Increased expression of the nme1 gene is associated with metastasis in epithelial ovarian cancer

The genetic events involved in the development of metastases of epithelial ovarian cancer are largely unknown. One gene postulated to play a role in tumour metastasis suppression is NMEI (nm23-HI), and an inverse relationship between NMEI expression and metastatic potential has been observed for some solid tumours. In this study we have investigated the levels of mRNA expression of the 2 isoforms of the NME gene, NMEI and NME2. A maximum of 45 tumour samples from 33 patients were available for Northern blot analysis. We observed variable levels of expression of NMEI and NME2 mRNA. The average level of NMEI, but not NME2, mRNA expression was statistically higher in metastatic biopsies when compared with primary tumour biopsies. To examine the possible tumour suppressor gene role of NMEI in ovarian tumours, 76 patients were investigated by Southern blot analysis to determine the rate of allelic deletion. Allele loss at 5 other chromosome 17 loci (D17S5, TP53, NF1, D17S74, D17S4) was also evaluated for many of these 76 patients. Allele loss was observed in 22/30 (73%) informative patients at the NME1 locus. We also observed high rates of allele loss at the other loci evaluated. No correlations with clinical stage, histological subtype or patient survival were observed in either mRNA or DNA analyses. We have established that tumour progression in ovarian cancer is accompanied by over-expression of the NME1 gene; however, despite high rates of allele loss at the NME1 locus, the concept that NME1 may be a candidate tumour suppressor gene in ovarian cancer cannot be confirmed by this study. © 1995 Wiley-Liss, Inc.     

Silencing of the thrombomodulin gene in human malignant melanoma

The loss of thrombomodulin (TM) expression is associated with tumour growth, infiltration and lymph node metastasis in human tumours. In melanoma cell lines, TM is reported to mediate cell adhesion, and its introduction into TM-negative melanoma cell lines suppresses their growth. In this study, we analysed TM expression in surgical melanoma specimens and the role of its promoter methylation in the loss of its expression. In 15 (75%) of the 20 specimens (five from a primary site and 15 from metastatic sites), melanoma cells lacked TM immunoreactivity. Methylation of the TM promoter region was detected in 10 (67%) of the 15 TM-negative specimens by methylation-specific polymerase chain reaction, whereas methylation was detected in two (40%) of the five TM-positive specimens. In cell lines, complete methylation of the TM promoter CpG island was detected in six (46%) of 13 melanoma cell lines, whereas no methylation was detected in two cultured normal melanocytes. There was a good correlation between the methylated status of the CpG island and the loss of TM messenger RNA (mRNA) expression. Treatment of melanoma cell lines with a demethylating agent, 5-aza-2'-deoxycytidine, induced demethylation of the promoter CpG island and the restoration of mRNA and protein expression. These findings suggest that most human melanomas lack TM expression, and that methylation of the promoter CpG island is one of the mechanisms responsible.     

DMBT1 is frequently downregulated in well-differentiated gastric carcinoma but more frequently upregulated across various gastric cancer types

Well-differentiated gastric carcinomas are considered to represent a distinct entity emerging via specific molecular changes different from those found in other gastric carcinoma types. The gene deleted in malignant brain tumours 1 (DMBT1) at 10q25.3-q26.1 codes for a protein presumably involved in cell differentiation and protection and has been proposed as a candidate tumour suppressor for brain and epithelial cancer. One study reported a loss of DMBT1 expression in 12.5% (5/40) of gastric cancer samples. Here, we examined in more detail DMBT1 protein and mRNA expression in 78 primary gastric tumour samples and corresponding normal gastric mucosa. DMBT1 was expressed in all non-tumour gastric mucosa tissues. Eleven out of 71 (15%) gastric tumours were negative for the DMBT1 protein in immunohistochemical analyses. Lack of DMBT1 expression was significantly more frequently found in well-differentiated gastric tumours (6/18 well-differentiated tumours vs. 5/53 other subtypes; P=0.025). Quantitative RT-PCR revealed a downregulation of the DMBT1 mRNA for 8/21 (38%) cases, while the remaining 13 cases (62%) displayed a substantial upregulation. Our data suggest that a loss of DMBT1 expression may preferentially take place in well-differentiated gastric carcinoma. However, an upregulation of DMBT1 expression is more frequently found across all gastric cancer types.     

