Methods of detecting an EML4-ALK fusion tumor biomarker

The present disclosure is a continuation-in-part application of the international application number PCT/CN2018/086201 filed on May 9, 2018 and claiming priority of CN application number 201810190175.1 filed on Mar. 8, 2018, the contents of all of which are incorporated herein by reference.

This application contains a sequence listing as an ASCII text file identified by the file name “Revised Sequence Listing P1100834.txt” created on Apr. 19, 2022 and having a size of 76,058 bytes which is incorporated herein by reference

The present disclosure relates to the field of biotechnologies and medicine, in particular to a tumor biomarker, application (use) and a tumor detection kit.

Lung cancer is currently the tumor with the highest mortality and incidence in the world, and over 85% is non-small cell lung cancer (NSCLC). Due to limited diagnostic methods, Most NSCLC patients are diagnosed at an advanced stage when first diagnosed, thereby missing the opportunity for surgery. Traditional chemotherapy has limited effect in the treatment of NSCLC, and the median survival is often less than one year after diagnosis. In recent years, researches on the molecular mechanism of lung cancer have made great progress, which can achieve satisfactory therapeutic effect on NSCLC by determining the molecular type of the lung cancer, and selecting a targeted drug adapted to target. Gene fusion is a chimeric gene consisting of two or more genes whose coding regions are connected end to end and placed under the control of the same set of regulatory sequences (including promoters, an enhancers, ribosome binding sequences, terminator, etc.). The echinoderm microtubule associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion gene has been found in NSCLC. The EML4-ALK fusion gene is a cancer-promoting gene mutation, accounting for 4-5% of the incidence of NSCLC. EML4-ALK causes abnormal expression of tyrosine kinase, causing malignant transformation of cells. In clinical NSCLC patients with EML4-ALK fusion gene positive, the use of crizotinib can significantly prolong the overall survival of these patients. The incidence of SL34A2-ROS1 fusion gene in NSCLC is approximately 1.0%-3.4%, and the incidence in EGFR/KRAS/ALK—negative individuals is 5.7%, the main pathological type is adenocarcinoma. The SL34A2-ROS1 genes lose extracellular domain, when fusion occurs, and retain transmembrane and intracellular tyrosine kinase domain, with main fusion sites in exons 32, 34, 35, and 36 of ROS1 gene. ROS1 receptor tyrosine kinase is involved in the activation of multiple downstream signal transduction pathways, including RAS-MAPK/ERK, PI3K/AKT/mTOR, JAK/STAT3, PLC/IP3 and SHP2/VAV3 pathways, thereby regulating of tumor cell growth, proliferation, cell cycle, differentiation, metastasis and migration. The ROS1 and ALK gene have 49% homology in the sequences of tyrosine kinase domain, the ATP binding site in the kinase catalytic domain has a homology of 77%, so the ALK inhibitor crizotinib has shown significant efficacy in the treatment of ROS1 fusion gene positive NSCLC. The ROS1 fusion gene provides a new treatment options for individualized treatment of lung cancer, and it is of important significance for clinical practice to determine the positive rate of the ROS1 fusion gene in lung adenocarcinoma, understand clinical pathological characteristics of positive patients and deduce the vulnerable population.

Researches have find that fusion mechanism of EML4 and ALK is that paracentric inversion on the short arm of chromosome 2, which in turn generates a fusion gene, and usually the fusion between different exons of EML4 and exon 20 of ALK generates different fusion types. Types of EML4-ALK fusion gene include E13-A20, E6a/b-A20, E20-A20, E15-A20, E14-A20, E18-A20, E2-A20, E17-A20, etc. The common types are E13-A20 (V1) and E6alb-A20 (V3a/b). The EML4 and ALK gene fusion generates oncogenic activity by activating of ALK kinase. Currently, crizotinib, a targeted drug for fusion protein EML4-ALK, has been approved by the US Food and Drug Administration, and is listed in the guideline as first-line drug for patients with positive ALK rearrangement (NCCN version 3. 2017). Meanwhile, the second-generation ALK inhibitors ceritinib and alectinib have been launched, and approved by the US Food and Drug Administration for use in crizotinib-resistant ALK positive NSCLC. With the successful development and clinical use of drugs targeting ALKand ROS1 positivity, the following question is what technical approach to screening patients with explicit gene fusion mutations? At present, the methods conventionally used are as follows: 1. Fluorescence in situ hybridization (FISH) technology, which is gold standard for detecting fusion genes at present, but the inability to distinguish between different variants and the high cost, which limits the wide application of this method. 2. Reverse transcription PCR (RT-PCR), which has the characteristics of rapid diagnosis and high sensitivity, but it requires high quality of RNA in samples, and there are blind spot for the detection of variation types, which makes clinical popularization difficult; 3. Immunohistochemistry (IHC), which determines the presence or absence of fusion by detecting expression of a specific antigen, characterized by low cost and relative simplicity, but relatively low detection sensitivity, and it is also incapability of distinguishing variation types of fusion. In addition, all of the above methods require a large amount of high-quality clinical tissue samples, which are often difficult to obtain. Therefore, there is an urgent clinical need to establish a simple and convenient method for the detection of EML4-ALK and SLC34A2-ROS1 fusion genes with sensitivity and specificity.

