A library construction method for detecting uterine inflammatory myofibroblastic tumor alk fusion based on RNA single-sided primer

Abstract

This invention discloses a library construction method for detecting ALK fusions in inflammatory myofibroblastic tumors of the uterus based on RNA one-sided primers, belonging to the biomedical field. The invention first utilizes a reverse transcriptase with terminal nucleotidyltransferase activity, specific capture primers, and template-converting oligonucleotides to introduce a universal anchoring sequence at the 3' end of the first strand of cDNA during synthesis. Subsequently, using single-tube one-step PCR, multiple downstream specific primers targeting exon 17-20 of the ALK gene, one upstream universal primer complementary to the anchoring sequence, and primer pairs carrying Illumina sequencing adapters are used to simultaneously complete target sequence enrichment and sequencing adapter introduction, ultimately preparing a high-throughput sequencing library. This method uses RNA samples and can comprehensively detect both known and unknown ALK fusions, offering advantages such as low RNA input, high sensitivity, multiplex detection capability, and rapid workflow.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for constructing a library for detecting ALK fusion in inflammatory myofibroblastoma of the uterus based on RNA one-sided primers. Background Technology

[0002] Uterine inflammatory myofibroblastic tumor (UIMT) is a rare, low-grade malignant mesenchymal tumor, a specific manifestation of inflammatory myofibroblastic tumor (IMT) in the uterus. The proportion of UIMT among uterine mesenchymal tumors is unclear, but recent studies suggest it may be underestimated. Studies indicate that the actual proportion of UIMT in cases preoperatively diagnosed as "uterine leiomyomas" is approximately 0.1%; however, this proportion can rise to 10% in "leiomyomas" removed during pregnancy; and in "smooth muscle tumors of uncertain malignant potential" (STUMP), the proportion of UIMT can reach 14%. Therefore, accurate identification of UIMT is of great clinical significance in the differential diagnosis of mesenchymal tumors of the uterus, but its preoperative diagnosis faces multiple challenges: 1) Lack of specificity in imaging: Ultrasound, MRI or CT examinations can usually only indicate uterine space-occupying lesions and cannot distinguish UIMT from other mesenchymal tumors of the uterus; 2) Overlapping histological morphology: UIMT can present morphological features similar to those of various tumors such as uterine leiomyosarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, and perivascular epithelioid cell tumor of the uterus.

[0003] Against this backdrop, the detection of ALK gene fusions is of great significance in the detection of UIMT, mainly in terms of its diagnostic and treatment guidance value. 1) Diagnostic biomarker: Due to the extensive histological overlap between UIMT and other uterine mesenchymal tumors, the detection of ALK gene status becomes a core basis for differential diagnosis. The incidence of ALK fusions in UIMT ranges from 50% to 96.3% (most literature reports above 90%), making it a specific biomarker for UIMT, while ALK rearrangements are rare in uterine leiomyosarcoma and endometrial stromal sarcoma. Therefore, for morphologically atypical uterine mesenchymal tumors, ALK detection results can directly guide the diagnostic direction. 2) Targeted therapy: Multiple case reports have confirmed that UIMT patients carrying ALK fusions show significant treatment responses to ALK-TKIs (such as crizotinib, alectinib, etc.). Approximately 25% of UIMT patients may experience local recurrence or distant metastasis, and these patients have limited sensitivity to conventional chemotherapy. ALK-TKIs provide an effective treatment option for these patients.

