BACKGROUND
one of the most significant tenet of tumor immunology is the assumption that the immune system is able to discriminate between normal and neoplastic tissues.
as this distinction is based on the overexpression of taas, the discovery of taas and their molecular and genetic characterization is important in both tumor immunodiagnosis and immunotherapy.
the progressive elucidation of the nature of taas recognized by antibodies and t lymphocytes coupled with the elaboration of methods for their isolation and synthesis of their peptides is leading towards new formulations of antitumor vaccines.
despite of the fact that long lists of well-characterized taas recognized by t cells  <cit>  and by antibodies  <cit>  are currently available, the attempts to use them to arouse an immune response able to cure tumor patients has met with very little success, at least so far  <cit> .
towards this goal significant obstacles are posed by the patient's state of immunosuppression and the numerous mechanisms to evade the immune response that a tumor progressively acquires  <cit> .
this two order of obstacles might suggest that active immunotherapy is not very appropriate for patients with advanced tumors.
a more modern endeavor is to exploit the potential of the immune response elicited by a vaccine against more plausible targets, such as pre-neoplastic lesions.
in this case, the immune response may be addressed towards taas that are already present in early lesions and that remain overexpressed during the progression of the tumor  <cit> .
identification of taas on preneoplastic cells will provide the opportunity to trigger the immune system against transformed cells at their earliest manifestation  <cit> .
the availability of animal models of autochthonous carcinogenesis offers an unprecedented opportunity to identify this kind of taas and to test the efficacy of the immune control of preneoplastic lesions  <cit> .
a preneoplastic lesion formed by few cells characterized by an indolent proliferation both displays a limited genetic instability and is more permeable to an immune attack than a large tumor  <cit> .
in addition it is possible that early expressed taas are more suitable target for tumor immune prevention than most of the already defined taas.
the characterization of taas expressed in early lesions is a prerequisite for setting up protocols for tumor prevention instead of for tumor cure  <cit> .
the protein product of the oncogene her <dig>  <cit> , igf-r  <cit>  and cyclin b <dig>  <cit>  are significant examples of early expressed taas causally involved in the progression of the lesions  <cit> .
mouse strains transgenic for oncogenes or knock-out for oncosuppressor genes that consequently develop defined kind of tumors are being used for preclinical assessment of the potential of preventive vaccines  <cit> .
by combining the information provided by the genome sequence and the expression profiles of entire transcriptomes , these mouse models can also lead towards the identification of taas expressed during progressive stages of carcinogenesis.
microarrays are an attractive way of identifying new taas since their procedure is now firmly established  <cit>  and they are well suited to the intrinsic limitations of the biological material to be examined .
this paper describes our application of meta-analysis to two balb-neut mice transcription profiling studies  <cit>  to evaluate the possibility of identifying, within the tumor modulated genes, a set of new putative targets to be used as taas instead of or in conjunction with her <dig>
RESULTS
our previous demonstration of persistent inhibition of autochthonous preneoplastic lesions in a mouse model of her <dig> mammary carcinogenesis by a combined dna and cell vaccine  <cit> , and the curing of her <dig> transplantable tumors by dna vaccination  <cit>  indicates that efficient immunological inhibition of her <dig> carcinogenesis can be achieved.
we have also shown that two transcriptional profiling studies based on different immunization protocols in balb-neut mice  <cit>  are integrable  <cit> .
since an important issue of dna vaccination is the definition of a set of target genes other that her <dig> in order to broaden its effective coverage, we decided to use these data to identify a set of new taas to be used in dna vaccination.
datasets
seven prototypic situations were evaluated.
mammary glands from:  6-week-old mice , displaying atypical hyperplasia;  10-week-old mice , showing atypical hyperplasia and some foci of in situ carcinomas;  15-week-old mice , with diffuse hyperplasia and in situ carcinomas;  19-week-old mice , displaying lobular invasive carcinomas;  22-week-old mice , with palpable carcinomas;  26-week-old mice , with large lobular and metastasizing carcinomas,  2-week-pregnant  wild type balb/c mice displaying marked pregnancy-related hyperplasia  <cit> .
wk6nt, wk15nt, wk19nt and wk26nt gland expression profiles were derived from astolfi  <cit> , wk10nt, wk22nt and wk2prg gland profiles from quaglino  <cit> .
selection of genes whose expression is linearly correlated with the increase of the tumor
the number of neoplastic cells in the mammary tissue of balb-neut mice increases constantly during aging, and at 19– <dig> weeks a tumor mass becomes palpable in all their mammary glands.
previously  <cit>  we have used pca  to show that the transcriptional profiles of balb-neut mammary glands are linearly correlated with age .
