Application of gene probes and kits for alveolar soft tissue sarcoma

A soft tissue sarcoma and gene probe technology, which is applied in the application field of fluorescence in situ hybridization probes, can solve the problems of time-consuming, unsuitable, complicated technology and difficult diagnosis, and achieves accurate, reliable and simple diagnosis, strong fluorescence signal, and high accuracy. high effect

Inactive Publication Date: 2017-12-05
THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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Problems solved by technology

Alveolar soft tissue sarcoma with typical histological morphology is easier to diagnose, and some tumor tissues lack typical morphological manifestations, which need to be associated with paraganglioma, granular cell tumor, XP11.2 translocation / TFE3 gene fusion-related renal cancer and melanoma Differentiate different phases of tumors, especially in cases of rare disease sites, metastatic lesions, and few fine-needle aspiration biopsy tissues
Acinar soft tissue sarcoma forms an ASPL-TFE3 fusion gene, resulting in the overexpression of TFE3 protein. Therefore, TFE3 immunohistochemical detection has certain specificity for acinar soft tissue sarcoma, but in other tumors such as XP11.2 translocation / TFE3 gene Positive results of TFE3 immunohistochemistry can also be found in tissues such as fusion-related renal carcinoma, granulosa cell tumor, adrenocortical carcinoma, perivascular epithelioid cell tumor, and high-grade myxofibrosarcoma, so TFE3 immunohistochemistry can only be used as a diagnostic marker for glandular tumors. Auxiliary basis for vesicular soft tissue sarcoma
[0004] For alveolar soft tissue sarcoma, its specific gene change type is an important basis for its diagnosis. Only RT-PCR and cell karyotype analysis can achieve the purpose of diagnosis more accurately, but the karyotype analysis must be performed after tumor resection. After the tumor cells are cultured to the mitotic stage, the karyotype analysis of the tumor cells is performed using techniques such as G-banding. This method is cumbersome and time-consuming and is not suitable for clinical application.
Similarly, considering the rarity of alveolar soft tissue sarcoma and the complexity of the PCR test process, it is impossible to routinely use RT-PCR to detect the ASPL-TFE3 fusion gene in alveolar soft tissue sarcoma tissue. In sarcoma, RNA can only be extracted from paraffin sections. RNA degradation and many other factors limit the application of RT-PCR, and the technology is complicated and not suitable for wide application.
[0005] These limitations lead to some patients with suspected alveolar soft tissue sarcoma cannot be accurately diagnosed, affecting clinicians' prognosis and postoperative management of patients with alveolar soft tissue sarcoma

Method used

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  • Application of gene probes and kits for alveolar soft tissue sarcoma
  • Application of gene probes and kits for alveolar soft tissue sarcoma
  • Application of gene probes and kits for alveolar soft tissue sarcoma

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Experimental program
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Embodiment 1

[0039] Step 1: Preparation of Gene Probes:

[0040] Find the bacterial artificial chromosome (BAC clone) corresponding to the X chromosome TFE3 gene and ASPL gene through http: / / genome.ucsc.edu / , select the corresponding BAC clone fragments respectively, and control the complete coverage of the BAC clone fragments selected on the same side relative to the gene , and the fragments on the same side have a certain sequence overlap with each other. According to the above requirements, the BAC clone fragments on the ASPL gene were selected as RP11-634L10 (chr17: 79796813-79969288, the fragment length is about 172Kb), RP11-51H16 (chr17: 79931578-80097452, the fragment length is about 166Kb) and RP11-475F12 ( chr17: 80032268-80216871, the fragment length is about 185Kb); the BAC clone fragment on the TFE3 gene is CTD-2311N12 (chrX: 48713289-48923401, the fragment length is about 210Kb), CTD-2522M13 (chrX: 48420425-48602847, The fragment length is about 182Kb), RP11-416B14 (chrX: 485...

Embodiment 2

[0052] Example 2: Diagnostic kit for alveolar soft tissue sarcoma

[0053] Gene probe kit for alveolar soft tissue sarcoma, the kit is composed of probe hybridization solution and 4', 6-diamidino-2-phenylindole counterstaining agent, characterized in that:

[0054] (1) The combination of RP11-634L10, RP11-51H16, and RP11-475F12 located on the ASPL gene on chromosome 17, marked with a red fluorescent signal; Combination of CTD-2312C1 and CTD-2248C21, labeled as green fluorescent signal;

[0055] (2) The probe hybridization solution is prepared by mixing the probe with Human Cot-1 DNA, hybridization buffer, and purified water in proportion, and it needs to be stored at -20°C in the dark;

[0056] (3) 4′,6-diamidino-2-phenylindole counterstain is mainly used for nuclear staining.

[0057] (4) The combination of RP11-634L10, RP11-51H16 and RP11-475F12 located on the ASPL gene of chromosome 17 described in the Gene Probe Kit for Acinar Soft Tissue Sarcoma, the three clone fragmen...

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Abstract

The invention relates to a gene probe of acinar soft tissue sarcoma and the application of the kit. The cloned fragments selected by the gene probe of the present invention are the combination of RP11-634L10, RP11-51H16 and RP11-475F12, and the combination of CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-2248C21. The present invention overcomes the cumbersome and time-consuming RT-PCR and karyotype analysis methods, RNA degradation and many other factors that limit the application of RT-PCR, cannot obtain accurate diagnosis, and affect prognosis and postoperative treatment. The invention has high accuracy, high specificity, high success rate, strong fluorescent signal, simple operation, can be applied to paraffin sections, expands the scope of detection specimens, and establishes a new method for accurate, reliable and simple diagnosis of acinar soft tissue sarcoma. Created a precedent for the use of FISH to detect acinar soft tissue sarcoma.

Description

technical field [0001] The invention belongs to the application field of fluorescent in situ hybridization probes, in particular to gene probes for alveolar soft tissue sarcoma and the application of kits thereof. Background technique [0002] Alveolar soft tissue sarcoma is a rare malignant soft tissue sarcoma of unknown origin that occurs in children and young adults. The main symptom is a soft tissue mass that grows slowly. Balanced translocation, namely: der(17)t(X;17)(p11.2;q25), resulting in translocation of the TFE3 telomeric side gene at Xp11.2 to 17q25 after duplication, and with the centromere of the 17q25ASPL gene Lateral fusions form the ASPL-TFE3 fusion gene. Most patients with alveolar soft tissue sarcoma are prone to distant metastasis after discovery and treatment, and are not sensitive to radiotherapy and chemotherapy. The overall prognosis is poor. The 5-year, 10-year, and 20-year survival rates are 60%, 38%, and 15%, respectively Therefore, accurate diag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 甘卫东陈显成樊祥山屈峰曾浩郭宏骞
Owner THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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