https://scholars.lib.ntu.edu.tw/handle/123456789/144478
標題: | 早期蕈狀肉芽腫之T細胞受體 g 基因群落變化 Study of T cell Receptor g Gene Clonality in Early Mycosis Fungoides |
作者: | 蕭百芬 Hsiao, Pa-Fan |
關鍵字: | 鏈鎖反應;蕈狀肉芽腫;聚合酶;T細胞受體g基因重組;mycosis fungoides;T cell receptor g gene rearrang | 公開日期: | 2004 | 摘要: | 蕈狀肉芽腫 (mycosis fungoides, MF)是最常見的一型皮膚T細胞淋巴瘤(cutaneous T cell lymphoma,CTCL)。MF 病灶依疾病發展可分為斑期、板塊期及腫瘤期。其中早期病例所佔比例最高,診斷上又最為困難,不容易和一般發炎性皮膚疾病做區分。目前雖然有不少學者提出一些病理變化來幫助早期診斷,但沒有單一項病理變化是百分之百存在,且各項變化均有程度上的不同,亦參雜著判讀醫師個人的差異性,並不客觀。病人往往需要經過長時間的追蹤、反覆切片才能確定診斷。由於MF愈早期診斷出來,治療上才比較有痊癒的機會,因此其他輔助診斷的方法如T細胞受體 (TCR) 基因重組的偵測亦成為研究上的重點。 本研究的目的在評估偵測單株TCR g 基因重組是否有助於MF的早期診斷,進一步探討此結果和病理變化間的相關性。另一方面針對同一個病人治療前後的病灶,看此項檢查結果的演變是否有助於病程的追蹤。 我們回溯性收集台大醫院皮膚部近八年來診斷為MF的病人,排除病理變化不明顯及蠟塊品質不佳的切片後,共9例病人(20個檢體)納入本研究。我們萃取蠟塊的DNA後,先做internal control確認萃取到的DNA後,再分別以兩組不同引子的multiplex聚合酶鏈鎖反應(Mix 1 及 Mix 2)偵測是否有單株TCR g 基因重組。我們並將結果和病理變化做比對及分析,嘗試找出哪一項變化在PCR陽性及陰性兩組之間有最大差異。病理診斷上則依確定程度將檢體區分為確定診斷、可以診斷、建議診斷、無法診斷四組。 實驗結果發現PCR陽性率在斑期為53% (8/15)、板塊期為100% (2/2)、腫瘤期為100% (3/3)。在早期階段(斑及板塊期)陽性率為59%。以病理診斷來區分,確定診斷組為50% (3/6)、可以診斷組為50% (1/2)、建議診斷組為71% (5/7)、無法診斷組為50% (1/2)。分析7項病理變化指標,發現PCR陰性組發炎現象較為厲害,較多出現真皮纖維化,然而只有後者在PCR陽性及PCR陰性兩組之間的差異達到統計上的顯著意義(p=0.01)。我們有嘗試用laser capture microdissection的方法,改變單株T細胞和反應性T細胞數目的比例,發現有助於偵測表皮內的單株T細胞。將其中一個病例治療後的檢體,其PCR產物作進一步cloning、定序後,發現經過治療之後臨床上認為已經治癒的病灶還是有原來的單株T細胞存在,但量已有減少(5/12)。對於這種少量殘餘疾病(minimal residual disease),其臨床上的意義仍有待進一步釐清。 結論發現PCR檢查對於早期MF病理診斷最棘手的建議診斷這一群病例幫助最大。當病理變化出現中等至厲害的發炎現象及真皮纖維化時,PCR的結果較有可能為陰性。我們認為單株T細胞和反應性T細胞數目的比例是影響實驗偵測一個重要因素,而真皮纖維化主要是反應病灶時間的長短及發炎的嚴重程度。此外單株TCR g 基因重組的序列具有病人專一性,適合作為病程追蹤上的指標。 Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma. The lesions of MF can be divided into patch, plaque and tumor stages as the disease progresses. Most cases are detected in early stages, when it is most difficult to distinguish the skin lesions from common inflammatory skin diseases. Although several pathologic criteria for early diagnosis have been suggested, none are 100% sensitive, and each criterion can vary in severity. The reading of pathologic slides is also subject to interobserver variability. Patients are often followed for long periods, with a definite diagnosis reached only after repeat biopsies. However, the earlier MF is diagnosed, the better the chance for a cure. Therefore, other adjunct diagnostic tools such as detection of TCR gene rearrangement are studied. The purpose of this study was to evaluate monoclonal TCR γ gene rearrangement as a method for early detection of MF. We also compared the association between detection of T-cell monoclonality and histologic paramenters for diagnosis of MF. In addition, we investigated serial biopsies of patients before and after treatment to see whether results of the test would be helpful in following patients’ clinical course. We retrospectively collected skin biopsy specimens from patients diagnosed with MF in the past 8 years in the Department of Dermatology of National Taiwan University Hospital. After excluding uninterpretable and poor quality slides, 20 specimens from 9 patients were included in the study. DNA was extracted from formalin-fixed, paraffin sections, confirmed with an internal control, and then subjected to two cycles of multiplex PCR with different primers (Mix 1 and Mix 2) to detect monoclonal TCR γ gene rearrangement. We compared the results of PCR with the histologic parameters, in attempt to find the histologic difference between PCR-positive and PCR-negative groups. The histologic diagnoses were categorized as diagnostic, consistent, suggestive, and non-diagnostic. The PCR was positive in 53% (8/15) of specimens in the patch stage, 100% (2/2) in the plaque stage, and 100% (3/3) in the tumor stage. The test was thus positive in 59% (9/17) of cases of early MF (that is, patch and plaque stages). The PCR was positive in 50% (3/6) specimens considered pathologically diagnostic, 50% (1/2) in the consistent group, 71% (5/7) in the suggestive group, and 50% (1/2) in the non-diagnostic group. Analyzing the seven pathologic parameters, we found that in PCR-negative specimens, inflammation was more severe and dermal fibrosis was commonly present, although only the latter differed significantly between PCR positive and negative groups (p = 0.01). We used laser capture microdissection to change the ratio of monoclonal T cells and reactive T cells to see if it was helpful in increasing the sensitivity of detection. We found that this method was helpful in detecting epidermal clonal T cells. In one patient, we futher cloned and sequenced the PCR product of a lesion that responded completely to treatment and found that the original T cell clone still existed, although in a decreased amount (5/12). The clinical significance of the minimal residual disease remains to be clarified. The results of PCR are more helpful in the pathologically consistent group, the group that presents the greatest diagnostic dilemma. The PCR is more likely to be negative when there is moderate to severe inflammation and particularly with dermal fibrosis. We suggest the ratio of monoclonal to reactive T cells is the most important factor in detection. Dermal fibrosis is probably related to chronicity and dependent on the degree of inflammation. The sequences of monoclonal T cell gene rearrangement are patient-specific and can thus serve as a marker for following the disease. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/55527 | 其他識別: | zh-TW |
顯示於: | 臨床醫學研究所 |
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