we identified  <dig> probe sets by linear fitting each gene expression with respect to age .
only one probe set was linked to an unmapped est , whereas the others were linked to annotated mouse genes.
investigation of specific gene ontology  class  over-representation within the annotated  <dig> genes revealed significant enrichments in functions associated with the extracellular compartment .
pathway analysis showed the presence of  <dig> genes involved in the integrin signal transduction pathway  linked to her <dig> action  <cit> .
evaluation of the expression of tumor-related genes in normal mouse tissues
if a taa is to be used as a vaccination target, its expression must be low in normal tissues.
to evaluate the expression of the  <dig> putative taas in normal tissues, we used transcription profiles recently published by su  <cit> .
the mouse atlas  was generated by using a custom affymetrix array which interrogates  <dig>  annotated mouse genes:  <dig> of the putative taas are present in gnfm, twelve of them  characterized by low expression  in normal mouse tissues.
expression of tumor related genes in normal and cancer human tissues
the  <dig> genes identified as taas were expressed in all the stages of breast carcinogenesis in balb-neut mice and characterized by low expression in normal mouse tissues.
to determine whether these characteristics were also present in the corresponding human genes, we cross-validated the expression of the  <dig> taas within the human tissue expression atlas published by yanai  <cit> .
since her <dig> is expressed at very low levels in normal human tissues, we used it as the "ideal reference" of low expression.
rtn <dig>  irf <dig>  sel1l were discarded since their expression levels in some human tissues were higher than those of her <dig> .
tes, rcn <dig>  rnf <dig>  cradd, galnt <dig>  clca <dig>  cdcp <dig>  socs <dig> and spred <dig> were expressed at levels similar to her <dig>
we also assessed the expression of these  <dig> taas in human breast specimens.
of the breast cancer transcriptional profiling data sets available  <cit> , that proposed by van't veer  <cit>  is ideal because its experimental structure allows evaluation of the expression level distribution within the specimens.
fold change variation of each specimen is measured with respect to a reference pool composed by mixing the same amount of all rna of the sporadic patients  <cit> .
the  <dig> taas and her <dig> were represented in the van't veer set  <cit> , her <dig> was highly expressed, while tes, rcn <dig>  rnf <dig>  cradd and galnt <dig> were medium expressed.
clca <dig>  cdcp <dig>  socs <dig> and spred <dig> were low expressed group  and since their expression was  <dig> times lower than that of her <dig> they were discarded as putative vaccination targets.
even though tes, rcn <dig>  rnf <dig>  cradd and galnt <dig> were less expressed than her <dig>  they were less scattered than her <dig> .
the highest expressed is tes, which maps to a fragile site on chromosome 7q <dig>   <cit>  and may have a tumor suppression activity  <cit> .
it is homogeneously expressed in all the  <dig> cancer specimens in the van't veer set  <cit> .
the function of rcn <dig>  reticulocalbin, has not been characterized.
it is implicated in tumor cell invasiveness, as it is expressed in the highly invasive breast cancer cell lines, but not in poorly invasive ones  <cit> .
rnf <dig> is a ring-finger protein which acts as a transcription regulator  <cit> , it is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters  <cit> .
it is expressed at very high levels in testis and at much lower levels in several other tissues.
when ectopically expressed, it inhibits the proliferation of both somatic and germ cell tumor-derived cells  <cit> .
cradd encodes a death domain -containing protein and induces apoptosis  <cit> .
galnt <dig> encodes udp-galnac transferase  <dig>  a member of the galnac-transferases family.
in gallbladder cancer, the presence of diffuse-type localization of galnac-t <dig> in the subserosal layer is correlated with aggressiveness  <cit> .
furthermore, galnt <dig> expression is associated with the differentiation and aggressiveness of ductal adenocarcinoma of the pancreas  <cit> .
CONCLUSIONS
this study identified  <dig> putative taas.
three were discarded because they were expressed in some normal human tissues and four because their expression was too low in human cancer specimens.
tes, rcn <dig>  rnf <dig>  cradd and galnt <dig> were retained for further studies.
this work was based on in silico analysis of published transcriptional profiling studies.
its results must now be referred back to the in vivo pre-clinical experimentation.
we shall select the most immunogenic domains of the  <dig> candidate taas and use them in vaccination protocols in balb-neut mice  <cit> .
other interesting taas may have been missed in this analysis since it integrates two independent experiments and this increases the experimental noise.
a further limitation could be the partial coverage of the mouse transcriptome of the arrays employed.
a new transcription profiling study covering the same experimental points, but using full genome-wide mouse arrays is now in progress.

