Sudhir Bahadur

Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry

Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3σ); YWHAZ (14-3-3ζ); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin α (PTHA); l-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

Stromelysin 3, Ets-1, and Vascular Endothelial Growth Factor Expression in Oral Precancerous and Cancerous Lesions: Correlation with Microvessel Density, Progression, and Prognosis

Purpose: Identification of molecular changes characteristic of development and progression of oral cancer are of paramount importance for effective intervention. Stromelysin 3 (MMP11) is a unique matrix metalloproteinase shown to have dual function during cancer progression. The transcription factor Ets-1 and vascular endothelial growth factor (VEGF) are important proangiogenic factors in cancer. This study was designed to test the hypothesis that concomitant expression of stromelysin 3, Ets-1, and/or VEGF affects the development, progression, and prognosis of oral cancer. Patients and Methods: Immunohistochemical analysis of stromelysin 3, Ets-1, VEGF, and platelet/endothelial cell adhesion molecule 1 (a marker for intratumoral microvessel density) was carried out in serial paraffin embedded tissue sections of 220 oral squamous cell carcinomas (OSCC), 90 precancerous lesions (59 hyperplasias and 31 dysplasias), and 81 matched histologically normal oral tissues. Results: Ets-1, VEGF, and stromelysin 3 expression independently correlated with increased intratumoral microvessel density in precancerous lesions (P = 0.05, 0.001, and 0.026, respectively) as well as in SCCs (P = 0.005, 0.01, and 0.031, respectively). Logistic regression analysis revealed that concomitant expression of stromelysin 3 and Ets-1 (stromelysin 3+/ Ets-1+ phenotype; odds ratio, 3.7; P = 0.001) was the most significant predictor for transition to precancerous stage, whereas dual expression of stromelysin 3 and VEGF (stromelysin 3+/ VEGF+ phenotype; odds ratio, 2.07; P = 0.004) was the most important predictor for progression from precancerous stage to frank malignancy. Intriguingly, Ets-1 expression was significantly associated with VEGF expression and stromelysin 3 expression in precancerous tissues as well as OSCCs. Follow-up data for 144 patients for a maximum period of 115 months showed that VEGF [hazards ratio (HR), 4.532; P = 0.004] and Ets-1 (HR = 2.182; P = 0.049) expression significantly correlated with reduced disease-free survival in univariate analysis. In bivariate analysis, patients harboring Ets-1+/VEGF+ phenotype had the worst survival (median disease-free survival, 50 months; HR, 2.943; P = 0.003). Multivariate analysis using Coxs proportional hazards model showed that increased VEGF expression was the most significant adverse prognosticator in OSCC patients (HR, 4.470; P = 0.004). Conclusions: In conclusion, this study provides the first evidence of concomitant expression of stromelysin 3, VEGF, and Ets-1 in clinical specimens in different stages of development of oral cancer. In early stages, concomitant expression of stromelysin 3 and Ets-1 favors the development of a precancerous state, whereas dual expression of stromelysin 3 and VEGF is associated with progression from precancerous to cancerous state. VEGF expression is an adverse prognosticator for disease-free survival.

Alterations of rb pathway components are frequent events in patients with oral epithelial dysplasia and predict clinical outcome in patients with squamous cell carcinoma.

