Studies on tumor virus epidemiology

The causal relationship between several virus infections and human cancers are well established. However, it is also possible that additional cancers may be caused by known or yet unknown viruses. The present thesis has sought to both further elucidate known relationships between virus and cancer as well as to provide a basis for further exploration in the area of infections and cancer. Infections during pregnancy have been suspected to be involved in the etiology of childhood leukemias. However, no specific infectious agent is yet linked to the etiology of these diseases. As a basis for further studies in this area, we applied high-throughput next generation sequencing (NGS) technology to describe the viruses most readily detectable in serum samples of mothers to leukemic children. The most common viruses found were TT viruses, including several previously not described TT viruses. Merkel cell polyomavirus (MCV) is found in Merkel cell carcinoma (MCC), a rare and aggressive neuroendocrine tumor of the skin. To explore whether MCV infection might be associated with additional cancers, we investigated whether MCC patients are at excess risk of other cancers, using population-based Nordic cancer registries. Bidirectional evaluation of excess risk of other diseases among MCC patients revealed that they are at increased risk of other skin cancers as a second cancer, compared to the general Nordic population. Shared causative factors, such as exposure to ultraviolet light and/or MCV infection are among the possible explanations. Also, impact of increased surveillance of the skin should be noted as an explanation of the excess risk. Cutaneous human papillomaviruses (HPV) are suspected to be involved in the etiology of non-melanoma skin cancer (NMSC). To evaluate whether there are any consistent association between cutaneous HPV infections and skin cancer, we conducted a systematic review and meta-analysis of studies that investigated HPV prevalences among cases of skin lesions and their healthy controls. We found that HPV species Beta-1, Beta-2, Beta-3 and Gamma-1 were more frequently detected in squamous cell carcinoma (SCC) compared to healthy controls. To provide clues about possible carcinogenicity of 47 mucosal HPV types, out of which 12 are established as causes of cervical cancer, we also investigated the prevalence of 47 mucosal HPV types across the entire range of cervical diagnoses from normal to cervical cancer. To investigate diversity of HPVs in skin lesions with increased sensitivity, different sample types from different skin lesion were subjected to high-throughput NGS after PCR amplification. Conventional molecular detection methods such as PCR are biased towards the primers used. Thus they might miss viruses that are divergent from the primer sequences. We also investigated whether NGS technology can be used to assess presence of virus DNA in an unbiased manner, both in skin lesions as well as in condylomas that were classified as “HPV negative” by conventional PCR methods. Unbiased sequencing identified two putatively new HPV types that were missed by NGS after PCR amplification. The advantage of unbiased sequencing over conventional molecular detection methods was further demonstrated in the study of “HPV negative” condylomas. We found several known as well as several putatively novel HPV types in condylomas that were previously found to be HPV negative by PCR. In conclusion, we have used registry linkage studies, systematic reviews and metaanalyses and modern NGS technology applied to biobanked specimens to extend our knowledge of the epidemiology of cancer-associated viruses and to provide a basis for further exploration in this area

Viruses in case series of tumors: Consistent presence in different cancers in the same subject.

Studies investigating presence of viruses in cancer often analyze case series of cancers, resulting in detection of many viruses that are not etiologically linked to the tumors where they are found. The incidence of virus-associated cancers is greatly increased in immunocompromised individuals. Non-melanoma skin cancer (NMSC) is also greatly increased and a variety of viruses have been detected in NMSC. As immunosuppressed patients often develop multiple independent NMSCs, we reasoned that viruses consistently present in independent tumors might be more likely to be involved in tumorigenesis. We sequenced 8 different NMSCs from 1 patient in comparison to 8 different NMSCs from 8 different patients. Among the latter, 12 different virus sequences were detected, but none in more than 1 tumor each. In contrast, the patient with multiple NMSCs had human papillomavirus type 15 and type 38 present in 6 out of 8 NMSCs

Molecular methods for identification and characterization of novel papillomaviruses.

