Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas

DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22-q21.3), GARP (11q13.5-q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate genes are likely to have distinct functional roles in the carcinogenesis and progression of CIN- and MIN-type sporadic CRCs and may be involved in the differential response of CIN- and MIN-type tumor cells to (adjuvant) therapy, such as 5-fluorouracil.     

Molecular genetic alterations in hamartomatous polyps and carcinomas of patients with Peutz-Jeghers syndrome

AIM: To investigate whether mutations in the STK11/LKB1 gene and genes implicated in the colorectal adenoma-carcinoma sequence are involved in Peutz-Jeghers syndrome (PJS) related tumorigenesis. METHODS: Thirty nine polyps and five carcinomas from 17 patients (from 13 families) with PJS were analysed for loss of heterozygosity (LOH) at 19p13.3 (STK11/LKB1 gene locus), 5q21 (APC gene locus), 18q21-22 (Smad4 and Smad2 gene locus), and 17p13 (p53 gene locus), and evaluated for immunohistochemical staining of p53. In addition, mutational analysis of K-ras codon 12, APC, and p53 and immunohistochemistry for Smad4 expression were performed on all carcinomas. RESULTS: LOH at 19p was seen in 15 of the 39 polyps and in all carcinomas (n = 5). Interestingly, six of the seven polyps from patients with cancer had LOH, compared with nine of the 31 polyps from the remaining patients (p = 0.01). In one polyp from a patient without a germline STK11/LKB1 mutation, no LOH at 19p or at three alternative PJS candidate loci (19q, 6p, and 6q) was found. No LOH at 5q was observed. However, mutational analysis revealed an APC mutation in four of the five carcinomas. LOH at 17p was not seen in polyps or carcinomas; immunohistochemistry showed expression of p53 in one carcinoma and focal expression in three polyps. At subsequent sequence analysis, no p53 mutation was found. One carcinoma had an activating K-ras codon 12 mutation and another carcinoma showed 18q LOH; however, no loss of Smad4 expression was seen. CONCLUSIONS: These results provide further evidence that STK11/LKB1 acts as a tumour suppressor gene, and may be involved in the early stages of PJS tumorigenesis. Further research is needed to see whether LOH in PJS polyps could be used as a biomarker to predict cancer. Differences in molecular genetic alterations noted between the adenoma-carcinoma sequence and PJS related tumours suggest the presence of a distinct pathway of carcinogenesis.     

Ki-67 MIB1 labelling index and the prognosis of primary TaT1 urothelial cell carcinoma of the bladder

AIMS: To evaluate whether ki-67 labelling index (LI) has independent prognostic value for survival of patients with bladder urothelial tumours graded according to the 2004 World Health Organisation classification. METHODS: Ki-67 LI was evaluated in 164 cases using the grid counting method. Non-invasive (stage Ta) tumours were: papilloma (n = 5), papillary urothelial neoplasia of low malignant potential (PUNLMP; n = 26), and low (LG; n = 34) or high grade (HG; n = 15) papillary urothelial carcinoma. Early invasive (stage T1) tumours were: LG (n = 58) and HG (n = 26) carcinoma. Statistical analysis included Fisher and chi2 tests, and mean comparisons by ANOVA and t test. Univariate and multivariate survival analyses were performed according to the Kaplan-Meier method with log rank test and Cox's proportional hazard method. RESULTS: Mean ki-67 LI increased from papilloma to PUNLMP, LG, and HG in stage Ta (p<0.0001) and from LG to HG in stage T1 (p = 0.013) tumours. High tumour proliferation (>13%) was related to greater tumour size (p = 0.036), recurrence (p = 0.036), progression (p = 0.035), survival (p = 0.054), and high p53 accumulation (p = 0.015). Ki-67 LI and tumour size were independent predictors of disease free survival (DFS), but only ki-67 LI was related to progression free survival (PFS). Cancer specific overall survival (OS) was related to ki-67 LI, tumour size, and p27kip1 downregulation. Ki-67 LI was the main independent predictor of DFS (p = 0.0005), PFS (p = 0.0162), and cancer specific OS (p = 00195). CONCLUSION: Tumour proliferation measured by Ki-67 LI is related to tumour recurrence, stage progression, and is an independent predictor of DFS, PFS, and cancer specific OS in TaT1 bladder urothelial cell carcinoma.     