Objective: This study was designed to test the hypothesis that alterations in expression of G1/S modulators cyclin D1, p16 and pRb occur in patients with oral epithelial dysplasia, considered to be at increased risk for malignant transformation. In addition, the analysis of expression of all three markers in the same set of oral cancer patients would provide a unique opportunity to determine whether these alterations have cooperative or synergistic effects on oral cancer development and prognosis. Patients and Methods: A prospective study was undertaken to carry out immunohistochemical analysis of cyclin D1, p16 and pRb proteins in serial paraffin-embedded tissue sections of 220 oral squamous cell carcinomas (OSCCs), 90 potentially malignant lesions (52 oral hyperplastic lesions, 38 dysplasias) and 81 matched histologically normal oral tissues and correlated them with clinicopathological parameters. Ninety-eight OSCC patients were followed up for a maximum period of 94 months with overall median survival of 21 months. Results: Seventy-five of 90 (83%) potentially malignant lesions and 198 of 220 (90%) OSCCs showed altered expression of at least one of the proteins in the pRb pathway, while 10 of 90 (11%) patients with potentially malignant lesions and 40 (18%) of 220 OSCC patients showed all three alterations. Loss of p16 was the earliest event in oral tumorigenesis. In a multivariate model, loss of pRb was associated with transition from hyperplasia to dysplasia (OR = 3.727, p = 0.005). The transition of potentially malignant lesions to malignant stage was associated with pRb–/cyclin D1+ phenotype (OR = 2.294, p = 0.001) and p53+ phenotype (OR = 2.230, p = 0.002). Loss of pRb and accumulation of p53 (pRb–/p53+) phenotype was associated with histologic progression of the tumors and acquisition of invasive potential. Multivariate analysis using Cox’s proportional hazards model revealed that pRb–/p53+ phenotype was the most significant adverse prognosticator for disease-free survival (hazards ratio, (HR) = 2.642, p = 0.004). Conclusions: Deregulation of the p16/pRb/cyclin D1 pathway is an early event in acquisition of dysplasia, but deregulation of both pRb and p53 pathways is associated with malignant transformation and adverse prognosis in oral tumorigenesis.

Weight loss during radiotherapy for head and neck malignancies: what factors impact it?

Radiotherapy (RT) is an important treatment modality in head and neck cancers. Loss of weight during RT due to various factors is a matter of concern. This study was conducted to see the pattern of weight loss and the causative factors involved. One hundred forty patients with head and neck cancer treated with radical RT, concurrent chemoradiation, or postoperative RT were retrospectively studied. A dose of 70 Gy was given in the radical and in the chemoradiation schedule. In postoperative RT, a dose of 60-64 Gy was delivered. During the weekly review of the patients, serial recording of their weight was done along with measurement of other parameters. Analysis was done to see which factors were causative in patients having a weight loss of >5 kg and of >10%. Three variables were found to be significant for the >5-kg weight loss. These were a low initial Karnofsky performance score (KPS; <0.001), use of chemoradiation (P < 0.001), and a total dose of >60 Gy (P = 0.04). For the >10% weight loss, the significant factors were low initial KPS (P < 0.001) and use of chemoradiation (P < 0.001). Therefore, it is important to take care of the nutrition of those patients who have a low KPS, are on chemoradiation, or will be delivered a dose of >60 Gy. The role of prophylactic Ryles tube placement or agents such as megestrol acetate in such patients should be further investigated.

Mandibular involvement in oral cancer

It is difficult to predict the tumour invasion into the mandible by oral cancer pre-operatively, and consequently the decision to preserve or sacrifice the mandible is largely individualistic. The present study of 44 cases analyses the reliability of pre-operative parameters to assess mandibular involvement. Clinical, radiological and scintigraphic features have been compared with the detailed histology of the bone. This study confirms the usefulness of superior marginal resections in lesions which are close to but not involving the mandible, as well as for superficial lesions which are actually seen to involve the mandible but their CT and bone scans are negative for tumour invasion.

COMBINED EVALUATION OF EXPRESSION OF TELOMERASE, SURVIVIN, AND ANTI-APOPTOTIC BCL-2 FAMILY MEMBERS IN RELATION TO LOSS OF DIFFERENTIATION AND APOPTOSIS IN HUMAN HEAD AND NECK CANCERS

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers, and it accounts for 5% of all adult cancers worldwide. Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumor progression. Little is known about the distribution of inhibitors of apoptosis in HNSCC or how they correlate with other biomarkers of malignancy, such as telomerase, an enzyme that plays a critical role in cellular immortalization. The objective of this study was to assess the protein expression of anti‐apoptotic members of Bcl‐2 family and survivin and correlate them with telomerase activity.