[0004] Currently, various methods are available for detecting ALK gene fusions, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), and next-generation sequencing (NGS). FISH is considered one of the "gold standards" for detecting gene rearrangements, using specific probes to label the regions flanking the ALK gene breakpoint. Its limitations include low throughput and high cost, inability to identify unknown fusion partners, and stringent requirements for sample quality. IHC offers advantages such as low cost, high speed, and widespread availability of technology platforms. However, IHC methods have significant drawbacks: insufficient specificity, inability to identify fusion partners, and difficulties in standardizing interpretation. While RT-PCR has high sensitivity, it can only detect known, pre-designed specific fusion variants. ALK fusion partners in UIMT exhibit high diversity (common ones include IGFBP5, THBS1, TIMP3, RANBP2, RRBP1, etc., as well as some less common ones such as TNS1, FN1, DCTN1, LBH, NRP2, PPP1CB, SEC31, SYN3, TNC, etc.). NGS is currently the most comprehensive detection method, divided into DNA sequencing and RNA sequencing. However, DNA-based NGS cannot directly confirm whether rearrangement events were successfully transcribed into functional fusion mRNA at the RNA level. RNA-based NGS can directly detect fusion transcripts and is considered the detection method with the highest functional relevance. It can directly capture breakpoints, identify fusion partners, and is unaffected by large intron regions. However, RNA is extremely prone to degradation in FFPE samples and its stability is far inferior to DNA, resulting in a higher detection failure rate and extremely stringent requirements for the quality of the RNA sample. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a library construction method for detecting ALK gene fusions in UIMT using one-sided primer fusion detection technology based on high-throughput sequencing. This method uses RNA samples to achieve single-tube PCR library construction for the detection of unknown ALK fusions, and can comprehensively cover all known and unknown ALK fusion types with common ALK breakpoints on introns in a single operation. Furthermore, it has advantages such as low RNA input, high sensitivity, multiplex detection capability, and rapid workflow, which has significant clinical significance and application value for the accurate diagnosis and targeted therapy of UIMT.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a primer composition for detecting ALK gene fusions in inflammatory myofibroblastic tumors of the uterus, the primer composition comprising: (i) Template-converting oligonucleotides, the sequences of which are shown in SEQ ID NO.1; (ii) A specific capture primer targeting the ALK gene exon 17-20 region, the sequence of which is at least one of SEQ ID NO.2-SEQ ID NO.5; (iii) A downstream gene-specific primer for the ALK gene, the sequence of which is at least one of SEQ ID NO.6-SEQ ID NO.9; (iv) The upstream universal primer, the sequence of which is shown in SEQ ID NO.10.

[0007] Furthermore, the last guanosine of the template-converting oligonucleotide is modified with locked nucleic acid, and the second to last and third to last guanosines are riboguanosine.

[0008] Furthermore, the primer composition further includes a capture primer for an internal control gene and a downstream gene-specific primer for the internal control gene; the internal control gene is at least one of LMNA, LRP1, MYC, and PUM1.

[0009] Furthermore, the capture primer sequence of the internal control gene is at least one of SEQ ID NO.11-SEQ ID NO.14, and the downstream gene-specific primer sequence of the internal control gene is at least one of SEQ ID NO.15-SEQ ID NO.18.

[0010] The second objective of this invention is to provide a library construction method for detecting ALK fusion in inflammatory myofibroblastic tumors of the uterus based on RNA one-sided primers, wherein the library construction method is performed using the primer composition.

[0011] Furthermore, the library construction method includes the following steps: (a) Using the specific capture primer, the template conversion oligonucleotide, and a reverse transcriptase with terminal nucleotide transferase activity, the RNA template is reverse transcribed to synthesize first-strand cDNA; (b) Using the downstream gene-specific primers and the upstream universal primers, perform a first round of PCR amplification on the cDNA template obtained in step (a) to obtain the first amplification product; (c) Using a pair of primers carrying complete sequencing adapters, perform a second round of PCR amplification on the first amplification product obtained in step (b) to obtain the library amplification product.

[0012] Furthermore, the primers carrying the complete sequencing adapter include a forward primer and a reverse primer; the sequence of the forward primer is shown in SEQ ID NO.19, and the sequence of the reverse primer is shown in SEQ ID NO.20, where NNNNNNN are all index sequences.

[0013] Furthermore, the reverse transcription reaction solution in step (a) contains 5× reverse transcription buffer, dNTPs, DTT, RNase inhibitor and the reverse transcriptase; the PCR amplification in steps (b) and (c) is performed continuously in a single reaction tube.

[0014] A third objective of this invention is to provide the application of the primer composition or the library construction method in the preparation of products for detecting ALK gene fusions in inflammatory myofibroblastic tumors of the uterus.

[0015] The fourth objective of this invention is to provide a product for detecting ALK gene fusions in inflammatory myofibroblastic tumors of the uterus, the product comprising a kit containing the primer composition.

