SLIT2 promoter methylation analysis in neuroblastoma, Wilms' tumour and renal cell carcinoma

The 3p21.3 RASSF1A tumour suppressor gene (TSG) provides a paradigm for TSGs inactivated by promoter methylation rather than somatic mutations. Recently, we identified frequent promoter methylation without somatic mutations of SLIT2 in lung and breast cancers, suggesting similarities between SLIT2 and RASSF1A TSGs. Epigenetic inactivation of RASSF1A was first described in lung and breast cancers and subsequently in a wide range of human cancers including neuroblastoma, Wilms' tumour and renal cell carcinoma (RCC). These findings prompted us to investigate SLIT2 methylation in these three human cancers. We analysed 49 neuroblastomas (NBs), 37 Wilms' tumours and 48 RCC, and detected SLIT2 promoter methylation in 29% of NB, 38% of Wilms' tumours and 25% of RCC. Previously, we had demonstrated frequent RASSF1A methylation in the same tumour series and frequent CASP8 methylation in the NB and Wilms' tumour samples. However, there was no significant association between SLIT2 promoter methylation and RASSF1A or CASP8 methylation in NB and RCC. In Wilms' tumour, there was a trend for a negative association between RASSF1A and SLIT2 methylation, although this did not reach statistical significance. No associations were detected between SLIT2 promoter methylation and specific clinicopathological features in the tumours analysed. These findings implicate SLIT2 promoter methylation in the pathogenesis of both paediatric and adult cancers and suggest that further investigations of SLIT2 in other tumour types should be pursued. However, epigenetic inactivation of SLIT2 is less frequent than RASSF1A in the tumour types analysed.     

Wilms' tumor and renal cell carcinoma in retroperitoneal teratoma.

A patient with a malignant retroperitoneal teratoma that contained Wilms' tumor and renal cell carcinoma is described. The rarity of renal tissue in teratoma, and the possible mode of development of Wilms' tumor and renal cell carcinoma in such a tumor are discussed.     

Recurrent renal cell carcinoma arising in Wilms' tumor.

A 19-month-old black girl had a radical nephrectomy for a Wilms' tumor that contained areas of epithelium indistinguishable from renal cell carcinoma. She was treated with chemotherapy but subsequently had pulmonary metastases develop and massive abdominal recurrence. The recurrent tumor was histologically renal cell carcinoma with no identifiable Wilms' tumor elements. The child died with recurrent and metastatic tumor 13 months after nephrectomy. Pathologic, immunoperoxidase, and flow cytometric studies of this unusual case are presented.     

Distinctive glycolipid patterns in Wilms' tumor and renal cell carcinoma.

Glycolipid patterns were analysed chromatographically in Wilms' tumor and renal cell carcinoma tissues and compared with those of uninvolved tissue. Ganglioside GM3 was found to be increased in both cancer tissues, whereas sulfatides accumulated only in renal cell carcinoma, as reported earlier. Neolactotetraosylceramide was detected in both cancer tissues, but not in the uninvolved kidney tissues. In four cases of Wilms' tumors, only a low level of sulfotransferase towards galactosylceramide was found in one case, while no activity was detected in the three other cases. Present results show that the increased sulfatide(s) in the renal cell carcinoma and the deficiency of the sulfatides in Wilms' tumors appear to be biochemical characteristics of histologically different carcinomas.     

Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer

The 3p21.3 tumour suppressor gene (TSG) RASSF1A is inactivated predominantly by promoter methylation and rarely by somatic mutations. Recently we demonstrated that epigenetic inactivation of RASSF1A is frequent in both clear cell and papillary adult renal cell carcinomas (even though 3p21.3 allele loss is rare in papillary tumours). Wilms' tumour is the most common childhood kidney tumour, but relatively little is known about its molecular pathogenesis. Thus TSGs such as WT1, p16(CDKN2a) and p53 are inactivated in only a minority of cases. In view of the involvement of RASSF1A in adult renal cancers we investigated RASSF1A as a candidate Wilms' TSG. We detected RASSF1A hypermethylation in 21 of 39 (54%) primary Wilms' tumours. 3p21.3 allele loss was not detected in nine informative Wilms' tumours (five with RASSF1A methylation). In contrast to RASSF1A, only a minority (10.3%) of Wilms' tumours demonstrated p16 promoter methylation. As chromosome 3p allele loss is frequent in colorectal cancer, we proceeded to investigate RASSF1A promoter methylation in colorectal cancer and detected RASSF1A methylation in 80% (4/5) colorectal cancer cell lines and 45% (13/29) primary colorectal cancers. There was no correlation between RASSF1A and p16 methylation in colorectal cancer. We have demonstrated that RASSF1A inactivation is the most frequent genetic or epigenetic event yet reported in Wilms' tumourigenesis and that allelotyping studies may fail to identify regions containing important TSGs.     

