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CA-3068122-C - TARGET-ENRICHED MULTIPLEXED PARALLEL ANALYSIS FOR ASSESSMENT OF TUMOR BIOMARKERS

CA3068122CCA 3068122 CCA3068122 CCA 3068122CCA-3068122-C

Abstract

The invention provides methods for assessment of tumor biomarkers in biological samples using target-enriched multiplexed parallel analysis. The methods of the invention utilize TArget Capture Sequences (TACS) to thereby enrich for target sequences of interest, followed by massive parallel sequencing and statistical analysis of the enriched population. The methods can be used with DNA samples from a patient, such as a tissue biopsy or plasma sample (liquid biopsy), for detection of the presence of tumor biomarkers, e.g., for purposes of diagnosis, screening, therapy selection and/or treatment monitoring. Kits for carrying out the methods of the invention are also provided.

Inventors

  • George KOUMBARIS
  • Marios Ioannides
  • Elena KYPRI
  • Acilleas Achilleos
  • Petros MINA
  • Alexia Eliades
  • Charalambos Loizides
  • Philippos Patsalis

Assignees

  • NIPD GENETICS PUBLIC COMPANY LIMITED

Dates

Publication Date
20260505
Application Date
20180706
Priority Date
20170707

Claims (20)

  1. 71 What is claimed is: 1. A method of detecting one or more tumor biomarkers in a DNA sample from a subject having or suspected of having a tumor, the method comprising: (a) preparing a sequencing library from the DNA sample; (b) hybridizing the sequencing library to a pool of double-stranded TArget Capture Sequences (TACS) that bind to one or more tumor biomarker sequences of interest, wherein: (i) each member sequence within the pool of TACS is between 100-500 base pairs in length, each member sequence having a 5’ end and a 3’ end; and (ii) the GC content of the pool of TACS is between 19% and 80%, as determined by calculating the GC content of each member within the pool of TACS; (c) isolating members of the sequencing library that bind to the pool of TACS to obtain an enriched library; (d) amplifying and sequencing the enriched library; and (e) performing statistical analysis on the enriched library sequences, optionally utilizing only fragments of a specific size range, to thereby detect the tumor biomarker(s) in the DNA sample.
  2. 2. The method of claim 1, wherein each member sequence binds to the tumor biomarker sequence of interest at least 50 base pairs away, on both the 5’ end and the 3’ end, from regions harboring Copy Number Variations (CNVs), Segmental duplications or repetitive DNA elements.
  3. 3. The method of claim 1 or 2, wherein the pool of TACS comprises a plurality of TACS families each directed to a different tumor biomarker sequence of interest, wherein each TACS family comprises a plurality of member sequences, wherein each member sequence binds to the same tumor biomarker sequence of interest but has different start and/or stop positions with respect to a reference coordinate system for the tumor biomarker sequence of interest.
  4. 4. The method of claim 3, wherein each TACS family comprises at least 3 member sequences.
  5. 5. The method of claim 3 or 4, wherein the pool of TACS comprises at least 5 different TACS families. 72
  6. 6. The method of any one of claims 3 to 5, wherein the start and/or stop positions for the member sequences within a TACS family, with respect to a reference coordinate system for the genomic sequence of interest, are staggered by at least 3 base pairs.
  7. 7. The method of any one of claims 3 to 5, wherein the start and/or stop positions for the member sequences within a TACS family, with respect to a reference coordinate system for the genomic sequence of interest, are staggered by at least 5 base pairs.
  8. 8. The method of any one of claims 1 to 7, wherein each member sequence within the pool of TACS is at least 160 base pairs in length.
  9. 9. The method of any one of claims 1 to 8, wherein the GC content of the TACS is between 19% and 46%.
  10. 10. The method of any one of claims 1 to 9, wherein amplification of the enriched library is performed in the presence of blocking sequences that inhibit amplification of wild-type sequences.
  11. 11. The method of any one of claims 1 to 10, wherein members of the sequencing library that bind to the pool of TACS are partially complementary to the TACS.
  12. 12. The method of any one of claims 1 to 11, wherein the pool of TACS is fixed to a solid support.
  13. 13. The method of any one of claims 1 to 12, wherein the TACS are biotinylated and are bound to streptavidin-coated magnetic beads.
  14. 14. The method of any one of claims 1 to 13, wherein the DNA sample comprises cell free tumor DNA (cftDNA).
  15. 15. The method of any one of claims 1 to 13, wherein the DNA sample is selected from the group consisting of a plasma sample, a urine sample, a sputum sample, a cerebrospinal fluid sample, an ascites sample and a pleural effusion sample from the subject having or suspected of having a tumor. 73
  16. 16. The method of any one of claims 1 to 13, wherein the DNA sample is from a tissue sample from the subject having or suspected of having a tumor.
  17. 17. The method of any one of claims 1 to 16, wherein the statistical analysis comprises a segmentation algorithm.
  18. 18. The method of claim 17, wherein the segmentation algorithm is selected from the group consisting of likelihood-based segmentation, segmentation using small overlapping windows, segmentation using parallel pairwise testing, and combinations thereof.
  19. 19. The method of any one of claims 1 to 16, wherein the statistical analysis comprises a score based classification system.
  20. 20. The method of any one of claims 1 to 16, wherein sequencing of the enriched library provides a read-depth for the genomic sequences of interest and read-depths for reference loci and the statistical analysis comprises applying an algorithm that tests sequentially the read-depth of the loci of the genomic sequences of interest against the read-depth of the reference loci, the algorithm comprising steps for: (a) removal of inadequately sequenced loci; (b) GC-content bias alleviation; and (c) genetic status determination.