Expression of amphiregulin and epidermal growth factor receptor in human breast cancer: analysis of autocriny and stromal-epithelial interactions

Amphiregulin (AR) and its receptor, epidermal growth factor receptor (EGFR), were evaluated by dual immunostaining in a series of 84 invasive ductal breast carcinoma specimens, 33 of which were from locally advanced inflammatory (T4d) cancer. Co-expression of AR and EGFR was always found in non-malignant breast tissues adjacent to tumours (24/24). Alternatively, expression of AR and EGFR was found in invasive epithelial tumour cells in 50% and 17.8% of specimens, respectively. In tumour stroma, 59.5% and 30.9% of specimens, respectively, were positively stained. By univariate analysis, AR and EGFR expression in invasive carcinomas was correlated with large tumour size, inflammatory carcinoma, node involvement, Bloom-Richardson (SBR) grade III, and absence of oestrogen receptor. EGFR expression in stromal cells was correlated with non-inflammatory carcinoma. A putative autocrine loop with AR and EGFR expression in invasive carcinoma was detected in 14.3% of cases. Stromal expression of AR and EGFR expression in invasive tumour cells was detected in 11.9% of cases and related to poor prognostic parameters. By multivariate analysis, AR expression in invasive tumour was strongly related to inflammatory carcinoma (p=0.005) and marginally related to SBR grade III (p=0.07). EGFR expression in invasive tumour and stromal cells was correlated with absence of oestrogen receptor and non-inflammatory carcinoma (p=0.002 and p=0.015, respectively).     

喉癌 STK15基因异常与中心体扩增的研究

目的　研究喉癌中 STK15基因的异常及中心体扩增情况。方法　应用逆转录 -聚合酶链反应方法 ,检测 6 2例喉鳞状细胞癌和人喉鳞状细胞癌细胞系 Hep- 2中 STK15基因 m RNA表达水平 ;应用聚合酶链反应 -单链构象多态性检测上述组织及细胞中 STK15基因第 6、7外显子突变 ;以 Hep- 2细胞系为代表应用免疫荧光方法检测中心体扩增情况。结果　在 39例 ( 6 3% )喉癌及 Hep- 2细胞系中检测到了STK15基因的高表达 ;在上述组织和细胞中均未检测到 STK15基因第 6、7外显子的突变 ;Hep- 2细胞系有明显的中心体扩增 :单个细胞中心体数目变化范围为 1～ 7,有中心体扩增的细胞数为 11%～ 2 3%。结论　在 Hep- 2细胞系中发现了 STK15基因高表达与中心体扩增 ,提示 STK15基因高表达引起中心体扩增 ,可能是导致该细胞系染色体不稳定的重要因素 ;在喉癌组织中观察到了STK15基因高表达 ,提示它可能是导致喉癌发生的多因素之一     

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.     

Immunohistological localisation of human FAT1 (hFAT) protein in 326 breast cancers. Does this adhesion molecule have a role in pathogenesis?

AIMS: To examine the immunohistological expression in human breast cancers of human FAT1 (hFAT) protein, a recently described member of the cadherin superfamily, and its correlation with histological type and grade. METHODS: A total of 326 cases of invasive and in situ breast cancer representing a broad spectrum of histological subtypes were immunostained with affinity-purified rabbit antibodies produced to the cytoplasmic region of hFAT using a standard avidin-biotin system. Staining intensity was arbitrarily graded on a scale of 0 to 3. RESULTS: All tumours showed diffuse staining for hFAT. Immunoexpression of the protein was generally strong in both lobular (LCIS, n = 2) and ductal in situ carcinoma (DCIS, n = 55). hFAT was also strongly immunoexpressed in all types of invasive carcinoma. Grade 3 DCIS displayed the highest hFAT intensity compared with lower grade tumours, with significant differences between grade 1 and 3 (p = 0.015) and grade 2 and 3 (p = 0.047). With invasive ductal carcinomas (n = 128) the difference was not as clear-cut, as most tumours showed moderate (n = 63) or strong staining (n = 49), although grade 3 IDC revealed significantly decreased immunoexpression compared with grade 1 IDC (p = 0.03). CONCLUSIONS: The results illustrate that hFAT1 does not display the pattern of expression seen with the E-cadherin-ss-catenin adhesion complex; however, its over-expression and diffuse expression in both in situ and invasive carcinoma strongly suggests a role in carcinogenesis. From the known functions of FAT1 it is suggested that the concurrent loss of classical cadherins from cell-cell junctions accompanied by increased FAT1 expression contributes to loss of duct formation, and increased cell migration and invasion.     