Papillomaviruses (PV) are a remarkably heterogeneous family of small DNA viruses that infect a wide variety of vertebrate species and are etiologically linked with the development of various neoplastic changes of the skin and mucosal epithelia. Based on nucleotide similarity, PVs are hierarchically classified into genera, species and types. Novel human PV (HPV) types are given a unique number only after the whole genome has been cloned and deposited with the International HPV Reference Center. As of March 09, 2015, 200 different HPV types, belonging to 49 species, had been recognized by the International HPV Reference Center. In addition, 131 animal PV types identified from 66 different animal species exist. Recent advances in molecular techniques have resulted in an explosive increase in the identification of novel HPV types and novel subgenomic HPV sequences in the last few years. Among PV genera, the Gamma-PV genus has been growing most rapidly in recent years with 80 completely sequenced HPV types, followed by Alpha- and Beta-PV genera that have 65 and 51 recognized HPV types, respectively. We reviewed in detail the contemporary molecular methods most often used for identification and characterization of novel PV types, including polymerase chain reaction, rolling circle amplification and next generation sequencing. Furthermore, we present a short overview of 12 and 10 novel HPV types recently identified in Sweden and Slovenia, respectively. Finally, an update on the International Human Papillomavirus Reference Center is provided

Deep sequencing extends the diversity of human papillomaviruses in human skin.

Most viruses in human skin are known to be human papillomaviruses (HPVs). Previous sequencing of skin samples has identified 273 different cutaneous HPV types, including 47 previously unknown types. In the present study, we wished to extend prior studies using deeper sequencing. This deeper sequencing without prior PCR of a pool of 142 whole genome amplified skin lesions identified 23 known HPV types, 3 novel putative HPV types and 4 non-HPV viruses. The complete sequence was obtained for one of the known putative types and almost the complete sequence was obtained for one of the novel putative types. In addition, sequencing of amplimers from HPV consensus PCR of 326 skin lesions detected 385 different HPV types, including 226 previously unknown putative types. In conclusion, metagenomic deep sequencing of human skin samples identified no less than 396 different HPV types in human skin, out of which 229 putative HPV types were previously unknown

Classification of viral reads.

<p>Classification of viral reads.</p

Taxonomic assignment of sequencing reads.

<p>Taxonomic assignment of sequencing reads.</p

Does human papillomavirus-negative condylomata exist?

Condylomata acuminata is caused by human papillomavirus (HPV). PCR with consensus primers will typically detect HPV in >96% of condylomata. Metagenomic sequencing has found that some "HPV-negative" condylomata do indeed contain HPV. We wished to perform a renewed evaluation of the "HPV-negative" condylomata using deeper metagenomics sequencing. Sequencing of whole genome amplified DNA from 40 apparently "HPV-negative" condylomata detected HPV in 37/40 specimens. We found 75 different HPV types, out of which 43 represented novel putative HPV types. Three types were cloned and established as HPV types 200, 201 and 202. Molluscum contagiosum virus was detected in 24 of the 40 samples. In summary, deep sequencing enables detection of HPV in almost all condylomata. "HPV-negative" condylomata might largely be explained by clinical misdiagnosis or the presence of viral variants, distantly related HPV types and/or low viral loads

Viremia during pregnancy and risk of childhood leukemia and lymphomas in the offspring: Nested case-control study.

A possible role for infections of the pregnant mother in the development of childhood acute leukemias and lymphomas has been suggested. However, no specific infectious agent has been identified. Offspring of 74,000 mothers who had serum samples taken during pregnancy and stored in a large-scale biobank were followed up to the age of 15 years (750,000 person years) through over-generation linkages between the biobank files, the Swedish national population and cancer registers to identify incident leukemia/lymphoma cases in the offspring. First-trimester sera from mothers of 47 cases and 47 matched controls were retrieved and analyzed using next generation sequencing. Anelloviruses were the most common viruses detected, found in 37/47 cases and in 40/47 controls, respectively (OR: 0.6, 95% CI: 0.2-1.9). None of the detected viruses was associated with leukemia/lymphoma in the offspring. Viremia during pregnancy was common, but no association with leukemia/lymphoma risk in the offspring was found