Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5–50 fg·mL–1 range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400 000 enzyme labels and ∼100 000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linke...

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Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array.

Pakistan, India, Sri Lanka, Bangladesh, Nepal and Bhutan, with their total population of more than 1,500 million, make up the subcontinent of South Asia. Despite massive diversity across the region, there are sufficient similarities to warrant a collective approach to chronic disease control, including development of cancer control programs. Cancer is already a major problem and there are general similarities in the prevalence patterns. In males, oral and lung cancer are either number one or two, depending on the registry, with the exceptions of Quetta in the far north, Larkana and Chennai. Moderately high numbers of pharyngeal and/or laryngeal cancer are also consistently observed, with prostate cancer now becoming visible in the more developed cities. Breast and cervical cancer share first and second place except in Muslim Pakistan, where oral cancer generally follows breast. The ovary is often included in the five most prevalent types. Markedly increasing rates for breast cancer and distribution shifts in other cancers suggest that, despite improvement in cervical and oral rates, the overall burden will only become heavier over time, especially with increasing obesity and aging of what are still youthful populations. Coordination of activities within South Asia is a high priority for cancer control in the region.

Cancer epidemiology in South Asia - past, present and future.

Squamous cell carcinoma (SCC) is by far the most common malignant neoplasm of the oral cavity. A number of etiologic factors have been implicated in its development. During the past few decades, a particular focus has been placed on the investigation of valid biomarkers predictive of cancer behavior and cervical lymph node metastasis in head and neck Squamous cell carcinoma (HNSCC).The present study was designed to investigate the expression of epidermal growth factor in these tumors in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis. Immunohistochemical (IHC) evaluation of epidermal growth factor receptor (EGFR) expression in 40 retrospective OSCC specimens and its correlation with proliferating cell nuclear antigen (PCNA), antiapoptotic antibody (P53), vascular endothelial growth factor (VEGF), and D2-40 monoclonal antibodies (Mab), in relation to the clinicopathological parameters. Data revealed positive EGFR immunoreactivity in 35(87.5%) cases. There was a statistically significant correlation regarding EGFR extent score with respect to intratumoral lymphatic vessel density (ILVD) (r = 0.35) as well as EGFR intensity score with respect to ILVD and peritumoral lymphatic vessel density (PLVD) (r = 0.33, r = 0.36 respectively). EGFR expression was not correlated with the clinicopathological parameters. Conclusions: EGFR is expressed by most of the cases. EGFR correlation with D2- 40 positive lymphatic vessels suggests a higher tendency of OSCC for lymphatic dissemination. Lack of correlation among the studied markers suggests their independent effect on tumor behavior.

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Immunohistochemical expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis.

PURPOSE. To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS. Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS. Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P 0.001; ZK991 by 53%, P 0.001) and lymphangiogenesis (PTK/ZK by 70%, P 0.001; ZK991 by 71%, P 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P 0.001; ZK991, P 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P 0.02). CONCLUSIONS. Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation. (Invest Ophthalmol Vis Sci. 2008;49: 1836‐1842) DOI:10.1167/iovs.07-1314

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation.

Squamous cell carcinoma of the oral cavity (OCSCC) is the most frequently observed form of head-and-neck cancer in Southeast Asia and is the sixth most common cancer worldwide. Most cases of this preventable disease are caused by alcohol consumption, smoking and betel nut chewing. The survival rates of patients with advanced OCSCC have not increased significantly in recent years. While treatments for OCSCC are similar worldwide, survival rates differ by geographical area. The various genetic profiles and individual genetic susceptibility for carcinogens may account for this discrepancy. In some respects, molecular alteration or accumulation affects tumor progression and the clinical outcomes among patients with OCSCC. Clarifying the tumor behavior of oral cancer, with regard to pathological features or molecular aspects, could help clinicians to judge, tailor and adopt more effective therapeutic strategies to treat oral cancer.

Clinical, pathological and molecular determinants in squamous cell carcinoma of the oral cavity

Background: Nodal metastasis is a major prognostic indicator for oral squamous cell carcinoma (OSCC) progression. Recently, it has been revealed that lymphangiogenic growth factor VEGF-C and its receptor Flt-4 play an important role for invasion and metastasis in cancer cells. Aim: To examine VEGF-C expression and its correlation with lymphatic status, including the number of lymph vessels and lymphatic invasion, tumour invasion and metastasis in OSCC. Methods: Intratumoural and peritumoural lymphatic vessels were examined using D2-40 in 54 OSCC cases and correlated with VEGF-C expression and clinicopathological findings. The histological pattern of invasion and pathological findings were compared. Results: High expression of VEGF-C was frequently observed in OSCC and was associated with increased number of lymph vessels and lymphatic invasion. VEGF-C was well correlated with invasion pattern and metastasis. Conclusions: Results suggest that VEGF-C may play an important role for lymphangiogenesis and invasion in the metastatic process and can be a strong predicting factor for metastasis of OSCC.

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VEGF-C is associated with lymphatic status and invasion in oral cancer.

Background: Oral squamous cell carcinoma (OSCC) most commonly occurs in the middle-aged and older individuals. The purpose of the present study was to evaluate the histopathological and immunohistochemical differences of the younger (<40 years) and the older (more than 50 years) groups.



Methods:  The histopathological parameters of 112 patients (younger 56 and older 56) were compared according to three grading systems (Broder JAMA 1920; 74: 656, Anneroth et al. Scand J Dent Res 1987; 95: 229, Bryne et al. J Pathol 1992; 166: 375) and as individual histopathological parameters. Further, the expression of p53 and Proliferative Cell Nuclear Antigen (PCNA) index was also compared.



Results:  Although there was no significant difference between two groups regarding the three grading systems, a significantly higher number of nuclear aberrations was found in younger group (P < 0.001). Interestingly, higher number of mitoses (P < 0.05) and lymph node metastasis (P < 0.05) were observed in the older group (P < 0.05). Furthermore, significantly a higher PCNA index was found in the older group (P < 0.005).



Conclusions:  Although tumours of the young patients showed more nuclear aberrations, OSCC of the older patients is proliferative and showed higher metastatic rate.

Analysis of histopathological and immunohistochemical differences of oral squamous cell carcinoma in young and old patients in Sri Lanka.

Background. Developing histological prediction models that estimate the probability of developing metastatic deposit will help clinicians to identify individuals who need either radical or prophylactic neck dissection, which leads to better prognosis. Identification of accurate predictive models in oral cancer is important to overcome extensive prophylactic surgical management for neck nodes. Therefore, accurate prediction of metastasis in oral cancer would have an immediate clinical impact, especially to avoid unnecessary radical treatment of patients who are at a low risk of metastasis. Methods. Histologically confirmed OSCC cases with neck dissection were used. Interrelation of demographic, clinical, and histological data was done using univariate and multivariate analysis. Results. 465 cases were used and presence of metastasis and extracapsular invasion were statistically well correlated with level of differentiation and pattern of invasion . Multivariate analysis showed level of differentiation, pattern of invasion, and stage as predictors of metastasis. Conclusions. The proposed predictive model may provide some guidance for maxillofacial surgeons to decide the appropriate treatment plan for OSCC, especially in developing countries. This model appears to be reliable and simple and may guide surgeons in planning surgical management of neck nodes.

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M N G P K Udagama

Papers

VEGF-C is associated with lymphatic status and invasion in oral cancer.

Analysis of histopathological and immunohistochemical differences of oral squamous cell carcinoma in young and old patients in Sri Lanka.

A Predictive Model to Determine the Pattern of Nodal Metastasis in Oral Squamous Cell Carcinoma