Abstract
Recently finalized regulatory guidance documents concerned with the identification of immunotoxicity (CPMP: Note for Guidance on Repeated Dose Toxicity; FDA: Guidance for Industry, Immunotoxicology Evaluation of Investigational New Drugs; ICH S8) state that immunotoxicity testing should be performed on all new investigational drugs or medicinal products. In addition, all documents clearly identify gross and microscopic examination of lymphoid tissues as necessary and pivotal first steps in the assessment of new xenobiotics for immunotoxic potential. However, as is true for the evaluation of other organs systems, there are numerous approaches to the histopathologic examination of lymphoid tissues. To assist in a more uniform and consistent histopathologic assessment of the immune system, the Society of Toxicologic Pathology (STP), has recently prepared “best practice” recommendations concerning the collection, interpretation and reporting of organ weights, gross and microscopic observations, and other pathology data relevant to the immune system. The STP recommendations are intended to provide a scientifically sound and well-considered guidance document for routine pathology evaluation of the immune system. This presentation will consider the implications of this “best practice” document and place these recommendations in the context of normal animal tissue variability.
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Over the last several years there has been a great interest in potential immunotoxicity of pharmaceutical compounds and, subsequently, the methods by which immunotoxicity may be detected. The potential for unintended immunosuppression with new therapeutics has been heightened by recent observations of recrudescent tuberculosis and secondary infections in patients treated with novel rheumatoid arthritis compounds. Regulatory guidances have been written, and continue to evolve to assist pharmaceutical companies with the consistent and systematic evaluation of new chemical entities (NCE) for potential unintended immunosuppression.
A review of the FDA Guidance for Industry, Immunotoxicology Evaluation of Investigational New Drugs, the CPMP Note for Guidance on Repeated Dose Toxicity and more recently, the ICH S8 guidance reveals a logical progression of carefully considered procedures and endpoints selected to be included in standard 28-day safety assessment studies. The regulatory agencies, fully aware of the ever increasing time and cost associated with developing NCEs have set out to maximize the utility of presently existing testing procedures rather than casually adding studies and/or assays. A recurring theme identified in all of the relevant guidances is that of the central importance of identifying immunotoxicity using an integrative analysis of multiple endpoints from multiple studies. Thus, as with any toxicity endpoint, the accurate identification of drug-induced alteration of the immune system requires a careful analysis of effects over multiple doses (dose-related effects), given over different dosing regimens, using multiple assays and in multiple species. Likewise, attention to species differences in metabolism of administered compounds is essential in putting observations suggesting immunotoxicity into proper context.
The regulatory guidelines have also uniformly recognized the high value that a thorough histologic evaluation of the immune system provides in the initial identification of immunotoxicity. However, it is also clear that the approach used by pathologists in the examination, identification and reporting of effects of the immune system may be variable as a result of differences in experience, local practice, terminology, and training. Such variability may translate into differences in opinion as to a lesions severity and/or biological significance; we make the assumption here that if a lesion is present, any qualified pathologist will identify it. This in no way suggests that such variability in approach inherently translates into inaccuracies; it does not. What it does argue for is a need for greater uniformity of approach and terminology to minimize confusion.
Therefore, because pathologists increasingly work in a global environment it behooves them to attempt to harmonize their terminology, processes and procedures to maximize clarity concerning the observations they make, not only for the immune system, but also for all tissues. In the end, it matters little if the pathologist makes the most accurate of diagnoses, but fails to accurately and clearly communicate his results to his/her clientele. To this end, the Society of Toxicologic Pathology (STP) recognized as part of its responsibility the writing of a best practice document concerning the pathologic evaluation of the immune system. The STP established an Immunotoxicity Working Group (IWG) comprised of anatomic and clinical pathologists from the chemical and pharmaceutical industries along with private consultants and pathologists from the NIEHS. The efforts of the STP IWG culminated in the publication of the Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (P. Haley (chair), R. Perry (cochair), D. Ennulat, S. Frame, C. Johnson, J.-M. Lapointe, A. Nyska, P. Snyder, D.Walker, G. Walter; Toxicol. Pathol. 33:404–407, 2005).
