in 1911 , peyton rous used cell - free filtered extract of a chicken sarcoma to establish an association between cancer and an infectious agent  the rous sarcoma virus . almost 100 years later , developments in the techniques used to detect microbial genomes and investigate their biological properties have led to a definitive role for viral , bacterial and parasitic infection in human carcinogenesis . recent estimates indicate that the average proportion of malignancies worldwide that could be avoided in the absence of an infectious agent is 17.8% , with this figure being higher in developing countries , at 26.3% . the development of cancer is a complex multistage process that , as outlined by hanahan and weinberg , can be grouped into " six essential alterations to the cell physiology " ( figure 1 ) . these changes most likely occur by a progressive , almost evolutionary mechanism of successive genetic changes . the main mechanisms by which chronic infection promotes cancer do not usually involve direct mutagenesis , but instead are due to the complex interactions that occur between host and pathogen . in order for viruses to replicate and persist , they often promote cell survival , drive cellular proliferation , and evade the immune system . together , these processes , especially if present over a long period of time , can lead to tumorigenesis , often by influencing the same pathways that are involved in the development of cancer in the absence of infection ( figure 1 ) . continued persistent infection by a pathogen ( outer circle ) requires host - cell survival ( red ) , host - cell proliferation ( yellow ) , and evasion of the immune system by the pathogen ( blue ) . these pathogen - driven processes are achieved via various mechanisms that interfere with normal cell physiology and are outlined in figure 2 . alterations in these normally highly regulated pathways can lead to transforming events that have been described as the ' hallmarks of cancer ' ( inner circle ) . cancer is not , however , an outcome that has been specifically selected by evolution to aid pathogen survival . rather , it is more likely an unfortunate coincidence of pathogen capabilities selected to enable successful infection . therefore , certain infections may not necessarily cause the infected individual to develop cancer , but may be an associated risk factor ( figure adapted from ) . indeed , several of the key mediators of pathways and networks proposed by hanahan and weinberg were discovered through the study of viruses . many oncogenes , for example ras and myc , were identified originally in cancer - causing retroviruses . similarly , the study of dna virus proteins , such as sv40 large t - antigen , was instrumental in discovering tumor suppressor genes such as p53 . understanding the biology of oncogenic infections will most likely continue to inform cancer biology in general . one challenge is that of identifying potentially oncogenic agents and proving their causal connection with cancer . a recent paper in bmc medical genomics by duncan et al . describes the new computational technique of digital karyotyping microbe identification ( dk - microbe ) , and its application to identifying pathogens . their paper further exemplifies both the potential of molecular and bioinformatics methods for identifying pathogen dna in tumor samples , but underlines the necessity of establishing a causal association rather than just a pathogen presence . the major mechanisms by which infectious agents can promote and maintain tumor formation can be divided broadly into three main categories ( figure 2 ) . the first is the induction of chronic inflammation as a result of a continuing immune response to a persistent infection . this occurs , for example , in the case of hepatitis c virus ( hcv ) , associated with liver cancer , which continually replicates in the liver , setting up a chronic state of inflammation there . similarly , the blood fluke schistosoma haematobium and the gram - negative bacterium helicobacter pylori can both directly contribute to cancer formation through persistence within the host causing chronic inflammation . h. pylori is a good example of this category , and was classified by the world health organization as a class 1 carcinogen in 1994 . there is a high prevalence of persistent infection with h. pylori : worldwide , 75% of people are infected , with prevalence being higher in sub - saharan africa , where h. pylori is associated with 63.4% of all stomach cancers . however , the fact that not all people infected with h. pylori develop gastric cancer clearly shows that the infectious agent is a risk factor , but that other environmental and genetic influences are involved in cancer formation . these can be broadly divided into : chronic inflammation , which drives abnormal levels of cell proliferation ( yellow ) ; direct virus - induced transformation of infected cells , leading to increased cell survival ( red ) ; and immunosuppression , which allows the pathogen to evade the immune system and persist ( blue ) . chronic inflammation leads to the production of inflammatory cytokines as well as reactive oxygen and nitrogen oxide species ( ros and rnos ) by phagocytes at the site of infection , which can lead to dna