Different expression of lysosome-associated membrane protein-1 in human melanomas and benign melanocytic lesions

Lysosome-associated membrane protein-1 is a protein with a significant content of beta1,6-branched N-glycans. It is thought that enhanced expression of lysosome-associated membrane protein-1 in tumour cells may promote invasion by influencing both adhesion to extracellular matrix and perhaps also binding to endothelial cells. The present study was aimed at examining levels of lysosome-associated membrane protein-1 in human melanomas and benign pigmented lesions to evaluate whether this protein might be considered a potential molecular marker of melanoma progression. The expression of lysosome-associated membrane protein-1 was for the first time determined immunohistochemically in formalin-fixed paraffin-embedded specimens comprising 42 primary cutaneous melanomas, 15 lymph node melanoma metastases (11 correlated with primary tumours), three melanoma recurrences (correlated with both primary and metastatic melanomas), 27 nevi and four epithelial tumours (two seborrhoeic keratoses and two basal cell carcinomas). Our results demonstrate that development and progression of melanoma are associated with changes of the lysosome-associated membrane protein-1 level. The expression was strongest in melanoma recurrences and lymph node metastases, weaker in primary cutaneous melanomas and not detectable in melanocytes of pigmented nevi. Nodular melanomas expressed lysosome-associated membrane protein-1 at higher level than superficially spreading melanomas.     

Loss of nonclassical MHC molecules MIC-A/B expression during progression of uveal melanoma

Uveal melanoma differs from cutaneous melanoma with respect to aetiology, metastatic behaviour and immune biology. The notion that loss of classical MHC class I molecules in uveal melanoma lesions is associated with an improved prognosis suggests that NK cells act as the predominant cells responsible for immune surveillance of this tumour. Consequently, immune escape mechanisms of uveal melanoma should impair the innate immunity. To this end, expression of the ligand for the NK receptor NKG2D, that is, MIC-A/B was expressed by 50% of primary tumours, but none of the metastatic lesions. MIC+ tumours were characterised by a NKG2D+ infiltrate, which was absent in MIC- lesions subsequent to chemoimmune therapy. Strikingly, MIC-A/B expression in metastatic lesions was observed subsequent to chemotherapy with fotemustine in one case. In summary, MIC/NKG2D interactions seem to be involved in the immune surveillance of primary uveal melanomas, whereas for metastatic tumours this ligand/receptor system seems not to be relevant, thus, suggesting an immune selection of MIC negative tumour cells.     

Deletions and altered expression of the RIZ1 tumour suppressor gene in 1p36 in pheochromocytomas and abdominal paragangliomas

Pheochromocytomas and abdominal paragangliomas are rare catecholamine-producing tumours arising from neural crest-derived chromaffin cells. Frequent deletions of several distinct regions on the short arm of chromosome 1 suggest their involvement in the tumourigenesis process. The RIZ1 tumour suppressor encoded by the RIZ gene in 1p36.21 represents an attractive candidate target for the distal 1p deletions in these tumours. A panel of 18 pheochromocytomas (14 benign, and 4 malignant) and 11 abdominal paragangliomas (4 benign, and 7 malignant) were characterised for somatic deletions and mRNA expression status of RIZ1 using loss of heterozygosity (LOH) analysis and real-time quantitative PCR, respectively. Furthermore, we evaluated the methylation status of the RIZ1 promoter utilising methylation-specific PCR (MSP). Intragenic LOH at the RIZ locus was detected in 10 of 16 informative cases (62%), including 8 of 12 pheochromocytomas (67%) and 2 of 4 paragangliomas (50%). RIZ1 mRNA appeared to be significantly under-expressed in the tumour samples compared to normal adrenal controls (mean 0.6 vs. 1.0, p<0.001). This was not associated with RIZ1 promoter methylation in any of the samples, indicating that promoter hypermethylation is unlikely to be the underlying cause of the frequent expressional silencing. The recurrent inactivation of the tumour suppressor RIZ1 suggests that this event may be a significant contributing factor to tumour development in pheochromocytomas and abdominal paragangliomas.     

Allele loss from 5q21 (APC/MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer.

Thirty-four primary, untreated sporadic breast cancers were examined for loss of heterozygosity (LOH) at tumour suppressor loci involved in colorectal cancer: APC/MCC at 5q21 and DCC at 18q21. LOH was identified in 28% informative patients at 5q21 and 31% at 18q21. LOH at 5q21 and 18q21 was compared with allele loss at 17p13 and concurrent LOH at two or more of the loci was noted in 24% of tumours. Expression of a 12 kb DCC mRNA was demonstrated in 14/34 (42%) of the cancers and in all five tumours with LOH at the DCC locus there was an additional 11 kb DCC mRNA. Abnormalities of three loci involved in colorectal cancer (5q21, 17p13 and 18q21) therefore also occur in sporadic breast cancer. The accumulation of such genetic abnormalities may confer a growth advantage important in the development of breast cancer.     

Disruption of imprinted genes at chromosome region 11p15.5 in paediatric rhabdomyosarcoma

Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH) at chromosome region 11p15.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2), H19, and p57^KIP2. We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI) of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3-FKHR or PAX7-FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57^KIP2 was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57^KIP2 expression, there was no evidence for inactivating mutations, suggesting that p57^KIP2 is not a tumour suppressor in rhabdomyosarcoma.     

