Loss of heterozygosity at 10q in tumors of the upper respiratory tract is associated with poor prognosis.

Frequent loss of a specific chromosomic region in cancers is often associated with inactivation of a tumor-suppressor gene. The long arm of chromosome 10 is deleted in several types of tumor, among them squamous-cell carcinomas of the head and neck (HNSCC). To determine the role of 10q deletions in the tumorigenesis of the upper respiratory tract, 47 HNSCCs were examined for loss of heterozygosity (LOH) at 10q: 43% of the cases analyzed showed LOH at 10q, and 2 distinct hot spots of deletion were identified, at 10q22-23 and 10q25-26. The possible involvement of pTEN/MMAC1, a tumor-suppressor gene mapped at 10q23, was also evaluated. No mutation, homozygous deletion or loss of expression of pTEN/MMAC1 was detected, indicating that inactivation of this gene plays a minor role in HNSCC development. Interestingly, the frequency of deletion at 10q was greater in invasive carcinoma than in adjacent carcinoma in situ, and a significant association between LOH and poor prognosis was observed. Taken together, our results suggest the presence in the long arm of chromosome 10 of (a) tumor-suppressor gene(s) other than pTEN/MMAC1 and presumably involved in the malignant progression of tumors of the upper respiratory tract. Int. J. Cancer (Pred. Oncol.) 84:432-436, 1999.

Microsatellite instability in squamous cell carcinomas of the head and neck related to field cancerization phenomena.

Patients affected by squamous cell carcinoma of the head and neck (HNSCC) show frequent occurrence of multiple cancers and widespread precancerous lesions in the mucosa of the upper respiratory tract, a phenomenon known as field cancerization. In this study, we investigated the role of genetic instability in the development of HNSCC and in particular in tumour multiplicity phenomena of the upper respiratory tract. For this purpose, we analysed microsatellite instability (MI) and loss of heterozygosity (LOH) at 20 loci mapping on five chromosomal arms in 67 HNSCC patients, 45 of whom had a single cancer and 22 had multiple primary tumours. The possible involvement of the hMLH1 gene in genetic instability and as a potential target of 3p21 deletion phenomena in head and neck cancers was also investigated. Our data indicate that mismatch repair-related genetic instability plays a minor role in the carcinogenesis of HNSCC and in tumour multiplicity of the head and neck region. Moreover, our results exclude a role for the hMLH1 gene as a determinant of MI and as a specific gene target of deletion at 3p21 in HNSCC. We conclude that presumably other genetic mechanisms, such as those hypothesized for MI-negative hereditary non-polyposis colorectal cancer patients, may play a major role in the carcinogenesis of the mucosa of the upper respiratory tract.

Molecular aberrations of the G1-S checkpoint in myxoid and round cell liposarcoma.

Myxoid and round cell liposarcoma represents a morphological spectrum in which tumor progression from low-grade myxoid to high-grade round cell areas is frequently observed. A distinctive t(12;16)(q13;p11) reciprocal translocation rearranges the CHOP gene localized to 12q13 in most cases. Data concerning the occurrence of cell cycle aberrations in this subset of mesenchymal malignancies are very limited. Therefore, we analyzed a histologically homogeneous series of 21 cases of myxoid and round cell liposarcoma. The p53 pathway was studied by investigating the TP53 gene and protein, mdm2 protein, and p21Waf1 protein. The Rb-cyclin D pathway was analyzed by studying the pRb protein, the p16MTS1 gene, cyclin D1, cyclin D3, p27Kip1, cdk4, and cdk6 proteins. In contrast with the rare involvement of the TP53 gene in well differentiated liposarcoma, aberrations of the TP53 gene were observed in approximately 30% of cases of myxoid and round cell liposarcoma. Notably, mdm2 overexpression was seen in 56% of cases and correlated with histological grade, therefore indicating a possible role in tumor progression. Abnormalities involving the Rb-cyclin D pathway were observed in more than 90% of cases. pRb loss was present in one-third of cases and, at variance with that observed in other subsets of sarcoma, overexpression of cyclin Ds represented a rare event. Interestingly, upregulation of either cdk4 or cdk6 was demonstrated in 85% of cases.