Distant metastasis in malignancies of the head and neck

It is a well known fact that disease in head and neck cancer remains confined above the clavicle in a majority of cases for a considerable length of time. The causes of death in head and neck cancer patients are known to be due to lymph node metastasis, fungation, asphyxia, cachexia, invasion of major vessels and infection. Distant metastasis occurs late in the disease. However, many reports have appeared in the literature which indicate a high incidence of distant metastasis in head and neck malignancies. Merino et al. (1977), in a clinical study, have indicated an incidence of 10.9 per cent. Studies based on autopsy findings, however, quote a much higher figure (30 per cent, Papac, 1984; 57 per cent, Gowen and Dessuto Nagy, 1963). Dennington and Caster (1980) reported that at least seven per cent of patients with head and neck cancer have distant metastasis when first seen. The present study was designed to find out the incidence of distant metastasis in our head and neck cancer patients.

SIGNIFICANCE OF PROMOTER HYPERMETHYLATION OF p16 GENE FOR MARGIN ASSESSMENT IN CARCINOMA TONGUE

Loss of p16 expression by promoter hypermethylation has been reported as an early event in the development of oral cancer. The aim of our study was to explore the prognostic implications of presence of promoter hypermethylation of p16 gene in surgical margins in carcinoma tongue.

Over-expression of 14-3-3zeta is an early event in oral cancer

BackgroundThe functional and clinical significance of 14-3-3 proteins in human cancers remain largely undetermined. Earlier, we have reported differential expression of 14-3-3ζ mRNA in oral squamous cell carcinoma (OSCC) by differential display.MethodsThe clinical relevance of 14-3-3ζ protein in oral tumorigenesis was determined by immunohistochemistry in paraffin embedded sections of oral pre-malignant lesions (OPLs), OSCCs and histologically normal oral tissues and corroborated by Western Blotting. Co-immunoprecipitation assays were carried out to determine its association with NFκB, β-catenin and Bcl-2.ResultsIntense immunostaining of 14-3-3ζ protein was observed in 61/89 (69%) OPLs and 95/120 (79%) OSCCs. Immunohistochemistry showed significant increase in expression of 14-3-3ζ protein from normal mucosa to OPLs to OSCCs (ptrend < 0.001). Significant increase in expression of 14-3-3ζ protein was observed as early as in hyperplasia (p = 0.009), with further elevation in moderate and severe dysplasia, that was sustained in OSCCs. These findings were validated by Western blotting. Using Co-immunoprecipitation, we demonstrated that 14-3-3ζ protein binds to NFκB, β-catenin and Bcl-2, suggesting its involvement in cellular signaling, leading to proliferation of oral cancer cells.ConclusionOur findings suggest that over-expression of 14-3-3ζ is an early event in oral tumorigenesis and may have an important role in its development and progression. Thus, 14-3-3ζ may serve as an important molecular target for designing novel therapy for oral cancer.

Concomitant chemoradiation versus radical radiotherapy in advanced squamous cell carcinoma of oropharynx and nasopharynx using weekly cisplatin: a phase II randomized trial

BACKGROUND To know the effectiveness and tolerance of weekly cisplatin added to radiotherapy (RT) in advanced carcinoma of oropharynx and nasopharynx. PATIENTS AND METHODS Stage II-IV cancer patients were randomly assigned to either radical RT, 70 Gy/35 fractions over 7 weeks (RT arm), or chemoradiotherapy (CRT), cisplatin 40 mg/m² weekly for seven doses plus RT. Primary end points were (i) the responses, (ii) toxicity profile, and (iii) overall survival (OS) in two groups. Study period was from June 2003 to July 2005. RESULTS One hundred and fifty-three patients were randomly allocated to the study, 76 in RT arm and 77 in CRT arm. Seventy-one in each arm completed the planned treatment; complete response (CR): 67.1% versus 80.5% in RT and CRT arms (P = 0.04). Grade III and IV toxicity were 16% and 40% in RT and CRT arms, respectively (P = 0.01). There were frequent treatment interruptions (9.3% versus 28.9%; P = 0.003) and hospitalization (20% versus 40.8%) in the CRT group. OS was superior in the CRT arm (P = 0.02): 27 months [95% confidence interval (CI) 15.2-36.8] for RT versus not reached for CRT. Three-year OS was 42% for RT and 62% for CRT group. CRT and CR were independent prognostic factors. CONCLUSION This trial on Indian head and neck squamous cell carcinoma patients confirms that the use of weekly cisplatin is safe and CRT is superior to RT alone resulting in higher OS.