Colorectal carcinoma (CRC) can be sporadic, familial or inherited. The sporadic form represents about 70% of CRC cases. DNA alterations detected in CRC somatic cells are due to several factors. Different cancerogenic agents are involved in the CRC onset/progression, including tumor viruses, such as herpes-, adeno-, pox-, papilloma-, hepatitis-, retro- and polyoma-viruses. Among the oncogenic Polyomaviruses, JC polyomavirus (JCPyV) was found to be associated with CRC cases. JCPyV virus is a polyomavirus identified in 1971 as the causative agent of progressive multifocal leukoencephalopathy (PML). In recent years, several studies reported on the association between JCPyV and different human cancers, mainly brain and colorectal tumors. However, confliting data were published. Indeed, several investigations found the association between colorectal cancer and JCPyV, whereas other studies reported negative results. On this ground, in this study I investigated the association between CRC and JCPyV with new technical approaches. To this purpose, JCPyV DNA sequences were investigated (i) in CRC and the adjacent healthy mucosa (HM) biopsies; (ii) in primary cell cultures derived from CRC; (iii) in serum samples from CRC patients and controls, represented by healthy subjects (HS) with the same median age and gender of the patients. Then, (iv) the prevalence of JCPyV-antibodies was investigated in serum samples of the two cohorts, CRC and HS. JCPyV sequences were detected in 22/53, 41.5%, of CRC biopsies and in 11/53, 21%, of HM tissues, being the different prevalence statistically significant. It is interesting to note that 11 CRC samples tested JCPyV-positive, whereas the matched HM sample were found to be JCPyV-negative. Furthermore, JCPyV sequences are more prevalent in the cecum and in the splenic flexure samples, both in tumor and normal mucosa biopsies. This data suggests an association between CRC and JCPyV with a particular focus in these two anatomical districts. It is known that B- and T-lymphocytes represent vehicles for JCPyV to reach different tissues of the host. To verify if JCPyV-positive CRC tumors are due to the presence of the virus in the cancer cells, or to the lymphocytes infiltrating the tumor in vivo, primary cultures derived from CRC cells were set up. Indeed, B- and T-lymphocytes do not grow in vitro during the cell culture passages, thus allowing the transformed epithelial cells to multiply in monolayers. Primary CRC cell cultures (n=13) from the biopsies were set up and characterized. Among these 13 randomly chosen biopsies, 4/13 (31%) CRC tested JCPyV-positive. The 4 cell samples found JCPyV-positive and the 9 other cell cultures tested JCPyV-negative had the same results when analyzed as biopsies. This result suggests 2 that the presence of JCPyV sequences in human colorectal cancer biopsy is not due to the lymphocytes infiltrating the tumor. In recent studies, circulating JCPyV DNA sequences were detected in human sera. However, in my investigation among 53 CRC and 89 HM sera only one sample, from CRC patient, tested JCPyVpositive. Subsequently, the prevalence of serum IgG antibodies against JCPyV was comparatively investigated in CRC tumor patients and HS controls. Mimotopes from JCPyV structural peptides were tested to investigate for specific reactions to human sera antibodies. An indirect ELISA with synthetic peptides from JCPyV viral capsid protein 1 (VP1) was set up and employed to test 53 CRC serum samples and 89 healthy subject. Data from immunologic assays indicate that the overall prevalence of JCPyVVP1 antibodies in CRC patients is 26%, whereas in HS is 51%. As a control of the data obtained by indirect E.L.I.S.A., hemagglutination inhibition assay (H.I.A.) was used. Data from H.I.A. overlapped the results obtained by the indirect ELISA with synthetic peptides. In conclusion, the new indirect ELISA is reliable, faster, sensitive, specific and with affordable costs. This investigation found an association between colorectal cancer and JCPyV infection. Indeed, JCPyV sequences were detected in CRC samples with a higher prevalence than that revealed in HM of the same patients. Interestingly, the majority of sera from CRC patients, tested JCPyV-positive, did not react with JCPyV VP antigens. It is possible that these CRC patients, as oncologic patients, were at least partially immunodepressed or non-responders and therefore they were unable to neutralize the JCPyV infection and consequently its oncogenic ativity. This data are innovative in this field and they may represent a starting point to investigate further the putative role of JCPyV in CRC onset/progression and its mechanism of transformation.

