Abstract B25: Emerging role of heterogeneous ribonucleoproteins (hnRNPs) as early predictive marker and prognosticator for head and neck oral squamous cell carcinoma.
Authors: Tripathi Satyendra C. , Kumar Manish , Kaur Jatinder , Chauhan Shyam S. , Shukla Nootan K. , Thakkar Alok , Duggal Ritu , Gupta Siddhartha D. , Ralhan Ranju , Siu KW Michael 
Published: June 2012

Introduction: Recently using iTRAQ-tagging and multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified a panel of proteins differentially expressed in head and neck/oral squamous cell carcinoma (HNOSCC) as compared to the non-malignant tissues. Heterogeneous ribonucleoprotein K (hnRNPK) and Heterogeneous ribonucleoprotein D (hnRNPD) are among few of the proteins identified in this panel. These are nuclear protein belonging to the RNA binding protein family. Here, we report the verification of these proteins in an independent set of clinical samples by immunohistochemistry, and investigate their potential as a prognostic biomarker for HNOSCC. The present study also explores their clinical relevance and potential as a biomarker in identification of oral lesions in early stages of malignancy.

Experimental Design: Two hundered HNOSCCs, 100 leukoplakias and 100 non-malignant archived tissues were retrieved from the research tissue bank to determine the expression of hnRNPK using immunohistochemistry (IHC). The IHC data were subjected to statistical analyses using the SPSS 15.0 software (Chicago). Receiver operating characteristic (ROC) analysis was used to determine the sensitivity and specificity as a biomarker. The expression in oral lesions was further validated by immunoblotting and RT-PCR analyses in the same tissue and serum samples as used for IHC.

Results: hnRNPK and hnRNPD expression was found to be significantly increased from normal mucosa to leukoplakia with or without dysplasia to HNOSCC (ptrend&lt;0.001). Abberant cytoplasmic expression of hnRNPK was also observed in HNOSCCs. ROC curve showed high potential of these proteins as a biomarker for HNOSCC. Cytoplasmic hnRNPK overexpression was significantly associated with dedifferentiation of tumors whereas nuclear hnRNP D was significantly associated with tumor size.

In univariate analysis, nuclear as well as cytoplasmic localization of hnRNPK and nuclear hnRNPD were found to be associated with poor survival. There was no correlation between hnRNPK and hnRNPD expression, however, significant reduced disease free survival was obtained for the patients harboring hnRNP D+hnRNP K (p = 0.005; median survival = 11 months) as compared with median disease-free survival of 55 months in the patients showing varied hnRNP D+hnRNP K expression. Cox regression model confirmed that this combination can be a better prognosticator for HNOSCC patients as revealed by multivariate analysis (p=0.013; HR = 2.2, 95% CI = 1.2–4.1). RTPCR and western blotting confirmed our IHC results. hnRNPK was also detected in serum samples of HNOSCC patients.

Conclusion: This is the first large scale study that suggests overexpression of hnRNPK and hnRNPD as an early event in development of HNOSCC. Furthermore, their subcellular localization suggests that they may be associated with increased risk of transformation of oral premalignant lesions and recurrence in HNOSCC. As detected in biological fluid, the potential of hnRNPK as a biomarker should be verified on a large scale. The enhanced performance of the combination of hnRNP D and hnRNP K versus either protein individually, in prognosticating the clinical outcome of HNOSCCs needs to be further explored for its implementation in patient care.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B25.