摘要
背景：皮肤相关性淋巴样组织(SALT)是人体重要的免疫器官，皮肤是淋巴网状组织之一，原发于皮肤的淋巴瘤约占结外淋巴瘤的第3位，仅次于胃肠和口咽环。原发性皮肤淋巴瘤(PCL)包括皮肤T细胞淋巴瘤(CTCL)和皮肤B细胞淋巴瘤(CBCL)，其具体分类复杂，不同类型预后差别显著。如果诊断时未进行具体分析，将导致对部分患者进行过度治疗引起较大副作用，或未能积极治疗耽误病情。怎样准确而相对简单地进行分类是临床医生最关心的。找寻一些能反映皮肤淋巴瘤恶性程度且操作较简单、成本较低、特异性和敏感性均较满意的指标是皮肤科工作者面临的难题。
目的：通过比较蕈样肉芽肿和非蕈样肉芽肿CTCL的临床、病理和免疫组化特点，初步探讨CTCL的分类及其免疫组化特点，为临床诊断和治疗提供一定帮助。
方法：在我科1980.1~2002.6考虑皮肤淋巴瘤的存档蜡块中结合临床、病理及免疫表型确诊CTCL，参照Willemze等提出的原发性皮肤淋巴瘤新分类法(简称新分类法)，分为MF组、CD30+CTCL组及CD30-CTCL组。应用图像分析系统定量检测并比较p53、肿瘤坏死因子α(TNF-α)、Ki-67的免疫组化表达、核异型性及核仁组成区相关嗜银蛋白(AgNOR)在CTCL各类型的差异，并随访患者了解其病程变化。阴性对照用PBS代替单抗，正常对照选用慢性皮炎病例，阳性对照选用大肠癌组织。
结果：1. 按照Willemze新分类法对34例CTCL进行了分类，分为MF和非MFCTCL两大类，其中MF19例，非MF15例(CD30+CTCL 6例，CD30-CTCL 9例)。2. 检测了p53，TNF-α，Ki-67在各组病例中的表达，通过统计分析，显示了这三个指标在不同类型的皮肤T细胞淋巴瘤的表达具有不同程度的统计学差异。3. AgNOR在各组间也具有显著性差异。4. 肿瘤细胞核异型性检测显示除MF组与CD30+CTCL组外，其它不同类型之间细胞核的平均光密度、积分光密度及形状因子有统计学差异。
结论：1. Willemze新分类法对CTCL的临床诊断、分类及指导治疗意义较大。2.图像分析能较客观地反映免疫组化表达强度，可对进一步分析CTCL的恶性程度提供参考。3. p53、Ki-67的AOD、IOD值在各组间表达强度有差异，随着CTCL的恶性程度增加而增高，对CTCL的诊断、鉴别诊断及判断恶性程度有不同程度的意义，并有一定程度的相关性，而TNF-α的差异不明显。4. AgNOR的定量检测能较好反映肿
瘤的演进过程，为CTCL的诊断、分类、评估预后提供了较为客观的依据。5. 肿瘤细胞核的异型性检测对CTCL的诊断、鉴别诊断及分类有一定意义
Background: The skin-associated lymphoid tissue(SALT) is an important immune part in humans. Primary cutaneous lymphoma (PCL) is the third most common group of extranodal non-Hodgkin lymphomas just after the groups of primary gastrointestinal and oropharynx lymphomas. The classification of PCL is very complex，which generally is classified into cutaneous T-cell lymphoma(CTCL)and cutaneous B-cell lymphoma (CBCL).
It is showed that there is considerable variation in histology,phenotype and prognosis in PCL.Patients with PCL would be treated based on a correct classification. So it is very important that we investigate some indices to reflect maliganant degrees of of PCL.
objective:By comparing the histological, immunohistochemical and prognostic features in mycosis fungoides(MF) and non-MF of CTCL,it provides some evidences to make diagnosis and therapy of CTCL.