The Best Practice Guideline for the Routine Evaluation of the Immune System discusses the merits of collection of lymphoid organ weights (i.e., which lymphoid organs should be weighed and why), addresses lymphoid tissue histomorphology (what are the limitations), and finally, makes recommendations concerning application of harmonized terminology to descriptions of alterations of the immune system. Clinical pathology is recognized in the Best Practice document as an important component of the evaluation of the immune system, but one that is beyond its scope; and it is anticipated that a separate best practice document on clinical pathology will be forthcoming.
There are basic concepts that are delineated by the Best Practice guideline including (a) each animal should receive a thorough macroscopic examination of the spleen, thymus and lymph nodes, (b) the thymus, spleen, draining lymph nodes, bone marrow in situ, and any gross lesions of a lymphoid organ represent the minimum tissues for routine evaluation of the lymphoid system, and (c) the most proximal regional lymphoid tissues that drain the drug application site can and should be examined microscopically. For orally given drugs, the Peyer's patches and mesenteric lymph nodes fulfill this node set. Therefore, in cases of cutaneous, subcutaneous, or intradermal application the most proximal draining peripheral lymph nodes, (i.e., brachial, axillary, or popliteal, etc.) are appropriate.
The STP IWG spent a great deal of time considering the relative merits of the evaluation of different lymphoid tissues for the identification of immunotoxicity. Considering the strengths and limitations of specific lymphoid organ collection, weighing, and histologic examination the STP IWG recommends the following:
Recording and evaluating thymic and splenic weights;
Interpretation of lymphoid organ weights should only be done in the context of all other clinical, histopathology, and clinical pathology data from the study;
Alterations of spleen and thymus weights when accompanied by histopathologic observations are reasonable indicators of systemic immunotoxicity; and
Spleen and thymus weights are likely to be more reliable indicators of immunotoxicity than are chan ges in the weight of peripheral lymph nodes.
Importantly, and a point of much discussion concerning the utility of collection of peripheral lymph nodes other than those that drain the site of drug application collection, the STP IWG contends that the normal histology of peripheral lymph nodes can be highly variable, often overlaps with that of altered node morphology, and cannot be unequivocally used as an indicator of systemic immunotoxicity. This inherent variability is exacerbated by minor differences in collection, embedding, and sectioning. Moreover, as a result of this review, that included a careful analysis of the available literature, the STP IWG felt that alterations of spleen, thymus, and bone marrow histology are likely to be more reliable indicators of systemic immunotoxicity than those of distal peripheral lymph nodes. Therefore, the STP IWG does not recommend the routine collection and examination of peripheral lymph nodes that do not drain the site of xenobiotic application.
The Best Practice guideline goes to some length to describe and define the concept of “enhanced histopathology” as a “a semi-quantitative description of changes in compartments and/or microenvironments of specified lymphoid organs.” The definition and approach is based on the premise that (1) each lymphoid organ has separate compartments that support specific immune functions, (2) these compartments can and should be evaluated individually for changes, and (3) descriptive, rather than interpretative terminology, should be used to characterize changes within these compartments. Thus the definition reinforces two primary concepts: first, that of the compartmentalized nature of individual lymphoid organs, and second that descriptive, rather than diagnostic or interpretive terms are the best for clearly describing and communicating associated changes. Importantly, the STP IWG attempted in this guideline to underscore the premise that the histologic examination of lymphoid tissue is not different than any other tissue. Virtually every tissue has compartments, and any time a lesion or change can be attributed to a specific compartment, it is in the best interest of the pathologist to do so, so as to communicate the nature of the change with the greatest clarity.
The document goes on to state that specialized techniques such as lymphoid tissue immunohistochemistry, blind scoring of lymphoid tissues, morphometry of lymphoid tissues, and flow cytometry of lymphoid tissue cell suspensions should only done AFTER the initial histologic assessment shows that a change has occurred, and after it has been determined that the nature of change might be clarified by the use of such specialized procedures.
