| Background and purposeNasal-type NK/T-cell lymphoma, which was previously termed as "midline malignant reticulocytosis","Angio-centric lymphoma","midline lethal granuloma" and so on, is a special entity of Non-hodgkin’s lymphoma, frequently affecting the orbits, nasal cavity, paranasal sinuses, palate, oral cavity, larynx and other facial midline sites, and still some other extranasal sites such as skin, gastrointestinal, lung, brain and testicle will also be involved. This disease always causes the granulomatous type lesion on tissue, gives rise to mucosa hyperemia, swelling, uncurable ulcer in the affected sites and brings out massive dry crest and necrotic tissue. Under the light telescope, the tumor cells growing in angio-centric pattern have broad size spectrum, lack of typical cytological character and often mix with massive necrotic tissue and inflammatory cells. Therefore, the diagnosis of nasal-type NK/T-cell lymphoma is very hard to make.The traditional diagnostic scheme is based on the triplet of clinical manifestations, morphological features and immunohistochemistry stain results, although it is efficient in most cases, when encounters with complicated and variant cases, failure of diagnosing will be not unusual. Recently, as the molecular biological technology is progressing and genetic research keeps deepening, the diagnostic approaches of nasal-type NK/T-cell lymphoma become more integrated. Since it was reported that some nasal-type NK/T-cell lymphoma cases were positive for TCR gene monoclonal rearrangement, the detection with polymerase chain reaction (PCR) of T-cell receptor (TCR) gene rearrangement has been clinically widespread and playing a significant role in diagnosis of nasal-type NK/T-cell lymphoma.However, it is impractical to demand fresh-frozen tissue, which is agreed as the most ideal subject of this detection, from every suspicious case, therefore, if the availability of this detection to routine paraffin-embedded tissue can be achieved, the clinical procedure and diagnostic efficiency will obtain a great improvement. Setting the detecting results of fresh-frozen tissue as reference, this study aims to investigate the statistical difference between the detecting results of fresh-frozen tissue and correspondent paraffin-embedded tissue, in order to find out whether the paraffin embedded tissue is reliable to TCR gene rearrangement detection of nasal-type NK/T-cell lymphoma.Materials and Methods1. Material and groupsMost of the suspicious nasal-type NK/T-cell lymphoma cases biopsied in Eyes, Ears, Nose and Throat hospital of Fudan University during from October,2009to December,2010were collected. Every biopsy was halved with one part for paraffin-embedding and the other for fresh-freezing. The paraffin-embedded tissue were cut to make HE and immunohistochemistry stain, the results of which eventually helped30cases with definite diagnosis of nasal-type NK/T-cell lymphoma. The paraffin-embedded specimen and their correspondent fresh-frozen specimen were grouped separately.10cases of nasal polyps were employed as control group.2. MethodsThe paraffin-embedded tissue and fresh-frozen tissue of nasal-type NK/T-cell lymphoma with clear-cut diagnosis were organized as experimental samples, grouping in their own type. Common cutting was employed for Specimen in paraffin-embedded group while scraping cutting for specimen in fresh-frozen group. The genomic DNA from specimen were extracted according to the protocol of QIAamp DNA Micro Kit, classic primers of TCRP and TCRy were selected from published documents, all the genomic DNA amplified with Hot-start PCR were given electrophoresis on2%agarose gel. The results were observed under ultra-violet light. The paired Χ2test were employed to compare the difference between the results of paraffin embedded group and fresh frozen group of nasal-type NK/T-cell lymphoma. Results1. Hemaxylene-eosin (HE) stainUnder the light telescope, the tumor tissue were observed with angio-centric growth pattern, the tumor cells have broad size spectrum and atypical karyotype, mixing with massive coagulative necrotic tissue and inflammatory cells.2. Immunohistochemistry stainAmong all30cases of nasal-type NK/T-cell lymphoma,19/30cases (63.3%) were positive for T lymphocyte intracellular antigen-1(TIA-1),29/30cases (96.7%) were positive for plasmic CD3,29/30cases (96.7%) were positive for CD56,23/30cases (76.7%) were positive for granzyme B (GB),30/30cases (100%) were positive for Leukocyte Common Antigen (LCA),30/30cases (100%) were positive for UCHL-1(CD45RO).3. results of TCR gene rearrangement detectionAfter amplified with human being β-globin primers, all the genomic DNA from specimen of30cases of nasal-type NK/T-cell lymphoma and10cases of nasal polyps showed the correspondent gene fragment. The results of30cases of nasal-type NK/T-cell lymphoma in the detection of TCR gene monoclonal rearrangement are as followed:15/30(50.0%) cases are positive in paraffin-embedded specimens,17/30(56.7%) cases are positive in fresh-frozen specimens. Among all30cases,14cases are positive in both fresh-frozen specimens and paraffin-embedded specimens;3cases are positive in fresh-frozen specimens while negative in paraffin-embedded specimens;1case is positive in paraffin-embedded specimen while unexpectedly negative in fresh-frozen specimen. After amplified with the same primers, all10cases of nasal polyp showed negative result of TCR gene monoclonal rearrangement. The result of Χ2test shows P(=0.625)>0.05, which means the difference between the results of paraffin embedded group and fresh frozen group of nasal-type NK/T-cell lymphoma has no statistical significance.Conclusions In the detection of TCR gene monoclonal rearrangement of nasal-type NK/T-cell lymphoma, there is no significant difference between the results of paraffin-embedded tissue and fresh-frozen tissue. It is clinically feasible to make TCR gene rearrangement detection based on paraffin-embedded tissues as the routine auxiliary test in the diagnosis and differential diagnosis of nasal-type NK/T-cell lymphoma. On the other hand, the negative results of TCR gene monoclonal rearrangement can not rule out the diagnosis of nasal-type NK/T-cell lymphoma. Last but not least, the negative result of fresh-frozen tissue can’t deny the existence of TCR gene monoclonal rearrangement of tumor tissue. |
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