Peter Riegman

Biobanking for better healthcare

Translational cancer research is highly dependent of large series of cases including high quality samples and their associated data. Comprehensive Cancer Centers should be involved in networks to enable large‐scale multi‐center research projects between the centers [Ringborg, U., de Valeriola, D., van Harten, W., Llombart‐Bosch, A., Lombardo, C., Nilsson, K., Philip, T., Pierotti, M.A., Riegman, P., Saghatchian, M., Storme, G., Tursz, T., Verellen, D, 2008. Improvement of European translational cancer research. Collaboration between comprehensive cancer centers. Tumori 94, 143–146.]. Combating cancer knows many frontiers. Research is needed for prevention as well as better care for those who have acquired the disease. This implies that human samples for cancer research need to be sourced from distinct forms of biobanking. An easier access to these samples for the scientific community is considered as the main bottleneck for research for health, and biobanks are the most adequate site to try to resolve this issue [Ozols, R.F., Herbst, R.S., Colson, Y.L., Gralow, J., Bonner, J., Curran Jr., W.J., Eisenberg, B.L., Ganz, P.A., Kramer, B.S., Kris, M.G., Markman, M., Mayer, R.J., Raghavan, D., Reaman, G.H., Sawaya, R., Schilsky, R.L., Schuchter, L.M., Sweetenham, J.W., Vahdat, L.T., Winn, R.J., and the American Society of Clinical Oncology, 2007. Clinical cancer advances 2006: major research advances in cancer treatment, prevention, and screening: a report from the American Society of Clinical Oncology. J. Clin. Oncol. 25, 146–162.].

Biospecimen Reporting for Improved Study Quality (BRISQ)

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

Characterization of the human kallikrein locus.

The human kallikrein gene family is composed of three members: tissue kallikrein (KLK1), prostate-specific antigen (PA or APS), and human glandular kallikrein-1 (hGK-1 or KLK2). The three genes have previously been isolated and mapped to chromosome 19q13.2-q13.4. Further analysis of an area of 110 kb surrounding the kallikrein genes by CHEF electrophoresis and chromosome walking showed clustering of the three genes. The KLK1 gene is positioned in the opposite orientation of the APS and KLK2 genes in the order KLK1-APS-KLK2. The APS and KLK2 gene are separated by 12 kb; the distance between KLK1 and APS is 31 kb. A CpG island was detected in the region between KLK1 and APS. Preliminary data indicate that this CpG island is located directly adjacent to a gene that is unrelated to the kallikreins and seems to be ubiquitously expressed.

Cytogenetic, Molecular Genetic and Pathological Analyses in 126 Meningiomas

Abstract. In a series of 126 meningiomas, tumor and patient charactersitics were investigated and statistically analyzed. A combined cytogenetic and molecular genetic approach was used to study chromosomal abnormalities and loss of markers on chromosome 22q. This apprach was successfully applied to 93 meningiomas. In 66 cases, complete or partial loss of chromosome 22 was observed and in at least 12 of them this chromosome was involved in structural aberrations. In addition to chromosome 22 changes, chromosomes 1, 6, 11, 13, 14, 18, 19, X, and Y were also frequently involved in structural and numerical aberrations. Statistical analysis revealed a significant association between the number of choromosomal abnormalities and tumor grade. Complex karyotypes predominated in the group of grade II/III meningiomas. Furthermore, other variables showed statistically (or marginally statistically) significant differences. Meningiomas from the convexity were more ofthen grade II/III, displayed predominantly (partial) loss of chromosome 22 and had complex karyotypes more often. These fetures were frequently found in meningiomas from males. Base meningiomas, on the other hand, occurred more often in females; they were usually grade I, showed loss of (parts of) chromosome 22 less often and displayed fewer additional chromosomal abnormalities.