Frequent loss of SFRP1 expression in multiple human solid tumours: association with aberrant promoter methylation in renal cell carcinoma

Oncogenic wingless-related mouse mammary tumour virus (Wnt) signalling, caused by epigenetic inactivation of specific pathway regulators like the putative tumour suppressor secreted frizzled-related protein 1 (SFRP1), may be causally involved in the carcinogenesis of many human solid tumours including breast, colon and kidney cancer. To evaluate the incidence of SFRP1 deficiency in human tumours, we performed a large-scale SFRP1 expression analysis using immunohistochemistry on a comprehensive tissue microarray (TMA) comprising 3448 tumours from 36 organs. This TMA contained 132 different tumour subtypes as well as 26 different normal tissues. Although tumour precursor stages of, for example kidney, colon, endometrium or adrenal gland still exhibited moderate to abundant SFRP1 expression, this expression was frequently lost in the corresponding genuine tumours. We defined nine novel tumour entities with apparent loss of SFRP1 expression, i.e., cancers of the kidney, stomach, small intestine, pancreas, parathyroid, adrenal gland, gall bladder, endometrium and testis. Renal cell carcinoma (RCC) exhibited the highest frequency of SFRP1 loss (89% on mRNA level; 75% on protein level) and was selected for further analysis to investigate the cause of SFRP1 loss in human tumours. We performed expression, mutation and methylation analysis in RCC and their matching normal kidney tissues. SFRP1 promoter methylation was frequently found in RCC (68%, n=38) and was correlated with loss of SFRP1 mRNA expression (p<0.05). Although loss of heterozygosity was found in 16% of RCC, structural mutations in the coding or promoter region of the SFRP1 gene were not observed. Our results indicate that loss of SFRP1 expression is a very common event in human cancer, arguing for a fundamental role of aberrant Wnt signalling in the development of solid tumours. In RCC, promoter hypermethylation seems to be the predominant mechanism of SFRP1 gene silencing and may contribute to initiation and progression of this disease.     

p16INK4a inactivation is not frequent in uncultured sporadic primary cutaneous melanoma

In an attempt to examine whether the inactivation of p16INK4a is an important early event in the development of sporadic melanoma in vivo, we have systematically analysed 46 uncultured primary cutaneous melanomas. Loss of heterozygosity (LOH) of chromosome region 9p21-22 (where the p16INK4a resides) was detected in 11 tumours (24%) by PCR-based LOH analyses. Direct sequencing of all three exons of the p16INK4a gene in these 11 tumours revealed no somatic mutation although germline mutations which have not been reported previously as common polymorphisms were detected in two patients. Further sequencing analyses of the p16INK4a gene exon 2 in 19 additional tumours with no evidence of LOH on 9p21-22 identified only one heterozygous C- >T mutation at codon 81 altering a proline to a leucine. A sensitive methylation-specific PCR assay did not reveal de novo methylation of the 5'CpG island in exon 1 of the p16INK4a gene in any of the tumours showing 9p21-22 allelic loss or a heterozygous p16INK4a mutation. Complete loss of p16INK4a protein, most likely due to homozygous deletion of the p16INK4a gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections using two different anti-p16INK4a antibodies. The results show that inactivation of p16INK4a is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21-22. This study also emphasizes the importance of examining uncultured primary tumours rather than cell lines to define early events in tumorigenesis.     

High expression of immunotherapy candidate proteins gp100, MART-1, tyrosinase and TRP-1 in uveal melanoma.

In the treatment of cutaneous melanoma, provisional therapeutic strategies have been designed to combat tumour load using T cells that are sensitized with peptides derived from melanoma autoantigens, such as glycoprotein 100 (gp100), melanoma antigen recognized by T cells 1 (MART-1 or MelanA), tyrosinase and tyrosinase-related protein 1 (TRP-1). We recently found that gp100, MART-1 and tyrosinase are heterogeneously expressed in human cutaneous melanoma (De Vries et al (1997) Cancer Res 57: 3223-3229). Here, we extended our investigations on expression of these immunotherapy candidate proteins to uveal melanoma lesions. Cryostat sections from 11 spindle-type, 21 mixed and epithelioid tumours and four metastasis lesions were stained with antibodies specifically recognizing gp100, MART-1, tyrosinase and TRP-1. In addition, we used the DOPA reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results. High expression of gp100, MART-1 and tyrosinase was found in the uveal melanoma lesions: 80% of the lesions displayed 75-100% positive tumour cells. TRP-1 positivity was slightly less: approximately 65% of the lesions stained in the 75-100% positive tumour cell category. All uveal melanoma lesions were positive for the four markers studied, this being in contrast to cutaneous melanoma where 17% of the advanced primary lesions and metastases were negative. The presence of these antigens was a little lower in metastases. We conclude that uveal melanomas and their metastases express melanocyte-lineage immunotherapy candidate proteins very abundantly. Uveal melanomas differ in this respect from cutaneous melanoma, in which the expression of these immunotherapy antigens was much more heterogeneous. This makes uveal melanoma a suitable candidate tumour for immunotherapeutic approaches.