Description

TARGET-ENRICHED MULTIPLEXED PARALLEL ANAL VSIS FOR ASSESSMENT OF TUMOR BIOMARKERS Field of the Invention The invention is in the field of biology, medicine and chemistry, more in particular in the field of molecular biology and more in particular in the field of molecular diagnostics. Background of the Invention The identification of tumor biomarkers has been an important advance in the detection, diagnosis and treatment of a wide variety of cancers. Various methods of detecting tumor biomarkers are known in the art; however, additional methods are still needed, in particular methods that allow for detection of tumor biomarkers non-invasively, such as in a plasma sample (liquid biopsy). The identification of hereditary (germline) mutations in patients with cancer or high 15 risk individuals suspected of cancer-predisposing syndrome is a useful clinical tool that enables early medical intervention, prophylactic surgery and close monitoring. These germline mutations can be identified in an individual's healthy tissue (such as buccal swab or lymphocytes). Next generation sequencing (NGS) technologies have been implemented in the development of non-invasive prenatal testing (NIPT). In 2008, two independent groups 20 demonstrated that NIPT of trisomy 21 could be achieved using next generation massively parallel shotgun sequencing (MPSS) (Chiu, R. W. et al.(2008) Proc. Natl. Acad. Sci. USA 105:20458-20463; Fan, H.C. et al. (2008) Proc. Natl. Acad. Sci. USA 105:16266-162710). Large-scale clinical studies using NGS for NIPT have been described (Palomaki, G.E. et al. (2011) Genet. Med. 13:913-920; Ehrich, M. et al. (2011) Am. J. Obstet. Gynecol. 204:205e1-11; Chen, E.Z. et al. (2011) PLoS One 6:e21791; 25 Sehnert, A.J. et al. (2011) Clin. Chem. 57:1042-1049; Palomaki, G.E. et al. (2012); Genet. Med. 14:296-305; Bianchi, D.W. et al. (2012) Obstet. Gynecol. 119:890-901; Zimmerman, B. et al. (2012) Prenat. Diag. 32:1233-1241; Nicolaides, K.H. et al. (2013) Prenat. Diagn. 33:575-579; Sparks, A.B. et al. (2012) Prenat. Diagn. 32:3-9). Initial NIPT approaches used massively parallel shotgun sequencing (MPSS) NGS 30 methodologies (see e.g., US Patent No. 7,888,017; US Patent No. 8,008,018; US Patent No. 8,195,415; US Patent No. 8,296,076; US Patent No. 8,682,594; US Patent Publication 20110201507; US Patent Publication 20120270739). Thus, these approaches are whole genome-based. More recently, targeted-based NGS approaches for NIPT, in which only specific sequences of interest are 1 Date re~ue/Date received 2024-01-23 sequenced, have been developed. For example, a targeted NIPT approach using TArget Capture Sequences (TACS) for identifying fetal chromosomal abnormalities using a maternal blood sample has been described (PCT Publication WO 2016/189388; US Patent Publication 2016/0340733; Koumbaris, G. et al. (2016) Clinical chemistry, 62(6), pp.848-855.). Such targeted approaches 5 require significantly less sequencing than the MPSS approaches, since sequencing is only performed on specific loci on the target sequence of interest rather than across the whole genome. Additional methodologies for NGS-based approaches are still needed, in particular approaches that can target specific sequences of interest, such as for example tumor biomarkers, thereby greatly reducing the amount of sequencing needed as compared to whole genome-based 10 approaches, as well as increasing the read-depth of regions of interest, thus enabling detection of low signal to noise ratio regions. In particular, additional methodologies are still needed that allow for genetic aberrations present in diminutive amounts in a sample to be reliably detected, such as for example in the early detection of cancer. 15 Summary of the Invention This invention provides improved methods for enriching targeted genomic regions of interest to be analyzed by multiplexed parallel sequencing, wherein the enriched sequences are tumor biomarker sequences and the DNA sample used in the method is from a subject having or suspected of having a tumor. Accordingly, the methods allow for detection of tumor biomarkers in 20 a variety of biological samples, including liquid samples, such as plasma samples (liquid biopsy), thereby providing non-invasive means for tumor detection and monitoring. The methods of the invention utilize a pool of TArget Capture Sequences (TACS) designed such that the sequences within the pool have features that optimize the efficiency, specificity and accuracy of genetic assessment of tumor biomarkers. The methods of the invention can be used, for example, in cancer 25 diagnosis, cancer screening, cancer treatment regimen selection and/or cancer therapy monitoring. Accordingly, in one aspect, the invention pertains to a method of detecting one or more tumor biomarkers in a DNA sample from a subject having or suspected of having a tumor, the method comprising: (a) preparing a sequencing library from the DNA sample; (b) hybridizing the sequencing library to a