Aberrant antigenic expression in extranodal NK/T-cell lymphoma: a multi-parameter study from Thailand

<p>Abstract</p> <p>Background</p> <p>Extranodal NK/T-cell lymphoma, nasal type (ENKTL) is not common worldwide, but it is the most common T- and NK-cell lymphomas in many Asian countries. Immunophenotypic profiles were studied based on limited series. The authors, therefore, studied on ENKTL according to characterize immunophenotypic profiles as well as the distribution of EBV subtype and LMP-1 gene deletion.</p> <p>Methods</p> <p>By using tissue microarray (TMA), immunohistochemical study and EBV encoded RNA (EBER) in situ hybridization were performed. T-cell receptor (TCR) gene rearrangement, EBV subtyping, and LMP-1 gene deletion were studied on the available cases.</p> <p>Results</p> <p>There were 22 cases eligible for TMA. ENKTL were positive for CD3 (91%), CD5 (9%), CD7 (32%), CD4 (14%), CD56 (82%), TIA-1 (100%), granzyme B (95%), perforin (86%), CD45 (83%), CD30 (75%), Oct2 (25%), and IRF4/MUM1 (33%). None of them was positive for βF1, CD8, or CD57. TCR gene rearrangement was negative in all 18 tested cases. EBV was subtype A in all 15 tested cases, with 87% deleted LMP-1 gene. Cases lacking perforin expression demonstrated a significantly poorer survival outcome (p = 0.008).</p> <p>Conclusions</p> <p>The present study demonstrated TIA-1 and EBER as the two most sensitive markers. There were a few CD3 and/or CD56 negative cases noted. Interestingly, losses of CD45 and/or CD7 were not uncommon while Oct2 and IRF4/MUM1 could be positive in a subset of cases. Based on the present study in conjunction with the literature review, determination of PCR-based TCR gene rearrangement analysis might not be a useful technique for making diagnosis of ENKTL.</p

Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis

<p>Abstract</p> <p>Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma.</p> <p>Introduction</p> <p>Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation <abbrgrp><abbr bid="B3">3</abbr><abbr bid="B4">4</abbr></abbrgrp>. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis <abbrgrp><abbr bid="B5">5</abbr><abbr bid="B6">6</abbr></abbrgrp>.</p> <p>GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost <abbrgrp><abbr bid="B7">7</abbr></abbrgrp>. Therefore, rapid and more cost effective technique are being sought.</p> <p>LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye <abbrgrp><abbr bid="B8">8</abbr><abbr bid="B9">9</abbr></abbrgrp>. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product <abbrgrp><abbr bid="B10">10</abbr><abbr bid="B11">11</abbr></abbrgrp>. Therefore, current investigations using melting curve analysis are being developed <abbrgrp><abbr bid="B12">12</abbr><abbr bid="B13">13</abbr></abbrgrp>.</p> <p>In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.</p

An A91V SNP in the perforin gene is frequently found in NK/T-cell lymphomas

NK/T-cell lymphoma (NKTCL) is the most frequent EBV-related NK/T-cell disease. Its clinical manifestations overlap with those of familial haemophagocytic lymphohistiocytosis (FHLH). Since PERFORIN (PRF1) mutations are present in FHLH, we analysed its role in a series of 12 nasal and 12 extranasal-NKTCLs. 12.5% of the tumours and 25% of the nasal-origin cases had the well-known g.272C>T(p.Ala91Val) pathogenic SNP, which confers a poor prognosis. Two of these cases had a double-CD4/CD8-positive immunophenotype, although no correlation was found with perforin protein expression. p53 was overexpressed in 20% of the tumoral samples, 80% of which were of extranasal origin, while none showed PRF1 SNVs. These results suggest that nasal and extranasal NKTCLs have different biological backgrounds, although this requires validation

Factors Affecting Unfavourable Results from a Sinonasal Inverted Papilloma Surgery

Objective: Sinonasal inverted papilloma (SNIP) is the most common nasal benign tumor, but locally invasive. The standard treatment is to identify origins of the tumor and total removal. Unfavourable results are finding postoperative residual or recurrent tumors. The aim of this study is to determine factors affecting postoperative residual or recurrent tumors and a rate of getting postoperative residual or recurrent tumors from SNIP surgeries. Methods: A retrospective study in patients with SNIPs was conducted. Relationships between demographic data, tumor sites, tumor stages by Krouse classification, surgical approaches, surgeons’ experience, using microdebrider assisted surgery, operative time, intraoperative blood loss, histopathology, Epstein Barr virus (EBV), human papillomavirus (HPV) infection, time to detect tumor after surgery and unfavourable results were evaluated. HPV and EBV were detected by in situ hybridization. Results: 73 patients were included in this study. Unfavourable results were found in 27 patients (36.99%). 50% of patients received unfavourable results after postoperative duration of 115 months. 5 years of a disease-free survival rate was 64.3% (95% CI: 51.9% to 76.7%). The patients with external surgical approaches got worse results than those with endoscopic sinus surgery (p = 0.01, a hazard ratio of 3.88, 95% CI: 1.39 to 10.87). The patients operated without using microdebrider assisted surgery got worse results than those with using the device (p < 0.001, an adjusted hazard ratio of 5.09, 95% CI: 2.08 to 12.45). The patients with abnormal pathological changes (tissue dysplasia and malignant transformation) had worse results than those without changes (p = 0.02, an adjusted hazard ratio of 3.42, 95% CI: 1.24 to 9.38).   Conclusion: Non-endoscopic nasal surgery, non-using microdebrider assisted surgery, and abnormal pathological changes may be some of the causes of unfavourable results from SNIP surgeries. Long postsurgical surveillance should be done, because of 36.99% of patients received unfavourable results from SNIP surgeries