Overexpression of CDC25A and CDC25B in head and neck cancers.

The deregulation of several cell cycle-related genes participates in neoplastic transformation. Cell cycle progression is driven by cyclin-dependent kinases, which are positively regulated by association with cyclins and negatively regulated by binding to inhibitory subunits. The activity of cyclin-dependent kinases is also regulated by the phosphorylation status, which is controlled by the antagonistic action of wee1 kinase and CDC25 phosphatases. Three CDC25 genes are present in human cells: CDC25A, CDC25B, and CDC25C. These three genes function at different phases of the cell cycle. Whereas CDC25A and CDC25B are expressed throughout the cell cycle, with peak expression in G1 for CDC25A and in both G1-S-phase and G2 for CDC25B, CDC25C is predominantly expressed in G2. Several lines of evidence suggest a role for CDC25s as oncogenes. CDC25A and CDC25B cooperate with Ha-ras or loss of Rb1 in the oncogenic transformation of rodent fibroblasts. Moreover, they are transcriptional targets of c-myc, and CDC25A in particular plays an important role as a mediator of myc functions. On the basis of the evidence that CDC25 phosphatases can act as oncogenes, we analyzed the expression of CDC25A, CDC25B, and CDC25C genes in 20 squamous cell carcinomas of the head and neck by quantitative reverse transcription-PCR. Our results show that whereas CDC25C is expressed at a low level with no relevant differences between neoplastic tissue and normal mucosa, CDC25A and CDC25B are overexpressed in a large fraction of tumors.

Rarity of microsatellite genomic instability in B-cell non-Hodgkin's lymphomas in hepatitis C virus-infected patients.

Several groups have emphasized the likely implication of the hepatitis C virus (HCV) in a fraction of B-cell non-Hodgkin's lymphomas. Since only a minority of patients with HCV infection and monoclonal mixed cryoglobulinaemia develop overt lymphoma, the identification of predisposing factors has relevant clinical implications. The replication error phenotype (RER+), as revealed by widespread microsatellite instability, is caused by defects in DNA mismatch repair genes, and has been frequently disclosed in subsets of B-cell lymphomas with underlying infection and chronic inflammation. We therefore investigated the occurrence of the RER+ phenotype in a series of eight consecutive B-cell NHLs in patients with chronic infection by HCV. A polymerase chain reaction-based assay was used to analyse an extended panel of 15 microsatellite loci. Microsatellite instability was not observed in six tumour samples in any locus; the two remaining cases showed instability at only one locus. Therefore genetic instability by defects in DNA mismatch repair genes should not represent the general mechanism predisposing to overt lymphoma in HCV-infected patients. Although a clearer definition of HCV-related B-cell disorders should better address future studies on genetic instability in larger series, we recommend additional oncogenetic pathways as the target of further research.

Human non-Hodgkin's lymphomas overexpress a wild-type form of p53 which is a functional transcriptional activator of the cyclin-dependent kinase inhibitor p21.

A large fraction of non-Hodgkin's lymphomas (NHLs) accumulate a wild-type form of the p53 tumor suppressor protein at the nuclear level. In normal cells, p53 induction is associated with a temporary cell growth arrest at the G1-S boundary of the cell cycle. This activity of p53 as a G1 checkpoint molecule is strictly dependent on its ability to induce the transcription of the inhibitor of the cyclin dependent kinase, p21. To verify the functionality of the wild-type p53 protein accumulated in NHL cells, 70 cases were comparatively analyzed for p53 and p21 expression and status of the respective genes. Overexpression of the wt p53 protein was associated with the accumulation of p21, indicating that p53 is functional with respect to p21 induction in these tumors. The coaccumulation of p53 with Ki-67 antigen indicates that wt p53-positive cells and p21-positive cells, as well, are actively proliferative elements, supporting the notion that p53-induced, p21-mediated growth arrest is somehow overridden in NHL cells. No p21 mutation or particular allele variant was shown to correlate with p21 protein accumulation, thus excluding a role for p21 structural abnormalities. Taken together, our data suggest the existence in NHL of a peculiar mechanism of functional inactivation of the p53 G1 checkpoint pathway occurring downstream of the CDK inhibitor p21.

p16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression.