 one of the most significant tenet of tumor immunology is the assumption that the immune system is able to discriminate between normal and neoplastic tissues. as this distinction is based on the overexpression of taas, the discovery of taas and their molecular and genetic characterization is important in both tumor immunodiagnosis and immunotherapy. the progressive elucidation of the nature of taas recognized by antibodies and t lymphocytes coupled with the elaboration of methods for their isolation and synthesis of their peptides is leading towards new formulations of antitumor vaccines. despite of the fact that long lists of well-characterized taas recognized by t cells and by antibodies are currently available, the attempts to use them to arouse an immune response able to cure tumor patients has met with very little success, at least so far. towards this goal significant obstacles are posed by the patient's state of immunosuppression and the numerous mechanisms to evade the immune response that a tumor progressively acquires. this two order of obstacles might suggest that active immunotherapy is not very appropriate for patients with advanced tumors. a more modern endeavor is to exploit the potential of the immune response elicited by a vaccine against more plausible targets, such as pre-neoplastic lesions. in this case, the immune response may be addressed towards taas that are already present in early lesions and that remain overexpressed during the progression of the tumor. identification of taas on preneoplastic cells will provide the opportunity to trigger the immune system against transformed cells at their earliest manifestation. the availability of animal models of autochthonous carcinogenesis offers an unprecedented opportunity to identify this kind of taas and to test the efficacy of the immune control of preneoplastic lesions. a preneoplastic lesion formed by few cells characterized by an indolent proliferation both displays a limited genetic instability and is more permeable to an immune attack than a large tumor. in addition it is possible that early expressed taas are more suitable target for tumor immune prevention than most of the already defined taas. the characterization of taas expressed in early lesions is a prerequisite for setting up protocols for tumor prevention instead of for tumor cure. the protein product of the oncogene her, igf-r and cyclin b are significant examples of early expressed taas causally involved in the progression of the lesions. mouse strains transgenic for oncogenes or knock-out for oncosuppressor genes that consequently develop defined kind of tumors are being used for preclinical assessment of the potential of preventive vaccines. by combining the information provided by the genome sequence and the expression profiles of entire transcriptomes, these mouse models can also lead towards the identification of taas expressed during progressive stages of carcinogenesis. microarrays are an attractive way of identifying new taas since their procedure is now firmly established and they are well suited to the intrinsic limitations of the biological material to be examined. this paper describes our application of meta-analysis to two balb-neut mice transcription profiling studies to evaluate the possibility of identifying, within the tumor modulated genes, a set of new putative targets to be used as taas instead of or in conjunction with her  our previous demonstration of persistent inhibition of autochthonous preneoplastic lesions in a mouse model of her mammary carcinogenesis by a combined dna and cell vaccine, and the curing of her transplantable tumors by dna vaccination indicates that efficient immunological inhibition of her carcinogenesis can be achieved. we have also shown that two transcriptional profiling studies based on different immunization protocols in balb-neut mice are integrable. since an important issue of dna vaccination is the definition of a set of target genes other that her in order to broaden its effective coverage, we decided to use these data to identify a set of new taas to be used in dna vaccination. datasets seven prototypic situations were evaluated. mammary glands from: 6-week-old mice, displaying atypical hyperplasia; 10-week-old mice, showing atypical hyperplasia and some foci of in situ carcinomas; 15-week-old mice, with diffuse hyperplasia and in situ carcinomas; 19-week-old mice, displaying lobular invasive carcinomas; 22-week-old mice, with palpable carcinomas; 26-week-old mice, with large lobular and metastasizing carcinomas, 2-week-pregnant wild type balb/c mice displaying marked pregnancy-related hyperplasia. wk6nt, wk15nt, wk19nt and wk26nt gland expression profiles were derived from astolfi, wk10nt, wk22nt and wk2prg gland profiles from quaglino. selection of genes whose expression is linearly correlated with the increase of the tumor the number of neoplastic cells in the mammary tissue of balb-neut mice increases constantly during aging, and at 19– weeks a tumor mass becomes palpable in all their mammary glands. previously we have used pca to show that the transcriptional profiles of balb-neut mammary glands are linearly correlated with age. we identified probe sets by linear fitting each gene expression with respect to age. only one probe set was linked to an unmapped est, whereas the others were linked to annotated mouse genes. investigation of specific gene ontology class over-representation within the annotated genes revealed significant enrichments in functions associated with the extracellular compartment. pathway analysis showed the presence of genes involved in the integrin signal transduction pathway linked to her action. evaluation of the expression of tumor-related genes in normal mouse tissues if a taa is to be used as a vaccination target, its expression must be low in normal tissues. to evaluate the expression of the putative taas in normal tissues, we used transcription profiles recently published by su. the mouse atlas was generated by using a custom affymetrix array