Il carcinoma al colon-retto (CRC) può essere di tipo sporadico, familiare o ereditario. La forma sporadica, comprende circa il 70% dei casi di CRC, mentre le alterazioni del DNA nelle cellule somatiche sono causate da diversi fattori. Tra i diversi agenti cancerogeni coinvolti nell'insorgenza del cancro del colon-retto sono inclusi anche i virus oncogeni. Alcuni esempi sono herpes-, adeno-, pox-, papilloma-, epatite-, retro- e polioma-virus. Tra i virus polioma oncogeni, JCPyV è stato trovato associato al CRC. Il virus JC è un virus polioma identificato nel 1971 come agente causale della leucoencefalopatia multifocale progressiva (PML). Negli ultimi anni, diversi studi hanno riportato l’associazione tra JCPyV e diversi tumori umani, come tumori cerebrali e del colon-retto. Tuttavia, i dati della letteratura sono conflittuali. Per questo motivo è in atto un forte dibattito sull’associazione tra il cancro del colon-retto e JCPyV nella comunità scientifica del settore. Infatti, alcuni studi hanno dimostrato l'associazione tra CRC e JCPyV, mentre altre ricerche non hanno confermato questa associazione. Nel mio studio ho indagato l'associazione tra CRC e JCPyV con nuovi approcci scientifici e tecniche innovative. A questo scopo ho analizzato la presenza delle sequenze di DNA di JCPyV (i) nelle biopsie di CRC e della mucosa sana adiacente (HM); (ii) in colture cellulari primarie derivate da CRC; (iii) in campioni di siero di pazienti affetti da CRC e controlli, rappresentati da soggetti sani (HS) con la stessa età media e genere dei pazienti. Poi, ho analizzato (iv) la presenza e prevalenza di anticorpi anti-JCPyV in campioni di siero delle stesse due coorti, CRC e HS. Le sequenze genomiche di JCPyV sono state rilevate nel 41,5% (22/53) delle biopsie di CRC e nel 21% (11/53) dei tessuti HM. La diversa prevalenza è statisticamente significativa. È interessante notare come in 11 campioni di CRC risultati JCPyV-positivi, il corrispondente tessuto sano di HM è risultato JCPyV-negativo. Inoltre, la prevalenza delle sequenze di JCPyV è risultata tra i CRCJCPyV-positivi, maggiore nei campioni di cieco e flessura splenica, sia nelle biopsie di tumore che nella mucosa sana. Questi dati nell’insieme indicano un'associazione tra CRC e JCPyV con una particolare attenzione a questi due distretti anatomici. È noto che i linfociti B e T rappresentano un veicolo per JCPyV per raggiungere i diversi tessuti dell'ospite. Per verificare se la positività di JCPyV riscontrata nelle biopsie di tumore è dovuta alla presenza del virus nelle cellule tumorali o nei linfociti infiltranti il tumore in vivo, sono state allestite e caratterizzate le colture cellulari primarie derivate dalla biopsia di CRC (n=13). Infatti, i linfociti B 2 e T non crescono in vitro durante i passaggi di coltura cellulare, permettendo così alle cellule epiteliali trasformate di moltiplicare in monostrati. Tra 13 biopsie di CRC scelte casualmente, il 31% (4/13) sono risultate JCPyV-positive. I 4 campioni di cellule JCPyV-positivi e le altre 9 colture cellulari JCPyV-negative hanno avuto gli stessi risultati quando analizzati come biopsie. Questo risultato suggerisce che la presenza di sequenze di JCPyV nella biopsia di carcinoma colorettale umano non è dovuto ai linfociti infiltranti il tumore. In studi recenti, sono state rilevate sequenze circolanti di DNA di JCPyV nel siero umano. Tuttavia, nella mia indagine tra 53 sieri di CRC e 89 di HM solo campione, di un paziente affetto da CRC, è risultato JCPyV-positivo. Successivamente, è stata indagata la prevalenza di anticorpi IgG contro JCPyV nel siero di pazienti affetti da CRC e controlli HS. In particolare, sono stati disegnati due peptidi sintetici (VP1 K e VP1 N) che mimano gli epitopi della proteina strutturale VP1 di JCPyV. È stato allestito un ELISA indiretto con l’uso di questi due peptidi sintetici, utilizzati come antigeni, per testare 53 campioni di siero di pazienti affetti da CRC e 89 di HS. I dati provenienti dai test immunologici indicano che la prevalenza di anticorpi anti-JCPyV in pazienti affetti da CRC è del 26% (14/53), mentre nel gruppo di controllo HS è del 51% (45/89). Come controllo dei dati ottenuti con il test E.L.I.S.A. indiretto, è stato impiegato il saggio di inibizione dell’emoagglutinazione (H.I.A.). I dati provenienti da questo saggio sono sovrapponibili ai risultati ottenuti con il test ELISA indiretto con peptidi sintetici. In conclusione, il nuovo ELISA indiretto è affidabile, veloce, sensibile, specifico ed economico. Questa indagine ha trovato un'associazione tra il cancro del colon-retto e l'infezione da JCPyV. Infatti, le sequenze di JCPyV sono state rilevate in campioni di biopsia di CRC con una prevalenza superiore a quella rivelata nella biopsia HM degli stessi pazienti. È interessante notare che la maggior parte dei sieri di pazienti affetti da CRC, risultati JCPyV-positivi, non hanno reagito con gli antigeni di JCPyV. È possibile che questi pazienti oncologici siano almeno parzialmente immunodepressi o nonresponder, quindi non siano in grado di neutralizzare l'infezione di JCPyV e di conseguenza la sua attività oncogenica. Questi dati sono innovativi in questo campo e possono rappresentare un punto di partenza per indagare ulteriormente il ruolo putativo di JCPyV nell’insorgenza/progressione di CRC.

