Browsing by Author "Lokuhetti, M.D.S."

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    Development of a quantitative PCR assay to evaluate HER2 status of Gastric carcinoma in a cohort of Sri Lankan patients
    (Sri Lanka Medical Association, 2016) Kannangara, D.K.S.; Subasinghe, D.; Lokuhetti, M.D.S.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.
    INTRODUCTION AND OBJECTIVES: Human epidermal growth factor receptor2(HER2) protein overexpression and/or HER2gene amplification is linked to dismal outcome of Gastric carcinoma(GCa). Immunohistochemistry(IHC) and fluorescence in situ hybridization(FISH) are key-methods to identify patients for HER2 targeted therapy. Drawbacks of both methods warrant novel tests. The study aimed to determine whether quantitative Polymerase Chain Reaction (qPCR) could serve as a supplementary-method to evaluate HER2 status of GCa in a cohort of Sri Lankan patients and investigate correlation between HER2 assessed by different methods and clinic-pathological features. METHOD: Twenty GCa-patients with known IHC-HER2 scores were evaluated. qPCR was performed for HER2gene and Ameloid precursor protein (reference gene) in Formalin fixed paraffin embedded GCa tissue. Threshold values(Ct) were analyzed using Pfaffl-method to detect HER2gene amplification. RESULTS: HER2positivity by IHC(protein) and qPCR(gene) were 20% and 35% respectively. Sensitivity and specificity of qPCR was 67% and 76% respectively and results were reproducible. HER2protein positivity was correlated with Tumour TNM-stage and Lauren-histological types(P<0.05). Positive expression of HER2gene was correlated with depth of tumour invasion, differentiation and Lymph node-status(P<0.05). Diagnostic consistency between IHC and qPCR(κ=0.146) was slightly agreeable(0.01
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    Development of modified mismatch PCR-RFLP to screen mutations in codon 12 and 13 of K- ras gene of colorectal (CRC) patients in Sri Lanka
    (Sri lanka Medical Association, 2015) Dhilhani, M.F.F.; de Zoysa, M.I.M.; Chandrasekharan, N.V.; Gunawardene, Y.I.N.S.; Lokuhetti, M.D.S.; Dassanayake, R.S.
    INTRODUCTION AND OBJECTIVES: Mutations in K-ras codon 12, 13 of exon 2 are known to affect prognosis and impart resistance to anti EGFR monoclonal antibody therapy in CRC. Although several diagnostic tools have been developed for K-ras mutation testing, these procedures are too expensive or time consuming. Oufaim was to develop an effective, reliable and inexpensive method for the detection of K-ras mutations in codons 12 and 13 of exon 2 in CRC patients in Sri Lanka, and to relate the mutational status to liver metastasis, METHOD: The mismatch PCR-RFLP was developed and used to screen mutations in codon 12 and 13 for DMA isolated from paraffinized tumour tissue of 30 CRC patients followed up for 5 year after surgery to detect liver metastasis. Cross-tabulations were generated between K-ras mutations and the metastatic status. The Chi Square test was used to indicate statistical significance of the association. RESULTS: Analysis of banding pattern obtained from restriction digestion of PCR amplified region containing codon 12 and/or 13 of KRAS gene of 14(46.6%) CRC patients revealed the presence of mutations. Of the 30 patients, 13(43.3%) had developed liver metastases. There was a significant association between the presence of a K-ros mutation and the occurrence of liver metastasis (X2=4.693, p=0.003). CONCLUSION: This mismatch PCR-RFLP protocol is a suitable method to screen codon 12 and 13 mutation of K-ros gene to predict liver metastasis. Presence of these mutations is associated with the occurrence of liver metastasis during the first 5 years after surgery.