RNAscop (TM) HPV assay for determining HPV status in head and neck cancers and cervical lesions

A head and neck cancer, HPV-16 technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as unpredictable effects, difficult assay methods, unclear high-specificity properties, etc.

Inactive Publication Date: 2013-12-25
ADVANCED CELL DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Second, the variety of oligonucleotide (oligo) target probes required to detect high-risk HPV subtypes allows the design of RNAscope assay is difficult
The performance of such a large number of oligonucleotides has never been tested in a single in situ hybridization experiment, and its effect is difficult to predict
Prior to the present invention, it was not clear that existing RNAscope Whether assays can be adapted to simultaneously detect multiple highly homologous HPV subtypes while maintaining the highly specific nature of such assays

Method used

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  • RNAscop (TM) HPV assay for determining HPV status in head and neck cancers and cervical lesions
  • RNAscop (TM) HPV assay for determining HPV status in head and neck cancers and cervical lesions
  • RNAscop (TM) HPV assay for determining HPV status in head and neck cancers and cervical lesions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] RNAscope Protocol for HPV Assay

[0082] For each tumor sample, 5 micron tissue sections were cut and mounted on glass slides. Perform the following steps for each tumor sample:

[0083] 1. Obtain a sample from the subject. The sample may be a non-preserved tissue sample obtained from a patient or a tissue sample in a formalin-fixed paraffin-embedded tissue section.

[0084] 2. Optionally if the sample is in a FFPE tissue section, the sample can be treated first by heating the FFPE tissue section in citrate buffer followed by protease digestion.

[0085] 3. Perform RNAscope on the sample HPV assay, which includes the following tests:

[0086] i) a negative control using a target probe against the bacterial gene dapB;

[0087] ii) using a target probe for the human gene UBC to carry out a positive control;

[0088] iii) Assays are performed using HPV target probe sets containing pooled high-risk HPV subtypes or individual target probe sets specific to these subt...

Embodiment 2

[0091] RNAscope of E6 / E7 mRNA of HPV subtypes in subtype-specific cell lines detection

[0092] To confirm RNAscope Feasibility of HPV Assays to Detect and Differentiate Different HPV Subtypes Experiments were performed to detect HPV subtypes in cell lines known to contain only specific HPV subtypes. Using the RNAscope described in Example 1 Protocol for HPV assay. Target probe sets specific to E6 / E7 mRNA of HPV-16, HPV-18 and HPV-45 were designed respectively. Hybridization of the probe set to its target is detected using an alkaline phosphatase-conjugated signal amplification system, followed by development with Fast Red, which produces a red fluorescent precipitate. Such as figure 2 As shown, the HPV-16 target probe set produced positive signals only in SiHa and CaSki cells, two cell lines bearing only the HPV-16 subtype. The HPV-18 target probe set produced a positive signal only in the HeLa cell line, which carries only the HPV-18 subtype. The HPV-45 target pro...

Embodiment 3

[0094] RNAscope of HPV subtypes in head and neck cancer tissue samples detection

[0095] The following experiments were performed to detect E6 / E7 mRNA of HPV high-risk subtypes in FFPE tissue samples obtained from HNC patients. In this experiment, the RNAscope described in Example 1 was used Protocol for HPV assay. Two sets of target probe sets were prepared. In one set, the target probe set contained only target probes that bound only to HPV-16. In another set, the target probe set contained target probes that bind to HPV-18, 31, 33, 35, 52, and 58 (referred to as the "HR-HPV probe set"). Multiple HNC patient samples diagnosed with cervical lesions were tested using each of the aforementioned panels of target probe sets. image 3 Exemplary results are shown. The results showed that positive E6 / E7 mRNA of HPV high-risk subtypes were detected in two patients. Two patients were known to have HPV-associated HNC. Therefore, the experiment confirmed that RNAscope with sp...

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Abstract

The present invention provides a method and a kit for determining whether a head and neck cancer is HPV-related. In one embodiment, an RNAscope (TM) HPV assay was designed to detect the presence of E6 / E7 mRNA of certain high-risk HPV subtypes related to head and neck cancer. The present invention also provides a method and a kit for determining whether a cervical lesion is a benign lesion or a cervical intraepithethial neoplasm lesion. In one embodiment, an RNAscope (TM) HPV assay was designed to detect the presence of E6 / E7 mRNA of certain high-risk HPV subtypes related to cervical cancer. The present invention further provides a method for determining the progression of cervical intraepithethial neoplasm based on the spatial pattern and levels of the E6 / E7 mRNA of certain high-risk HPV subtypes. The present invention also provides a method for determining the risk of developing cervical cancer in a human diagnosed with cervical intraepithethial neoplasm based on presence and absence of the certain subgroups of high-risk HPV subtypes.

Description

[0001] Cross References to Related Applications [0002] This application requires a filing date of January 28, 2011, and the title of the invention is "RNAscope for Determination of HPV Status in Head and Neck Cancer" TM Priority and benefit to U.S. Provisional Application No. 61 / 437,337 for HPV". [0003] The entire content and disclosure of the aforementioned applications are incorporated by reference into this application. field of invention [0004] Provided herein are methods and kits for determining whether head and neck cancer is associated with human papillomavirus (HPV) based on the presence or absence of E6 / E7 mRNA of high-risk HPV subtypes. This article also provides a method and a kit for diagnosing whether a subject's cervical lesions are benign according to the presence or absence of E6 / E7 mRNA of high-risk HPV subtypes. Also provided herein are methods for determining the progression of cervical intraepithelial neoplasia (CIN) based on the spatial pattern an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
CPCC12Q1/6841C12Q1/6886C12Q1/708C12Q2600/112C12Q2600/118
Inventor 晓-君·马约翰·弗拉纳根罗宇龄
Owner ADVANCED CELL DIAGNOSTICS INC
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