Study Of Novel Biomarkers For Early Diagnosis And Treatment Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumor in southern China and Southeast Asia,which has posed great threats to human health as well as economic stability.Because of the complicated anatomical structure of nasopharynx,NPC usually is not suitable for surgery,and even more NPC is not sensitive to chemotherapy,so radiation therapy(RT)becomes the mainstay treatment for NPC.With the development of modem medical technology,the outcomes of early stage NPC patients have improved greatly.However,many patients have advanced NPC before therapy,missed the optical timing for treatment,and most patients have cervical lymph node and distant metastases,resulting in a five-year survival rate decreased significantly.On the other hand,after primary treatment with radiation therapy or chemo-irradiation,some patients will relapse with either locoregional recurrence or distant metastases.For recurrent NPC,traditional treatments offer limited local control,more seriously,the re-treatment frequently induce severe late complications,and affect the quality of life in almost all the patients.Based on this reasons,this thesis composed of three parts,including development of early diagnostic reagents,individualized diagnostic reagents and therapeutic antibodies for NPC.The first study aimed to developmpment of serologicl early diagnostic reagents and histological detection reagents specific for NPC.Currently,available diagnostic reagents such as EBNA1/IgA and VCA/IgA,have good sensitivity for early detection of NPC,but the specificity can't meet the requirements for clinical detection of NPC.Evidence accumulated so far indicates that Epstein-Barr virus(EBV)is present in virtually all poorly differentiated and undifferentiated non-keratinizing NPC,leading to the strong etiological association between NPC and EBV latent infection.All EBV-associated cancers involve the virus's latent cycle,and the NPC has been associated with Latency ?.So,based on the EBV gene expression of Latency ?,development of new reagents for early diagnosis of NPC has great significance.In this study,LMP1,LMP2A and BARF1 were chosen as the research object.Different hydrophilic fragments of LMP1 and LMP2A and full-length of BARF1 recombinant antigen were prepared and used to detect the serum levels of IgA and IgG for both NPC patients and healthy controls.The result showed that,LMP1-EX3 and LMP2A-EX5 can serve as a good target for NPC screening,because LMP1-EX3/IgA and LMP2A-EX5/IgA could differentiate NPC patients and healthy controls clearly.However,BARF 1/IgA and BARF1/IgG have no difference between NPC patients and healthy controls.By using the LMP1-C.terminal and LMP2A-N.terminal recombinant antigens,BALB/c mouse were immunized,25 anti-LMP1 monoclonal antibodies(mAbs)and 16 anti-LMP2A mAbs were prepared,respectively.Results of Western blot and indirect immunofluorescence assay(IFA)showed that anti-LMP1 mAb 14B6 and 5H8,and anti-LMP2A mAb 13A12 and 12A2 have good reactivity with LMP1 or LMP2A plasmid transfected 293T cells.Then,anti-LMP1 representative mAb 14B6 and anti-LMP2A representative mAb 13A12 were chosen and used to establish LMP1 and LMP2A immunohistochmical(IHC)detection reagents,respectively,and this two IHC detection reagents have good reactivity and specificity for NPC tissue sections.Our results revealed that LMP1-EX3 and LMP2A-EX5 are good target to develop screening reagents for NPC.By using anti-LMP1-C.terminal and anti-LMP2A-N.terminal mAbs,the LMP1 and LMP2A IHC detection reagents were established,this two reagents provide good tools for LMP1 and LMP2A related research in NPC.The second studye aimed to explore the individualized diagnostic target for detection of the recurrent and metastatic NPC in the early stage.Even though the outcomes in patients with primary NPC have improved greatly mainly because of advances of radiation therapy and comprehensive chemotherapy strategies.However,15-58%of NPC patients will experience recurrent disease and must undergo re-treatment.As for recurrent NPC,which still represents a clinical