which interrogates annotated mouse genes: of the putative taas are present in gnfm, twelve of them characterized by low expression in normal mouse tissues. expression of tumor related genes in normal and cancer human tissues the genes identified as taas were expressed in all the stages of breast carcinogenesis in balb-neut mice and characterized by low expression in normal mouse tissues. to determine whether these characteristics were also present in the corresponding human genes, we cross-validated the expression of the taas within the human tissue expression atlas published by yanai. since her is expressed at very low levels in normal human tissues, we used it as the "ideal reference" of low expression. rtn irf sel1l were discarded since their expression levels in some human tissues were higher than those of her. tes, rcn rnf cradd, galnt clca cdcp socs and spred were expressed at levels similar to her we also assessed the expression of these taas in human breast specimens. of the breast cancer transcriptional profiling data sets available, that proposed by van't veer is ideal because its experimental structure allows evaluation of the expression level distribution within the specimens. fold change variation of each specimen is measured with respect to a reference pool composed by mixing the same amount of all rna of the sporadic patients. the taas and her were represented in the van't veer set, her was highly expressed, while tes, rcn rnf cradd and galnt were medium expressed. clca cdcp socs and spred were low expressed group and since their expression was times lower than that of her they were discarded as putative vaccination targets. even though tes, rcn rnf cradd and galnt were less expressed than her they were less scattered than her. the highest expressed is tes, which maps to a fragile site on chromosome 7q and may have a tumor suppression activity. it is homogeneously expressed in all the cancer specimens in the van't veer set. the function of rcn reticulocalbin, has not been characterized. it is implicated in tumor cell invasiveness, as it is expressed in the highly invasive breast cancer cell lines, but not in poorly invasive ones. rnf is a ring-finger protein which acts as a transcription regulator, it is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters. it is expressed at very high levels in testis and at much lower levels in several other tissues. when ectopically expressed, it inhibits the proliferation of both somatic and germ cell tumor-derived cells. cradd encodes a death domain -containing protein and induces apoptosis. galnt encodes udp-galnac transferase a member of the galnac-transferases family. in gallbladder cancer, the presence of diffuse-type localization of galnac-t in the subserosal layer is correlated with aggressiveness. furthermore, galnt expression is associated with the differentiation and aggressiveness of ductal adenocarcinoma of the pancreas.  this study identified putative taas. three were discarded because they were expressed in some normal human tissues and four because their expression was too low in human cancer specimens. tes, rcn rnf cradd and galnt were retained for further studies. this work was based on in silico analysis of published transcriptional profiling studies. its results must now be referred back to the in vivo pre-clinical experimentation. we shall select the most immunogenic domains of the candidate taas and use them in vaccination protocols in balb-neut mice. other interesting taas may have been missed in this analysis since it integrates two independent experiments and this increases the experimental noise. a further limitation could be the partial coverage of the mouse transcriptome of the arrays employed. a new transcription profiling study covering the same experimental points, but using full genome-wide mouse arrays is now in progress. 
the five taas identified are those whose expression is linearly related to the tumor mass increase in balb-neut mammary glands. although the number of putative taas identified is limited, this pilot study suggests that meta-analysis of expression profiles produces results that could assist in the designing of pre-clinical immunopreventive vaccines. identification of overexpressed genes provides an unprecedented opportunity to address the immune system against antigens typical of defined stages of neoplastic transformation. moreover, they have a low expression in normal tissues and are generally expressed in human breast tumors, though at a lower level than her 
 neoplastic transformation is a multistep process in which distinct gene products of specific cell regulatory pathways are involved at each stage. identification of overexpressed genes provides an unprecedented opportunity to address the immune system against antigens typical of defined stages of neoplastic transformation. her-2/neu/erbb oncogene is a prototype of deregulated oncogenic protein kinase membrane receptors. mice transgenic for rat her were studied to evaluate the stage in which vaccines can prevent the onset of her driven mammary carcinomas. as her is not overexpressed in all mammary carcinomas, definition of an additional set of tumor associated antigens expressed at defined stages by most breast carcinomas would allow a broader coverage of vaccination. to address this question, a meta-analysis was performed on two transcription profile studies to identify a set of new taa targets to be used instead of or in conjunction with her  the five taas identified are those whose expression is linearly related to the tumor mass increase in balb-neut mammary glands. moreover, they have a low expression in normal tissues and are generally expressed in human breast tumors, though at a lower level than her  although the number of putative taas identified is limited, this pilot study suggests that meta-analysis of expression profiles produces results that could assist in the designing of pre-clinical immunopreventive vaccines. 17– march italian society of bioinformatics: annual meeting milan, italy 