Methods:The specimens were collected from the cases of PCL which were diagnoised clinically and histologically from Jan,1980 to June,2002 in our department. With immunohistochemical staining, we classified the PCL into CTCL and CBCL.Then CTCL were classified into MF,non-MF(including CD30+CTCL and CD30-CTCL). The immunohistochemistry，quantitative image analysis of p53, tumor necrosis factor α (TNF-α), Ki-67, silver stained nucleolar organizer region (AgNOR) and the nucleus of pathological morphology were studied in CTCL. The negative control was used phosphate buffered saline (PBS) to replace monoclonal antibody. The normal control included the specimen from the cases of chronic dermatitis.
Results:
1. According to the clinical characteristics, pathology and immunohistochemistry, we divided 34 cases of CTCLs into the MF , non-MF CD30+CTCL and non-MF CD30-CTCL groups. Among them, MF was 19 cases, non-MF CD30+CTCL 6 cases and non-MF CD30-CTCL 9 cases.
2. The expression of p53,TNF-α ,Ki-67 was examined in all cases respectively, and was analyzed through statistic, showing the significant difference among various types.
The expression of p53 in CTCL increased significantly (p<0.01). The average optical density(AOD) and integrated optical density(IOD)of p53 were higher in MF group than in normal group(p<0.01),and the similar situation happened between non-MF CD30-CTCL and non-MF CD30+CTCL groups.But there was not statistic difference between MF group and CD30+ CTCL group.
The AOD and IOD of TNF-α in MF group were higher than those in normal group(p<0.01),but there was little difference between MF and CD30+CTCL,CD30+CTCL and CD30-CTCL, or MF and CD30-CTCL respectively.
There was significant difference of AOD and IOD of Ki-67 between normal and MF groups, MF and CD30-CTCL groups,CD30+CTCL and CD30-CTCL groups (p<0.01 or p<0.05).
3. The AgNORs/cell ,AOD and IOD of AgNOR in four groups showed distinctive difference.They increased according to the malignant degrees of CTCL.But there was little difference in AgNORs/cell and AOD between MF and CD30+CTCL groups.
4. The AOD,IOD, and form factor (FF) of the nucleus between MF and normal groups showed the statistical difference,while between CD30+CTCL and CD30-CTCL groups.only the IOD of the nucleus was significantly different.
Conclusion: At present study, we successfully completed classification of the CTCL,and analysed the difference of p53,TNF-α,Ki-67,AgNOR and nucleus measure among different groups of CTCL.The results suggested that: the differences of p53,Ki-67,AgNORs count and nucleus measure reflected the malignant degrees of CTCL. The methods of immunohistochemistry,silver stain and nucleus measure were simple and cheap.They could be applied for the clinical diagnosis and guideline of treatment
引文
1. Willemze R，Meijer CJ. EORTC classification for primary cutaneous lymphomas: a comparison with the R.E.A.L. Classification and the proposed WHO Classification. Ann-Oncol, 2000,11 Suppl 1: 11~15.
2. Willemze R, Meijer CJ.Rationale of a new classification for the group of primary cutaneous lymphomas. Semin Cutan Med Surg, 2000 Jun,19(2): 71~77.
3. Willemze R, Meijer CJ.EORTC classification for primary cutaneous lymphomas: the best guide to good clinical management. European Organization for Research and Treatment of Cancer. Am J Dermatopathol,1999 Jun,21(3): 265~273.
4. Willemze R, Kerl H, Sterry W,et al.EORTC classification for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer. Blood, 1997 Jul 1,90(1): 354~371.
5. Bruin PC,Gruss HJ,Valk P, et al. CD30 expression in normal and neoplastic lymphoid tissue: biological aspects and clinical implications. Leukemia,1995 Oct,9(10): 1620~1627.
6. Grange F,Hedelin G,Joly P, et al.Prognostic factors in primary cutaneous lymphomas other than mycosis Fungoides and the Sezary Syndrome.Blood,1999,93(11):3637 ~3642.
7. Iggo R, Gatter K, Bartek J,et al.Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet,1990,335(8691):675.
8. 张士发,许静,王亚坤,等.蕈样肉芽肿皮损p55及p75肿瘤坏死因子受体表达.中国皮肤性病学杂志,1999,13(2):81~82.
