The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

Biobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation-a nationwide network and registry of histo- and cytopathology in the Netherlands-was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA's nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use. We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA's nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (- 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA's national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands

Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence

Biomarker discovery for head and neck cancer, a proteomics approach

The prognosis of head and neck squamous cell carcinomas (HNSCC) has only moderately improved during the last decades. Major causes for this are the high number of patients that present with advanced stage of disease and the high frequency of second primary tumors. This emphasizes the need for early diagnosis of both primary and secondary tumors. Head and neck tumors arise in a field of premalignant cells, i.e. a precursor field. Early detection of these fields, in particular those with a high risk of progression, might improve the overall prognosis of HNSCC patients. Proteomics is the field of research that aims to study all proteins present in a cell or tissue and thereby enables the identification of protein biomarkers, i.e. those proteins, present in tissue or body fluids, that are indicative of the presence, state or behavior of a tumor or precancerous lesion. The development of protein biomarkers is characterized by different phases, starting with an initial discovery of the possible protein candidates, to the final thorough clinical validation of these candidates in prospective multicenter trials. The studies described in this thesis report the discovery and initial clinical validation of protein biomarkers that can predict malignant progression of HNSCC precursor fields. We used genetically characterized normal, precursor and tumor tissue from eight patients to identify proteins with a different expression level. We demonstrated that expression of four proteins, keratin 4, keratin 13, cornulin and small proline-rich protein 3, was not only significantly decreased in tumors, but also in an independent series of severe dysplasias, mucosal tissue at a high risk of malignant progression. This suggested that we found potential promising candidate biomarkers, and we decided to investigate whether these proteins might predict malignant progression of precancerous tissue in different patient groups. Our data revelaed that these candidate biomarkers could not be used to discriminate progressing from non-progressing leukoplakias, i.e. white lesions in the mouth that carry a potential risk of malignant progression. However, keratin 4 and cornulin expression in surgical margins of patients treated for HNSCC strongly correlated with the occurrence of local recurrence. In the future, patients at high risk for local recurrence can be considered for increased surveillance or tertiary treatment to eradicate precursor fields. Taken together, the work in this thesis describes different approaches to discover and validate protein biomarkers for head and neck cancer and has provided new input for the early detection of head and neck cancer

Differential proteomics identifies protein biomarkers that predict local relapse of head and neck squamous cell carcinomas

Purpose: The 5-year survival rates of head and neck squamous cell carcinomas (HNSCC) remain disappointing. HNSCCs develop in precursor fields of genetically altered cells that are often not completely resected when the tumor is excised, causing local relapse. These precursor fields are mostly recognized as dysplasia, but histologic grading cannot reliably predict malignant transformation. Our aim was to discover and validate protein biomarkers that can detect precursor fields and predict local relapse in HNSCC using immunostaining of surgical margins. Experimental Design: We compared paired and genetically characterized normal, precursor, and tumor tissues of eight patients by proteome analysis to identify differentially expressed proteins. The prognostic value of candidate protein biomarkers was evaluated by immunohistochemical analysis of 222 surgical margins of 46 HNSCC patients who developed local relapse or remained disease free. Significant associations were determined by Kaplan-Meier survival analysis and Cox-proportional hazards models. Results: Forty proteins showed significant differential expression (false discovery rate-corrected P < 0.05). Most discriminative markers suited for immunostaining were keratin 4 and cornulin. Low expression in the surgical margins of keratin 4 (hazard ratio, 3.8; P = 0.002), cornulin (hazard ratio, 2.7; P = 0.025), and their combination (hazard ratio, 8.8; P = 0.0005) showed a highly significant association with the development of local relapse. Dysplasia grading had no prognostic relevance. Conclusions: Immunohistochemical assessment of keratin 4 and cornulin expression in surgical margins of HNSCC patients outperforms histopathologic grading in predicting the risk for local relapse. These markers can be used to initiate more frequent and lifelong surveillance of patients at high risk of local relapse, and enable selection for adjuvant treatment or tertiary prevention trials