malignant tumors of the nasal cavity and paranasal sinuses account for 0.2% of all human primary malignant neoplasms , with an incidence of 0.11.4 new cases / year/100,000 inhabitants [ 13 ] . adenocarcinomas account for 1020% of all primary malignant neoplasms of the sinonasal tract [ 4 , 5 ] . many of these have salivary gland origin , while others have histologic patterns resembling those of colon adenocarcinoma . this second type of sinonasal adenocarcinoma has been named intestinal - type adenocarcinoma ( itac ) and is responsible for less than 4% of the total malignancies of this region . itacs of the nasal cavity and paranasal sinuses can occur sporadically or are associated with occupational exposure to hardwood and leather dusts . exposure to wood and leather dusts increases the risk of adenocarcinoma by 500-fold [ 8 , 9 ] . findings from several studies have suggested clinical differences between itac arising in individuals with occupational dust exposure and itac arising sporadically . in fact tumors related to occupational exposure affect men in 8590% of cases , showing a strong tendency to arise in the ethmoid sinuses [ 1012 ] . itacs are aggressive tumors characterized by frequent local recurrences , low incidence of distant metastases , and an overall mortality of approximately 53% . itac seems to be preceded by intestinal metaplasia of the respiratory mucosa , induced by hardwood dust , leather dust , and other unknown agents , which is accompanied by a switch to an intestinal phenotype . the molecular mechanisms involved in metaplastic transformation of terminally differentiated epithelium to a phenotypically different epithelium are largely unknown . the morphological appearance of these tumors is variable , and they may resemble conventional colorectal adenocarcinoma . the similarities between itac and intestinal adenocarcinoma involve their ultrastructural and immunohistochemical aspects [ 1417 ] . numerous studies have shown that mutations of the k - ras and tp53 genes are common in colorectal adenocarcinomas [ 18 , 19 ] . in comparison , there is very little information about the molecular events involved in the pathogenesis of itac [ 16 , 1926 ] , in contrast to even more increasing information about the molecular mechanisms involved in the pathogenesis of head and neck squamous cell carcinomas ( hnscc ) [ 2732 ] . tumor necrosis factor ( tnf)-related apoptosis - inducing ligand ( trail ) is a tnf - family member , found in a variety of tissues   and with a conditional expression in several immune effector cells [ 34 , 35 ] . to date , five different receptors have been identified to interact with trail : trailr1 ( dr4 ) , trail - r2 ( dr5 ) , trail - r3 ( dcr1 ) , trail - r4 ( dcr2 ) , and osteoprotegerin . dr4 and dr5 are two different death domain - containing membrane receptors , whereas dcr1 and dcr2 are two decoy receptors that compete for trail binding with dr4 and dr5 [ 3639 ] . the final effect of the trail signaling is the induction of apoptosis by the intrinsic death pathway , recruiting the inactive form of caspase-8 . expression of trail and its receptors has been detected in various human tumors , suggesting that trail signaling pathway is involved in endogenous tumor surveillance , but the mechanism of how trail and its receptors contribute to carcinogenesis remains unknown . to gain further insight into the phenotype and possible mechanisms of ethmoidal itac , we investigated the expression of trail , correlating with clinicopathological data . the samples of 23 primary ethmoidal intestinal - type adenocarcinomas were retrospectively retrieved from the archives of the institute of pathology , marche polytechnic university , ancona , italy . all the tumors in which the diagnosis of itac was confirmed were subsequently subtyped , as papillary , colonic , solid , mixed , or mucinous types , as described by barnes . inclusion criteria were complete clinical data , uniformity of histological differentiation throughout the tumor sample , and the availability of sufficient material from the primary tumor for investigations . patients with previous or synchronous second malignancies or with previous radiation therapy or chemotherapy were excluded from the study . the follow - up time ranged from 2 to 10 years ( mean 4.8 years ) . four - micrometer serial sections from formalin - fixed , paraffin - embedded blocks of tumour representative areas were cut for each case . sections were deparaffinized in two changes of xylene for 10 minutes each , then , were rehydrated through graded alcohols , and immersed in 0,3% hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity . the tissue sections were placed in a microwave oven ( philips , cooktyronic m720 , 700  w ) in a plastic coplin jar filled with 10  mm sodium citrate buffer ( ph 6,0 ) at 5-minute interval , the fluid level in the coplin jar was removed from the microwave oven and allowed to cool . slides were incubated overnight with a 1  :  50 dilution of the primary mouse antihuman trail monoclonal antibody ( dako do-7 , glostrup , denmark ) . slides were washed twice with pbs and incubated with the linking reagent ( biotinylated anti - immunoglobulins ) for 15 minutes , at room temperature . after rinsing in pbs , the slides were incubated with the peroxidase - conjugated streptavidin label for 15 minutes at room temperature . the sections were again rinsed in pbs and incubated with diaminobenzidine for 10 minutes , in the dark . after chromogen development , slides were washed in two changes of water and counterstained with a 1  :  10 dilution of hematoxylin . trail expression and location were evaluated on histological section using a leitz orthoplan microscope equipped with a x 400 objective . the percentage of trail positive cells was evaluated from a minimum of 1,000 cells in each case . only nuclear staining of epithelial cells was observed , and the nuclei with a clear brown color , regardless of staining intensity , were regarded as trail positive . a negative control for trail immunostaining was performed in all cases by omitting the primary antibody , which , in all instances , resulted in negative immunoreactivity . both the histological diagnoses and evaluation of the positivity for trail were carried out indipendently by two of the authors ( c.r . and a.z . ) . immunohistochemical labeling for trail was classified as positive , when more than 5% of nuclei or cells were stained . the following pathological and clinical parameters were analyzed : tumour grade of differentiation ( g1,g2,g3 ) , tumour extension ( t1,t2,t3,t4 ) , tumor stage ( i , ii , iii , iv ) , and trail immunoreactivity . differences in trail immunoreactivity between the different groups were compared by the nonparametric kruskal - wallis test . statistical significance was set at p < 0.05 . all statistical analyses were performed using the spss statistical package ( spps inc . , chicago , il ) . there were 21 males and 2 females , with a mean age of 66.3 years ( range 5477 ) . there were 8 grade i , 8 grade ii , and 7 grade iii adenocarcinomas . 