Molecular profiling and classification of sporadic renal cell carcinoma by quantitative methylation analysis.

PURPOSE: Preoperative histologic classification of solid renal masses remains limited with current technology. We determine the utility of molecular profiling based on quantitative methylation analysis for characterization of sporadic renal cell carcinoma. EXPERIMENTAL DESIGN: Primary renal cell carcinomas representing three different histologic subtypes were obtained from a total of 38 patients who underwent radical nephrectomy for suspected malignant disease. Genomic DNA was isolated from tumors and was subjected to sodium bisulfite modification. The normalized index of methylation (NIM) for each sample was determined by quantitative real-time methylation-specific PCR at 17 different gene promoters. Hierarchical cluster analysis was performed by using an unsupervised neural network with binary tree topology. RESULTS: The majority of gene promoters that were analyzed in this study demonstrated very low levels of methylation (NIM <1.0). The RASSF1A gene promoter, however, was methylated in 30 of 38 (79%) cases. The frequency of RASSF1A methylation in papillary, clear-cell, and oncocytoma subtypes was 100, 90, and 25%, respectively. The highest levels of RASSF1A methylation were observed in the papillary (mean NIM = 78.9) and clear-cell (mean NIM = 13.4) subtypes. The vast majority of oncocytomas were completely unmethylated, and none demonstrated >1% methylation (mean NIM = 0.11). Hierarchical cluster analysis based on quantitative methylation levels resulted in stratification of sporadic renal cell carcinomas into their discrete histologic subtypes. CONCLUSIONS: Classification of sporadic renal cell carcinomas into histologic subtypes can be accomplished via multigene quantitative methylation profiling. Validation of this approach and selection of appropriate methylation markers may ultimately lead to use of this technology in the preoperative assessment of suspicious renal masses.     

Metastatic renal cell carcinoma without evidence of a primary renal tumour

Although metastases are common in patients with renal cell carcinoma (rcc), it is extremely rare for patients to present with metastatic rcc (mrcc) without evidence of a primary mass in the kidney. Two cases of mrcc with no detectable primary renal mass are reported here. Both patients had bilateral native kidneys in situ and no significant prior urologic history. The first patient presented with a hip fracture and was found to have multiple radiologic bony and lung metastases. Biopsy of a mass involving the pubic bone demonstrated clear cell mrcc. Multiple scans by computed tomography (ct) and confirmatory imaging by magnetic resonance demonstrated no renal mass. This first patient had disease stabilization for 18 months on sunitinib and was still alive at last follow-up. The second patient was diagnosed with clear-cell mrcc after thickened synovium was discovered and biopsied during a knee arthroplasty. Multiple scans by ct in this second patient demonstrated no primary renal mass. Sunitinib and radiotherapy to the knee lesion were initiated, but unfortunately, the patient deteriorated clinically and passed away from disease progression shortly after diagnosis. Because of the rare nature of these cases, a standardized course of action has not yet been established. However, we hypothesize that it is reasonable to manage metastases in these patients by following established mrcc protocols.     

Multigene methylation analysis for detection and staging of prostate cancer.