[0016] The core advantage of this invention lies in establishing a highly efficient, accurate, and economical detection scheme for unknown ALK gene fusions at the RNA level, which has the following beneficial effects compared with existing technologies: 1) Unique and efficient technical principle: Utilizing the template conversion properties of reverse transcriptase with TdT activity, a uniform anchoring sequence is added to the 3' end of cDNA during synthesis. This design cleverly transforms the problem of capturing unknown sequences into the amplification of known adapters, thereby achieving efficient acquisition of fusion partner genes. 2) Detection results are closer to biological reality: Detecting fusion at the RNA level directly identifies successfully expressed fusion mRNA, avoiding false positives that may occur with DNA-level detection, resulting in more intuitive and accurate results. 3) Flexible and economical workflow: The library preparation process is greatly simplified (one-step single-tube PCR, library preparation time ≤ 3 hours), saving time and costs. The targeting range of the detection panel can be freely customized, offering strong scalability and being friendly to precious samples, ensuring a high success rate even with low initial RNA amounts. 4) Easy to add or remove the target detection range, making panel settings more flexible. 5) Precise and sensitive detection: Relying on a next-generation sequencing platform and combined with specific primer amplification, high sensitivity and high specificity can be synergistically optimized. 6) It provides efficient and reliable technical support for the accurate differential diagnosis of uterine inflammatory myofibroblastic tumor (UIMT) and the decision-making of ALK targeted therapy. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the library construction principle in this invention.

[0018] Figure 2 The image shows the quality control results of five sample library fragments from this invention. The main peaks of the five samples were located between 180-500 bp, as expected, and primer dimers were not present.

[0019] Figure 3 This is a diagram showing the fusion result of the S9 sample IGFBP5 E1-ALK E18 IGV in this invention.

[0020] Figure 4 This is a diagram showing the fusion result of the S9 sample IGFBP5 E1-ALK E17 IGV in this invention.

[0021] Figure 5 This is a diagram showing the fusion result of sample S10, FN1 E32-ALK E19 IGV, in this invention.

[0022] Figure 6 This is a diagram showing the fusion result of sample S11 THBS1 E7-ALKE19 IGV in this invention. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0024] This invention aims to address the shortcomings of existing technologies for detecting ALK gene fusions in patients with uterine inflammatory myofibroblastic tumors (UIMTs). To enable single-tube PCR library construction for detecting unknown ALK gene fusions, this invention provides a one-step rapid library construction method based on RNA one-sided primers for detecting ALK gene fusions in uterine inflammatory myofibroblastic tumors.

[0025] The principle of the library construction method of this invention is to first perform reverse transcription of RNA using specific capture primers, template-changing oligonucleotides (TSO) with specific adapters, and reverse transcriptase with terminal nucleotide transferase (TdT) activity. This step not only synthesizes the first strand of cDNA, but also precisely introduces a universal anchoring sequence at the 3' end of the cDNA through the template-changing mechanism.

[0026] A one-step PCR amplification process is then performed: multiple downstream gene-specific primers (GSPs) complementary to the target region and carrying partial adapter sequences, along with one upstream universal primer complementary to the template transition adapter, are used to precisely amplify the cDNA template, achieving efficient enrichment of the target sequence. Next, a pair of primers carrying complete Illumina sequencing adapters are introduced to amplify the enriched product, thus completing the introduction of the sequencing adapter and preparing the final library suitable for high-throughput sequencing. The entire sequencing library construction process requires only one round of PCR.

[0027] This technology combines specific one-sided primer reverse transcription with the use of highly specific 5×RingCap Mix enzyme. By guiding reverse transcription with specific one-sided primers, the target RNA region is efficiently converted into cDNA. High-throughput sequencing libraries of the target sequence in RNA are then constructed on a standard PCR platform, enabling accurate and rapid detection of all known and unknown fusion types of common ALK breakpoints in introns. The ALK primers designed in this invention target exon 17-20, covering over 90% of known ALK fusion breakpoints in UIMT. For rare upstream breakpoints, this method can rapidly expand the detection range by supplementing with specific primers for the corresponding exons.

[0028] Table 1. Details of the primer sequences designed and used in this invention.

[0029] In the Ill-iXX-P5 and Ill-iXX-P7 sequences, "NNNNNNN" represents an index. This index can be replaced with the corresponding index sequence, and the primer names should be changed to the corresponding numbers, such as Ill-i01-P5 and Ill-i01-P7. The last G base in ill-TSO-LNA1 is modified with locked nucleic acid (LNA), and the second-to-last and third-to-last G bases are riboguanosine (rG). LMNA, LRP1, MYC, and PUM1 are internally controlled genes.

[0030] The implementation of this invention includes the following steps: 1. Using the optimized reverse transcription reaction solution (refer to Tables 2 and 3), reverse transcribe the target sequence using the RNA sample as a template according to the procedure in Table 4 to obtain cDNA products.

[0031] Table 2 Basic Formulation of Reverse Transcription Reaction Solution

[0032] Table 3. MIX formulations for capturing primers

[0033] Table 4 Reverse transcription reaction procedure

[0034] 2. The reverse transcription product was purified using magnetic beads to obtain purified cDNA.

