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Novel FISH assay detects PTEN and tumor suppressor gene deletions with improved accuracy, reducing false positives in cancer diagnostics

Background

Fluorescence in situ hybridization (FISH) is a valuable technique for detecting genetic alterations in cancer samples. However, nuclear truncation artifacts in fixed and preserved samples can lead to false positive results when detecting tumor suppressor gene deletions. Current methods using minimum thresholds for apparent deletion frequency can reduce sensitivity. There is also a need to distinguish between small and large deletions, for example homozygous deletions in phosphatase and tensin homolog (PTEN) may indicate increased metastatic potential. PTEN is a crucial tumor suppressor gene involved in regulating DNA repair, genomic stability, stem cell self-renewal, cellular senescence, and cell migration. Deletions of PTEN are common in various cancers, particularly prostate cancer, and are associated with poor prognosis. Accurate detection of PTEN deletions is clinically important for diagnosis, prognosis, and treatment decisions.

Technology Overview

This invention introduces a novel FISH-based assay for detecting tumor suppressor gene deletions, particularly PTEN, with significantly improved accuracy and sensitivity. The key innovation lies in the strategic design of a four-color probe set that effectively distinguishes true deletions from truncation artifacts, a common source of false positives in traditional FISH methods.

Key features of the technology include:

• Advanced probe design: The assay utilizes a four-color probe set strategically positioned around the PTEN gene:
  • PTEN Test probe (Orange): Hybridizes to the PTEN gene
  • Probe A (Green): Hybridizes to WAPAL/BMPR1A, centromeric to PTEN
  • Probe B (Aqua): Hybridizes to FAS, telomeric to PTEN
  • Centromere 10 probe (Red): Detects chromosome 10 copy number changes
• Boundary zone targeting: Flanking probes are positioned near genomic architecture features such as segmental duplications and copy number variations, optimizing detection accuracy.
• Quantification of artifactual deletions: The method incorporates control samples to establish baseline artifactual deletion frequencies, enhancing result interpretation.
• Size-based deletion discrimination: The assay can distinguish between small and large deletions, providing valuable prognostic information, particularly for prostate cancer.
• Optimization for FFPE samples: The probe set is specifically designed for use with formalin-fixed paraffin-embedded tissues, enabling retrospective studies on archived samples.

This technology significantly reduces false positives from truncation artifacts while maintaining high sensitivity, potentially improving cancer diagnostics and prognostics, particularly in prostate cancer.

Further Details

• Vidotto T, Melo CM, Lautert-Dutra W, Torrezan GT, Scapulatempo-Neto C, Petersen JFO, Reis RMV, Squire JA, Carraro DM, Barros-Filho MC, Reis RM. Pan-cancer genomic analysis shows hemizygous PTEN loss tumors are associated with immune evasion and poor outcome. Scientific Reports 13(1):5049, 2023.
• Vidotto T, Melo CM, Castelli E, Koti M, Dos Reis RB, Squire JA. Emerging role of PTEN loss in evasion of the immune response to tumours. British Journal of Cancer 122(12):1732-1743, 2020.
• Yoshimoto M, Ludkovski O, Good J, Pereira C, Gooding RJ, McGowan-Jordan J, Boag A, Evans A, Tsao MS, Nuin P, Squire JA. Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections. Lab Invest 98(4):403-413, 2018.
• Vidotto T, Tiezzi DG, Squire JA. Distinct subtypes of genomic PTEN deletion size influence the landscape of aneuploidy and outcome in prostate cancer. Mol Cytogenet 11:1, 2018.
• Picanço-Albuquerque, C.G., Morais, C.L., Carvalho, F.L.F. et al. In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading. Virchows Arch 468, 607–617, 2016.
• Yoshimoto M, Ludkovski O, DeGrace D, Williams JL, Evans A, Sircar K, Bismar TA, Nuin P, Squire JA. PTEN genomic deletions that characterize aggressive prostate cancer originate close to segmental duplications. Genes Chromosomes Cancer 51(2):149-60, 2012.
• Squire JA. TMPRSS2-ERG and PTEN loss in prostate cancer. Nature Genetics 41(5):509-10, 2009.
• Yoshimoto M, Cunha IW, Coudry RA, Joshua A, Fonseca FP, Zielenska M, Soares FA, Squire JA. Absence of TMPRSS2:ERG fusions and PTEN losses identifies prostate cancer genomic grade with favorable outcome. Modern Pathology 21(12):1451-6, 2008.
• Yoshimoto M, Cunha IW, Coudry RA, Fonseca FP, Torres CH, Soares FA, Squire JA. FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome. British Journal of Cancer 97(5):678-85, 2007.
• Yoshimoto M, Cutz JC, Nuin PAS, Joshua AM, Bayani J, Evans AJ, Zielenska M, Squire JA. Interphase FISH Analysis of PTEN in Histologic Sections Shows Genomic Deletions are Present in 68% of Primary Prostate Cancer and 23% of High-Grade Prostatic Intra-Epithelial Neoplasia. Cancer Genetics and Cytogenetics 169:128-37, 2006.

Benefits

• Reduced artifactual deletion frequency, especially in fixed and preserved samples
• Improved ability to detect and characterize true tumor suppressor gene deletions
• Can differentiate between hemizygous and homozygous deletions
• Enhanced sensitivity and specificity for cancer diagnostics and prognostics

Applications

• Cancer diagnostics and research applications in cancer biology
• Potential for development into clinical diagnostic assays
• Prostate cancer research and other PTEN-associated cancers
• Detection of tumor suppressor gene deletions (e.g., PTEN, p16, RB1, p53)
• Characterization of deletion size and zygosity
• Analysis of fixed and preserved samples, including FFPE tissues

Opportunity

This technology offers improved accuracy and capabilities for FISH-based tumor suppressor gene deletion assays. Additional licensing opportunities are available for companies and research institutions outside of North America. Potential licensing opportunities may be available for companies in the fields of cancer diagnostics, cytogenetics, or molecular pathology.

Patents

• Granted Canadian Patent CA2696545 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”
• Granted European Patent EP2547809 (validated in France, UK, Germany, Sweden, Italy, Denmark, Netherlands, Switzerland)
• Granted Japanese Patent JP5976550 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”
• Granted Brazilian Patent BR112012022764 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”
• Granted Hong Kong Patent HK1181086 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”
• Granted Chinese Patent CN102892934 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”
• US Patent Application US17/124214 “Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization”

IP Status

• Patented
• Patent application submitted

Seeking

Licensing

Posted/updated

December 6, 2024/March 19, 2025 (Technology Overview)