Aberrant Expression of Proteins Involved in Signal Transduction and DNA Repair Pathways in Lung Cancer and Their Association with Clinical Parameters

Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues.We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery.Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers

Granulomatous Slack Skin Presenting as Acquired Ichthyosis and Muscle Masses

We describe a case of granulomatous slack skin in a 31-year- old woman with an unusual presentation of acquired ichthyosis and muscular masses involving four limbs over 3 years. Vesicles and ulcerative skin nodules first appeared only 3 months prior to diagnosis. The diagnosis was confirmed after sequential biopsies of muscle, skin lesions, and lymph nodes, together with molecular genetic studies. The patient responded poorly to various therapies, including thalidomide, and died of doxorubicin-related cardiomyopathy

Study of T cell Receptor g Gene Clonality in Early Mycosis Fungoides

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.一、中文摘要 1 二、緒論 3 研究背景 3 文獻回顧 6 研究問題及重要性 11 研究假說 11 三、研究方法 2 病例收集 2 陽性對照組 12 陰性對照組 13 內部控制引子 13 實驗用引子 13 自Jurkat T cell line 萃取DNA 13 自蠟塊中萃取DNA 14 DNA定量 14 PCR 14 聚丙烯醯胺電泳 15 PCR靈敏度測試 16 Laser capture microdissection 16 洋菜膠電泳 17 DNA定序 17 DNA選殖 17 四、結果 19 病人資料分析 19 PCR靈敏度測試結果 19 第一部份 19 PCR結果 19 病理變化分類及統計分析 20 第二部份 21 LCM及PCR結果 21 第三部份 21 病人有多個檢體的PCR結果 21 DNA定序結果 22 選殖結果 22 五、討論 23 蠟塊檢體的限制 23 Multiplex PCR的設計 23 PCR的靈敏度 24 病理變化和PCR偵測之間的關係 24 Laser capture microdissection 26 病人的追蹤指標 27 蕈狀肉芽腫及乾癬的關係 28 六、展望 29 七、英文摘要 30 八、參考文獻 32 九、圖 38 圖一 PCR靈敏度 38 圖二 以內部控制引子測試檢體 39 圖三 檢體A-H進行Mix 1及Mix 2 PCR 40 圖四 檢體I-O進行Mix 1及Mix 2 PCR 41 圖五 檢體P-R進行Mix 1及Mix 2 PCR 42 圖六 檢體S、T進行Mix 1及Mix 2 PCR 43 圖七 真皮發炎細胞浸潤 44 圖八 以LCM取表皮內之Pautrier's microabscess 45 圖九 以LCM取表皮、真皮交界處之淋巴球 46 圖十 以LCM取真皮血管周圍之淋巴球 47 圖十一 檢體D、B、A以LCM取得之細胞進行Mix 1及Mix 2 PCR 48 圖十二 脫色素蕈狀肉芽腫病人治療前後檢體之病理變化 49 圖十三 脫色素蕈狀肉芽腫病人治療後檢體只取其表皮部份進行Mix 1及 Mix 2 PCR 50 圖十四 脫色素蕈狀肉芽腫病人治療後檢體其Mix 1 PCR產物挑選的12個clones比對結果 51 圖十五 12個clones中有5個clones和治療前的單株TCR序列相同 52 圖十六 12個clones中有2個clones完全相同 52 圖十七 12個clones中有3個clones類似 53 圖十八 檢體C及F分別經一次及兩次Mix 1 PCR 54 圖十九 檢體C及F分別經一次及兩次Mix 2 PCR 54 十、表 55 表一 蕈狀肉芽腫的TNMB分類 55 表二 蕈狀肉芽腫的臨床階段分類系統 56 表三 國內各醫學中心蕈狀肉芽腫佔皮膚T細胞淋巴瘤的比例 57 表四 幫助診斷蕈狀肉芽腫的病理變化條件 58 表五 蕈狀肉芽腫的病理變化分類標準 59 表六 實驗用引子 60 表七 蕈狀肉芽腫病人的臨床資料及實驗結果 61 表八 蕈狀肉芽腫病人的臨床階段 62 表九 蕈狀肉芽腫病人的病灶數目 63 表十 蕈狀肉芽腫病人的臨床表現 64 表十一 蕈狀肉芽腫病人的實驗結果分析(1) 65 表十二 蕈狀肉芽腫病人的實驗結果分析(2) 66 表十三 蕈狀肉芽腫病人的病理變化及實驗結果的比對 67 表十四 蕈狀肉芽腫病人的病理變化及實驗結果 68 表十五 exocytosis 與PCR結果的比較 69 表十六 Pautrier's microabscess 與PCR結果的比較 70 表十七 single basal lymphocyte 數目與PCR結果的比較 71 表十八 haloed lymphocyte 數目與PCR結果的比較 72 表十九 hyperconvoluted lymphocyte 數目與PCR結果的比較 73 表二十 dermal inflammation與PCR結果的比較 74 表二十一 fibrosis與PCR結果的比較 75 表二十二 病人有多個檢體的實驗結果及治療前後的變化 76 表二十三 脫色素蕈狀肉芽腫病人治療後檢體其Mix 1 PCR產物選殖結果 77 十一、附錄 一 一例成人T細胞淋巴瘤 78 二 一例大板塊類乾癬 80 三 一例親毛囊及親汗管蕈狀肉芽腫 8

Histopathologic-Molecular Correlation in Early Mycosis Fungoides Using T- Cell Receptor Gama Gene Rearrangement by Polymerase Chain Reaction with Lsaer Capture Microdissection

