Expressions Of Human Papillomavirus 16 E7 DNA And E7 Oncoprotein In Primary Colorectal Adenocarcinoma

Background: Colorectal carcinoma is the third most common form of cancer and the second leading cause of death among cancers in the world. The correlation between human papillomavirus (HPV) infection and the occurrence and development of tumors has been a focus of study in recent years. The HPV E6 and E7 genes can produce oncoproteins that alter cell growth regulation. Specifically, E6 oncoprotein inactivates the tumor suppressor gene p53, and the oncoprotein produced by E7 inactivates pRb (retinoblastoma). Previous studies have reported that HPV viral genome and antigen, especially the HPV 16, have been found in the colorectal tumors. There have been significantly different results in different studies. The reasons may be as follows: different methods of detection; different primers or probes; different sources or numbers of specimens. The correlation between HPV infection and colorectal carcinoma is still not clear now. Does the whole large bowle or only the cancer area get HPV infection? Does HPVparticipate the cancerous occurrence in colorectal cancer or only a chaperonage infection? We conducted a prospective case control study with a magnitude of samples to clarify the relationship between HPV 16 infection and the locations, TNM stages, differentiation degrees of colorectal adenocarcinoma.Objectives: The relationship between Human papillomavirus (HPV) infection and the natural course of colorectal adenocarcinoma has not been fully defined. In this study, HPV 16 E7 DNA and E7 oncoprotein were detected in 106 patients with primary colorectal adenocarcinoma to study the correlation between HPV 16 infection and colorectal carcinoma. Material and Methods:1. The Clinical samples: One hundrerd and six consecutive patients, median age 56years (range 26-81); male/female, 62/44, with primary colorectal adenocarcinoma were enrolled, from August to December 2004, in a prospective case control study from the Department of General Surgery, West China Hospital. All the patients had not been treated with chemotherapy, radiation therapy and immunotherapy before operations. Samples were taken from both the tumors and the adjacent normal mucosa (10 cm away from the tumor border) in each patient. All of the 212 clinical samples were collected during primary surgery. Immediately following the resection, both cancer tissues (2 samples) and normal mucosa tissues (2 samples) were rinsed with sterile 0.9%NaCl solution. One tissue sample, used for HPV PCR analysis, was frozen at -70°C before experiments. The other tissue sample, used for IHC and H&E staining, was formaldehyde-fixed and paraffin-embedded.Tissue sections from all the patients were reviewed by one pathologist, and the diagnoses of all the 212 specimens were reconfirmed histopathologically (primary colorectal adenocarcinoma or normal mucosa) .2. Polymerase Chain Reaction (PCR): DNA was extracted from tissue specimens by a routine procedure of proteinase K digestion and a standard phenol-chloroform-isoamyl alcohol extraction technique. The HPV-16 E7 specific primer pairs were used (forward primer 5'-CAC GTA GAG AAA CCC AGC TGT AA-3'; reverse primer 5'-GCA GGA TCA GCC ATG GTA GAT T-3') . These primers should yield 297-bp fragments for the HPV 16 E7 sequences. 35 cycles of PCR were performed on a MJ PTC-100 thermal cycler. PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. The UV-illuminated gels were photographed with Polaroid negatives (type 665). DNA from the CaSki cell lines was used as a positive PCR control to assess the success of the amplification. PCR reagents lacking DNA (no sample added) served as a negative control. The amplified products were cut out and subsequently purified. The DNA sequence were analyzed and determined by the FacturaTM and Sequence Navigator version 2.0 (PerkinElmer; Applied Biosystems).3. Immunohistochemistry (IHC): Immunohistochemistry was performed on 4-um sections with the streptavidin-biotin-peroxidase technique. Tissue sections were incubated with a mouse monoclonal antibody against HPV16 E7 oncoprotein (Sc-6981, Santa Cruz, USA) at a 1:100 dilution. We examined HPV16-containing cervical carcinoma specimens as a positive control, and used phosphate buffered saline instead of primary antibody as the negative control. Brown staining of the nucleus andperinucleus indicated positivity for HPV 16 E7 oncoprotein. If the brown stained nuclei account for more than 10% on each of the 5 amplification fields (200X ), the result is positive. The results were semi-quantitatively analyzed by image analyze system. Results: 1. The expression of HPV16 E7 DNA1.1 The results of gel electrophoresis of PCR products showed a clear band of about 300 bp. DNA sequence analysis indicated that PCR product was 297 bp. It is the equivalent of 562-858th pairs in the HPV 16 primitive sequences.1.2 PCR-test