[0035] 3. Using the optimized library construction reaction solution (refer to Tables 5 and 6), the purified cDNA sample was used as a template to amplify the sequencing library according to the procedure in Table 7.

[0036] Table 5 Basic Formulation of Reaction Solution for One-Step Library Construction

[0037] Table 6 Primer MIX Formulation

[0038] Table 7 Enrichment reaction amplification procedure

[0039] 4. Purify the PCR product with magnetic beads to obtain the sample library.

[0040] 5. Use the library for the next step of high-throughput sequencing instrument detection to obtain the target sequence information, i.e., the FASTQ file.

[0041] 6. The FASTQ files underwent the following bioinformatics analysis: 1) Data preprocessing: The FASTQ file was processed using the fastp software to remove adapter sequences, low-quality bases, and low-quality sequences.

[0042] 2) Sequence alignment: The preprocessed FASTQ file was aligned to the human hg19 (GRCh37) reference genome using STAR software to generate a bam file.

[0043] 3) Quality control statistics: Statistics were compiled on indicators such as Q30, alignment rate, primer efficacy, and number of qualified internal control genes.

[0044] 4) Fusion Analysis: The STAR-Fusion software was used to analyze and obtain the breakpoint locations of the fusion, and annotation filtering was performed to finally obtain the fusion result.

[0045] The method described above for constructing a library for detecting ALK fusions in inflammatory myofibroblastic tumors of the uterus based on RNA one-sided primers includes primer design and optimization of the experimental procedure. The library uses Illumina platform adapters as an example; however, different sequencing adapter sequences can be used to achieve compatibility with different sequencing platforms, including BGI Genomics and Ion Torrent, depending on the primer design approach.

[0046] The technical solution of the present invention (including the invention content and advantages) will be further described in detail below with reference to the embodiments.

[0047] Example 1 Sequencing library quality control is crucial for ensuring the accuracy of sequencing results and is divided into pre-sequencing library quality control and post-sequencing data quality control. Pre-sequencing quality control includes library concentration and fragment quality control; post-sequencing library data quality control includes indicators such as Q30, alignment rate, validity rate, and the number of qualified internal control genes. To verify that our library preparation method has good quality control data, RNA from five extracted FFPE samples was analyzed, and the library concentration, fragment size, and sequencing quality control data results were statistically analyzed.

[0048] The above method includes the following steps: 1. Using the optimized reverse transcription reaction solution (refer to Tables 2 and 3), amplify the target gene sequence according to the procedure in Table 4 using the RNA sample as a template to obtain the reverse transcription product.

[0049] 2. The reverse transcription product was purified using magnetic beads to obtain purified cDNA.

[0050] 3. Using the optimized library construction reaction solution (refer to Tables 5 and 6), amplify the PCR products according to the procedure in Table 7 using the cDNA sample as a template.

[0051] 4. Purify the PCR product with magnetic beads to obtain the sample library.

[0052] 5. Use the library for the next step of high-throughput sequencing instrument detection to obtain the target sequence information, i.e., the FASTQ file.

[0053] 5. The FASTQ files were processed using bioinformatics analysis to obtain the final results.

[0054] Quality control of library fragments constructed using the technique of this invention for 5 FFPE samples is as follows: Figure 2 As shown in Table 8, the quality control statistics for sequencing libraries and sequencing data are presented. Figure 2 Capillary electrophoresis fragment quality control of the libraries showed that the main peaks of the five samples were between 180-500 bp, consistent with the expected library fragment sizes. Furthermore, the libraries did not exhibit small fragment peaks due to primer dimers, indicating the rationality of the primer design. Table 8 shows the quality control statistics for sequencing libraries and sequencing data, indicating that the library concentrations were all greater than 2 ng / μL, meeting the requirements for library loading. In the quality control of the sequencing data, the alignment rate, Q30, validity rate, and number of qualified internal control genes all showed excellent performance.

[0055] Table 8. Quality control statistics of sequencing libraries and sequencing data from 5 samples.

[0056] The quality control results of the five sample libraries and sequencing data show that the library construction technology of this invention performs well in library quality control and sequencing data quality control, and can provide quality control assurance for ALK gene fusion detection.

[0057] Example 2 To verify the feasibility of using the single-sided primer of this invention to detect unknown ALK gene fusions, an RNA pseudovirus containing two ALK fusions (fusion forms: IGFBP5 E1-ALK E19, THBS1 E4-ALK E19) was synthesized. RNA was extracted and fragmented from the pseudovirus, and library construction and detection were performed, following the procedures outlined in Example 1.