dilemma because of an incomplete understanding of the mechanism,lack of adequate early stage detection reagents,high radioresistance and serve late complications.Thus,development of an early detection reagents for recurrent NPC and take highly individualized measures have great importance for NPC therapy.In this study,transcripts A73 of BARTs region in Latency II was chosen as the research object.Firstly,rGST-A73-NP9 recombinant antigen was prepared in E.coli,and was used to detect the serum levels of IgA and IgG for NPC patients and healthy controls.The result showed that,A73/IgG was negative in both NPC patients and healthy controls,but A73/IgA could differentiate some NPC patients from others,indicating that A73/IgA could be a target for NPC classification.Secondly,rGST-A73-NP9 was used to immunize BALB/c mouse and 18 anti-A73 specific mAbs were prepared.Results of Western blot and IFA showed that,mAb 6A2 has good reactivity with A73 plasmid transfected 293T cells.In order to identify whether A73 protein was expressed in EBV-associated cells and tissues,EBV infected lymphocyte cell line B95?8 and epithelial cell line C666-1 were detected with Western blot and IFA by using anti-A73 mAb 6A2.The result showed that A73 protein was not expressed neither for B95-8 nor C666-1 cells.However,IHC result showed that A73 ptotein could be detected in some NPC tissue sections,implying that A73 protein could be an IHC target for NPC classification.Thirdly,exosomes from supernatant of C666-1 cell culture and serum samples of NPC patients were isolated,and A73 mRNA was identified in EBV-associated exosomes for the first time.This result implied that high level of A73 mRNA is likely to be transported out of EBV infected cells,which may have some relationship with metastasis of NPC.In addition,more research should be needed to identify to the relationship of serum level of A73 mRNA with recurrent and metastatic NPC.Our results showed that serum level of anti-A73 antibody,the expression of A73 in NPC tissues and serum level of A73 mRNA all could be used for NPC classification,but whether the target could be used for early detection of recurrent and metastatic NPC needs more depth research.The third study aimed to explore the LMP1 and LMP2A as therapeutc antibody targets for NPC.At present,there are still no good therapy strategies for recurrent and distant metastatic NPC,and excessive re-treatment will induce a lot of side effects.The use of mAbs as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer due to the advantages of high specificity and low side effects of mAbs.EBV encoded LMP1 and LMP2A are latent membrane proteins expressed in NPC cells,which play a critical role in development of tumors.In addition,LMP1 and LMP2A which are not expressed in any normal tissues are good targets for development of therapeutic antibodies for NPC.In this study,LMP1 and LMP2A were chosen as the research object.Firstly,expressing LMP1 or LMP2A along with luciferase stable NPC cell lines CNE-2Z-Luc-LMP1 and CNE-2Z-Luc-LMP2A were constructed.Secondly,by immunizing of BALB/c mouse with cross reacting material A displayed LMP1-EX3 and LMP2A-EX5 peptides,25 anti-LMP1-EX3 and 6 anti-LMP2A-EX5 mAbs were prepared,respectively.Results of Western blot and IFA showed that anti-LMP1-EX3 mAb 14H5 amd 12A1,anti-LMP2A-EX5 mAbs 5E9 and 7H3 could bind with natural LMP1 or LMP2A proteins.Baed on anti-LMP2A-EX5 mAb 7H3,a linear epitope 382SLSSTEFIP390 located in LMP2A-EX5 was identified.Furthermore,anti-LMP1-EX3 mAb 2B8,5D4 and 7G9,and anti-LMP2A-EX5 mAb 5E9 and 7H3 showed high growth inhibitory activity for NPC cells,implied that LMP1 and LMP2A could be used to development therapeutic antibodies for NPC.In short,based on the latent gene transcripts of Epstein-Barr virus,LMP1-EX3 and LMP2A-EX5 were identified as good targets for early diagnosis and treatment of nasopharyngeal carcinoma,and A73 was identified as a potential target for individualized diagnosis of nasopharyngeal carcinoma,which paved the way for development of specific early diagnosis reagents and therapeutic antiboides for nasopharyngeal carcinoma.