Correlation of Oncotype DX Recurrence Score with the Expression of Insulin Receptor Substrate Proteins in Estrogen Receptor + Breast Cancer

Insulin receptor substrate (IRS) 1 and 2 are downstream signaling proteins that influence breast pathophysiology. IRS-1 promotes carcinoma cell proliferation; whereas IRS-2 regulates cell motility, invasion, and glycolysis. Our lab has shown that distinct cellular localization of IRS-2 also plays a role in carcinoma cell function. Oncotype DX (Genomic Health) (ODX) is a 21-gene expression profile used to classify carcinomas with low, intermediate, and high risk recurrence scores (RS). Our aim is to correlate expression and cellular localization of IRS proteins in breast carcinomas with their ODX RS. 97 breast carcinomas sent for ODX testing from 2006-2009 were collected and grouped according to their RS (low, intermediate or high). Immunohistochemistry for IRS-1/-2 was performed. Specific criteria were used to evaluate IRS staining patterns. Follow-up data, ranging from 3-6 years, was available. Statistical analysis was performed to correlate staining patterns of IRS-1/-2 with the three RS groups. IRS-1 staining, predominantly nuclear, did not significantly correlate with RS (P=.5645). IRS-2 expression patterns did show statistical significance amongst the three RS groups (P=.0371). Tumors with intermediate and low RS were more likely to exhibit punctate and diffuse cytoplasmic expression of IRS-2, and cell membrane expression was uncommon in this group. Expression and cellular localization of IRS proteins play an important role in breast cancer cell biology, and expression patterns for IRS-2 do demonstrate a significant correlation with ODX RS. Further studies are required to elucidate the significance of cellular localization of IRS-1/-2 proteins in breast carcinoma cells and their relationship to ODX scores. DOI : 10.14302/issn.2572-3030.jcgb-13-369 Corresponding author : Ashraf Khan M.D. , Department of Pathology, University of Massachusetts Medical School Biotech Three, One Innovation Drive, Worcester, MA 01605 Telephone: 508-793-6142; Fax: 508-793-6110; E-mail: ashraf.khan@umassmemorial.org Running Title: Oncotype DX Recurrence Score and IRS Expression Patterns


Introduction :
The insulin receptor substrate (IRS) adaptor proteins (IRS-1 and IRS-2) are expressed relatively ubiquitously in human tissues and in many cancers. 1 They act as signaling intermediates, downstream to multiple cytokine and growth factor receptors which are involved in the regulation of breast carcinoma cell function, including the insulin receptor (IR) and insulinlike growth factor receptors (IGF-1R). 1-8IRS-1 and IRS-2 are expressed in normal mammary epithelium, as well as in benign and malignant breast lesions. 3Despite considerable sequence and structural homology, IRS-1 and IRS-2 have been shown to serve divergent functions. 2[11][12] A recent study from our lab examined IRS-1 and IRS-2 expression patterns in approximately 300 invasive breast tumors. 13Though most tumors exhibited nuclear expression of IRS-1 consistent with previous studies, expression of IRS-2 was variable; exhibiting diffuse cytoplasmic, punctate cytoplasmic, and cell membrane staining patterns. 13Multivariate analysis revealed a statistically significant decrease in overall survival associated with IRS-2 expression at the cell membrane. 13The progesterone receptor (PR) negative subset with cell membrane expression demonstrated significantly worse overall survival compared to all other subtypes. 13Though larger studies with longer follow-up are needed, we concluded that IRS-2 expression patterns in breast tumors, particularly in the setting of PR negativity, could be helpful in determining prognosis and guiding treatment. 13mor grade, receptor expression, nodal status,  (ER) positive breast carcinomas; a subset for which the test has been well-validated. 14,15Studies have shown that patients with high RS tend to benefit more from chemotherapy. 15This was validated in a retrospective analysis using data from the National Surgical Adjuvant Breast and Bowel Project Clinical Trial Study B14 and B20. 14,15Oncotype DX testing is also discussed in the For many patients, follow-up data on recurrence-free survival, overall survival (OS), metastases, and therapy were also available.

Immunohistochemistry
Immunohistochemical studies were performed on 5-um sections of formalin-fixed, paraffin-embedded tissue.Antigen retrieval was carried out with 0.01M citrate buffer at pH 6.0 for slides to be stained with IRS- The sections were counterstained with hematoxylin, dehydrated, and coverslipped with permanent media.
We used the following histologic criteria for IRS-1 and IRS-2 staining patterns which are outlined in a previous study. 13For IRS-1, a nuclear pattern was defined as diffuse nuclear staining (FIGURE 1).IRS-2 staining patterns were defined using the following criteria: Diffuse cytoplasmic staining was defined as evenly distributed cytoplasmic reactivity (FIGURE 2); punctate cytoplasmic staining was defined as clearly demarcated puncta of staining within the cytoplasm (FIGURE 3); and membrane staining was defined as focal or diffuse membranous staining (FIGURE 4).

Sections of normal pancreas and normal breast tissue
were used for positive and negative controls.The pathologists assessing staining patterns were blinded to the tumor recurrence score and all other data at the time of assessment.

Imaging
Light microscopy was performed using a Nikon Eclipse E400 microscope (Nikon Inc., Melville, NY).
Photomicrographs were obtained using a SPOT Insight Color Model 3.2.0microscope camera with SPOT Basic 5.1 imaging software (SPOT Imaging Solutions, Diagnostic Instruments Inc., Sterling Heights, MI).