[0058] The pseudovirus was tested at its original concentration and diluted 5-fold and 25-fold. The quality control of sequencing libraries and sequencing data for different pseudovirus gradients is shown in Table 9. The sequencing quality control of samples with different pseudovirus gradients (different fusion frequencies) was satisfactory, and the target fusion sequence was detected in all samples, indicating that the library construction technology of this invention can effectively detect ALK gene fusions.

[0059] Table 9. Quality control statistics of pseudovirus sequencing libraries and sequencing data

[0060] Example 3 To verify the accuracy of the library construction technology of this invention in detecting ALK gene fusions, three clinical FFPE samples with ALK fusions verified by transcriptome sequencing were selected (two of which were initially diagnosed as uterine fibroids, and one was initially diagnosed as submucosal spindle cell tumor of the uterus, which was subsequently confirmed as UIMT by transcriptome sequencing) and three negative clinical FFPE samples verified by transcriptome sequencing were used for library construction and detection. The experimental procedure was as described in Example 1.

[0061] The test results of 6 clinical samples are shown in Table 10 below. The positive concordance rate between this technology for detecting ALK fusion and transcriptome sequencing for detecting ALK fusion was 100% (3 / 3) (see schematic diagram of IGV fusion). Figure 3-6 This embodiment verifies the accuracy of this technology by comparing it with transcriptome sequencing results, showing a 100% positive and 100% negative concordance rate.

[0062] Table 10. Quality control statistics of clinical sample sequencing libraries and sequencing data

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer composition for detecting ALK gene fusion in uterine inflammatory myofibroblastic tumor, characterized by, include: (i) Template-converting oligonucleotides, the sequences of which are shown in SEQ ID NO.1; (ii) A specific capture primer targeting the ALK gene exon 17-20 region, the sequence of which is at least one of SEQ ID NO.2-SEQ ID NO.5; (iii) A downstream gene-specific primer for the ALK gene, the sequence of which is at least one of SEQ ID NO.6-SEQ ID NO.9; (iv) The upstream universal primer, the sequence of which is shown in SEQ ID NO.

10.

2. The primer composition of claim 1, wherein The last guanosine in the template-converting oligonucleotide is a locked nucleic acid modified, and the second to last and third to last guanosines are riboguanosines.

3. The primer composition according to claim 2, characterized in that, The primer composition further includes a capture primer for an internal control gene and a downstream gene-specific primer for the internal control gene; the internal control gene is at least one of LMNA, LRP1, MYC, and PUM1.

4. The primer composition according to claim 3, characterized in that, The capture primer sequence of the internal control gene is at least one of SEQ ID NO.11-SEQ ID NO.14, and the downstream gene-specific primer sequence of the internal control gene is at least one of SEQ ID NO.15-SEQ ID NO.

18.

5. A method for constructing a library for detecting uterine inflammatory myofibroblastic tumor ALK fusion based on RNA single-sided primer, characterized in that, It is carried out using the primer composition according to any one of claims 1-4.

6. The library construction method of claim 5, wherein, The library construction method includes the following steps: (a) Using the specific capture primer, the template conversion oligonucleotide, and a reverse transcriptase with terminal nucleotide transferase activity, the RNA template is reverse transcribed to synthesize first-strand cDNA; (b) Using the downstream gene-specific primers and the upstream universal primers, perform a first round of PCR amplification on the cDNA template obtained in step (a) to obtain the first amplification product; (c) Using a pair of primers carrying complete sequencing adapters, perform a second round of PCR amplification on the first amplification product obtained in step (b) to obtain the library amplification product.

7. The library construction method of claim 6, wherein, The primers carrying complete sequencing adapters include forward primers and reverse primers; the sequence of the forward primer is shown in SEQ ID NO.19, and the sequence of the reverse primer is shown in SEQ ID NO.20, where NNNNNNN are all index sequences.

8. The library construction method of claim 6, wherein, The reverse transcription reaction solution in step (a) contains 5× reverse transcription buffer, dNTPs, DTT, RNase inhibitor and the reverse transcriptase; the PCR amplification in steps (b) and (c) is performed continuously in a single reaction tube.

9. The use of the primer composition according to any one of claims 1-4 or the library construction method according to any one of claims 5-8 in the preparation of a product for detecting ALK gene fusions in inflammatory myofibroblastic tumors of the uterus.

10. A product for detecting ALK gene fusion in uterine inflammatory myofibroblastic tumor, characterized by, The product includes a kit containing the primer composition according to any one of claims 1-4.

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