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 language | English (US) |
|---|---|
| Pages (from-to) | 108-115 |
| Number of pages | 8 |
| Journal | Growth Hormone and IGF Research |
| Volume | 22 |
| Issue number | 3-4 |
| DOIs | |
| State | Published - Jun 2012 |
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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 journal › Article
}
TY - JOUR
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
UR - http://www.scopus.com/inward/record.url?scp=84863774566&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84863774566&partnerID=8YFLogxK
U2 - 10.1016/j.ghir.2012.04.001
DO - 10.1016/j.ghir.2012.04.001
M3 - Article
C2 - 22551578
AN - SCOPUS:84863774566
VL - 22
SP - 108
EP - 115
JO - Endocrinology and Metabolism, Supplement
JF - Endocrinology and Metabolism, Supplement
SN - 1096-6374
IS - 3-4
ER -