9. Beljaards RC,Meijer CJLM,Scheffer E,et al.Prognostic significance of CD30 (Ki-1/Ber-H2) expression of primary cutaneous large-cell lymphomas of T-cell origin.A clinicopathologic and immunohistochemical study in 20 patients.Am J Pathol,1989,135:1169.
10. 赵辩主编.临床皮肤病学,第3版.南京:江苏科学技术出版社.2001:1204.
11. 邱丙森.非MF/SS皮肤恶性淋巴瘤及其治疗原则.临床皮肤科杂志,2002,31(5)：335~336.
12. Willemze R,Beljaards RC,Rijlaarsdam U.Classification of primary cutaneous large cell lymphomas,Dermatol Clin,1994,12(2):361~373.
13. Lennert K.Non-Hodgkin Lymphoma:Prizipien anwendung under Kiel classification,Verh Dtsch Ges Path 1992,76:1.
14. Sander CA,Flaig MJ,Kaudewitz F,et al.The revised European-American classification of lymphoid neoplasma(REAL):A preferred approach for the classification of cutaneous lymphomas.Am J Dermatopathol.1999,21(3):274~278.
15. Harris NL,Jaffe ES,Stein H,et al.A revised European-American classification of lymphoid neoplasmas:a proposal from the International Study Group.Blood, 1994,84(5):1361~1392.
16. Notton AJ.Classification of cutanoeus lymphoma a critical appraisal of recent proposals.Am J Dermatopathol.1999,21(3):279~287.
17. Willemze R, Kerl H, Sterry W,et al. EORTC classification for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer. Blood,1997 Jul 1,90(1): 354~371.
18. 邱丙森,皮肤恶性淋巴瘤的分类:近代提案的分析和评价.临床皮肤科杂志,2000,29(4):242~244.
19. 邱丙森.皮肤恶性淋巴瘤的新分类.临床皮肤科杂志,1998,27(2):133~135.
20. Beljaards RC,Kaudewitz P,Berti E,et al.Primary cutaneous CD30-positive large cell lymphoma :Definition of a new type of cutaneous lymphoma with a favorable prognosis.A European multicenter study on 47 cases.Cancer,1993,71:2097.
21. Beljaards RC,Meijer CJLM,Putte SCJ,et al.Primary cutaneous T-cell lymphomas.Clinicopathologic features and prognostic parameters of 35 cases other than mycosis fungoides and CD30-positive large cell lymphoma. J Pathol,1994,172:53.
22. Lim SM,Dim L,Smith R,et al.Morphmoetric analysis of nucleolar organizer regions in benign and malignant peritoneal effusion using backscattered electron microscopy.J Pathol,1992,166:53~59.
23. Rao J, Seligson D, Hemstreet GP.Protein expression analysis using quantitative fluorescence image analysis on tissue microarray slides.Biotechniques,2002 Apr,32(4): 924~932.
24. 王树党.图像分析仪在形态计量学研究中的使用体会.中国体视学与图像分析,2001,6(4):253~255.
25. 杨建茹.平均光密度在医学图像分析中的定量.中国医学影像技术,1999,15(4):322~323.
杨建茹,陈跃,管曾伟,等.对乳腺癌细胞形态定量分析中形状因子参数的研究.中国医
26. 学影像技术,2000,16(2):130~131.
27. Castagnetta LA, Traina A.Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy.Anal Quant Cytol Histol,1999 Feb,21(1): 59~62.
28. Roels S, Tilmant K, Van-Daele A,et al.Proliferation, DNA ploidy, p53 overexpression and nuclear DNA fragmentation in six equine melanocytic tumours. J Vet Med A Physiol Pathol Clin Med.,2000 Sep,47(7):439~448.
29. Liu T J,EL-Naggar A K,Modonnell T J,et al.Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck[J].Cancer Reseach,1995,55:3117~3122.
30. Wang Y K,Ji X L ,Gu Y G,et al. p53 and PCNA expression in glandular dilatation of gastric mucosa .China Natl New Gastroenterol,1996,2(2):106~108.