Circular RNA (circRNA), a kind of endogenous non-coding RNA, possesses the potential of disease marker, which has the characteristics of conservativeness, richness and tissue specificity. Studies have find that the circRNA has correlation with tumors, and is enriched in blood exosomes. However, the traditional clinical tumor biomarkers, such as CEA, which is common in gastrointestinal tumors, have the problem of poor tissue specificity; tissue biopsy and immunohistochemistry involve invasive procedures, and often require multiple times of puncturing so as to obtain a definite pathological result. Therefore, circRNA can act as a novel biomarker for tumors.

The object of the present disclosure includes, but is not limited to, providing a tumor biomarker, which is circRNA having a base sequence as represented by any one of SEQ ID NOS. 1-6.

Another object of the present disclosure includes, but is not limited to, providing use of the above biomarkers.

Another object of the present disclosure includes, but is not limited to, providing a tumor detection kit.

The present disclosure is realized as follows:

A tumor biomarker, which is a circRNA selected from the group consisting of SEQ ID NOS. 1-6.

It should be noted that sequences represented by SEQ ID NOS. 1-6 are linear forms of the circRNA.

Use of a primers or a probes for detecting the above biomarkers in the preparation of tumor detection kits.

Further, in some embodiments of the present disclose, the tumor is lung cancer.

Further, in some embodiments of the present disclose, the lung cancer is NSCLC.

Further, in some embodiments of the present disclosure, the NSCLC is associated with the fusion of echinoderm microtubule associated protein-Like 4 (EML4) gene and Anaplastic lymphoma kinase (ALK) gene (EML4-ALK), or the NSCLC is associated with the fusion of solute carried family 34 member 2 gene and ROS proto-oncogene 1, receptor tyrosine kinase gene (SLC34A2-ROS1).

In the above, SEQ ID NOS. 1-4 are markers for fusion of two genes, EML4 and ALK, by fusion types V3a and V3b, and SEQ ID NOS. 5-6 are markers for the fusion of two genes of SLC34A2 and ROS1.

Further, in some embodiments of the present disclose, the primer is selected from the group consisting of SEQ ID NOS. 7-371.

Further, in some embodiments of the present disclosure, the primer includes: any one selected from the group consisting of SEQ ID NOS. 7-58 and any one selected from the group consisting of SEQ ID NOS. 59-110; alternatively, the primer includes: any one selected from the group consisting of SEQ ID NOS.111-242 and any one selected from the group consisting of SEQ ID NOS. 243-371.

It should be noted that, the person skilled in the art, on the premise that the circRNA shown in SEQ ID NOS. 1-6 is disclosed in this disclosure, it could quite easily select any one of the primers from SEQ ID NOS. 7-58 and any one of the primers from SEQ ID NOS. 59-110, and combine them for detecting the circRNA represented by SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 2, SEQ ID NO. 4; accordingly, it would be easy for those skilled in the art to select any one of the primers from SEQ ID NOS. 111-242 and any one of the primers from SEQ ID NOS. 243-371, and combine them for detecting the circRNA as represented by SEQ ID NO. 5 or 6.

Further, in some embodiments of the present disclosure, the primer includes a combination of SEQ ID NO. 7 and SEQ ID NO. 59 or the primer includes a combination of SEQ ID NO. 8 and SEQ ID NO. 60.

Further, in some embodiments of the present disclosure, the primer includes a combination of SEQ ID NO. 113 and SEQ ID NO. 243, or the primer includes a combination of SEQ ID NO. 114 and SEQ ID NO. 243.

Further, in some embodiments of the present disclosure, the primer includes a combination of SEQ ID NO. 111 and SEQ ID NO. 243, or the primer includes a combination of SEQ ID NO. 112 and SEQ ID NO. 243.

A tumor detection kit, which includes a reagent for detecting a circRNA in a sample, wherein the circRNA has a base sequence as represented by any one of SEQ ID NOS. 1-6.

Further, in some embodiments of the present disclosure, the reagent is a primer, and the primer comprises any one selected from SEQ ID NOS. 7-58 and any one selected from SEQ ID NOS. 59-110; alternatively, the primer comprises any one selected from SEQ ID NOS. 111-242 and any one selected from SEQ ID NOS. 243-371.

In the above, a combination of any one of SEQ ID NOS. 7-58 and any one of SEQ ID NOS. 59-110 can detect the circRNA represented by SEQ ID NOS. 1-4, and a combination of any one of SEQ ID NOS. 111-242 and any one of SEQ ID NOS. 243-371 can detect the rcRNA represented by SEQ ID NOS. 5-6.

Further, in some embodiments of the present disclosure, the above reagent is a probe.