damage as well as cellular damage and increased cell cycling . virus - induced transformation is caused by the actions of pathogen - encoded oncogenic proteins as well as by integration into the host genome ( hpv ) . the transforming events outlined in this figure do not necessarily lead directly to cancer formation ; for example , despite encoding similar proteins , other infectious agents do not cause cancer . the fact that some pathogens have evolved to persist without causing tumorigenesis also highlights that persistence is maybe a prerequisite for , but is on its own insufficient for , oncogenesis in humans . immune evasion mechanisms include control of the adaptive and innate immune system , allowing avoidance of tumor surveillance . ebv , epstein - barr virus ; hbv , human hepatitis virus b ; hcv , hepatitis virus c ; hiv , human immunodeficiency virus ; hpv , human papillomavirus ; htlv-1 , human t - lymphotropic virus 1 ; kshv , kaposi sarcoma - associated herpesvirus . this is due to the persistence of the viral genome in a latent form in an infected cell , either without replication , as with epstein - barr virus ( ebv ) , which infects b lymphocytes , or through integration of the viral genome into a host - cell chromosome , as with human papillomavirus ( hpv ) , the cause of cervical cancer . ebv is frequently detected in childhood burkitt 's lymphoma , post - transplant b - cell lymphomas , non - hodgkin 's lymphoma , hodgkin 's disease and nasopharyngeal carcinoma . the transforming capability of this virus is exemplified further by its ability to transform resting b cells in vitro at high efficiency to obtain stable proliferating lymphoblastoid cell lines . this process is driven by ebv - encoded latent proteins that directly promote cell growth and survival  for example , lymphocyte membrane - associated protein-1 ( lmp-1 ) . the third mechanism is the chronic suppression of the immune system by the infectious agent , such as the immunodeficiency ( aids ) caused by hiv infection . the presence of natural mechanisms of immunosurveillance for cancer cells , which in the case of an infectious etiology will also involve immune mechanisms that routinely control the infection , suggests why pathogens with oncogenic potential do not rapidly cause malignancy . a compromised immune system can result in an increased incidence of infection - driven tumors by weakening the immune control . such an increase is seen , for example , in transplant patients , who are being treated with immunosuppressants , or in individuals with aids . pathogens associated with cancer exemplify many of these mechanisms ; persistent infection involves evading the immune response as well as chronic inflammation , which even in the immune - competent leads to chronic cell proliferation and a greater risk of oncogenic transformation . however , many non - oncogenic pathogens are equally adept at these processes , indicating that other factors must be involved . for example , the risk of an infectious agent causing cancer may also depend on the cell type infected , as certain cell lineages could be more ' prone ' to transformation than others . for example , the increased prevalence of lympho mas and leukemias in children and young adults suggests that lymphocytes are more susceptible to transformation . ' the harder you look the more you find ' seems to be true for infectious agents in disease . the ability of some agents to remain latent , as well as the existence of new and emerging infections , make detection and proving causality challenging . an infectious agent may trigger the initial events of oncogenesis but be absent in the final tumor , which adds timing of detection to the problem . however , once the causal agent has been unequivocally found , development of a preventive treatment can be relatively rapid , as has been the case for cervical cancer . human papilloma viruses , especially types hpv16 and hpv18 , are now firmly associated with cervical cancer after their discovery in 1983 , and this has led to the development and widespread use of an hpv vaccine within 25 years  a timescale not dissimilar to that of drug discovery . similarly , recovery of immune function in aids by inhibiting hiv replication by antiretroviral therapy can lead to a regression of kaposi 's sarcoma , an endothelial tumor caused by the herpesvirus kshv , and a decrease in incidence of other aids - related cancers . treating infection is therefore a valuable addition to antitumor therapy if the infectious agent can be identified . today , two main techniques are used to detect microbial genomes in disease , one based on hunting for acknowledged candidates and the other on removing ( either physically or computationally ) known human sequences to reveal any foreign nucleic acid . pcr and microarray - based strategies are limited by a finite number of probes and sequences available but can be very sensitive . subtractive methods include representational difference analysis ( rda ) , which was used to detect kshv in kaposi 's sarcoma in 1994 . they do , however , require isogenic controls , which are not always readily available . one new approach to finding pathogen genomes is that of duncan et al . , which applies computational subtraction to digital karyotyping to hunt for virus genomes in several primary colorectal cancer samples and metastases as well as in normal tissue . the technique of dk - microbe described by duncan et al .   