The p16/CDKN2(MTS1) gene encoding for the p16 inhibitor of cyclin D/CDK4 complexes is frequently mutated and deleted in a large fraction of melanoma cell lines, and p16 germline mutations have also been observed in familial melanomas. Moreover, a CDK4 gene mutation, responsible for a functional resistance of CDK4 kinase to p16 inhibitory activity, has been described to occur in some cases of familial melanoma. These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma. To shed light on the role of these alterations in the development and progression of sporadic melanoma, 12 primary melanomas and 9 corresponding metastases were analyzed for CDKN2 and CDK4 gene mutations. Of the 12 primary melanomas analyzed, 4 showed the presence of mutational inactivation of the p 16 protein and 2 carried silent mutations. No metastases showed the presence of CDKN2 mutations, indicating that mutations of this cyclin-dependent kinase inhibitor is not common in the progression of sporadic melanoma. On the other hand, the absence, in the metastases, of the CDKN2 mutation detected in the corresponding primary tumors suggests that 9p21 homozygous deletion may play a major role in the metastatic spreading of this type of tumor. None of the cases analyzed showed the presence of an Arg24Cys mutation, which functionally protects CDK4 from p16 inhibition. This indicates that CDK4 mutation plays a minor role in the development and progression of sporadic melanoma.

All-trans, 13-cis and 9-cis retinoic acids induce a fully reversible growth inhibition in HNSCC cell lines: implications for in vivo retinoic acid use.

Retinoids are a group of vitamin A analogues that have shown promise as chemopreventive and therapeutic agents in many types of malignancy and have been entered in clinical trials with some successful results. To better understand the mechanism that mediates retinoid action and the anti-proliferative effects, we treated 7 human oral squamous-cell carcinoma (SCC) cell lines (FADU, HEp-2, CCL-17, SCC-9, SCC-15, SCC-25 and HN-212) with 10(-6) M of all-trans retinoic acid (ATRA), 9-cis and 13-cis retinoic acid (RA) in continuous for different periods of time. We assessed the extent of growth inhibition, the stability of the anti-proliferative effect and the mRNA expression levels (by RT-PCR) of RA receptors (RARs), retinoid X receptors alpha (RXR alpha) and cytosolic RA-binding proteins (CRBP I and CRABP II) in treated cells compared with controls. The data obtained showed that all 3 RAs were able to inhibit the cellular growth of the tested cell lines, although to a different extent. The cis compounds were able to inhibit the proliferation of all cell lines, whereas ATRA was ineffective in inhibiting the proliferation of the CCL-17 cell line, which was naturally resistant to ATRA concentrations in the range between 10(-5) and 10(-6) M. All inhibitory effects were completely reversible since all cell lines restored their normal growth proliferation within few days after drug removal. RT-PCR analysis of the receptor and cell binding protein status of control and treated cells showed a good correlation between growth inhibition and induction of, or increase in, the expression levels of RAR beta in RA-treated cells. No differences were observed in RAR alpha and RXR alpha mRNA expression levels between control and treated cells. CRBP I, CRABP II and RAR gamma mRNA levels increased in some treated cell lines but not in all.

Chromosome 13q deletion mapping in head and neck squamous cell carcinomas: identification of two distinct regions of preferential loss.

Heal and neck squamous cell carcinomas show frequent cytogenetic alterations involving the long arm of chromosome 13. To define the extent of 13q deletions and to identify the minimal areas of chromosome loss, 48 primary squamous cell carcinomas of the head and neck were analyzed for loss of heterozygosity using 11 different polymorphic loci. About 67% of the tumors displayed loss of genetic material at 13q. Most of the cases showed loss of the entire long arm of the chromosome. However, the presence of partial deletions in 10 cases provided evidence of the existence of two preferential sites of chromosome loss at 13q32-ter and 13q14.2-q14.3. The colocalization of the 13q14 minimal region of deletion with the retinoblastoma (RB) gene, which has been proposed as an oncosuppressor in diverse tumor types, prompted us to verify the involvement of this gene in the development of head and neck cancer. No significant variation in RB protein or RB mRNA expression was detected, thus excluding a role for such a gene in the genesis of this type of tumor. Taken together, our data suggest the existence of two new tumor suppressor genes (one close to and one distal to RB), which play a role in the development and/or progression of head and neck squamous cell carcinomas.

