Method for the quantification of pd-l1 expression

a pdl1 and expression technology, applied in the field of pdl1 expression quantification, can solve the problems of t-cell exhaustion, ineffective response, and many patients who do not benefit from these therapies, and achieve the effect of reducing variations and increasing errors

Inactive Publication Date: 2019-03-21
PHARMASSIST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0029]In another aspect, step (v) of the method may further comprise a reference primer pair that hybridizes to a reference mRNA sequence of a reference gene in order to ensure that amplifiable material is present in the test samples and in order to avoid false negative results.

Problems solved by technology

Despite the recent development of targeted therapies in many types of cancer, many patients do not benefit from these therapies.
A basic hindrance for immunotherapeutic approaches is that the majority of mechanisms are active at the tumor site, which act together in order to balance effectively the anti-tumor immunity.
Despite the fact that an endogenous immune response to cancer has been observed in patients, this response seems to be ineffective, and the established cancers are tolerated by the immune system.
When PD-1 binds to tumor cells expressing PD-L1, T-cell activity and cytokine production are suppressed, leading to T-cell exhaustion.
Most importantly, these blood-based tests seem to be very challenging and highly important in case that tumor biopsies are not accessible (such as in the case of NSCLC).

Method used

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  • Method for the quantification of pd-l1 expression
  • Method for the quantification of pd-l1 expression
  • Method for the quantification of pd-l1 expression

Examples

Experimental program
Comparison scheme
Effect test

example 1

Development and Analytical Validation of the Assay

[0135]The experimental flowchart of the study is outlined in FIG. 4.

Protocol Optimization.

[0136]A RT-qPCR based methods for the quantification of PD-L1 and B2M expression were optimized in a number of experiments, using as positive control cDNA samples from peripheral blood mononuclear cells (PBMC) of healthy control samples and negative control of PCR reaction mix, with respect to: PCR annealing temperature, cycling parameters, primers and probe concentrations, Mg+2 and dNTPs concentrations.

[0137]Single RT-qPCR was performed for PD-L1 and B2M expression. Quantification is based on real-time monitoring during PCR of labelled specific hydrolysis probe for PD-L1. The cycle where the fluorescence signal rises above background noise (quantification cycle, Cq) is best quantified through the LightCycler software as the second derivative maximum of the curve. Real-time RT-PCR for PD-L1 mRNA was performed using the LightCycler system (Roche ...

example 2

RT-qPCR Quantification Using External Calibrators

[0140]Firstly, individual PCR amplicons specifically for PD-L1 and B2M were generated in order to be used as external quantification calibrators. For this purpose, total RNA was extracted from PBMC pool from normal samples since PBMC express as well PD-L1. cDNA was synthesized and served as a template for the amplification of PD-L1 and B2M by the above described RT-qPCR. PCR products were purified using MinElute PCR Purification Kit (Qiagen, Germany) and the amplicons were quantified in the Nanodrop-1000 spectrophotometer (NanoDrop, Technologies, USA).

[0141]DNA concentration was converted to copies / μL by use of the Avogadro number and the molecular weight of the amplicon number of bases of the PCR product multiplied by the mean molecular weight of a pair of nucleic acids which is 660. A standard stock solution corresponding to 1010 copies / μL for each gene transcript was prepared. Serial dilutions of this stock amplicon solution in DNa...

example 3

The Limit of Detection and Linearity of Assay

[0142]The limit of detection (LOD) of the developed RT-qPCR assay for both PD-L1 and B2M as copies / μL in the reaction was evaluated. To estimate LOD, the quantification calibrators containing a known number of copies / μL were prepared as described below in detail: For each gene target a calibration curve was generated using serial dilutions of these standards in triplicate for each concentration, ranging from 105 copies / μL to 10 copies / μL. The calibration curves showed linearity from 10 copies / μL up to 105 copies / μL, with correlation coefficients larger than 0.99 in both cases, indicating a precise log-linear relationship (FIG. 5). None of the primers and hydrolysis probes gave any signal for any of the gene target transcripts when 50 ng / μL of 10 different genomic DNAs was analyzed.

[0143]The LOD for both of these assays was found to be 3 copies / μL and the limit of quantification (LOQ) was found to be 9 copies / μL, (estimated according to th...

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Abstract

A highly sensitive for determining the expression of PD-L1 that is based on a RT-qPCR in a RNA sample of, for example, Circulating Tumor Cells (CTC) or fresh frozen primary tumor tissues. In particular, to a method for the detection of PD-L1 mRNA positive CTCs or primary tumor tissues (fresh frozen) based on the quantitative determination of the molecular marker (PD-L1) in biological samples of patients suffering from cancer. By using the method, detection can take place before, during or after the immune therapy or any other treatment in order to provide significant information concerning guiding or monitoring of the anti-PD-L1 agents effectiveness. This RT-qPCR assay could comprise a promising companion diagnostic test in order to evaluate the PD-L1 expressional status on CTC or tumor tissue, providing clinical applications, which could have an important impact on therapeutic interventions since the expression of PD-L1 is associated with response to immunotherapy.

Description

TECHNICAL FIELD[0001]The present invention relates to a highly sensitive, specific and reproducible real time RT-qPCR assay for the quantification of PD-L1 expression in a RNA sample, such as an isolated RNA sample from Circulating Tumor Cells (CTCs) in peripheral blood of patients with solid cancers or fresh frozen primary tumor tissues.BACKGROUND OF THE INVENTION[0002]The discovery of crucial molecular pathways that promote tumor growth and maintenance together with the development of drugs that specifically inhibit these pathways has changed much in the perennially limited panoply of options to treat many types of cancer. The promise of the emerging field of personalized medicine will increasingly be used to tailor therapeutics to defined sub-populations, and eventually, individual patients in order to enhance efficacy and minimize adverse side effects. However, the success of personalized medicine depends on having accurate, reproducible and clinically useful companion diagnosti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/6886C12Q1/6853
CPCC12Q1/6886C12Q1/6853C12Q2600/16C12Q2600/112C12Q2600/166C12Q2600/118C12Q2600/106C12Q2600/156
Inventor LIANIDOU, EVRYKLEIASTRATI, ARETI
Owner PHARMASSIST
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