Cholangiocarcinoma (CC) is a rare but fatal primary liver cancer and accounts for an estimated 15% of primary liver cancer worldwide. It is associated with high mortality due to the lack of established diagnostic approaches. Autoantibodies can be used clinically as diagnostic markers for early cancer detection of cholangiocarcinoma. Studies, indicating the presence of auto-antibodies (AAbs) in CC have not been reported yet. No immunological biomarker, correlated to the disease, has been identified. The objective of our study was to identify cellular proteins from liver tissues (tumoral and non tumoral) and cholangiocarcinoma cell lines which could be recognized by antibody of CC patients. We used serological proteome analysis (SERPA) technique which leads us to suggest some molecules as potential biomarkers for the early diagnosis of CC. Proteins from different origins were 2DE separated: CCSW1 and CCLP1 tumor cell lines, five different samples of hepatectomies for CC with respect to their tumoral and non-tumoral counterparts and a normal liver from amyloid neuropathy. Sera from 13 CC patients and a pool of 10 healthy subjects were probed on immunoblot performed with these different separations. Comparison of immunoblotting patterns given by patient's sera compared to patterns given by controls allowed to define immunoreactive spots of interest and those reacting with more than one-third of sera were identified by orbitrap type mass spectrometry. In this way we identified 10, 11, 9, 14 and 16 proteins from CCSW1, CCLP1, tumor part, non-tumor counterpart and normal liver antigenic extracts respectively. Different patterns of reactivity were observed according to sera on the same antigenic extract, and for a same serum, according to the antigenic extract, even though few common patterns were also observed. This widespread of reactivity is not unusual and reported earlier in several studies of this sort. It is indicated that a single AAb have an ability to identify only a small proportion of patient. For this reason, several antibodies in combination must be used to ensure sensitivity and specificity of assays used in the daily clinic.Identified proteins were then categorized by gene ontology analysis by which they fall into three main groups; biological process and molecular functions, protein class and molecular pathway and cellular component, according to the Panther classification. By Gene Ontology classification, two different patterns of targeted antigens were observed. The vast majority of targeted-proteins with catalytic activity were found in normal liver or non-tumor specimens. The second pattern was mainly represented by targeted proteins categorized as structural proteins extracted from CC cell lines and tumor tissues. Proteins identified with catalytic activity were: alpha-enolase, fructose biphosphate aldolase B and glyceraldedyde 3-phosphate dehydrogenase; which were reactive with more than 50% of CC sera. Proteins identified with structural activity, and detected with high rates by using cell lines and tumor tissues, were: vimentin, prelamine A/C, annexin A2 and actin; reactivity of each protein was higher than 62% with CC sera. Serotranferrin, identified under the category of transfer/carrier proteins, recognized by 100% of CC sera by using tumor tissues.High sensitivity and specificity is a prime requisite of AAbs that might be used as CC biomarkers for CC diagnosis. Most of the AAbs detected in this study had previously been reported in other cancers and auto-immune disorders. Hence it is essential to prove the specificity of antigenic proteins, a combination of various antigens therefore needs to be tested to enable the development of new biomarkers for the diagnosis and prognosis of CC.In conclusion, the proposed potential biomarkers need to be tested in a variety of different combinations with a panel of significant number of patients and using the most appropriate substrate defined during this study.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-01058149 |
| Date | 25 June 2014 |
| Creators | Mustafa, Mohammad Zahid |
| Publisher | Université Paris Sud - Paris XI |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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