Recurrences and second primary tumours in the head and neck region: differentiation by p53 mutation analysis.

BACKGROUND: Patients with head and neck cancer are at high risk of developing additional primary tumours in the aerodigestive tract as a result of field cancerization phenomena. In this context, the appearance of a new neoplasm often poses a problem of differential diagnosis between recurrence and new primary tumour. The differentiation between the two entities in essentially clinical and conventionally based on the histological and spatio-temporal relations between the two lesions; however, the validity of these criteria has still to be assessed. DESIGN: To evaluate whether field cancerization phenomena may affect the clinical diagnosis of relapse/metastasis in the head and neck region, p53 mutation pattern was analysed in a series of primary tumours and corresponding recurrences/metastases. The rationale was that, since p53 mutations are a very early and polymorphic phenomenon, a recurrence/metastasis must retain the same mutation as the the primary tumour, whereas independent tumours are likely to display a different p53 gene status. RESULTS: Molecular analysis provided conclusive results in 9 of 12 cases analysed. The clinical diagnosis of recurrence was confirmed by molecular analysis in 4 of these cases. In contrast, a differential p53 mutation pattern supported an independent origin for 3 presumed local recurrences and 2 lung lesions. CONCLUSIONS: The use of p53 mutation analysis as a clonality marker allowed us to ascertain the inadequacy of the current diagnostic criteria for the differentiation between a new independent tumour and recurrence/metastasis. These findings substantiate the relevance of field cancerization phenomena in the head and neck region and prompt the use of p53 mutation analysis as a fundamental tool to improve the diagnosis and management of head and neck cancers.

Spontaneous mutation of cell oncogenes plays a minor role in neoplastic transformation of virus-induced murine T-cell lymphomas.

Mink cell focus-forming viruses (MCF) are slow-transforming retroviruses that are able to accelerate the appearance of T-cell lymphomas when injected in newborn AKR mice. Activation of proto-oncogenes by proviral insertion is thought to be the major mechanism by which these viruses exert their oncogenic potential. However, molecular phenomena not strictly virus-determined, such as mutations in cellular oncogenes/tumor suppressor genes or chromosome aberrations, have been hypothesized to contribute to the achievement of the fully neoplastic phenotype in MCF-infected mice. To evaluate the role of spontaneous mutagenesis phenomena in murine virus-induced lymphomagenesis, we analyzed a series of 18 MCF247-induced thymic lymphomas and derived cell lines for the presence of p53 and c-ras gene mutations. Only 1 mutation at the p53 gene and 1 mutation at the ki-ras gene were detected in our study. Our results suggest that spontaneous mutagenesis plays a minor role in virus-induced lymphomagenesis and support the notion that multiple proviral insertions could be the prevalent mechanism of transformation in this experimental system.

MDM2 overexpression does not account for stabilization of wild-type p53 protein in non-Hodgkin's lymphomas.

p53 protein overexpression is a frequent finding in non-Hodgkin's lymphomas (NHL), being detected in over 25% of the cases. Moreover, some high-grade lymphomas and a large fraction of low-grade tumors show a pattern of scattered p53 accumulation in a limited percentage of neoplastic cells. In contrast, NHLs show a low frequency of p53 gene mutations. To investigate the molecular bases of p53 protein overexpression, a large series of NHLs was analyzed for p53 gene status. The analysis of the entire coding region of the gene (exons 2-11) and corresponding donor and acceptor splicing sites indicated that a significant proportion of p53-positive tumors overexpresses a wild-type form of p53 protein (wt-p53). To assess whether wt-p53 accumulation was related to the formation of inactive complexes with endogenous proteins, MDM2 oncogene expression and amplification were analyzed. MDM2 overexpression was detected only in one third of the wt-p53-positive cases, thus excluding that MDM2 accounts tout court for the accumulation of a normal p53 protein. However, the fact that MDM2 overexpression was detected in only the p53-positive cases and the observation that MDM2-positive cells were a subpopulation of p53-positive cells suggest a link between the two phenomena. In particular, our results indicate that the accumulation of a wt form of p53 protein could promote the overexpression of the MDM2 gene product. In addition, the prevalence of MDM2 positivity in intermediate/high-grade tumors together with the concordant expression of wt-p53 and MDM2 only in the high-grade component of a 'composite' lymphoma suggests that perturbation in the MDM2/p53 critical ratio could play a role in lymphoma progression.

