The PCR assay in the preclinical safety evaluation of nucleic acid medicines.

The polymerase chain reaction (PCR) is a highly efficient gene amplification procedure which is increasingly being applied to the safety assessment of nucleic acid (NA) medicines such as gene therapies and DNA vaccines. Although clinical experience is limited, a number of potential safety issues exist with these new compounds including toxicity associated with the expression of encoded gene products, autoimmunity due to the induction of anti-DNA antibodies and insertional mutagenesis. PCR enables these questions to be addressed and provides data on mRNA expression, biodistribution and integration. In this review the use of PCR methodologies in the preclinical safety evaluation of NA medicines is discussed. Particular consideration is given to the issues surrounding the use of PCR in regulatory toxicology, including sensitivity requirements, cross-contamination problems, tissue sampling procedures and good laboratory practice (GLP) compliance. In addition, the use of a PCR-based assay to demonstrate the integration of DNA vector into host DNA is described. As the use of PCR in the development of NA medicines will undoubtedly increase over the next few years, it is important that pathologists and toxicologists familiarise themselves with the principles and applications of this technique.

The role of the toxicologic pathologist in the preclinical safety evaluation of biotechnology-derived pharmaceuticals.

Biotechnology-derived pharmaceuticals, or biopharmaceuticals, represent a special class of complex, high molecular weight products, such as monoclonal antibodies, recombinant proteins, and nucleic acids. With these compounds, it is not appropriate to follow conventional safety testing programs, and the preclinical "package" for each biopharmaceutical needs to be individually designed. In addition to standard histopathology, the use of molecular pathology techniques is often required either in conventional animal studies or in in vitro tests. In this review, the safety evaluation of biopharmaceuticals is discussed from the perspective of the toxicologic pathologist, and appropriate examples are given of the use of molecular pathology procedures. Examples include the use of in situ hybridization to localize gene therapy vectors, the assessment of vector integration into genomic DNA by the polymerase chain reaction (PCR), and the use of immunohistochemistry to evaluate the potential cross-reactivity of monoclonal antibodies. In situ PCR techniques may allow for confirmation of the germ cell localization of nucleic acids and may therefore facilitate the risk assessment of germline transmission. Increased involvement with biopharmaceuticals will present challenging opportunities for the toxicologic pathologist and will allow for much greater use of molecular techniques, which have a critical role in the preclinical development of these compounds.

Expression of surfactant protein mRNA in normal and neoplastic lung of B6C3F1 mice as demonstrated by in situ hybridization.

The localization of surfactant protein (SP), A, B, C, and D mRNAs was examined in B6C3F1 mice in the normal lung, and in a range of spontaneous proliferative lung lesions using nonisotopic in situ hybridization (ISH). The aim was to develop diagnostic markers, and if possible, throw further light on the histogenesis of these lesions. Tissues from 21 animals were examined, the lesions studied were: 4 alveolar epithelial hyperplasias, 12 alveolar/bronchiolar (A/B) adenomas, and 5 A/B carcinomas. Lung metastases of hepatocellular carcinoma (HCC) were used as controls. In the nonneoplastic lung, staining for SP A, B, and C mRNA was observed in normal and hyperplastic type II cells but not in the bronchiolar epithelium. SP mRNAs were present in all lung tumors, with SPs A, B, and C being coexpressed in 10/12 (83%) of adenomas and 4/ 5 (80%) of carcinomas in both solid and tubulopapillary areas. No signals for SP D mRNA were noted in normal or neoplastic lung. Additionally, no staining for any SP transcript was observed in the HCC metastases examined. In summary, ISH for SP A, B, or C mRNA was a helpful aid in the diagnosis of proliferative lesions of the murine lung, enabling differentiation from hepatocellular metastases. Furthermore, this work provides strong support for the proposal that spontaneous lung tumors in B6C3F1 mice are of alveolar, not bronchiolar origin, and consistently show type II cell differentiation. We suggest that such tumors should be referred to as alveolar adenomas and carcinomas.

In situ hybridization demonstration of albumin mRNA in B6C3F1 murine liver and hepatocellular neoplasms.

In situ hybridization was used to detect albumin mRNA in normal liver and hepatocellular neoplasms in 20 male B6C3F1 mice between 17 and 24 months of age. Positive signals for albumin were observed consistently in the cytoplasm of hepatocytes in normal liver, particularly in periportal areas. No signals were observed in other cells, such as Kupffer's cells, mesenchymal cells, or bile duct epithelium. Of hepatocellular adenomas, 11/11 (100%) stained positively for albumin mRNA, whereas 14/15 (93%) of primary hepatocellular carcinomas showed positive expression. Albumin mRNA was also detected in extrahepatic metastases of hepatocellular carcinoma, including 9/15 (60%) of pulmonary neoplasms and 5/12 (42%) of metastases at other sites. The pulmonary metastases of hepatocellular carcinoma frequently exhibited a glandular, papillary, or sarcomatous histologic appearance. The presence of albumin in these tumors, lacking characteristic hepatocellular phenotype, is a potential determinant of hepatic lineage. We conclude that in situ hybridization for albumin mRNA in mice is a useful tool in the differential diagnosis of hepatocellular carcinoma, particularly in the case of pulmonary metastasis. This technique may also enable recognition of hepatocyte differentiation in glandular structures with phenotypic features of biliary cells, as seen in mixed hepatocellular-cholangial neoplasms.