Further, in some embodiments of the present disclosure, the above mentioned sample is blood or a blood product prepared therefrom, such as plasma.

Further, in some embodiments of the present disclose, the tumor is lung cancer.

Further, in some embodiments of the present disclose, the lung cancer is non-small cell lung cancer.

Further, in some embodiments of the present disclosure, the non-small cell lung cancer is associated with fusion of echinoderm microtubule associated protein-like4 gene and anaplastic lymphoma kinase gene (EML4-ALK), or the non-small cell lung cancer is associated with fusion of solute carrier family 34 member 2 and ROS1 proto-oncogene 1, receptor tyrosine kinase gene (SLC34A2-ROS1).

In the above, SEQ ID NOS. 1-4 are markers for fusion of two genes, EML4 and ALK, by fusion types V3a and V3b, and SEQ ID NOS. 5-6 are markers for the fusion of two genes of SLC34A2 and ROS1.

Further, in some embodiments of the present disclose, the reagent is a primer, and the primer is selected from the group consisting of SEQ ID NOS. 7-371.

An auxiliary diagnosis method for tumor, which includes: detecting whether a biological sample derived from a subject has a circRNA having a base sequence as represented by any one of SEQ ID NOS. 1-6.

If a detection result indicates that the circRNA represented by any one of SEQ ID NOS. 1-6 exists in the biological sample, it means that the subject with a high probability of having tumor (e.g., lung cancer, for example, NSCLC).

Further, in some embodiments of the present disclosure, the method comprises detecting EML4-ALK fusion gene, comprising: detecting whether a biological sample derived from a subject has a circular RNA having a base sequence as represented by any one of SEQ ID NOS. 1-4.

Further, in some embodiments of the present disclosure, the method comprises detecting SL34A2-ROS1 fusion gene, comprising: detecting whether a biological sample derived from a subject has a circular RNA having a base sequence as represented by SEQ ID NO. 5 or 6.

Further, in some embodiments of the present disclosure, the subject is a human.

Further, in some embodiments of the present disclosure, the biological sample is blood.

Further, in some embodiments of the present disclosure, the tumor is lung cancer.

Further, in some embodiments of the present disclosure, the lung cancer is NSCLC.

A method for detecting EML4-ALK fusion gene, which includes: detecting whether a biological sample derived from a subject has a circRNA having a base sequence as represented by any one of SEQ ID NOS. 1-4.

If the detection result indicates that the circRNA represented by any one of SEQ ID NOS. 1-4 exists in the biological sample, it shows that EML4 gene and ALK gene of the subject undergo fusion.

A method for detecting SL34A2-ROS1 fusion gene, which includes: detecting whether a biological sample derived from a subject has a circRNA having a base sequence as represented by SEQ ID NO. 5 or 6.

If a detection result indicates that the circRNA represented by SEQ ID NO. 5 or 6 exists in the biological sample, it shows that SL34A2 gene and ROS1 gene of the subject undergo fusion.

In order to more clearly illustrate the technical solutions of examples of the present disclosure, accompanying drawings which need to be used in the examples will be introduced below briefly, and it should be understood that the accompanying drawings below merely show some examples of the present disclosure, and therefore should not be considered as limitation to the scope, and a person ordinarily skilled in the art still could obtain other relevant accompanying drawings according to these accompanying drawings, without any inventive effort.

FIG. 1 shows a detection result of circRNA in plasma of patients with EML4-ALKV3a/3b fusion gene positive in Example 1 of the present disclosure (SEQ ID NO. 372 shown in panel B);

FIG. 2 shows a detection result of circRNA in plasma of patients with SLC34A2-ROS1 fusion gene positive in Example 2 of the present disclosure (SEQ ID NO. 373 shown in panel B; SEQ ID NO. 374 shown in panel D);

FIG. 3 shows a PCR result of determining the type of SLC34A2-ROS1 fusion gene by means of combining primers of SEQ ID NO. 111/SEQ ID NO. 243 and SEQ ID NO. 243/SEQ ID NO. 112 in Example 3 of the present disclosure;

FIG. 4 is a structural schematic diagram of positions and structures of the primers SEQ ID NO. 243, SEQ ID NO. 111 and SEQ ID NO. 112 corresponding to different fusion types of SLC34A2 and ROS1 in Example 3 of the present disclosure.

In order to make the objects, technical solutions and advantages of the examples of the present disclosure clearer, the technical solutions in the examples of the present disclosure will be described below clearly and completely. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by manufacturers. If manufacturers of reagents or apparatuses used are not specified, they are conventional products commercially available.

The features and performances of the present disclosure are further described below in detail in combination with examples.

the circRNA with the base sequence as represented by any one of SEQ ID NOS. 1-6 disclosed in the present disclosure can be used as a biomarker for tumor detection or tumor auxiliary diagnosis, or used as a marker to detect whether the EML4-ALK fusion gene or the SLC34A2-ROS1 fusion gene occurs, and the purpose of rapid and convenient detection can be realized using this marker.