TTV infection in colorectal cancer tissues and normal mucosa

Genetic modifications and environmental factors are presently seen as the key factors in the etiology of colorectal cancer,1, 2, 3 whereas a role for infections has been debatable.4, 5, 6, 7 A broad spectrum of TT virus types has been detected and isolated from cancer tissue, including an elevated level in colon cancer,8, 9 but several attempts to link these viruses to the etiology of a specific disease have failed.10, 11 We determined the presence of TT virus DNA in tumor biopsies and normal colon tissue from patients (n = 82) with colorectal cancer in comparison to colon samples taken from individuals (n = 40) with normal endoscopic and histopathological findings. This study was approved by the local ethics committee and informed consent was obtained from all patients. All persons provided personal information of behavioral (smoking and alcohol consumption) and dietary aspects (high fat, red meat consumption) (data not shown). DNA was extracted as previously described12 and samples were blinded for subsequent analyses. PCR amplification of TTV DNA in the highly conserved region of its control region13 was performed (100 ng DNA per sample) using primer combinations NG133-NG352 during the first round, followed by a nested reaction with primers NG249-NG351 (amplicon size 134 bp). All amplicons were cloned and at least 10 clones per sample sequenced. Sequences were compared to all TTV sequences available in the databanks. TT virus DNA was present in 72 of 82 (87.8%) malignant samples and 61 of 82 (74.4%) normal mucosa from cancer patients in comparison to 16 of 40 (40%) of the control group. This is an interesting difference in view of the ubiquitous nature of TT viruses as reported in a multitude of studies analysing blood samples. The difference in the presence of TTV DNA observed between the normal mucosa and tumor tissue from the same patient is summarized in Table I. The DNA sequence in the control region of the TT virus genome is highly conserved amongst TT virus types, in contrast to the remainder of the genomes being very heterogenous in nature. We amplified this conserved region in order to detect all known, as well as putative new TT viruses in these samples. We compared sequences obtained here (using FASTA) to TTV complete genomes available in the databanks. The exact identification of the specific TT type involved, can however only be ascertained after amplification and characterization of each complete genome. The number of different TTV types identified within 1 sample varied. A total of 55 DNA sequences from the samples were identical to 15 known TTV types, whereas 224 were related (summarized in Figs. 1a and 1b). The TTV type TA278 (accession no. AB017619) was considered as the prototype sequence. Multiple types were detected more often in the same tumor tissue (up to 6). On the other hand, the identical sequence was in some cases found in the matched normal mucosa. Interestingly, the majority of sequences varied in single nucleotides from that of the corresponding identical sequence albeit upholding the overall conserved pattern of the respective TTV type (Fig. 1b). TT viruses phylogenetically group into 6 larger clusters, each consisting of smaller clusters of closer related TTV types.9 Of note is the large number of sequences isolated from malignant and normal tissue from cancer patients, related to the TTV types saj-30 (n = 70), ja10 (n = 23), tchn-c1 (n = 28) and saa-10 (n = 24) (Figs. 1a and 1b). (a) Distribution and prevalence of known and related TT virus DNA sequences detected in all samples tested. More than half (54.8%) of the isolated sequences were either identical or related to TT viruses in group Gamma 6. (b) DNA sequences from samples tested in comparison to closest known TTV types (italic). TTV type TA278 (accession no. AB017619) was considered as the prototype sequence. Hyphen indicates nucleotide identical to prototype the TA278 DNA sequence. a—number of additional sequences identical to the respective sequence. cm—sequence obtained from malignant tissue. cn—sequence obtained from normal mucosa of cancer patient. cc—sequence obtained from normal mucosa of control patient (gray). Abbreviations of known TT viruses (full names and accession numbers): j10 (jt10 - acc. no af122919), ja20 (af122914), gh1 (af122913), hel32 (hel32-6a, ay034068), tchc1 (tchn-c1, af345523), jt34 (ab064607), ct44f (ab064605), tjn1 (tjn01, ab028668), ts1 (tus01, ab017610), tchb (tchn-b, af348409), saa-01 (ab060597), ir31 (ttvsan-ir031, ab038619), sa10 (saa-10, ab060594), sj30 (saj-30, ab060595), tth4 (aj620226). The complete genomes of more than 100 TTV genotypes have been isolated, in addition to the several hundreds of putative TT viruses which have been identified in the form of partial sequences. Therefore it is surprising that the DNA sequences obtained in the present study were mainly related to only 15 TTV types, despite the large number of TTV types presently known. An additional observation is that only 20% of the sequences were identical to known TTV types, whereas the others harboured single nucleotide modifications when comparing the same region of the genome of the respective known TTV type. Sequence analyses of a number of isolates from the spleen of a Hodgkin's disease patient revealed single nucleotide changes leading to major modifications in the genome organization, even resulting in the expression of both modified and new viral proteins.9 The identification of TT viruses as quasi-species was also confirmed in the same study, pointing to a continuous modification of a TT virus type within the same host. Nucleotide modifications within the highly conserved control region of the genome amplified however rarely occurred in these TT virus genomes9 Chronic inflammation, often caused by infectious agents, inevitably causes tissue damage, thereby modifying the levels of expressed cellular proteins.14 Chronic inflammatory disease such as ulcerative colitis, predispose patients to cancer development. Elevated TT virus titres under similar conditions15 indicate an ongoing increased virus replication process during which inflammatory cytokines may play a role in inducing mutations16 in the viral genome. On the other hand, nitroso-compounds which result from the high consumption of red meat and increased smoking, as reported by the patients with colorectal carcinoma in our study (data not shown), may induce mutations not only in cellular genes, but also in the TTV genomes already present in the host. Single mutational modifications may alter the viral genes to express proteins with an oncogenic potential. Additional in vitro studies are required to investigate this possibility. Our data do not provide direct proof for an etiological relationship between TTV infection and colorectal cancer, however are suggestive of a preferential presence of these viruses in such cancers. The identification of a high level of TT viruses in colon tissue may therefore indicate an increased risk for colorectal cancers. Yours sincerely, We thank Ms. Sonja Stephan, Ms. Imke Grewe, Ms. Romana Kimmel and Dr. Helene Rahn for technical assistance and Ms. Andreas Hunziker for sequencing (Deutsches Krebsforschungszentrum), as well as Dr. Miljan Ceranic (First Surgical Clinic, Medical School of Belgrade) for collection of the biopsies. Ethel-Michele de Villiers*, Milutin Bulajic , Christine Nitsch*, Dragutin Kecmanovic , Maja Pavlov , Annette Kopp-Schneider§, Matthias LÖhr¶, * Division for the Characterization of Tumorvirus, Deutsches Krebsforschungszentrum, Heidelberg, Germany, Department of Gastroenterology, Clinical Center Dr Dragisa Misovic, University Medical School Belgrade, Serbia and Montenegro, Department of Colorectal and Pelvic Surgery, First Surgical University Hospital, Clinical Center of Serbia, Belgrade, Serbia, § Central Unit Biostatistics, Deutsches Krebsforschungszentrum, Heidelberg, Germany, ¶ Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg and the Molecular Gastroenterology Unit, Deutsches Krebsforschungszentrum, Heidelberg, Germany.