aims to circumvent limitations on detection imposed by the different mechanisms by which pathogens contribute to disease . in dk - microbe , genomic dna from the tumor is digested enzymatically into fragments of less than 10 kb in size that are processed to yield 21-bp tags for amplification , concate nation and sequencing . human sequences are compu tationally removed ; the remaining unidentified ' pathogen ' tags are then studied further . however , dk - microbe in its present form can only detect the genomes of dna viruses . were able to detect the human herpesvirus 6 ( hhv6 ) genome in samples from tumor tissue , but the fact that they also identified the viral dna in healthy tissues well illustrates the difficulties of causally associating a particular virus with a particular cancer . another subtractive method known as digital transcript subtraction ( dts ) attempts to identify exogenous pathogen transcripts via high - throughput sequencing , and can thus potentially identify the presence of rna and dna viruses . this method involves developing a long serial analysis of gene expression ( l - sage ) library from the tumor cells by quantitatively joining 21-bp tags composed of cdna copied from the 3 ' end of mrnas . as all human tumor viruses to date express part of their genome in the transformed cells , this has proved effective in virus discovery . the pioneers of this technique , yuan chang and patrick moore , have validated dts by identifying sequences from kshv in the primary effusion lymphoma cell line bcbl-1 ( feng et al . ) . more recently , dts has been used to identify a new polyomavirus in an uncommon but aggressive human skin cancer , merkel cell carcinoma ( mcc ) . a fusion transcript between an unknown virus t - antigen and a human receptor tyrosine kinase was detected . the new virus was named merkel cell polyoma - virus ( mcv ) and was detected in 80% of mcc tumors and also in 16% of normal skin biopsies . in 75% of the mcv - related mccs viral dna mcc occurs predominantly in the elderly and immuno - suppressed , two of the key features that indicate an infectious etiological agent . the question of where to look for other cancer - causing infectious agents is partly answered by the example of mcc . cancers with increased incidence in hiv - infected individuals   or in transplant recipients are an ideal place to look . one example is squamous cell conjunctival carcinoma ( sccc ) , which has emerged with the aids epidemic and is common in parts of sub - saharan africa . papillomaviruses have been implicated , but the evidence from pcr - based studies and serology testing is controversial . .   attempted to identify viral transcripts using dts , finding 21 candidate sequences that did not align with the human genome . dts is a powerful tool , but it is important to realize that its sensitivity is governed by the depth of sequencing , that is , the number of sequence reads analysed by dts or dk microbe , which may have to be increased in tumor samples , in which the ratio of viral mrna to human mrna is low . nevertheless , the combination of carefully selected tumors with deep sequencing and computational identification of non - human sequences is set to uncover more tumor viruses . detection of a microbial genome in a tumor does not prove causation , but the classical koch 's postulates for proving the microbial cause of a disease can not be invoked in this setting . one can not , for example , infect a person with a suspected causal agent and then wait to see if they develop cancer . modified criteria for infection , namely hill 's nine criteria outlined in 1965 , provide an evaluation method that allows weight to be given to causative factors . these were further developed by fredricks and relman to take into account molecular evidence ( table 1 ) . these rules , when applied to the detection of pathogen genomes , can serve as a basis for the association of an infectious agent with a particular disease . new techniques will undoubtedly lead to the identification of new and existing infectious agents in disease tissues , including cancer . however , care must be taken not to overemphasize association without proper proof of causation . molecular guidelines for establishing microbial causation of disease koch 's postulates for proving a causal connection between a particular infectious agent and a disease can not be applied to many human diseases as it would be unethical to experimentally infect humans with a potentially lethal infectious agent . the development of molecular diagnostic technology has enabled the criteria for causality summarized here to be drawn up . we wish to thank robin a weiss for reading a draft manuscript and making helpful comments .
molecular techniques for identifying pathogens associated with cancer continue to be developed , including one reported recently in bmc medical genomics . 
 identifying a causal infectious agent helps in understanding the biology of these cancers and can lead ultimately to the development of antimicrobial drugs and vaccines for their treatment and prevention .