喉癌中STK15基因扩增及表达的研究

目的研究喉癌中5TK15基因扩增、mRNA和蛋白表达增高在喉癌发生、发展中的作用。方法应用差异PCR方法检测40例喉鳞状细胞癌及癌旁正常对照组织5TK15基因扩增的情况；应用逆转录-PCR方法检测同批标本STK15mRNA表达情况；用免疫组织化学方法分析STK15蛋白表达。结果肿瘤组织中5TK15基因扩增率为35％（14／40）；STK15 mRNA表达增高占67．5％（27／40）；STK15蛋白表达的阳性率为72．5％（29／40）；将扩增与表达的结果与喉癌患者的临床各项指标进行统计学分析，5TK15基因扩增、STK15mRNA表达增高与肿瘤的分化程度显著相关（P〈0．05）；STK15蛋白表达与肿瘤的分化程度和病理分级显著相关（P〈0．05）。结论喉癌中5TK15基因扩增、mRNA和蛋白表达水平增高，导致中心体复制异常、染色体不稳定。可能在喉癌的发生及恶性进展中起一定作用。     

p53 protein accumulation in oesophageal squamous cell carcinomas and precancerous lesions.

AIMS--To investigate the immunohistochemical expression of p53 protein in oesophageal squamous cell carcinomas and in dysplastic areas of the oesophageal mucosa surrounding the tumours. METHODS--Biopsy samples were obtained from 20 patients with an oesophageal squamous cell carcinoma. Blocks of the tumours and of the surrounding mucosa were immunostained with the monoclonal antibody DO-7. RESULTS--Fourteen of the 20 carcinomas were positive for p53 (70%). The frequency of p53 overexpression increased with the differentiation of the tumour. Nine out of 13 dysplastic specimens were positive for p53 (69%): eight cases with severe dysplasia and one case with moderate dysplasia. No p53 immunostaining was detected in normal oesophageal epithelium. All p53 positive dysplastic specimens were taken from the mucosa adjacent to tumours that were also immunostained. In moderate dysplastic mucosa the p53 positive cells were located in the proliferative basal zone, whereas in severe dysplasia the immunostained cells increased in number and spread to upper cell layers of the epithelium. CONCLUSION--This study supports the hypothesis that TP53 gene is frequently involved in the development of oesophageal squamous cell carcinoma and that p53 protein accumulation is an early event in human oesophageal carcinogenesis.     

Loss of heterozygosity in the clonal evolution of flat colorectal neoplasms

In contrast to invasive colorectal carcinomas that develop in typical exophytic adenoma-carcinoma sequences, some invasive cancers may evolve from flat mucosal dysplastic lesions. Despite their relatively small size, these flat colorectal lesions are often associated with high-grade dysplasia and may show an aggressive clinical course. To delineate the genetic pathways in the clonal evolution of these tumors, multiple foci were microdissected from 13 cases and the allelic deletions of 15 chromosomal arms were analysed. Loss of heterozygosity (LOH) was detected most frequently on 17p (77%), followed by 18q (69%), and 5q (54%). In five cases with concomitant low-grade adenomas, only one case showed LOH in low-grade adenoma foci. In high-grade dysplasia with/without submucosal invasion, early and homogeneous LOH of one to several chromosomal arms was detected. Overall, homogeneous and thus early LOH were most frequently detected on 17p (seven of 10 cases with 17p LOH), followed by 3p (two of three cases with 3p LOH), and 5q (four of seven cases with 5q LOH). In addition to homogeneous LOH, the LOH patterns observed in different portions of dysplasias and invasive cancers in individual cases identified several different genetic patterns of tumour progression, either with linear or branching (divergent) trees. Positive immunostaining for p53 was detected in 10 of the 13 cases; of these, five cases were concomitant with 17p LOH in all of the microdissected foci, four cases were concomitant with 17p LOH in a majority of foci and, one case showed retention of 17p. Except for the flat configuration and early 17p LOH, genetic heterogeneity in the flat high-grade dysplastic foci was found to be similar to genetic chaos in the late dysplastic and preinvasive stages of exophytic adenoma. These findings suggest a potentially aggressive course for these neoplasms.     

