Hamid Masoudi

Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

Drugs that target the insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (IR) are currently under investigation for a variety of malignancies including breast cancer. Although we have previously reported that IGF-IR expression in primary breast tumors is common, the activation status of this receptor has not been examined in relation to survival. Phosphorylated IGF-IR/IR (P-IGF-IR/IR) and its downstream signaling partner phospho-S6 (P-S6) were evaluated immunohistochemically in tumor tissue microarrays representing 438 cases of invasive breast cancer. P-IGF-IR/IR (n = 114; P = 0.046) and total levels of IR (n = 122; P = 0.009) were indicative of poor survival, whereas total IGF-IR (n = 112; P = 0.304) was not. P-IGF-IR/IR and P-S6 were coordinately expressed in primary breast tumors (likelihood ratio, 11.57; P = 6.70 x 10(-4)). Importantly, P-IGF-IR/IR was detected in all breast cancer subtypes (luminal, 48.1%; triple negative, 41.9%; and HER2, 64.3%). In vitro, the IGF-IR/IR inhibitor BMS-536924 decreased phospho-RSK and P-S6, and significantly suppressed the growth of breast cancer cell lines MCF-7, SUM149, and AU565 representing the luminal, triple negative, and HER2 subtypes, respectively, in monolayer and soft agar. BMS-536924 also inhibited growth in tamoxifen resistant MCF-7 Tam-R cells while having little effect on immortalized normal breast epithelial cells. Thus, we can determine which patients have the activated receptor and provide evidence that P-IGF-IR/IR is a prognostic factor for breast cancer. Beyond this, P-IGF-IR/IR could be a predictive marker for response to IGF-IR and/or IR-targeted therapies, as these inhibitors may be of benefit in all breast cancer subtypes including those with acquired resistance to tamoxifen.

Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors.

Redefining prognostic factors for breast cancer: YB-1 is a stronger predictor of relapse and disease-specific survival than estrogen receptor or HER-2 across all tumor subtypes

IntroductionGene expression analysis is used to subtype breast cancers such that the most aggressive tumors are identified, but translating this into clinical practice can be cumbersome. Our goal is to develop a universal biomarker that distinguishes patients at high risk across all breast cancer subtypes. We previously reported that Y-box binding protein-1 (YB-1), a transcription/translation factor, was a marker of poor prognosis in a cohort of 490 patients with breast cancer, but the study was not large enough to subtype the cancers. We therefore investigated whether YB-1 identifies patients at risk for either reduced relapse free survival or decreased r breast cancer specific survival (BCSS) across all tumor subtypes by evaluating 4,049 cases.MethodsTumor tissue microarrays, representing 4,049 cases of invasive breast cancers with 20 years of follow up, were subtyped by the expression profiles of estrogen receptor, progesterone receptor, or HER-2. We then addressed whether YB-1 expression identified patients at higher risk for relapse and/or lower BCSS.ResultsWe found YB-1 to be a highly predictive biomarker of relapse (P < 2.5 × 10-20) and poor survival (P < 7.3 × 10-26) in the entire cohort and across all breast cancer subtypes. Patients with node-positive or node-negative cancer were more likely to die from the disease if YB-1 was expressed. This was further substantiated using a Cox regression model, which revealed that it was significantly associated with relapse and poor survival in a subtype independent manner (relapse patients, hazard ratio = 1.28, P < 8 × 10-3; all patients, hazard ratio = 1.45, P < 6.7 × 10-7). Moreover, YB-1 was superior to estrogen receptor and HER-2 as a prognostic marker for relapse and survival. For a subset of patients who were originally considered low risk and were therefore not given chemotherapy, YB-1 was indicative of poor survival (P < 7.1 × 10 -17). Likewise, YB-1 was predictive of decreased BCSS in tamoxifen-treated patients (P = 0.001); in this setting a Cox regression model once again demonstrated it to be an independent biomarker indicating poor survival (hazard ratio = 1.70, P = 0.022).ConclusionsExpression of YB-1 universally identifies patients at high risk across all breast cancer subtypes and in situations where more aggressive treatment may be needed. We therefore propose that YB-1 may re-define high-risk breast cancer and thereby create opportunities for individualized therapy.