Analysis of the p53 gene in relation to tobacco and alcohol in cancers of the upper aero-digestive tract.

To elucidate the relationship, if any, of p53 gene expression according to smoking and drinking habits, 135 subjects with cancer of the upper aero-digestive tract were sampled from a case-control investigation conducted in Pordenone province, Northeast Italy, between 1986 and 1991. Adequate pathological material for immunohistochemical analysis was available for 83 subjects. Level of p53 expression was classified according to intensity on immunoreaction and percentage of p53-positive cells. Mutations were analyzed by means of single-strand conformation polymorphism and sequencing in 8 p53-positive life-long non-smokers exposed to various levels of alcohol intake. The association between p53 expression and smoking status and alcohol intake was evaluated by means of odds ratios, and 95% confidence intervals. Significantly, more men (39/65) than women (6/18) had elevated p53 expression. No significant trend of increasing percentage of p53-positive cancers with increasing smoking and drinking level was detected, especially after allowance for gender. With respect to specific mutation pattern, in 8 life-long non smokers who drank alcohol, 6 mutations involved G:C base pairs. G-to-A transitions were identified in 4 cases. The present study does not support an association between elevated p53 expression and tobacco smoking or alcohol intake. It provides an example of a molecular biology study in the framework of a large epidemiological investigation.

p53 protein over-expression and p53 gene abnormalities in HIV-1-related non-Hodgkin's lymphomas.

Alteration of the p53 tumor-suppressor gene was studied in non-Hodgkin's lymphomas (NHLs) from HIV-1-infected patients. p53 protein was over-expressed in 10 out of the 45 (22%) cases analyzed, mainly clustering in the small-non-cleaved-cell (SNC) (5/19) and Ki-1+ anaplastic large-cell (ALC) (3/8) sub-types, according to previous findings on HIV-1-unrelated NHLs. p53-positive small-non-cleaved-cell lymphomas presented a diffuse or clustered pattern of p53-positive neoplastic cells consequent upon p53-gene mutations. In contrast, in Ki-1+ ALC lymphomas p53 immunohistochemical reactivity was limited to scattered tumor cells, and no p53-gene alterations could be detected. These results suggest that p53-gene alterations play a role in the lymphomagenetic process of a fraction of HIV-1-related SNC NHLs, however with a frequency no different from that observed in HIV-1-unrelated NHLs of the same sub-type. In HIV-1-related Ki-1+ ALC lymphomas, mechanisms different from gene alterations might be implicated in over-expression of p53 protein.

Three discrete regions of deletion at 3p in head and neck cancers.

Alteration of the short arm of chromosome 3 is one of the most consistent cytogenetic abnormalities found in human head and neck cancers. These alterations, composed of translocations and deletions, have been associated with the presence of a tumor suppressor gene(s), but no clear evidence of the location of this presumptive gene(s) was available. We performed a molecular analysis of the 3p region using a polymerase chain reaction-based approach. Twenty-eight of the 38 cases analyzed (74%) showed the presence of single or multiple areas of allelic loss. Three commonly deleted regions, tentatively mapped to 3p24-ter, 3p21.3, and 3p14--cen, were identified. Our results suggest that at least three oncosuppressor genes mapping on 3p may be involved in head and neck cancer development and support a common oncogenic pathway with squamous cell lung cancer, for which a similar pattern of 3p deletion has been described recently.