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Title: Proteomic tools in clinical tissues: unlocking the pathology archives
Authors: Faoro, Valentina
Supervisore/Tutore: Stanta, Giorgio
Issue Date: 28-Apr-2011
Publisher: Università degli studi di Trieste
Clinical proteomics aims to characterize the protein networks altered by pathologic processes or therapeutic treatment, and to develop biomarker profiling technologies to promptly detect diseases and treat them more effectively. The challenge of translating proteomic profiling to the bedside lies in applying technologies for the analysis of human tissues, which are routinely obtained by biopsy or surgery, without substantially modifying the clinical workflow. Formalin-fixed and paraffin-embedded (FFPE) tissues are the most widely available archive material suitable to discover new biomarkers or therapy targets, for their validation and for their implementation in clinical practice. However, the lack of standardised guidelines for protein analysis of archive tissues has hampered their regular use in the daily practice. Furthermore, methods to extract proteins and to identify and analyse them also quantitatively have only recently been developed.
This research was carried out to develop, standardise and apply qualitative and quantitative proteomic methodologies to archive tissues.
1. Development, standardisation and validation of protein extraction methods and protocols from fixed and paraffin-embedded tissues with different pre-analytical conditions.
2. Application of protein analysis in two different research settings:
a. at diagnostic level: screening of monoclonal antibodies against Human papillomaviruses (HPVs) in cervix uteri lesions suitable for early detection and progression risk assessment;
b. at translational research level: identification of a new therapy target for glioblastoma, deepening the functions of human RECQ1 helicase, an enzyme involved in the maintenance of chromosome stability.
1. In order to develop, standardise and validate protein extraction methods and protocols from fixed and paraffin-embedded tissues, lung, colon and breast cancer specimens were collected by three groups from the European consortium IMPACTS (Integration of Molecular Pathology and Cell and Tissue Structure). Each specimen was divided into two equivalent sets: one was fixed in formalin and paraffin-embedded; the other one was fixed in a new alcohol-based fixative (FineFix) and paraffin-embedded. In order to compare the fixation procedures and the protein extraction efficiency, a total of 81 protein lysates were prepared in five different laboratories of the consortium. Selected marker proteins were compared, at quantitative level, between the two fixation procedures using the reverse phase protein array technology. Tests were applied to determine whether any of the fixation methods caused differences in protein lysate microarray measurements. Pairwise differences between fixation methods were tested.
2. (a) In diagnostics, protein analysis was applied to 138 FFPE cervix uteri squamous lesions provided by a multicentric retrospective study. They were characterized at molecular level for the presence and type of HPV by using PCR-based systems or the Hybrid Capture assay. Thirty-nine monoclonal antibodies directed against the E7 viral oncoprotein were supplied by a private company. They were validated by generating different tissue microarrays in which multiple punches for each bioptic sample were done, taking different cervical lesions (from low grade to invasive squamous cancer). The antibody providing better staining and specificity results was selected and validated with other tissue microarrays, which included a total of 138 tissue cores from different cervical lesions, and with different HPV-types infections. HPV capsid protein L1, the surrogate biomarkers p16, hTERT, p53 and Ubiquitin, and the proliferative marker Ki67 were also tested in the same way. For the selected E7 antibody and the six biomarkers, one-way analysis of variance was performed to detect significant differences in the mean number of positive cells and in the mean staining intensity (evaluated by a score from 0 to 3+). The analysis was carried out among different types of cervical lesions, both at cytoplasmatic and nuclear level, by epithelial layer. We made use of repeated-measure analysis of variance to ascertain significant differences in the mean number of positive cells between E7 monoclonal antibody and each biomarker. Spearman’s rank correlation test and linear regression analysis were employed in order to detect correlations between E7 and the other biomarkers.
2. (b) The application of proteomics to FFPE tissues at translational research level concerned the expression of the RECQ1 helicase in glioblastoma and in perilesional brain tissue, and its expression in lung, colon and thyroid carcinomas and in their perilesional tissues. All specimens were submitted to immunohistochemistry. The expression pattern of RECQ1 was further analysed on a tissue microarray containing 63 glioblastomas and 19 perilesional brain tissues. Furthermore, to test the function of RECQ1 in glioblastoma growth, RNA interference experiments were performed in different glioblastoma cell lines and the effect of gene silencing on cell growth and proliferation was monitored. The Wilcoxon test for matched-samples was performed to evaluate differences in immunohistochemistry outcome measures, i.e. the proportion of positive cells for RECQ1, the intensity of the signal and the number of positive cells for Ki67, between tumour and perilesional tissues. Spearman’s rank correlation test was applied to assess the relationship between intensity score of RECQ1 and the number of positive cells in tumour specimens. Kruskal-Wallis test was carried out to investigate differences in the proportion of positive cells for RECQ1 and Ki67 among groups defined by patient gender and by RECQ1 intensity score.