Molecular Phenotyping of Thyroid Tumors Identifies a Marker Panel for Differentiated Thyroid Cancer Diagnosis

BackgroundCurrently, a large proportion of individuals undergo thyroidectomy as a diagnostic procedure for cancer. The objective of this work was to evaluate the molecular phenotype of differentiated thyroid cancer (DTC) and benign thyroid lesions to identify molecular markers that allow for accurate thyroid cancer diagnosis.MethodsTissue microarrays consisting of 100 benign and 105 malignant thyroid lesions, plus 24 lymph node samples, were stained for a panel of 57 molecular markers. Significant associations between marker staining and tumor pathology (DTC versus benign) were determined using contingency table and Mann-Whitney U (MU) tests. A Random Forests classifier algorithm was also used to identify useful/important molecular classifiers.ResultsOf the 57 diagnostic markers evaluated 35 (61%) were significantly associated with a DTC diagnosis after multiple testing correction. Of these, in DTC compared with benign thyroid tumors, 8 markers were downregulated and 27 upregulated. The most significant markers for DTC diagnosis were: Galectin-3, Cytokeratin 19, Vascular Endothelial Growth Factor, Androgen Receptor, p16, Aurora-A, and HBME-1. Using the entire molecular marker panel, a Random Forests algorithm was able to classify tumors as DTC or benign with an estimated sensitivity of 87.9%, specificity of 94.0%, and an accuracy of 91.0%.ConclusionEvaluation of the DTC and benign thyroid tumor molecular phenotype has allowed for identification of a marker panel, composed of both established and novel markers, useful for thyroid cancer diagnosis. These results suggest that further study of the molecular profile of thyroid tumors is warranted, and a diagnostic molecular marker panel may potentially improve patient selection for thyroid surgery.

HER-3 overexpression is prognostic of reduced breast cancer survival: a study of 4046 patients.

Introduction:Advances in molecular biology have led to the identification of potential markers of prognostic and therapeutic importance in human cancers. HER-2 testing and targeted therapy now represents a critical cornerstone in the management of breast cancer. The objectives of the current study were to determine the frequency and prognostic significance of HER-3 over-expression and HER-4 over-expression by invasive breast cancer. Methods:Tissue microarrays were constructed using clinically annotated formalin-fixed and paraffin-embedded tumor samples from 4046 patients diagnosed with invasive breast carcinoma with a median 12.5 years of follow-up. Type 1 growth factor receptor family members HER-1, HER-2, HER-3, and HER-4 expression levels were determined by immunohistochemistry, and HER-2 status was further resolved by fluorescent in-situ hybridization. The study cohort was randomly divided and analyzed as a core data set and a validation data set. Results:HER-3 over-expression was identified in 10.0% of tumors and was a significant marker of reduced patient breast cancer-specific survival on univariate analysis (P = 1.32 × 10−5). Furthermore, in tumors with normal expression levels of HER-1 and HER-2, the overexpression of HER-3 had a significant negative prognostic effect on disease-specific survival (HR: 1.541, 95% CI: 1.166–2.036, P = 2.37 × 10−3) independent of patient age at diagnosis, Estrogen receptor status, tumor grade, tumor size, nodal status, and the presence of lymphatic or vascular invasion by cancer. HER-4 overexpression was identified in 78.2% of breast cancers and was not a significant marker of patient survival (P = 0.214). Results of all statistical tests were positively confirmed in the validation data set analysis. Conclusions:HER-3 status is an important prognostic marker of disease-specific survival in patients with invasive breast cancer. Accordingly, evaluation of the HER-3 expression level may identify a subset of patients with a poor disease prognosis, and who could undergo further evaluation for the efficacy of HER-3 targeted anticancer agents.

Cell cycle regulators show diagnostic and prognostic utility for differentiated thyroid cancer.

BackgroundDifferentiated thyroid cancer (DTC) generally has a favorable outcome, but some patients develop local recurrence and/or distant metastases and ultimately die of their disease. Molecular markers that accurately predict tumor behavior are lacking. This study’s aim was to ascertain the role of cell cycle regulators in predicting malignant histology and tumor behavior in DTC.MethodsTissue microarrays consisting of 100 benign and 105 malignant thyroid lesions, plus 24 lymph node samples, were stained for p16, p21, p27, p53, p57, p63, cyclin D1, cyclin E, and mdm2. Statistical analysis was used to compare the expression of the markers in benign versus DTC lesions and correlate their expression with clinicopathologic characteristics.Resultsp16, p21, cyclin D1, and cyclin E showed significantly (P < .001) increased expression in DTCs compared with benign thyroid lesions (54.7% vs. 5%, 71.7% vs. 38%, 87.1% vs. 45.7%, and 72.3% vs. 37.4%, respectively). There was no significant difference in expression between benign lesions and DTC for the remaining markers. p16 expression correlated significantly with extrathyroidal tumor extension (P = .02) and the presence of cancer in lymph nodes (P = .03). A total of 73% vs. 45% of the cancers of patients with and without lymph node involvement, respectively, stained positive for p16 (P = .01).ConclusionsThere is a statistically significant difference in the expression of p16, p21, cyclin D1, and cyclin E between DTCs and benign thyroid lesions, and p16 expression correlates with clinicopathologic variables predicting poor outcomes for DTC. These results suggest that evaluation of cell cycle derangement in thyroid tumors may serve as a useful tool for both DTC diagnosis and prognosis.