3 patients were in i stage , 7 patients were in ii stage , 11 patients were in iii stage , and 2 patients were in iv stage . all the patients had a known history of occupational exposure to hardwood dust , and intestinal - type adenocarcinoma was localized in all cases in the ethmoid region as confirmed by endoscopic and imaging ( tc and/or mr ) evaluation . the main clinicopathologic features of the patients and the oncoprotein expression are summarized in table 1 . the cases classified as itac showed a variable cellular appearance and were composed of a mixture of tall columnar absorptive cells , atypical stratified cylindrical cells similar to the cells seen in conventional colorectal adenocarcinoma , goblet cells , and large round to polygonal nondescriptive epithelial cells . the percentage of neoplastic cells immunoreactive for trail ranged from 2% to 90% . in all cases , there was a relationship between trail overexpression and the histological grading . indeed , poorly differentiated cases ( g2 and g3 ) showed a higher percentage of trail expressing cells in comparison to well differentiated ( g1 ) cases . no correlation between trail expression and other clinicopathological parameters , including t , stage and relapses was found ( figures 1 and 2 ) . despite their histological similarity to colorectal carcinomas , there is very little information about the molecular events involved in the pathogenesis of itac . several crucial pathways of tumorigenesis have been identified in colorectal adenocarcinomas [ 18 , 19 ] . these pathways involve the mutation and inactivation of multiple oncogenes , tumor suppressor genes , and dna mismatch repair genes including k - ras , apc , p53 , mlh1 , and msh2 [ 1820 ] . working on the hypothesis that morphological similarities to colorectal adenocarcinomas might reflect equivalent genetic alterations , several authors have investigated the presence of activating mutations of ras oncogenes and tp53 mutations in itac [ 2125 ] . tp53 mutations were found in 1844% of mostly occupational itacs , whereas , k - ras mutations were found in 1015% of itacs [ 2125 ] . the results of these studies suggest that mutations of k - ras and other ras genes are relatively uncommon in itac , and similarly , tp53 mutations in itacs have not been widely demonstrated . other studies have shown that k - ras mutation and c - erb-2 expression could be associated with more aggressive itacs [ 16 , 26 ] . found the existence of two genetic itacs subgroups , defined by differences in tp53 mutational status or protein functionality , that strongly influence pathologic response to primary chemotherapy and , ultimately , prognosis . perez - ordonez et al . evaluated the possible role of dna mismatch repair ( mmr ) gene defects or disruptions of e - cadherin/-catenin complex in itac by investigating the immunohistochemical expression of the mmr gene products , e - cadherin , and -catenin in a group of sporadic itacs . the preserved nuclear expression of mlh1 , msh2 , msh3 , and msh6 suggested that mutations or promoter methylation of mmr genes do not play a role in the pathogenesis of itac . kennedy et al . found that sinonasal itacs have a distinctive phenotype , with all cases expressing ck20 , cdx-2 , and villin and most itacs also expressing ck7 , so that the expression pattern of ck7 , ck20 , cdx-2 , and villin positive may be useful in separating these tumors from other non - itac adenocarcinomas of the sinonasal tract . given these findings , to explore other pathways involved in the molecular pathogenesis of itacs , we immunohistochemically investigated the expression of the apoptosis - regulating protein trail in a group of 23 ethmoidal itac associated with occupational exposure . this immunohistochemical expression was also retrospectively correlated with the patient outcome to evaluate their independent prognostic relevance . to the best of our knowledge , there are no previous reports on the expression of trail protein in itacs . trail is an apoptosis - inducing protein and a molecule important in inhibiting cellular immunity [ 4446 ] . similarly , cancer cells may use trail to evade the antitumor immune response . in literature , there are many works that have detected the expression of trail and its receptors in several tumors , correlating this expression with clinicopathological analysis . trail - r1 was identified as an independent prognostic factor for disease - free survival in 128 patients with colon cancer . mccarthy et al . showed that high trail - r2 expression was significantly associated with decreased survival and lymph node involvement in patients with primary breast cancer . revealed that trail and dr4 expression were correlated with the pathological grading in patients with laryngeal squamous cell carcinoma , while the alteration in dr5 expression was correlated with the clinical staging . despite the several works , the specific mechanism of how trail and its receptors contribute to carcinogenesis remains unknown . the results of our immunohistochemical examination showed a relationship between trail upregulation and the increase of the histological tumor grading . no correlation was found with other clinicopathological parameters , including t , stage and relapses . our results suggest that mutations of trail expression , in combination with additional genetic events , could play a role in the pathogenesis of itac . however , this study showed that trail expression can not be considered as an independent prognostic factor in patients with itacs , because there was not sufficient statistical power to detect significant associations between immunohistochemical expression of this protein and the clinicopathologic parameters of the tumors , therefore , further investigations with a larger sample are needed .