Background/Purpose: Early mycosis fungoides (MF) is difficult to distinguish from other benign inflammatory dermatoses. We evaluated clonal T-cell receptor (TCR) gamma gene rearrangement by polymerase chain reaction (PCR) as a surrogate to histologic diagnosis in early MF Methods: Twenty paraffin-embedded skin biopsies from nine patients diagnosed with MF were included. Two multiplex PCR encompassing various V gamma and 1 gamma regions were used to detect TCR gamma gene rearrangements. Histologic diagnoses were categorized as &quot;diagnostic&quot;, &quot;consistent&quot;, &quot; suggestive&quot;, or &quot;nondiagnostic&quot;. We compared TCR gamma PCR results with histologic parameters to determine the differences between PCR-positive and PCR-negative groups. Results: TCR gamma PCR was positive in 53% (8/15) of the patch stage, in 100% (2/2) of the plaque stage, and in 100%( 3/3) of the tumor stage. TCR gamma PCR was positive in 50%( 4/8) of the specimens in both the diagnostic and consistent of MF groups, 71% (5/7) in the suggestive of MF group. We found that inflammation was more severe in PCR-negative specimens. Papillary dermal fibrosis was common, and differed significantly between PCR-positive and PCR-negative groups (p=0. 01). T-cell monoclonality was detected in one nondiagnostic lesion in a patient with psoriasis and ME Conclusion: TCR gamma PCR allows the diagnosis of MF in patients with lymphocyte-poor lesions, suggestive of MF pathologically. TCR gamma PCR is more likely to be negative with moderate to severe inflammation, particularly with papillary dermal fibrosis. We suggest that the ratio of malignant clonal to reactive T-cells is critical for MF diagnosis

早期蕈狀肉芽腫以聚合酶鏈鎖反應偵側T細胞受體基因重組與病理組織變化之相關性

Background: Early Mycosis fungoides (MF) is difficult to distinguish from other benign inflammatory dermatoses. We evaluated clonal T-cell receptor (TCR) gene rearrangement by polymerase chain reaction (PCR) as a surrogate to histological diagnosis in early MF. Methods: Twenty paraffin -embedded skin biopsies from 9 patients diagnosed with MF were included. Two multiplex PCR encompassing various V and J讪 regions were used to detect TCR讪 gene rearrangements. Histological diagnoses were categorized as “diagnostic”, “consistent”, suggestive” ,or “non-diagnostic”. We compared TCR PCR results with histological parameters to determine the differences between PCR-positive and PCR-negative groups. Results: TCR PCR was positive in 53% (8/ 15) of the patch stage, in 100% (2/2) of the plaque stage, and in 100% (3/ 3) of the tumor stage. TCR讪 PCR was positive in 50% (4/8) of the specimens in both the diagnostic and consistent of MF groups; 71% (5/7) in the suggestive of MF group. We found that inflammation was more severe in PCR-negative specimens. Papillary dermal fibrosis was common, and differed significantly between PCR-positive and PCR-negative groups ( P = 0.01). T-cell monoclonality was detected in one non- diagnostic lesion in a patient with psoriasis and MF

Unilesional Folliculotropic/Syringotropic Cutaneous T-Cell Lymphoma Presenting as an Indurated Plaque on the Nape

Folliculotropic and syringotropic cutaneous T-cell lymphoma( CTCL) are rare variants of CTCL characterized by exclusive infiltration of a malignant clone of T cells into follicular and eccrine epithelium without T-cell epidermotropism or Pautrier microabscess formation in the interfollicular epithelium. We report a case of combined folliculotropic/ syringotropic CTCL presenting as a solitary lesion mimicking scleredema. A 58-year-old man who was generally well and without signs of infection presented with an ill-defined indurated plaque with focal erythema and skin-colored papules on the nape of the neck of an approximate 2-week duration. The indurated plaque was 60 mm × 100 mm in area ( Fig. 1). Initial clinical impression was scleredema, but there was no history of diabetes mellitus. A skin biopsy revealed dense lymphocytic infiltration exclusively around the follicles and eccrine coils as well as invasion into the follicular and eccrine epithelium, but no epidermotropism ( Fig. 2a). Infiltrating lymphocytes were of various sizes and some showed atypia and perinuclear halos (Fig. 2b,c). Polymerase chain reaction for T-cell receptor γ gene rearrangement showed a clonal T-cell population within the specimen. Complete blood cell count and biochemistry tests revealed no abnormalities. Chest X-ray and abdominal computed tomographic scans were normal. showed a clonal T- cell population within the specimen. Complete blood cell count and biochemistry tests revealed no abnormalities. Chest X -ray and abdominal computed tomographic scans were normal. Psoralen photochemotherapy, radiotherapy, and systemic chemotherapy have been reported to be effective. 1, 2 Other topical therapies were limited in efficacy owing to limited penetration into deeper adnexal structures. Radiotherapy proved effective and well tolerated in our case with only mild radiation dermatitis found within the treatment area. In a previous study, syringotropism and folliculotropism were noted in 30% and 57% of mycosis fungoides specimens, and might be more prominent than the intensity of epidermotropism. 5 Occasionally, mycosis fungoides can present only with adnexotropism without the typical epidermotropism, as in our case. We did not find follicular mucinosis or syringohyperplasia, which have been reported with adnexotropism. Our patient presented with an unusual indurated plaque that could be easily misdiagnosed without deep biopsy examination. We confirm a previously reported suggestion that radiotherapy is the treatment of choice for folliculotropic/ syringotropic CTCL