The Distribution of M2 Macrophage and Treg in Nasopharyngeal Carcinoma Tumor Tissue and the Correlation with TNM Status and Clinical Stage.

OBJECTIVE: This study aimed to identify the distribution of M2 macrophage and Treg in Nasopharyngeal Carcinoma (NPC) tumor tissue samples. The presence of these two groups of cells was further correlated to clinical stage, tumor size, the lymphatic node involvement, and metastasis. METHODS: The total of 50 formalin-fixed paraffin-embedded (FFPE) NPC tissue samples was collected retrospectively (27 samples) and prospectively (23 samples). Samples were FFPE tissue slices. Immunohistochemistry was done on the FFPE tissue slides using anti-CD-163 and anti-FoxP-3 antibodies for M2 macrophage and Treg detection, respectively. The M2 macrophage interpretation was performed by eye-balling method and the score was divided into 0 (negative), 1 (scant), 2 (focal), and 3 (abundant). The average number of Treg FOXP3+ cells in 5 high power fields (HPF) was calculated. The relationship of M2 macrophage and Treg was tested with Spearman's correlation. The relationship between M2 macrophage and Treg with clinical stage, tumor size, node involvement and metastasis was tested by chi square, with p<0.1. RESULTS: M2 macrophage and Treg were positive correlated (r=0.469, p<0.001). The presence of M2 macrophage and regulatory T cell (Treg) was significantly correlated to tumor size (p= 0.091 for M2 macrophage and p=0.022 for Treg) and clinical stage (p= 0.030 for M2 macrophage and p= 0.002 for Treg), but did not correlate with lymphatic node involvement and metastasis. CONCLUSIONS: In Epstein-Barr virus related NPC tumor microenvironment, the presence of M2 macrophage was correlated with Treg, and both types of the cells were correlated with tumor size and clinical stages.

BCR-ABL Gene Transcript Types of Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia.

The aim of this study was analyzing the BCR-ABL transcript types of patients with chronic myeloid leukemia (CML) in Dr Sardjito General Hospital, Yogyakarta, Indonesia. This study is very relevant because the data concerning BCR-ABL gene transcript types is very limited in Indonesia. Furthermore, it is important for patient's management, particularly in defining the tyrosine kinase inhibitors (TKIs) therapy and monitoring after therapy. The introduction of TKIs has become a major advance in the management of patients with CML, especially in the chronic phase (CML-CP), in which most patients are diagnosed. METHODS: One hundred eighty five (185) of 370 recruited patients were included in this study (2010-2014). RNA samples were isolated from mononuclear cells of peripheral blood of the subjects taken at primary diagnosis. Detection of BCR-ABL gene transcript types was done using multiplex reverse transcriptase PCR (multiplex RT-PCR) and/or nested PCR following the cDNA synthesis. When the first PCR set failed to amplify the BCR-ABL gene, RT-conventional PCR and/or nested PCR would be applied. The proportion of each transcript type was calculated among the BCR-ABL positive CML patients. RESULTS: Approximately 99% (183/185) of CML patients are BCR-ABL positive, with the most common type is major b3a2 (136/183; 74.3%), followed by major b2a2 (41/183; 22.4%). Two samples (1.1%) showed co-expression of b3a2 and b2a2; 1 sample showed co-expression of b3a2 and fragment at 500bp; and 3 samples showed uncommon fragments. CONCLUSION: Ninety nine percent (99%) of CML patients in Yogyakarta, Indonesia are BCR-ABL positive, with 74.3% have b3a2 transcript, 22.4% have b2a2 trascript, 1.1% have co-expression of b3a2 and b2a2 transcript, and the rest (2.2%) have uncommon bands that still need to be confirmed.

New Genetic Variation in BCR gene of Major B3a2 Breakpoint BCR-ABL Fusion Gene in Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia

Background: Polymorphic bases in several exons of the BCR gene have been found in several studies of the BCR-ABL fusion gene . Most of the polymorphisms do not have any implications for the primary structure of the BCR-ABL protein. Nucleotide changes are often located in the area close to the fusion region, and therefore may influence primer annealing. Our previous work failed to amplify 15 of 200 samples from BCR-ABL positive chronic myelogenous leukemia (CML) patients using multiplex PCR, the standard method to detect BCR-ABL transcripts used in our institution. The failure was considered due to problems in primer annealing caused by sequence variations. Sequence analysis of BCR-ABL fusion gene breakpoint types in CML patients has never been hitherto performed in Indonesia. Therefore, the aim of this study was to perform sequence analysis of several samples that did not show amplification using the standard method. Methods: Fifteen samples were qualitatively amplified by two-step PCR using inner primers in the 2nd PCR to determine the breakpoint type of the BCR-ABL fusion gene. The 2nd PCR products were used as templates to perform sequence analysis, and the results were compared to those in genbank. Result: Seven and 5 of 15 samples were confirmed as major b3a2 and major b2a2, respectively. One sample featured a combination of b3a2 and b2a2, and 2 samples a combination of b3a2 and b2a2 with an additional fragment at 500bp. Sequence analysis showed 3 sequence variations in the major b3a2 breakpoint. One had been reported earlier (c.3296T>C) but the others (c.3245C>T and c.3359T>C) were novel. Fragments at 500bp were confirmed as b3a2 and similar sequence b3a2 in genbank. Conclusion: This study found two new genetic variations in the BCR gene in BCR-ABL fusion cases.