantitatively and absolutely in frozen breast cancer tissues with QDB method using clinically validated antibodies for IHC (IHC antibodies) 15 .…”

Section: Introductionmentioning

confidence: 99%

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

Zhang²,

Zhang³

et al. 2020

Sci Rep

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Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

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Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers

Cited by 17 publications

References 8 publications

Combined use of absolutely quantitated cyclin D1 and Ki67 protein levels to improve prognosis of Luminal-like breast cancer

Combined use of absolutely quantitated cyclin D1 and Ki67 protein levels to improve prognosis of Luminal-like breast cancer

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

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

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