p53 Protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene

We performed an immunohistochemical analysis to investigate the expression of p53 protein in a panel of 18 laryngeal squamous cell carcinomas, 15 primary tumours and three in relapse, previously analysed by us for the presence of p53 gene mutations. Dysplastic and/or normal surrounding mucosa was evaluated in 15 different tumours. The results of our study are the following: (1) expression of p53 protein was observed in one out of five tumours positive for p53 gene mutations (20%) and in 10 out of 13 (80%) negative cases; (2), p53 protein over-expression was frequently observed in normal and/or dysplastic mucosa surrounding either wild-type (7/11) or mutated p53 tumours (2/4); (3), p53 immunoreactive cells showed a pattern of distribution in normal and mildly/moderately dysplastic mucosa (basal layers), different from that in severely dysplastic mucosa (whole thickness). These data further support the hypothesis that p53 protein over-expression may be a marker of the earliest phases of multistep tumorigenesis in laryngeal squamous cell carcinoma.     

STK 15基因沉默导致胃癌细胞分裂停滞

目的探讨丝氨酸／苏氨酸激酶15（serine／threonine kinase15,STK15）基因在胃癌细胞有丝分裂中的作用。方法应用RNA干扰技术（RNAi）抑制胃癌细胞株MKN45细胞STK15基因表达;real-time定量PCR及Western blot检测干扰前后STK15 mRNA及蛋白质表达的变化;倒置显微镜观察MKN45细胞形态的变化;流式细胞仪检测细胞周期的变化;MTT法检测细胞增殖速度的变化;免疫荧光结合激光共聚焦显微镜观察MKN45细胞微管及有丝分裂表型的改变。结果STK15基因沉默后。其mRNA及蛋白质表达明显下降,real-time PCR结果显示,转染后48h,STK15^-组STK15 mRNA水平较对照siRNA组的下降了89．54％;Western blot灰度比半定量检测显示,STK15蛋白水平较对照siRNA组降低了57．18％。较多MKN45细胞呈圆形改变并呈现G2期细胞的DNA含量,STK15^-组3个时间点的圆形细胞占18．95％,而对照siRNA组为8．34％,差异有统计学意义（P〈0．05）;流式细胞仪检测结果显示,STK15。组G2期DNA含量细胞平均值为26．13％,而对照siRNA组G2期DNA含量细胞平均值12．46％（P〈0．05）。细胞增殖速度减慢（P〈0．05）。细胞有丝分裂表型发生改变（P〈0．05）。结论STK15基因在MKN45细胞有丝分裂过程中可能起着关键作用,阻断其表达可导致MKN45细胞有丝分裂停滞。     

Clinical importance of p53 protein in gall bladder carcinoma and its precursor lesions.

AIMS--To study the expression and importance (if any) of p53 protein in gall bladder carcinoma and its precursor lesions. METHODS--Immunohistochemical staining was performed on formalin fixed, paraffin wax embedded histological sections with an anti-human p53 monoclonal antibody (DO-7; Dako Corporation M7001) (24 carcinomas, one adenocarcinoma in situ, six dysplasias, three adenomas and four cases of chronic cholecystitis). Invasive, in situ, and dysplastic areas as well as normal-looking epithelium were sought. Nuclear staining was assessed according to intensity and extent of positive cells. Both variables were graded on a scale of 1-3 and aggregate p53 scores were obtained (range: 0, 2-6). Only p53 scores of > or = 3 were regarded as significant. RESULTS--Clinically important amounts of p53 were expressed in 92% of invasive carcinomas, 86% of carcinoma in situ, and 28% of dysplastic areas. None of the adenomas contained clinically important amounts of p53. Normal epithelium, present in all the cases, did not express p53 except in one case of moderately differentiated adenocarcinoma (p53 score 3). In general, there was no difference in the prevalence of p53 protein expression between dysplasias associated with, and those unassociated with invasive disease. There was a tendency for higher grade carcinomas to express more p53 protein. CONCLUSIONS--The distribution of p53 protein in invasive carcinomas and the adjacent dysplastic and preinvasive lesions suggests that it is more commonly expressed than previously thought. The fact that p53 protein is also expressed in cases of dysplasia and carcinoma in situ unassociated with invasive malignancy lends further support to the contention that p53 gene mutations may have a role in the pathogenesis of gall bladder cancer. Expression of p53 protein may possibly be an indication of likely disease progression from dysplasia, to carcinoma in situ, to invasive disease.