31. Evan G,Littlewood T.A matter of life and cell death .Science,1998,281:1317~1322.
32. Pines J.p21 inhibitas cyclin shock.Name,1994,369:520~521.
33. Zhang GJ, Kimijima I, Abe R,et al.Correlation between the expression of apoptosis-related bcl-2 and p53 oncoproteins and the carcinogenesis and progression of breast carcinomas.Clin Cancer Res,1997 Dec,3(12 Pt 1):2329~2335.
34. Villuendas R,Piris MA,Orradre JL,et al.P53 protein expression in lymphomas and reactive lymphoid tissue,J Pathol,1992,166(3):235~243.
35. Kanavaros P,Ioannidou D,Tzardi M,et al.Mycosis Fungoides:expression of C-myc p62,p53,bcl-2 and PCNA proteins and absence of association with Epstein-Barr virus.Pathol Res Pract,1994,190(8):767~774.
36. Bodey B, Groger AM, Bodey B,et al. Immunohistochemical detection of p53 protein overexpression in primary human osteosarcomas.Anticancer Res,1997 Jan-Feb,17(1A): 493~498.
37. Masuda M.Prognostic significance of ki-67 labeling index in urothelial tumors of the renal pelvis and ureter.J urol,1996,155:1877~1884.
38. Berti E,Tomasini D,Vermeer MH,et al.Primary cutaneous CD8-positive epidermotropic cytotoxic T cell lymphomas.A distinct clinicopathological entity with an aggressive clinical behavior.Am J Pathol, 1999 Aug,155(2):483~492.
39. Edstrom,DW.Photodynamic therapy with topical 5-aminolevulinic acid for mycosis fungoides: clinical and histological response.Acta-Derm-Venereol,2001 Jun-Jul,81(3): 184~188.
40. Quintanilla-Martinez L,Thieblemont C,Fend F,et al.Mantle cell lymphomas lack expression of P27 kipl,a cyclin-dependant kinase inhibitor.Am J Pathol,1998,153:175~182.
41. Holte H,Suo Z,Smeland EB,et al.Prognostic value of lymphoma-specific S-phase fraction compared with that other cell proliferation markers.Acta Oncol,1999, 68(4) :495~503.
42. Logan TF,Gooding WE,Whiteside TL,et al.Biological response modulation by tumor necrosis factor alpha(TNF alpha) in a phase Ib trial in cancer patients.J Immunother,1997,20(5):387~398.
43. Nooijen PTGA,Mnusama ER,Eggermont AMM,et al.Synergistic effects of TNF-α and melphalan in an isolated limb perfusion model of rat sarcoma:a histopathological immunohistochemical and electron microscopical study.Br J Cancer,1996,74(12): 1908~1915.
44. Wong GH,Strategies for manipulating apoptosis for cancer therapy with tumor necrosis factor and lymphotoxin.J Cell Biochem,1996,60(1):56~60.
45. Zembara M,Siedar M,Ruggiero I,et al.The MHC class-II and CD44 molecules are involved in the induction of tumor necrosis factor(TNF) gene expression by human monocytes stimulated with tumor cells.Int J Cancer,1994,56:269.
46. Underwood JCE,Giri DD.Nuclear organizer regions as diagnostic discriminantis for malignancy .J Pathol,1988,155(2):95~97.
47. Derzini M,Romagnoli P,Mingazzim P,et al.Interphase nucleolar organize region distribution as a diagnostic paro meter to differentiate benign from malignant epithelial tumors of human intestine.Virehows Arch B cell Pathol,1998,54(6):334.
48. Arora DS,Ramsdale J,Lodge JP,et al.p53 but not bcl-2 is expressed by most cholangiocarcinomas:a study of 28 cases.Histopathology,1999,34:497~501.
49. Kastan MB,Onyekwere O,Sidransky D,et al.Participation of p53 protein in the cellular response to DNA damage.Cancer Res,1991,51:6304.
50. Hall PA,Crocker J,Watts A,et al.Acomparision of nucleolar organizer region staining and ki-67 immunostaining in non-Hodgkin's lymphoma.Histopathology ,1988,12:373.
51. Baak JP. The framework of pathology: good laboratory practice by quantitative and molecular methods.J-Pathol,2002,198(3):277~283.
