Differential Expression of the Insulin-Like Growth Factor Receptor among Early Breast Cancer Subtypes

<div><p>Introduction</p><p>We sought to determine the level of protein expression of the critical components of the insulin-like growth factor receptor (IGFR) pathway and to evaluate their prognostic significance across the different early breast cancer subtypes.</p><p>Patients and Methods</p><p>Archival tumor tissue from 1,021 women with early, node positive breast cancer, who were prospectively evaluated within two randomized clinical trials, was used to construct tissue microarrays that were stained for hormone receptors (HR), Ki67, HER2, epidermal growth factor receptor (EGFR) and cytokeratins 5/6, to classify tumors into five immunophenotypical subgroups. Immunohistochemical (IHC) expression of IGF1R-alpha and beta subunits, IGF2R and IGF-binding protein 2 (IGFBP2) was assessed using the immunoreactive score (IRS). Repeated internal cross-validation was performed to examine the statistical validity of the cut off points for all biomarkers.</p><p>Results</p><p>After a median follow-up time of 105.4 months, overall 370 women (36.2%) had relapsed and 270 (26.4%) had died. Tumors expressing IGF1R-alpha above the median IRS were significantly more frequently HR positive (luminal A+B+HER2), as compared to HER2-enriched and triple negative ones (p<0.001 for both comparisons). IGF2R was overexpressed significantly more frequently in HR negative tumors (p = 0.001) and had an inverse correlation with all other biomarkers. Patients with luminal A and B tumors with high IGF1R-alpha and negative EGFR expression (N = 190) had significantly higher 4-year survival rates, as compared to the rest (log-rank p = 0.046), as did patients with luminal A and B tumors with high IGF1R-alpha and low IGF2R expression, as compared to the rest (N = 91), (log-rank p = 0.035). After adjustment for significant variables, patients in the latter group had a relative 45% reduction in the risk of death, as compared to the rest (p = 0.035).</p><p>Conclusion</p><p>Aberrant expression of components of the IGF1R pathway is associated with better clinical outcomes in women with luminal A and B, node positive, early breast cancer.</p></div

Kaplan - Meier Curves for the IGF1R- alpha and IGF2R combination variable- DFS (patients of luminal A and luminal B subtype) in panel A.

<p>B: Kaplan- Meier Curves for the IGF1R- alpha and IGF2R combination variable- OS (patients of luminal A and luminal B subtype).</p

Protein expression detected by IHC from invasive breast carcinoma cases.

<p>(<b>A</b>) IGF1R-alpha moderate to strong membraneous staining in carcinoma cells; (<b>B</b>) IGF1R-alpha cytoplasmic staining; (<b>C</b>) IGF1R-beta intense cytoplasmic and membraneous staining; (<b>D</b>) IGF1R-beta cytoplasmic expression in neoplastic population; (<b>E</b>) IGF2R, predominantly granular cytoplasmic staining; (<b>F</b>) IGF2R expression limited to non-neoplastic epithelial remnants; (<b>G</b>) IGFBP2 moderate to intense cytoplasmic staining in neoplastic cells; (<b>H</b>) IGFBP2 cytoplasmic and dot-like pattern of staining. Scale Bar: 10 µm.</p

Clinicopathological characteristics of the whole patient cohort and by immunophenotypical subtypes.

<p>Note: The p-values correspond to Kruskal-Wallis test for age and Chi-square test for the other categorical variables. MRM: Modified radical mastectomy.</p

REMARK flow chart.

<p>REMARK flow chart.</p

Multivariate model for the IGF1R-alpha and IGF2R combined variable adjusted for clinical parameters (patients of luminal A and B subtype only).

<p>Multivariate model for the IGF1R-alpha and IGF2R combined variable adjusted for clinical parameters (patients of luminal A and B subtype only).</p

Selected patient and tumor characteristics according to breast cancer subtypes defined by immunohistochemistry.

*<p>BCP  = 75.9% of TNBC.</p><p>BCP, basal core phenotype; HT, hormonal therapy; MRM, modified radical mastectomy; N, number; RT, radiotherapy; TNBC, triple-negative breast cancer.</p>1<p>p = 0.004, ²p = 0.013, ³p<0.001, ⁴p<0.001,⁵p = 0.030, ⁶p = 0.031,⁷p<0.001, ⁸p<0.001, ⁹p<0.001/</p><p>Patients were classified as: luminal A (ER<i>-positive</i> and/or PgR-positive, <i>HER2-negative, Ki67^low</i>); luminal B (ER<i>-positive</i> and/or PgR-positive, <i>HER2-negative, Ki67^high</i>); luminal-HER2 (ER<i>-positive</i> and/or PgR-positive, HER2-positive); HER2-enriched (ER-negative, PgR-negative, HER2-positive); triple-negative (TNBC) (ER-negative, PgR-negative, HER2-negative); and basal core phenotype (BCP) (TNBC, CK5-positive and/or EGFR-positive).</p

Proteins, source and dilution of antibodies, staining procedures and patterns and interpretation analysis.

<p>DAB: 3,3′-Diaminobenzidine; C: Cytoplasmic; EGFR: epidermal growth factor receptor; ENZ: Proteolytic enzyme; ER1: Citric acid, pH 6.0;</p><p>ER2: ethylenediaminetetraacetate, pH 8.8; HP: Hot plate; HRP: Horseradish peroxidase; M: Membranous; N: Nuclear; SQ: semi-quantitative.</p><p>(1): Leica Biosystems, Newcastle, UK; (2): Dako, Glostrup, Denmark; (3): Invitrogen, Carlsbad, CA.</p>1<p>Scores 1+, 2+ and 3+ were considered to be positive; ²Score 3+ in >30% of tumor cells was considered to be positive.</p>*<p>Double IHC was performed for the following antibodies: CK5/p53, CK14/Ki67 and CK17/Vimentin in the TMAs of the HE10/97 trial.</p