results showed that HPV 16 E7 DNA expression was significantly higher in colorectal carcinoma, 45.28% (48/106), than that in adjacent normal mucosa, 6.60%(7/106), (PO.01) . Among them, 4 cases of rectal cancer and 3 cases of sigmoid colon cancer were HPV 16 positive in both the tumors and the adjacent normal mucosa.1.3 A correlation was found between HPV16 E7 DNA expressions and tumor locations, 23.53 % in the ascending colon carcinoma and 59.52% in the rectal carcinoma (P <0.05). Gradually increasing tendency from the cancer site of caecum to anal manifested itself.1.4 Higher HPV 16 E7 DNA expression was also associated with higher TNM stages (TNM stages between III and I , P <0.05) .1.5 The differentiation degrees of colorectal carcinoma had no relation to the positive rate of HPV 16 E7 DNA. There were two well-differentiated adenocarcinomas in our experiment. They were negative of HPV16 E7. Expressions of HPV 16 E7 in moderate and poorly differentiated colorectal carcinoma were 44.74% (34/ 76) and 50.00% (14/ 28) . There was nosignificant difference.2. The expression of HPV16 E7 oncoprotein2.1 HPV16 E7 oncoprotein immunohistochemistry expression in the nuclear and perinuclear of both tumor and normal mucosal cells confirmed the presence of HPV16. There was a correlation between the expression of E7 oncoprotein and E7 oncogene.2.2 HPV16 E7 oncoprotein expression was significantly higher in colorectal carcinoma, 33.02% (35/106), than that in adjacent normal mucosa, 1.89% (2/106), (P <0.01) . Among them, 2 cases of rectal cancer were HPV16 positive in both the tumors and the adjacent normal mucosa.2.3 A correlation was found between HPV16 E7 oncoprotein expressions and tumor locations. The expressions of E7 oncoprotein in The rectal carcinoma group (24/42, 57.14%) was significantly higher than that in the ascending colon group (2/17, 11.76%, P<0.01) , Descending/transverse colon group(2/20,10.00%, P<0.01) and Sigmoid colon group(7/27, 25.93%, PO.05). Gradually increasing tendency from the cancer site of caecum to anal manifested itself.2.4 Higher HPV16 E7 oncoprotein expression was also associated with higher TNM stages (TNM stages between III and I , P <0.05) .2.5 The differentiation degrees of colorectal carcinoma had no relation to the positive rate of HPV16 E7 oncoprotein.Conclusions:1. HPV 16 E7 gene PCR products were cut out and subsequently purified . The DNA sequence were analyzed and determined to be the HPV 16 style.2. Immunohistochemistry technique, in situ DNA hybridization (ISH)technique and PCR technique have been used in recent years to study the relationship between HPVs and colorectal carcinoma. Our results showed that PCR technique is more sensitivity than the IHC method.3. The expressions of HPV16 E7 DNA and E7 oncoprotein were relatively higher in the colorectal carcinoma than in the adjacent normal mucosa and existed in about half of the cases of colorectal adenocarcinoma, indicating that there was a correlation between colorectal adenocarcinoma and HPV 16 infection. The finding gave us a new perspective to produce vaccine against HPV16 E7 and to deal with colorectal adenocarcinoma in a way of targeted cancer therapy.4. The positive rate of HPV 16 E7 oncoprotein in rectum carcinoma group is significantly higher than in ascending colon carcinoma group, Descending/ transverse colon carcinoma group and Sigmoid colon carcinoma group. The closer to the anus the tumor exists, the higher the positive rate is. We estimate that large intestine infection with HPVs might be retroinfection since the infection of anus and reproduction organs with HPVs always results from sexual transmission. The positive rate of HPVs increases in inverse proportion to the distance to the anus. It is more likely for rectum and Sigmoid colon to be infected with HPVs than ascending colon, Descending/transverse colon, which results in higher rate of lower rectum cancer. The concordance of HPV 16 infection and tumor position also suggests that colorectal cancer is relevant to HPV16 infection. [The Draft of Rulebook for Surgical Treatment for lower-rectal cancer in China(2005.03) shows that the incidence rate and death rate from rectal cancer is rising, 80% of which exist in lower-rectalcancer, accounting for more than half of the case in carcinoma of large intestine.]5. The differentiation degrees of colorectal carcinoma were irrelevant to the positive rate of HPV16, while being relevant to TNM stages. Yet is it the outcome of HPV DNA integrating into host gene which results in the occurrence and development of colorectal cancer? Or is it an "accompanying infection" induced by HPV invading the damaged site of colorectal mucosal which was the outcome of the tumor tissue death and shedding during its development? Do HPVs matter only in the occurrence stage or accelerate the growth and metastasis of the tumor after the tumor tissue is infected with HPVs? Further studies are needed.