Statistical Analysis
Statistical correlation for IRS-1 and recurrence score was done using contingency analysis and t-test; and correlation for IRS-2 and recurrence score was done using contingency analysis and one-way analysis of variance (ANOVA).The REMARK criteria were used for this study. 20 The very low number of patients with significant followup precluded the achievement of statistical significance.

Discussion :
We present the first report of IRS expression patterns in relation to Oncotype DX in ER+ early breast cancer.IRS-1 expression, which was predominantly nuclear when present, did not correlate to RS.However, we did find a significant association between IRS-2 expression patterns and RS.Consistent with a previous study from our group demonstrating a poor prognosis in the setting of IRS-2 expression at the cell membrane, the membrane pattern was least likely to be seen in the low RS group. 13Unfortunately, due to a very low number of cases with high RS in our study, it is not possible to determine if the high RS group is more likely to demonstrate membrane expression, as expected based on our prior study. 13We conclude that IRS-2 expression patterns used in conjunction with Oncotype  4 Her2 score: Her2 evaluated using ASCO-CAP guidelines.FISH was performed on 24 cases, all of which were negative for over-expression.

Oncotype DX RS, no. (%)
Low (0-  It is not surprising that IRS-1 expression was not a significant factor in this study population.2][23][24][25][26] The high rates of IRS-1 negativity in this study are not surprising either, given a previous study showing highly variable nuclear and cytoplasmic expression of IRS-1 in invasive ductal carcinoma. 27This suggests that the level of IRS-1 expression may fall on a spectrum, and further studies might further elucidate how expression levels, rather than cellular localization, correlate with disease progression, perhaps in the setting of intermediate RS where overall prognosis is unclear.
While a prior study demonstrated a role for membrane expression of IRS-2 in disease progression, this expression pattern was relatively rare in our study. 13ere are two likely explanations for this finding.First, this study focused on cases with ER positivity, the population for which Oncotype DX is used in clinical practice, and ER activity upregulates PR expression. 28r previous study demonstrated that membrane expression of IRS-2 was statistically more likely in the setting of PR negativity, and so it is unlikely to occur in a predominantly hormone receptor positive study population. 13Second, there were relatively few high RS cases in this study, which based on our previous study, is the subgroup expected to have the highest rates of IRS-2 expression at the cell membrane. 13e high rate of punctate IRS-2 expression in our study was also unexpected.Previous work demonstrated that there was no significant relationship between the punctate expression pattern, hormone receptor status, or clinical outcomes. 13Electron microscopy could be used to determine the specific subcellular localization responsible for this expression pattern, and a larger study might explore this expression pattern in the setting of intermediate RS where it could potentially have more prognostic value than in the population as a whole.The diffuse pattern was also found more frequently than expected, likely due to the high rate of hormone receptor positivity in this study, as this pattern is more likely to be seen in hormone receptor positive tumors with a relatively better prognosis. 13rk from our group and others has shown a role for IRS-2 in breast carcinoma cell motility and invasion, and clinical stage are currently used to determine prognosis and management of patients with breast cancer.However, genomic assays have become more widely used and accepted in recent years.One such assay is Oncotype DX (Genomic Health, Redwood City, CA), a 21-gene expression profile which assesses 5 groups of 16 cancer-related genes compared to 5 reference genes.It calculates a recurrence score (RS) from a complex mathematical formula based on the expression of genes from these groups [RS= 0.47x (HER2 Group score) -0.34x(ER Group score) + 1.04x

1
and 0.001M EDTA at pH 8.0 for slides to be stained with IRS-2.The slides were stained on the Dako Autostainer (Dako Corporation, Carpinteria, CA) using EnVision+ (Dako) staining reagents.After an application of Dual Endogenous Block (Dako), followed by a buffer wash, the sections were incubated with rabbit polyclonal IRS-1 (cat.# sc-559, Santa Cruz, Santa Cruz CA) at a concentration of 1:400 and rabbit monoclonal IRS-2 (cat.# EP976Y, Epitomics, Burlingame CA) at a concentration of 1:400.Following a buffer wash, sections were then incubated with the EnVision+ Dual Link (Dako) detection reagent linked to horseradish peroxidase.The sections were then washed and treated with a solution of diaminobenzidine and hydrogen peroxide (DAB, Dako).After rinsing, a toning solution (DAB Enhancer, Dako) was used to enrich the final color.

TABLE 2 :
341][32][33]S-2 correlation with Oncotype DX Recurrence Scores (RS) both markers of aggressive disease.2,8,11,29,30Similarly,severalgenesincluded in the Oncotype DX assay have also been associated with invasiveness and tumor progression, including CD68, stromolysin 3, and cathepsin L2.[31][32][33]IRS-2 expression in certain subtypes of breast cancer might have prognostic value as well.In combination with Oncotype DX recurrence scores, we would expect even higher statistical power to predict recurrence for individual patients.For example, in a study using both Oncotype DX scores and Adjuvant!Online scores, the combination was more predictive of actual outcomes than either score alone.34ConclusionInsummary, we have shown for the first time that IRS-2 expression patterns in ER+ breast carcinoma correlate with Oncotype DX recurrence scores.In particular, membrane expression thought to be associated with worse outcome was least likely to be observed in the low RS group.Larger studies utilizing more cases with high RS and longer follow-up times are required to determine how IRS-2 expression might be used in conjunction with Oncotype DX recurrence scores to better predict outcomes and guide appropriate treatment.It might also give beneficial prognostic information in clinical settings where Oncotype DX testing is unavailable.