Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients.

BACKGROUND: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). PATIENTS AND METHODS: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFbeta1, TGFbeta2, TGFbeta3 in oral SCC. RESULTS: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFbeta isoforms, we found that Smad2 mRNA and TGFbeta1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFbeta1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. CONCLUSION: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFbeta signaling should be better clarified in the future.

Overexpression of Fos-related antigen-1 in head and neck squamous cell carcinoma.

The activating protein-1 (AP-1) family of transcription factors has been implicated in the control of proliferation and differentiation of keratinocytes, but its role in malignant transformation is not clear. The aim of this study is to assess the pattern of mRNA expression of jun-fos AP-1 family members in 45 samples of head and neck squamous cell carcinomas (HNSCC) and matched adjacent mucosa by means of Northern blot analysis. Transcripts of all family members were identified, except for JunB that was detected only by means of reverse transcription polymerase chain reaction. Neither c-Fos nor JunD or FosB mRNA differed between tumours and normal tissues. We observed a strong Fos-related antigen-1 (Fra-1) and Fra-2 expression, but only Fra-1 mRNA densitometric values were higher in tumour, compared to normal adjacent mucosa (t-test, P = 0.006). A direct relationship between the positive expression of Fra-1 mRNA, above tumour median, was associated with the presence of compromised lymph nodes (Fischer exact test, P = 0.006). In addition, Fra-1 protein staining was assessed in a collection of 180 tumours and 29 histologically normal samples adjacent to tumours in a tissue array. Weak reactivity, restricted to the basal cell layer, was detected in 79% of tumour adjacent normal tissues, opposed to the intense reactivity of cancer tissues. In the subgroup of oral cancers, we have observed a shift in Fra-1 immunoreactivity, as long as the number of patients in each category, cytoplasmic or nuclear/cytoplasmic staining, was analysed (Fischer exact test, P = 0.0005). Thus, Fra-1 gene induction and accumulation of Fra-1 protein may contribute to the neoplastic phenotype in HNSCC.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.