Association between colorectal carcinoma and the oncogenic polyomavirus JCPyV

PIETROBON, SILVIA
2016

Abstract

Colorectal carcinoma (CRC) can be sporadic, familial or inherited. The sporadic form represents about 70% of CRC cases. DNA alterations detected in CRC somatic cells are due to several factors. Different cancerogenic agents are involved in the CRC onset/progression, including tumor viruses, such as herpes-, adeno-, pox-, papilloma-, hepatitis-, retro- and polyoma-viruses. Among the oncogenic Polyomaviruses, JC polyomavirus (JCPyV) was found to be associated with CRC cases. JCPyV virus is a polyomavirus identified in 1971 as the causative agent of progressive multifocal leukoencephalopathy (PML). In recent years, several studies reported on the association between JCPyV and different human cancers, mainly brain and colorectal tumors. However, confliting data were published. Indeed, several investigations found the association between colorectal cancer and JCPyV, whereas other studies reported negative results. On this ground, in this study I investigated the association between CRC and JCPyV with new technical approaches. To this purpose, JCPyV DNA sequences were investigated (i) in CRC and the adjacent healthy mucosa (HM) biopsies; (ii) in primary cell cultures derived from CRC; (iii) in serum samples from CRC patients and controls, represented by healthy subjects (HS) with the same median age and gender of the patients. Then, (iv) the prevalence of JCPyV-antibodies was investigated in serum samples of the two cohorts, CRC and HS. JCPyV sequences were detected in 22/53, 41.5%, of CRC biopsies and in 11/53, 21%, of HM tissues, being the different prevalence statistically significant. It is interesting to note that 11 CRC samples tested JCPyV-positive, whereas the matched HM sample were found to be JCPyV-negative. Furthermore, JCPyV sequences are more prevalent in the cecum and in the splenic flexure samples, both in tumor and normal mucosa biopsies. This data suggests an association between CRC and JCPyV with a particular focus in these two anatomical districts. It is known that B- and T-lymphocytes represent vehicles for JCPyV to reach different tissues of the host. To verify if JCPyV-positive CRC tumors are due to the presence of the virus in the cancer cells, or to the lymphocytes infiltrating the tumor in vivo, primary cultures derived from CRC cells were set up. Indeed, B- and T-lymphocytes do not grow in vitro during the cell culture passages, thus allowing the transformed epithelial cells to multiply in monolayers. Primary CRC cell cultures (n=13) from the biopsies were set up and characterized. Among these 13 randomly chosen biopsies, 4/13 (31%) CRC tested JCPyV-positive. The 4 cell samples found JCPyV-positive and the 9 other cell cultures tested JCPyV-negative had the same results when analyzed as biopsies. This result suggests 2 that the presence of JCPyV sequences in human colorectal cancer biopsy is not due to the lymphocytes infiltrating the tumor. In recent studies, circulating JCPyV DNA sequences were detected in human sera. However, in my investigation among 53 CRC and 89 HM sera only one sample, from CRC patient, tested JCPyVpositive. Subsequently, the prevalence of serum IgG antibodies against JCPyV was comparatively investigated in CRC tumor patients and HS controls. Mimotopes from JCPyV structural peptides were tested to investigate for specific reactions to human sera antibodies. An indirect ELISA with synthetic peptides from JCPyV viral capsid protein 1 (VP1) was set up and employed to test 53 CRC serum samples and 89 healthy subject. Data from immunologic assays indicate that the overall prevalence of JCPyVVP1 antibodies in CRC patients is 26%, whereas in HS is 51%. As a control of the data obtained by indirect E.L.I.S.A., hemagglutination inhibition assay (H.I.A.) was used. Data from H.I.A. overlapped the results obtained by the indirect ELISA with synthetic peptides. In conclusion, the new indirect ELISA is reliable, faster, sensitive, specific and with affordable costs. This investigation found an association between colorectal cancer and JCPyV infection. Indeed, JCPyV sequences were detected in CRC samples with a higher prevalence than that revealed in HM of the same patients. Interestingly, the majority of sera from CRC patients, tested JCPyV-positive, did not react with JCPyV VP antigens. It is possible that these CRC patients, as oncologic patients, were at least partially immunodepressed or non-responders and therefore they were unable to neutralize the JCPyV infection and consequently its oncogenic ativity. This data are innovative in this field and they may represent a starting point to investigate further the putative role of JCPyV in CRC onset/progression and its mechanism of transformation.
MARTINI, Fernanda
CUNEO, Antonio
File in questo prodotto:
File Dimensione Formato  
Tesi_Silvia Pietrobon.pdf

accesso aperto

Tipologia: Tesi di dottorato
Licenza: PUBBLICO - Pubblico senza Copyright
Dimensione 1.52 MB
Formato Adobe PDF
1.52 MB Adobe PDF Visualizza/Apri

I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2403267
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact