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Identification and use of circulating nucleic acid tumor markers

A tumor and oligonucleotide technology, which is applied in biochemical equipment and methods, microbial determination/inspection, bioinformatics, etc., and can solve the problems of availability and inability to detect the existence of mutations in plasma samples.

Active Publication Date: 2016-04-20
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods rely on a relatively small number of known cancer genes, however, they do not provide any gene ordering in terms of effectiveness in detecting relevant mutations
Additionally, these methods cannot detect the presence of mutations in most actual cancer patient plasma samples

Method used

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  • Identification and use of circulating nucleic acid tumor markers
  • Identification and use of circulating nucleic acid tumor markers
  • Identification and use of circulating nucleic acid tumor markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0782] Example 1: Ultra-sensitive method for quantifying circulating tumor DNA in a wide range of patients

[0783] Circulating tumor DNA (ctDNA) represents a promising biomarker for noninvasive detection of disease burden and recurrence monitoring. However, existing ctDNA detection methods are limited in sensitivity, focus on a small number of mutations, and / or require patient-specific optimization. In order to solve these shortcomings, the Deep Sequencing Cancer Personalized Profiling Analysis (CAPP-Seq) has been developed, an economical and highly sensitive method for quantifying plasma ctDNA in almost every patient. We performed CAPP-Seq on non-small cell lung cancer (NSCLC), designed to identify> 95% of tumors are mutated, and point mutations, insertions / deletions, copy number variants and rearrangements are also detected. When the tumor mutation characteristics are known, we detected ctDNA in 100% stage II-IV NSCLC pretreated plasma samples and 50% stage I NSCLC samples, a...

Embodiment 2

[0881] Example 2: Design an individualized selector set

[0882] In some cases, it may be impractical to monitor the tumor burden in patients with known cancers using "existing" strategies. This strategy uses knowledge from a group of patients of the same tumor type and uses CAPP-Seq to select Sexually capture genomic regions frequently mutated in this tumor type. These conditions include, but are not limited to, the following cases, where (1) the tumor has an unknown original histology (for example, CUP); (2) the histology is known, but is too rare to have enough of the previously described tumor types The number of patients defines the average patient tumor somatic gene panorama (for example, subtype of soft tissue sarcoma); (3) the histology is known, but the average / median recurrence somatic damage in this tumor type is too large It is too low to reach the expected sensitivity level (for example, pediatric tumors, etc.); or (4) The histology is known, and the average / median ...

Embodiment 3

[0884] Example 3. Use of selector set in the diagnosis of cancer

[0885] Ingredients

Volume (μL)

cfDNA

1-75

Phosphorylation reaction buffer (10X)

10

T4DNA polymerase

5

T4 polynucleotide kinase

5

dNTPs

4

DNA polymerase I, large (Klenow)

1

139 --> Sterile H 2 O

-Add to a total volume of 100μL

[0886] The end repair reaction mixture was incubated in a thermal cycler for 30 minutes at 20°C.

[0887] The purification of the end-repaired cfDNA was performed by adding 160 μL (1.6X) of resuspended AMPureXP beads to the end-repair reaction mixture. Mix the AMPure beads on a vortex mixer or pipette up and down (for example, 10 times or more) into the solution. The reaction was incubated at room temperature for 5 minutes. The reaction was placed on a magnetic stand to separate the beads from the supernatant. After the solution is clear (about 5 minutes), the supernatant is removed and discarded. The beads were washed twice by adding 200 μL of 80% freshly prepared e...

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Abstract

Methods for creating a selector of mutated genomic regions and for using the selector set to analyze genetic alterations in a cell-free nucleic acid sample are provided. The methods can be used to measure tumor-derived nucleic acids in a blood sample from a subject and thus to monitor the progression of disease in the subject. The methods can also be used for cancer screening, cancer diagnosis, cancer prognosis, and cancer therapy designation.

Description

[0001] Government Support Statement [0002] The present invention is a government-funded project granted by the Ministry of National Defense under the grant number W81XWH-12-1-0285. The government has certain rights in the invention. Background of the invention [0003] Tumors constantly flow DNA into the circulation, where it can be easily obtained (Strounetal. (1987) EurJCancerClinOncol 23:707-712). Analysis of this type of cell-free DNA (cfDNA) derived from cancer has the potential to revolutionize the detection and monitoring of cancer. For solid tumors, non-invasive access to malignant DNA is particularly attractive, and solid tumors cannot be repeatedly sampled without invasive methods. For non-small cell lung cancer (NSCLC), PCR-based tests have been used to detect gene recurrence points in plasma DNA, such as KRAS or EGFR mutations (Taniguchietal. (2011) Clin. Cancer Res. 17:7808-7815; Gautschietal. (2007) CancerLett.254:265-273; Kuangetal.(2009) Clin.CancerRes.15:2630-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G16B30/10
CPCC12Q2600/156C12Q1/6806C12Q1/6827C12Q1/6855C12Q1/6886G16B30/00G16B30/10C12Q2525/179C12Q2525/191C12Q2535/122C12Q2537/159C12Q2537/165C12Q2563/131C12Q2563/179
Inventor M.迪恩A.A.阿利扎德A.M.纽曼S.V.布拉特曼
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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