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I Gusti Bagus Datasena
Vivo Research Initiative
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One Step Nucleic Acid Amplification (OSNA) Study in Indonesia Samuel J Haryono; Lenny Sari; Sony Sugiharto; I Gusti Bagus Datasena; Raymond Mulyarahardja; . Rudianto
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 48, No 4 (2016): SUPPLEMENT
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (92.981 KB) | DOI: 10.19106/JMedScieSup004804201625

Abstract

ABSTRACTSentinel lymph node (SLN) is defined as the first of a few selected lymphatic nodes, into which lymphatic fluid from a primary tumor drains. Streamlined processing of sentinel lymph nodes (SLN) for detection of lymph node metastasis involves the able command over methodical blocks of SLN identification, surgical removal of SLN and SLN analysis. One Step Nucleic Acid Amplification (OSNA) method, which relies on CK19 mRNA expression to detect intraoperatively  lymph node metastases in breast cancer cases, emerged as a plausible alternative to the current gold standard that uses histopathological node analysis. Sixty selected axillary sentinel lymph nodes from thirty breast cancer patients. Sentinel lymph nodes were directly bi-halved after collection using customized lymph node cutting device (Sysmex), or scalpel. The first halves were subjected to histopathological examination and were stored in specimen containers containing fresh formaldehyde prior to processing. The adjacent halves were weighed to comply with the required mass by OSNA detection in the range of 50 – 600 mg and wrapped in clean foils for storage in -80°C prior to OSNA analysis. 60 SLNs were same diagnosis using both methods. 25 SLNs were negative and 25 SLNs were positive using both methods. 3 SLNs were positive on OSNA but negative on histology. Other 7 SLNs were negative on OSNA but positive on histology, and these 1 nodes contained only micrometastasis lesion. These results suggest that OSNA is a useful for detecting SLNs metastasis, but a copy number of CK19 might be an indepedent factor from prediction and prognosis of breast cancer.   
High Resolution Melting (HRM) Analysis for Genetic Changes in BRCA1/2 gene Samuel J Haryono; I Gusti Bagus Datasena; Ariananda Hariadi; Raymond Mulyarahardj
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 48, No 4 (2016): SUPPLEMENT
Publisher : Journal of the Medical Sciences (Berkala Ilmu Kedokteran)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (92.955 KB) | DOI: 10.19106/JMedScieSup004804201623

Abstract

Conventional mutation analysis requires a separation step and include single-strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis, heteroduplex analysis, denaturing HPLC, and temperature gradient capillary electrophoresis These methods require separation of PCR products on a gel or other matrix, often take hours to perform, and increase the risk of contamination in future reactions because PCR products are exposed to the environment. High Resolution Melting (HRM) can simplify the mutation scanning  analysis in BRCA 1/2 gene. DNA from affected patients and family members were amplified with Real-Time PCR reaction and followed by Sanger Sequencing to reconfirm the mutation status if mutation obtained by HRM Method. HRM Method was able to show distinction in differential curves of mutated BRCA 2 gene c.4600T>C, with codon modification of CAT>TAT, when compared to wildtype. To determine point mutation in a sample, this method requires two groups of experimental standards and standard curves. The first standard produced by using samples without mutation (wildtype/negative control) and the second standard produced by using samples with mutation (positive control), that have been confirmed with Sanger Sequencing. The sequencing analysis of the affected patient and the family members showed that a mutation occurred (BRCA2 c.4600T>C) and was segregated in the family history. This mutation caused amino acid alteration in BRCA2 protein (p.H1458Y). HRM Method is an excellent tool to analyze genetic modification of BRCA1/2 genes, especially to investigate co-segregation of mutated genes among family members of affected patient. This method can provide more sensitive results to determine mutation in patient, before using Sanger Sequencing analysis.Keyword: BRCA, HRM, Gene Mutation
Co-Authors . Rudianto Ariananda Hariadi Lenny Sari Raymond Mulyarahardj Raymond Mulyarahardja Samuel J Haryono Samuel J Haryono Sugiharto, Sony