HLA-DR protein status predicts survival in patients with diffuse large B-cell lymphoma treated on the MACOP-B chemotherapy regimen

Loss of major histocompatibility class II (MHC class II) molecules on diffuse large B-cell lymphoma (DLBCL) has been associated with poor survival; however, none of these reports analysed a uniformly treated patient cohort. This study was designed to validate one MHC class II antigen, HLA-DR, as a prognostic marker in patients uniformly treated with the MACOP-B regimen. Immunostaining results were correlated with the international prognostic index (IPI) score and overall survival (OS). Of the 97 cases, 82 had interpretable staining. Of these, 52 expressed HLA-DR (median OS, 16.2 years) while 30 were negative (median OS, 4.2 years, P = 0.037). The IPI was also predictive of OS in the study group (P = 0.023). A Cox multivariate model established both IPI (P = 0.031) and HLA-DR (P = 0.04) as independent predictors of OS. This is the first demonstration of the prognostic relevance of HLA-DR in a uniformly treated DLBCL patient group.

β-Catenin (CTNNB1) gene amplification: A new mechanism of protein overexpression in cancer

β‐Catenin nuclear translocation is frequently observed in different types of malignancies, including gastric cancer. In gastric cancer, however, the molecular mechanisms leading to accumulation of this protein in the nucleus remain unknown. In this setting, β‐catenin (CTNNB1) mutations have been reported, but studies of mutation frequency have yielded conflicting results. Mutations or silencing of other partners of β‐catenin (i.e., APC and AXIN) are also considered rare genetic events in gastric tumorigenesis. Gene amplification is a common mechanism of activation and/or overexpression of oncogenes in gastric and other cancers. In this study, we investigated whether gene amplification is a possible mechanism of β‐catenin activation in gastric cancer by determining its presence in 49 patients with gastric cancer and two gastric‐derived cell lines (KATO III and ST2957). Using fluorescence in situ hybridization, we identified β‐catenin amplification in one of the tumor samples as well as in KATO III cells. β‐Catenin immunostaining revealed nuclear translocation of the protein in both cases. In the KATO III cells, β‐catenin overexpression was confirmed by quantitative real‐time PCR and Western blot analyses and β‐catenin gene amplification by Southern blot analysis and multiplex ligation probe amplification. In the KATO III cell line, no correlation was found between β‐catenin nuclear translocation and increased expression of the WNT1 target gene CCND1 (cyclin D1). Our data suggest that gene amplification is a possible mechanism of β‐catenin overexpression in cancer.

Clinical utility of type 1 growth factor receptor expression in colon cancer.

BACKGROUND To evaluate the expression pattern and prognostic significance of the type 1 growth factor receptor (T1GFR) family in colon carcinoma. METHODS Tissue microarrays were constructed using 127 tumor samples and 47 metastatic lymph nodes and T1GFR family expression was determined by immunohistochemistry. Univariate and multivariate analyses examined clinicopathologic variables for prognostic significance, and the correlation between primary and lymph node expression was determined by Spearman correlation. RESULTS Overexpression of HER-1, HER-2, HER-3, and HER-4 in tumor samples was 32%, 1%, 12%, and 37%, respectively, and 30%, 0%, 11%, and 24% in nodal samples, respectively. On multivariate analysis, positive margins, lymphatic invasion, and HER-3 expression were significant predictors of survival outcome. There was significant correlation between tumor and regional lymph node expression for the T1GFR family members. Tumor HER-3 expression was associated with lymphatic invasion and distant recurrence. CONCLUSIONS Tumor HER-3 expression has prognostic utility in individuals with colon carcinoma. Correlation between tumor and lymph node expression of T1GFR family members suggests that tumor receptor status may guide targeted therapy selection.

Evaluation of type 1 growth factor receptor family expression in benign and malignant thyroid lesions.

BACKGROUND The aim of this study was to evaluate the diagnostic and prognostic utility of the type 1 growth factor receptor family in the management of differentiated thyroid cancer (DTC). METHODS Tissue microarrays consisting of 100 benign thyroid lesions and 105 malignant thyroid lesions stained for HER1, HER2, HER3, and HER4 were evaluated. RESULTS HER1, HER2, HER3, and HER4 were expressed in 76%, 2%, 57%, and 73% of DTC cases, respectively. HER1 and HER3 showed significantly increased expression, and HER4 showed significantly decreased expression, in DTC compared with benign thyroid lesions. HER3 expression correlated with the presence of lymph node metastasis, tumor type, and higher N stage; the expression of HER4 correlated with lower T stage. A classifier targeting benign versus malignant status with all 4 markers as potential predictors displayed an accuracy, sensitivity, and specificity of 66.8%, 63.5%, and 70.0%, respectively. CONCLUSIONS For DTC, HER1, HER3, and HER4 have diagnostic and prognostic utility, and warrant further study as targets for cancer treatment.