Jan Klapwijk

In vivo mapping of Fractional Plasma Volume (FPV) and endothelial transfer coefficient (KPS) in solid tumors using a macromolecular contrast agent: Correlation with histology and ultrastructure

Contrast‐enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T1‐weigthed 3D pulse sequences were acquired before and after administration of Gd‐DTPA‐albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I–II) or in the core (zones III–IV) of the tumor itself. Significant differences (p<0.001) were found in the values of Kps and fpv of zones I–II with respect to zones III–IV. In the peripheral rim, permeability was significantly higher (p<0.01) in the muscle‐peripheral region (zone I) with respect to the skin‐peripheral region (zone II). In areas with high Kps, histological and ultrastructural examination revealed clusters of newly formed vessels and signs of intense permeability. Numerous vascular vesicular organs were visible in these areas. In the tumoral core, analysis of the microcirculatory parameters revealed regions with mild permeability (zone III) and regions with negligible permeability (zone IV). These 2 zones were discriminated by the average value of Kps (p<0.05), while their fpv was not significantly different. Upon histological examination, the tumoral core exhibited necrotic areas; CD31 immunocytochemistry exhibited that it was diffusely hypovascularized with large avascular areas. Upon ultrastructural examination, capillaries were rarely visible and exhibited signs of endothelial cell damage. The results suggest that segmentation based on microvascular parameters detects in vivo zones characterized by immunocytochemical and ultrastructural aspects of intense angiogenesis. The finding that a certain amount of contrast agent penetrates in the tumoral core suggests that high oncotic and hydrostatic pressure only partially hinders the penetration of macromolecules.

Spontaneous Cardiomyopathy in Young Sprague-Dawley Rats: Evaluation of Biological and Environmental Variability

Cardiovascular safety signals in nonclinical studies remain among the main reasons for drug attrition during pharmaceutical research and development. Drug-induced changes can be functional and/or associated with morphological alterations in the normal heart histology. It is therefore crucial to understand the normal variations in histology to discriminate test article–related changes from background lesions. Rodent progressive cardiomyopathy is probably the most commonly encountered change in control animals of nonclinical toxicity studies. A multisite study mimicking standard short-term toxicity studies using young male Sprague-Dawley rats was performed to better characterize this finding. Using an enhanced sectioning method for this research study, it was observed that the incidence of background cardiomyopathy was 100%. The vast majority of the microscopic findings were inflammatory in nature, with associated necrotic changes (defined as necrosis/inflammatory cell infiltrate) and these changes were mainly located in the myocardium of the mid region of the ventricles (the left side being predominantly affected). The monitored environmental factors in this study (multiple facilities, study duration, handling) did not have an effect on the incidence or severity of the spontaneous cardiomyopathy. In addition, cardiac-specific serum troponin levels were measured and were within the published control range.

Industry Survey of Approaches to Examination and Terminology of Spontaneous Changes in the Heart of Young Rats

Toxicologic pathologists are tasked with morphologic evaluation of tissues in animal toxicity studies to ascertain drug or chemical-related effects. These assessments are based on knowledge of the species and spectrum of morphologic changes that occur in the untreated control population. Within the rat heart, a number of morphologic changes have been observed as spontaneous events in control populations, one of the most common being myocardial degeneration or cardiomyopathy (Greaves 2000; King and Russell 2006; Kemi et al. 2000). Experience suggests this change can be observed with a highly variable incidence in very young rats and increases in severity with age; however, many classic literature descriptions identify this as a condition of aging rats. To gain better understanding of the industry’s approach to sampling the heart and terminology in common use in young rats, an informal survey was conducted in 2009 that focused on rat studies of 7 days’ to 28 days’ duration. The survey was sent to 89 individuals who represent the pharmaceutical (53), contract research organization (CRO) and consultant (35), or chemical industries (1). Responses were received from 36 contacts, for a 40.5% return rate. Respondents were asked if survey results could be published; all but one agreed. Sectors represented in the reported data include 20 pharmaceutical (13 North America, 5 Europe, 2 Japan), 9 CRO (6 Europe, 3 North America), 4 consultants (2 Europe, 2 North America), 1 chemical company (Japan), and 1 anonymous (unknown affiliation). A graphical summary of selected survey questions is presented in Figure 1A-J. The following general trends in short-term studies (less than 28 days) were noted:

Hyalinization of the Pyloric Stomach in CD-1 Mice Following Oral (Dietary) Administration of the Corticosteroid Agonists Mometasone Furoate, Budesonide, and Flunisolide

The purpose of this study was to compare the toxicity of three marketed corticosteroid receptor agonists (mometasone furoate, budesonide, or flunisolide) to the stomach of female CD-1 mice following oral administration via the diet for up to 52 weeks, with a 16-week recovery period (budesonide and flunisolide). A range of tissues was examined by light microscopy, accompanied by clinical pathology measurements to assess anticipated corticosteroid effects as a surrogate marker of systemic drug exposure. Microscopic changes seen in the stomach with each corticosteroid included pyloric hyalinization. This previously unreported finding was investigated using histochemical and immunohistochemical techniques and was found to consist of hyalinized collagen, in association with increased immunohistochemical signal for transglutaminase-2 and osteopontin. The significance of the osteopontin finding is unclear; however, the ability of transglutaminase-2 to facilitate the formation of degradation resistant protein bonds implies this protein may be involved in the pathogenesis of this change. Furthermore, published evidence that transglutaminase-2 may be induced by a corticosteroid agonist raises the possibility that pyloric stomach hyalinization may be a class effect of corticosteroids via the action of this enzyme.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2)

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist’s examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

Neoplasia—all you wanted to know but were afraid to ask

This presentation will give a brief outline of three important aspects of neoplasia: the role of genetic defects, prediction of tumour behaviour and how tumours cause morbidity and mortality. Neoplasia is a process of uncontrolled new growth of tissue resulting in a space-occupying neoplasm (or tumour). All cells within a neoplasm are thought to be clones of a single cell, which developed genetic defects that are then passed on to further generations of cells. Neoplasia can therefore be seen as a genetic disease. Particularly in human diagnostic pathology, prediction of the behaviour of a tumour is critical for providing a correct prognosis and treatment options. This has traditionally been performed by assessing the morphological characteristics of the tumour in a biopsy specimen. Combined with other clinico-pathological information, this allows staging or grading of the tumour. Whilst this is still an important part in the overall assessment of the likely behaviour of a tumour, many additional laboratory tests (on the neoplasm itself or on serum/plasma samples) are now also available as an adjunct. The ways in which tumours cause clinical signs are diverse; as well as acting as space-occupying lesions, they can release a number of factors that can lead to adverse effects in sites well removed from the mass.