PURPOSE: Aberrant gene promoter methylation profiles have been well-studied in human prostate cancer. Therefore, we rationalize that multigene methylation analysis could be useful as a diagnostic biomarker. We hypothesize that a new method of multigene methylation analysis could be a good diagnostic and staging biomarker for prostate cancer. EXPERIMENTAL DESIGN: To test our hypothesis, prostate cancer samples (170) and benign prostatic hyperplasia samples (69) were examined by methylation-specific PCR for three genes: adenomatous polyposis coli (APC), glutathione S-transferase pi (GSTP1), and multidrug resistance 1 (MDR1). The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. We further investigated whether methylation score (M score) can be used as a diagnostic and staging biomarker for prostate cancer. The M score of each sample was calculated as the sum of the corresponding log hazard ratio coefficients derived from multivariate logistic regression analysis of methylation status of various genes for benign prostatic hyperplasia and prostate cancer. The optimal sensitivity and specificity of the M score for diagnosis and for staging of prostate cancer was determined by receiver-operator characteristic (ROC) curve analysis. A pairwise comparison was employed to test for significance using the area under the ROC curve analysis. For each clinicopathologic finding, the association with prostate-specific antigen (PSA) failure-free probability was determined using Kaplan-Meier curves and a log-rank test was used to determine significance. The relationship between M score and clinicopathologic findings was analyzed by either the Mann-Whitney U test, Kruskal-Wallis test, or the Spearman rank correlation test. RESULTS: The frequency of positive methylation-specific PCR bands for APC, GSTP1, and MDR1 genes in prostate cancer samples was 64.1%, 54.0%, and 55.3%, respectively. In benign prostatic hyperplasia samples, it was 8.7%, 5.8%, and 11.6%, respectively. There was a significant correlation of M score with high pT category (P < 0.001), high Gleason sum (P < 0.001), high preoperative PSA (P = 0.027), and advanced pathologic features. For all patients, the M score had a sensitivity of 75.9% and a specificity of 84.1% as a diagnostic biomarker using a cutoff value of 1.0. In patients with low or borderline PSA levels (<10.0 ng/mL), the M score was significantly higher in prostate cancers than in benign prostatic hyperplasias (2.635 +/- 0.200 and 0.357 +/- 0.121, respectively). ROC curve analysis revealed that the M score had a sensitivity of 65.4% and a specificity of 94.2% when 1.0 was used as a cutoff value. For all patients, M score can distinguish organ-confined (< or =pT(2)) from locally advanced cancer (> or =pT(3)) with a sensitivity of 72.1% and a specificity of 67.8%. Moreover, considering patients with PSA levels of <10 ng/mL, the M score has a sensitivity of 67.1% and a specificity of 85.7%. The ROC curve analysis showed a significant difference between M score and PSA (P = 0.010). CONCLUSIONS: This is the first report demonstrating that M score is a new method for multigene methylation analysis that can serve as a good diagnostic and staging biomarker for prostate cancer.     

3号染色体短臂抑癌基因与肾透明细胞癌

人类3号染色体短臂(3p)上抑癌基因的异常改变(如缺失突变、转录失活或沉默等)被认为是包括肾癌尤其是肾透明细胞癌在内的人类多种肿瘤起始发生的关键步骤,它们与肿瘤的后续发展演变亦关联紧密.大量研究发现并证实诸如VHL、RASSF1A、SEMA3B、SETD2、PBRM1、NPRL2、GPX1等3p基因的缺失或下调在肾透明细胞癌的形成与发展中扮演了极为重要的角色,这为进一步阐释肾透明细胞癌分子形成机制奠定了基础,是当前研究的重要方向.关于其具体调控路径及作用机制的研究除部分基因已初步明晰外,余尚在继续进行中.     

抑癌基因甲基化在肾细胞癌研究中的新进展

抑癌基因启动子高度甲基化被认为是除突变和缺失以外的抑癌基因功能失活的关键机制,在肿瘤的发生和发展中起重要作用,已成为目前肿瘤病因学基础研究的热点.肾细胞癌是泌尿系统最常见的恶性肿瘤之一,近年来也有许多文献报道了肾细胞癌中抑癌基因的异常甲基化情况.肾细胞癌临床发病隐匿,出现症状时多为晚期,肾细胞癌相关基因异常甲基化检测有望为肾癌的早期无创诊断提供新的途径.针对肾癌对放、化疗效果均不敏感的特点,改变DNA甲基转移酶活性和抑癌基因甲基化状况可作为肾细胞癌辅助治疗的一种新思路.本文对抑癌基因甲基化与肾细胞癌关系的研究进展作一综述,介绍DNA甲基化在肿瘤发生过程中的作用及机理;总结近7年来肾细胞癌中抑癌基因甲基化的研究情况和肾细胞癌独特的甲基化谱,并着重介绍了新近报道的HOXB13,HAI2/SPINT2,CDH1,CTNNG/JUP四个基因启动子异常甲基化与肾细胞癌的关系;阐述DNA甲基化研究对肾细胞癌的临床意义.     