The role of the pathologist in tissue banking: European Consensus Expert Group Report

Human tissue biobanking encompasses a wide range of activities and study designs and is critical for application of a wide range of new technologies (-“omics”) to the discovery of molecular patterns of disease and for implementation of novel biomarkers into clinical trials. Pathology is the cornerstone of hospital-based tissue biobanking. Pathologists not only provide essential information identifying the specimen but also make decisions on what should be biobanked, making sure that the timing of all operations is consistent with both the requirements of clinical diagnosis and the optimal preservation of biological products. This document summarizes the conclusions of a Pathology Expert Group Meeting within the European Biological and Biomolecular Research Infrastructure (BBMRI) Program. These recommendations are aimed at providing guidance for pathologists as well as for institutions hosting biobanks on how to better integrate and support pathological activities within the framework of biobanks that fulfill international standards.

Molecular cytogenetic evaluation of gastric cardia adenocarcinoma and precursor lesions

Analyses of cancer incidence data in the United States and Western Europe revealed steadily rising rates over the past decades of adenocarcinomas of the esophagus and gastric cardia. Genetic information on gastric cardia adenocarcinoma and its preneoplasias is sparse. We have used comparative genomic hybridization to obtain a genome-wide overview of 20 archival gastric cardia adenocarcinomas and 10 adjacent preneoplastic lesions (4 metaplasias, 1 low-grade dysplasia, 5 high-grade dysplasias). Multiple genetic alterations were discriminated in all adenocarcinomas. Frequent loss (> or =25% of all tumors) was detected, in decreasing order of frequency, on 5q, 18q, 4q, 3p, 9p, 2q, 11q, 14q, 21q, 4p, 9q, 16q, 1p, and 8p. Frequent gain (> or =25% of all tumors) was disclosed, in decreasing order of frequency, on 20q, 7p, 8q, 1q, 7q, 20p, 17q, 13q, Xp, 6q, 8p, 19q, 5p, 6p, and Xq. Loss of the Y chromosome was found in 60% of male cases. High level amplification was frequently (>10% of all tumors) detected on 7q21, 8p22, 12p11.2, 17q12-q21, and 19q13.1-q13.2. The precursor lesions showed multiple aberrations in all high-grade dysplasias, whereas few genetic changes were discerned in LGD and metaplasias. High level amplifications were also found in high-grade dysplasias, ie, on 7q21, 8p22, and 17q12-q21. Moreover, the percentage of aberrations was not significantly different for invasive carcinomas or high-grade dysplasias. Approximately 70% of the precursor aberrations were also present in the adjacent carcinoma. Minimal overlapping regions in the preneoplasias included loss on 18q12-q21 and gains on 8q23 and 17q12-q21, suggesting involvement of genes residing in these regions. In conclusion, we have (i) created a map of genetic alterations in gastric cardia adenocarcinomas and (ii) provided evidence for the presence of a metaplasia-dysplasia-carcinoma sequence in this poorly understood type of cancer.

A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.

Proteomic Analysis of PAXgene-Fixed Tissues

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.

Biobanking for interdisciplinary clinical research.

Biobanking nowadays is mostly strongly determined by the specific aims of a research group in charge of the biobank, determining their own standards for the collection and annotation of samples. Often a long period is needed to build up the sample and data collections, especially when long-term follow-up data is required. Such collections need a long-term dedication and proper funding. Neglecting either sample number or annotation can result in insignificant or poor results. However, outcome of translational research does not only depend on the sample quality. In many cases it can also be improved to start the experimental design within a multidisciplinary team composed of clinicians including pathologists, molecular biologists, statisticians, bioinformaticians and tissue resource managers. Such a team, capable of careful evaluation of the numbers needed and which or what part of the samples are to be included, could help in obtaining far better results. Many lines of clinical research could benefit more efficiently from the wealth of information stored in well-preserved disease-oriented tissue sample collections with the proper annotations, when the infrastructure around biobanks and new collection build-up is well organized, standardized and streamlined. Future medical research will refine its scientific questions, demanding even further refinement of corresponding clinical information. In addition, larger sample collections are needed to study for instance multifactorial diseases. Today, the samples are collected for tomorrow, therefore, improvement is needed now in standardization, automated enrichment of annotations from hospital information systems and disease registries, insight in overlapping collections of different forms of tissue banking and cooperation in national and international networks.

Histological assessment of PAXgene tissue fixation and stabilization reagents.

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.