It was made clear to the STP IWG that some of the confusion surrounding the histologic examination of lymphoid tissue was the absence of entries in pathology reports or tables for specific lymphoid tissue compartments in which no change was identified. It has been interpreted that this lack of specific notation indicates that the compartment had not been examined. To resolve this the STP IWG strongly encourages that a statement be included in histopathology SOPS to the effect that “Histopathologic examination of all tissues will include a detailed examination of all tissue compartments and histopathologic abnormalities will be recorded according to the compartment in which they are identified.” Thus histopathologic examination of any tissue requires that all compartments of all tissues, including lymphoid tissue, be examined but that entries for specific compartments are only made if an abnormality is identified within that compartment. If all tissue compartments demonstrate changes considered to be within normal ranges of variability or within the variability of the control group, for that specific organ, an entry of “No Abnormality Determined” (NAD) is appropriate for the tissue as a whole; specific additional annotation for each separate compartment is not required.
One of the more challenging points for pathologists, particularly those working in an international setting, is the harmonization of terminology. What is true for pathologists within the same international company is even greater for the entire pathology community. The STP IWG has attempted to facilitate such harmonization by encouraging the use of more objective semi-quantitative descriptive terms, i.e., increased or decreased cells (specify). Simple questions that will guide the pathologist in this endeavor include the following:
Is the lymphoid organ grossly larger or smaller than normal: macroscopically increased or decreased in size?
Which compartment is specifically involved?
Is the change in size of the organ due to a change in components (e.g., cells, stroma, edema fluid) of a particular compartment?
Is this change in size due to a change in cell numbers in one or more compartments, i.e., microscopically increased or decreased number of cells and, if so, which cells are involved (lymphocytes, macrophages, stromal cells, etc.)?
Are the changes due to resident cells within the compartment, or due to passenger cells migrating through the compartment?
Does the weight, color or texture of lymphoid organs from dosed animals differ from normal background and/or control group organs macroscopically?
Once these questions have been resolved, the pathologist is encouraged to use the terms set forth in the guideline, for example, lymphocytes: increased/decreased; granulocytes: increased/decreased; mast cells: increased/decreased, and so forth. The reader is encouraged to review the non-exhaustive list of possible terms that the STP IWG considers more descriptive than interpretive. Moreover, the STP IWG feels that the interpretation is best reserved for the Discussion section of a pathology report rather than the Results section. However, the ultimate interpretation of the constellation of changes in a meaningful pathobiologic context is primarily the study pathologist's responsibility, following consultation with the peer review pathologist when appropriate.
While it may be of some debate as to with whom the specific authority for the final diagnoses rests, (i.e., who signs the pathology report) the intent of this last point is to reinforce the premise that when it comes to best practices for pathology, such determinations are made through the collaborative interaction of professional colleagues, rather than guidelines, so as to provide the most scientifically accurate conclusion possible. The specifics concerning the roles and responsibilities of the study pathologist and peer review pathologist, as well as sign-off procedures, should be defined by company SOPs, and not by these guidelines.
It should be apparent that these guidelines merely restate what the pathology community already knows to be appropriate practice, and what is in most cases, common sense. It is hoped that with the publication of these Best Practice guidelines, the routine procedures for pathology described therein will facilitate a more uniform and transparent approach to the pathologic evaluation of the immune system. Once detailed and appropriate histomorpholigic observations are made for a given lymphoid organ, then scientifically sound suggestions for the consequent selection of functional assays should follow; accurate and detailed histopmorphology is only the first step. Delineation of such observations are best made through the careful selection of additional functional tests and more focused experiments in which specialized histomorphologic procedures can be brought to bear on specific scientific questions.
I would like to acknowledge the very hardwork by the STP ImmunotoxicityWorking Group that included R. Perry (cochair), D. Ennulat, S. Frame, C. Johnson, J.-M. Lapointe, A. Nyska, P.W. Snyder, D.Walker, and G. Walter. Without their help, the Best Practices paper and this manuscript would not have been possible.