1. Quantitative comparison between fixatives for the tested antibody panel (β-Actin, E-cadherin, EGFR, HER2 and ER) yielded a higher immunostaining intensity for the FineFix lysates compared to the FFPE ones (p = 0.04). Signal intensities for EGFR (p = 0.007) and HER2 (p = 0.02) were significantly higher in the FineFix samples compared to the FFPE samples, whereas no differences were observed for β-Actin, E-cadherin and ER.
2. (a) The E7 immunostaining was similar to that of p16, which is the most widely-used surrogate marker in clinical practice for all types of cervical squamous lesions. Furthermore, in high grade squamous intraepithelial lesions (HSILs) E7 was similar to hTERT. In the detection of squamous cell carcinoma, E7 was similar to hTERT and Ubiquitin. A comparison between the mean number of E7 and L1 positive cells showed a significant difference in low grade squamous intraepithelial lesions (p = 0.002), among HSILs (p = 0.01) and in squamous cell carcinoma (p = 0.01). Correlation analysis between the two viral proteins, E7 and L1, allows to identify two possible groups of lesions, both in the low grade squamous intraepithelial lesions (LSILs) and in the HSILs. One group was charcharterized by high level of E7 and low level of L1, the other group was characterized by high level of both marker. This groups were also ri-evalueted at morphological level and lead us to hypothesize two possible models in the progression of LSILs: the viral replicative model and the cell proliferative one.
2. (b) RECQ1 was highly expressed both in the perilesional sections and in lesional sections of all tumours analyzed, with the exception of glioblastoma where its expression was significantly higher in tumour (p = 0.001). RECQ1 expression was confined in the nuclei of the tumour cells, thus suggesting that this enzyme might play an important role in glioblastoma growth. As expected, immunohistochemical analyses against Ki67 showed that protein expression was higher in glioblastoma than in the surrounding normal tissue (p = 0.0008). No correlation between the proportion of positive cells for RECQ1 and Ki67 was found (Spearman’s rho = 0.2, p = 0.2), suggesting that the high expression of RECQ1 in brain tumours is not simply related to the higher degree of proliferation of these cells. An essential role of RECQ1 in tumour growth and proliferation was confirmed by RNA interference experiments and clonogenic assay on cell lines.
During my doctorate, all the ojectives set were obtained. The goals achieved trough this research are the following:
 The standardization of a suitable protein extraction protocol from FFPE tissues. This study demonstrated that it is possible to harmonize protein analysis in archive tissues in different European laboratories by using the same protocols for tissue processing and protein extraction.
 The optimization of an appropriate protocol for molecular analysis of tissues fixed with new formalin-free fixative FineFix. The possibility of using proteomic approaches also on tissues fixed with reagents alternative to formalin has great potential for future pathology, when the use of formalin will be banned due to its carcinogenetic effects. Furthermore, FineFix has proven a suitable formalin substitute in clinical practice, because it preserves both specimens’ morphology and immunoreactivity.
 The validation of a new antibody against the E7 oncoviral protein for the detection f HPV in cervical squamous lesions. This leads us to conclude that E7 might be a suitable specific marker for the diagnosis of cervical lesions and could be used also for others HPV-linked diseases. The analysis of a battery of E7 monoclonal antibodies has highlighted their great usefulness in differential diagnosis.
 An in-depth knowledge of the function of RECQ1 in glioblastoma. RECQ1 expression has been studied in glioblastoma cell lines for the first time ever. This research has demonstrated its important role in glioblastoma growth and proliferation, and in maintaining genome stability.
 The recommendation of RECQ1 helicase as therapy target in glioblastoma, based on the evidence that the enzyme expression in this tumour type is higher than in perilesional normal tissues or in cancers of different origins. These findings lead us to consider RECQ1 a reliable target for the development of new anti-cancer therapies to eliminate proliferating tumour cells.
Finally, this research demonstrated that archive tissues could be a valuable source of material for proteomics research.
Ciclo di dottorato: XXIII Ciclo
Keywords: Fixed and Paraffin Embedded TissuesProteomicsMolecular DiagnosticsTranslational ResearchHPVGlioblastoma
Type: Doctoral
Language: en
Settore scientifico-disciplinare: MED/08 ANATOMIA PATOLOGICA
NBN: urn:nbn:it:units-9097
Appears in Collections:Scienze mediche

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