乳头状肾细胞癌的临床病理特征及预后

目的 探讨乳头状肾细胞癌(PRCC)的临床病理特点、免疫表型和预后.方法 回顾性分析19例PRCC患者的临床和病理资料,对肿瘤组织进行免疫组化染色并鉴定其免疫表型,对患者进行随访.结果 PRCC临床上症状多不明显,常在体检时发现.光镜下PRCC组织主要由多少不等的乳头状和管状结构组成,被覆单层立方或多层柱状肿瘤细胞,乳头轴心及间质内可见泡沫细胞、砂粒体沉积,部分肿瘤细胞胞浆内可见含铁血黄索.Ⅰ型12例,Fuhrman核分级均为1～2级;Ⅱ型7例,其中5例Fuhrman核分级为3～4级.Ⅰ型和Ⅱ型PRCC不同程度地表达vimentin、EMA、CKpan、CK7、CD10和p504s,但均不表达34βE12和CK20.16例获得随访的患者中,3例分别于术后3、8和9个月死于肿瘤转移,且均为Ⅱ型PRCC;2例死于其他疾病;其余11例患者均为无瘤生存.结论 PRCC的两种亚型在形态学、免疫表型和预后上有差别,与Ⅰ型PRCC比较,Ⅱ型较Ⅰ型预后不良.PRCC细胞核分级高、出现肉瘤样成分或有透明细胞癌结构可能提示肿瘤具有侵袭性,预后不良.     

Serological analysis of human renal cell carcinoma

Serological analysis of cDNA expression libraries (SEREX) has proven to be a useful technique in the quest to elucidate the repertoire of immunogenic gene products in human cancer. We have applied the SEREX method to human renal cell carcinoma (RCC) in order to identify associated immunogenic gene products. cDNA expression libraries were prepared from a RCC tumor, a RCC cell line and human testis. The 3 libraries were screened with sera from 35 RCC patients and 15 healthy controls. Approximately 4.5 x 10(6) phage plaques were screened resulting in 234 positive clones, which corresponded to 74 different gene products. The seroreactivity toward 49 of these antigens was assessed. Seroreactivity to 21 (43%) of the antigens was similar in RCC patients and healthy controls, 9 antigens (18%) elicited antibodies more frequently and 19 antigens (39%) solely in RCC patients. In the reverse setting, reactivity of RCC patients' sera was tested against a panel of 44 previously identified "tumor-associated" antigens via the SADA (serum antibody detection array) method; 6 antigens reacted with RCC patients' and healthy donors' sera, 8 were reactive only with RCC patients' sera. From the 27 antigens identified by SEREX and SADA, which did not react with sera from healthy controls, 10 antigens reacted with a significant proportion of RCC patients' sera and 77% of RCC patients' sera reacted at least with one of these antigens. Sera from patients with non-malignant renal diseases or an autoimmune disease did not react with these 10 antigens.     

Flow cytometric analysis of renal cell carcinoma

Forty cases of renal cell carcinoma were studied retrospectively by flow cytometry and DNA contents of the cancer cells were measured. The results indicated that the incidence of aneuploid tumor was 57.5% (23/40), diploid tumor or quasi-diploid tumor 42.5% (17/40). DNA ploidy was strictly correlated to histopathological grade, clinical stage, cancer cell type and survival time. Therefore, analysis of cellular DNA ploidy of renal cell carcinoma is of prognostic value for renal cell carcinoma.     

透明细胞乳头状肾细胞癌临床病理特征分析

目的:探讨透明细胞乳头状肾细胞癌（CCPRCC）的临床及病理学特征。方法:回顾性分析6例CCPRCC的临床特征、组织学特点及免疫表型,并复习相关文献。结果:6例肿瘤均位于肾皮质内,肿瘤边界清晰,有纤维性包膜,切面灰红或灰黄色,部分呈囊性改变。镜下肿瘤呈乳头状、管状、囊性及实性混合性生长,胞浆透明,细胞核远离基底膜,世界卫生组织（World Health Organization,WHO）/国际泌尿病理协会（International Society of Urological Pathology,ISUP)分级为1级或2级。免疫表型:6例肿瘤组织均表达CK7、PAX-8和34βE12,CAIX呈“杯状”或完全膜阳性表达。1例CD10部分阳性,其余5例CD10阴性。所有病例AMACR和TFE3均为阴性。结论:透明细胞乳头状肾细胞癌是一种少见的肾肿瘤,呈惰性生物学行为。形态上应与具有透明细胞和乳头状结构的肾细胞癌鉴别,可借助免疫组织化学和分子遗传学检测予以鉴别。