Quantifying insulin receptor isoform expression in FFPE breast tumors

Sean C. Harrington, Saravut (John) Weroha, Carol Reynolds, Vera Jean Suman, Wilma L. Lingle, Paul Haluska

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Background: The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER). +. and ER. - breast cancer tumors. Design: FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n = 83) and ER. - (n = 64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n = 61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. Results: All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER. - and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p < 0.05, HR ER+ p < 0.0005) and both groups had greater InsR-A expression when compared to ER. - tumors (ER+ p < 0.0005, HR ER+ p < 0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p < 0.0005) but higher than InsR-B (p < 0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p < 0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p < 0.0005). Conclusions: The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.

Original languageEnglish (US)
Pages (from-to)108-115
Number of pages8
JournalGrowth Hormone and IGF Research
Volume22
Issue number3-4
DOIs
StatePublished - Jun 2012

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Insulin Receptor
Paraffin
Formaldehyde
Protein Isoforms
Estrogen Receptors
Breast Neoplasms
Neoplasms
Biomarkers

Keywords

  • Breast cancer
  • Monoclonal antibodies therapeutic use
  • Protein kinase inhibitors
  • QPCR
  • Real-time
  • Receptor IGF Type 1
  • Receptor insulin

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Quantifying insulin receptor isoform expression in FFPE breast tumors. / Harrington, Sean C.; Weroha, Saravut (John); Reynolds, Carol; Suman, Vera Jean; Lingle, Wilma L.; Haluska, Paul.

In: Growth Hormone and IGF Research, Vol. 22, No. 3-4, 06.2012, p. 108-115.

Research output: Contribution to journalArticle

Harrington, Sean C. ; Weroha, Saravut (John) ; Reynolds, Carol ; Suman, Vera Jean ; Lingle, Wilma L. ; Haluska, Paul. / Quantifying insulin receptor isoform expression in FFPE breast tumors. In: Growth Hormone and IGF Research. 2012 ; Vol. 22, No. 3-4. pp. 108-115.
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abstract = "Background: The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER). +. and ER. - breast cancer tumors. Design: FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n = 83) and ER. - (n = 64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n = 61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. Results: All qPCR assays were highly reproducible with amplification efficiencies between 96-104{\%} over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90{\%} of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88{\%} of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82{\%} ER+, 68{\%} ER. - and 100{\%} HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p < 0.05, HR ER+ p < 0.0005) and both groups had greater InsR-A expression when compared to ER. - tumors (ER+ p < 0.0005, HR ER+ p < 0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p < 0.0005) but higher than InsR-B (p < 0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p < 0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p < 0.0005). Conclusions: The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.",
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T1 - Quantifying insulin receptor isoform expression in FFPE breast tumors

AU - Harrington, Sean C.

AU - Weroha, Saravut (John)

AU - Reynolds, Carol

AU - Suman, Vera Jean

AU - Lingle, Wilma L.

AU - Haluska, Paul

PY - 2012/6

Y1 - 2012/6

N2 - Background: The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER). +. and ER. - breast cancer tumors. Design: FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n = 83) and ER. - (n = 64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n = 61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. Results: All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER. - and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p < 0.05, HR ER+ p < 0.0005) and both groups had greater InsR-A expression when compared to ER. - tumors (ER+ p < 0.0005, HR ER+ p < 0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p < 0.0005) but higher than InsR-B (p < 0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p < 0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p < 0.0005). Conclusions: The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.

AB - Background: The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER). +. and ER. - breast cancer tumors. Design: FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n = 83) and ER. - (n = 64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n = 61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. Results: All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER. - and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p < 0.05, HR ER+ p < 0.0005) and both groups had greater InsR-A expression when compared to ER. - tumors (ER+ p < 0.0005, HR ER+ p < 0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p < 0.0005) but higher than InsR-B (p < 0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p < 0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p < 0.0005). Conclusions: The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.

KW - Breast cancer

KW - Monoclonal antibodies therapeutic use

KW - Protein kinase inhibitors

KW - QPCR

KW - Real-time

KW - Receptor IGF Type 1

KW - Receptor insulin

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