Humane Endpoints and Cancer Research

This is the authors' abstract. We don't add key points for this paper.

James Wallace, F.I.A.T., C.Biol., M.I.Biol., is a Fellow of the Institute of Animal Technology, Chartered Biologist, and Member of the Institute of Biology at The McElwain Laboratories Institute of Cancer Research, Surrey, United Kingdom. Cancer is the term used to describe a number of diseases that are characterized by uncontrolled, abnormal growth of cells in tissues or the blood. Cancer development may be localized to specific tissues or cells or may spread from the primary tumor to other parts of the body to produce secondary tumor growth (metastases). Cancer is a serious disease common to humans and many species of animals. The worldwide incidence of human cancer is increasing ( Boyle 1997 ), and the available therapies and outcome for certain malignancies, metastatic disease in particular, are less than satisfactory. Understanding the biologic processes that lead to the development of cancer, identifying potential carcinogens in the environment, and developing safe, effective treatments are major scientific and medical objectives. Experimental animal models of cancer provide a link between how cancer develops at the molecular or cellular level and how the condition manifests and may be treated in complex, living organisms. In vivo experimental models of cancer allow the collection of scientific and preclinical data that is not available from cell cultures or human subjects. To this end, a variety of experimental tumor systems have been developed, mainly using laboratory rodents as hosts, to study cancer biology and treatment ( Clarke 1996 ; Fidler 1986 ; Kallman 1987 ; Kerbel 1999 ; Welch 1997 ). The development of immunodeficient mouse models engrafted with human tumors has enabled anticancer drug/human tumor sensitivity and toxicity tests to be conducted. Genetically modified (transgenic) rodent models have been developed to promote the study of the molecular and genetic causes of the disease and to develop new therapeutic approaches ( Amundadottir et al. 1996 ; Lipkin 1997 ; Rosenberg and Bortner 1999 ; Rovigatti et al. 1998 ). Some experimental cancers can cause profound physiologic and metabolic changes that lead to an irreversible decline in the host. Laboratory animals may experience significant adverse effects as a result of experimentally induced cancers and the effects of investigative or treatment regimes ( Montgomery 1990 ; UKCCCR 1997 ; van Loo et al. 1997 ). The routine, or intentional, use of either death of the animals or evidence of significant pain or distress as an experimental endpoint in scientific studies is to be discouraged ( CCAC 1993 ; Hamm 1995 ; Olfert 1996 ; Tomasovic et al. 1998 ). In the Canadian Council on Animal Care document Ethics of Animal Investigation , it is stated, “toxicologic and biologic testing, cancer research and infectious disease investigation may, in the past, have required continuation until the death of animal. However, in the face of distinct signs that the processes are causing irreversible pain or distress, alternative endpoints should be sought that satisfy both the requirements of the study and the needs of the animal” ( CCAC 1989 , p. 2). The United Kingdom Coordinating Committee also recognizes that animals used in cancer research may experience pain and/or distress and recommends the implementation of humane endpoints in cancer studies. The objectives they describe in Guidelines for the Welfare of Animals in Experimental Neoplasia ( UKCCCR 1997 ) are quite clear; however, the practical methods to achieve them are less certain. Cancer is a complex condition, and the recognition and ranking of the severity of effects of experimental cancer on the animals can be problematic in practice. Development of universal scientific and humane endpoints for cancer studies is further frustrated by the number of scientific disciplines involved in cancer research, the range of scientific techniques and challenges employed, and the variety of tumor systems and animal models. However, a useful starting point is to encourage investigators to “seek the least traumatic techniques feasible that would allow the (scientific) question to be adequately evaluated” ( Goy 1982 , p. 346). Scientific and humane endpoints are inextricably linked and should be developed concurrently. There is little purpose in terminating experiments prematurely if this action confounds the experimental objectives and potentially wastes animals. It is equally unacceptable to extend animal distress beyond the point required to meet the scientific objectives. Scientific endpoints are criteria that determine the outcome of an experiment. Humane endpoints are finite, compassionate limits placed on the amount of pain or distress any experimental animal will be allowed to experience. Scientific and humane endpoints utilize data derived from observation of changes in the condition of the animals and laboratory-based assays. Both scientific and humane endpoints should promote the objective of establishing a scientifically valid answer to the experimental question with a minimum of harm to the animals. This situation requires that specific scientific and humane endpoints are developed for each project and are subjected to continuous adjustment and review. Clearly defined experimental objectives are essential to the development of both scientific and humane endpoints. The nature, intensity, and duration of the scientific challenge determine the potential consequences of the experiment for the animals. The experimental objective will determine the scientific approach, tumor system, animal model, and group sizes and will define the scientific and humane endpoints. for example, when the experimental objective is serial transplanation of tumors or determination of the oncogenicity of a cell line, the development of a single tumor of a predetermined size may act as both a scientific and humane endpoint. When the effects of therapy on the development of experimental tumors is to be evaluated, the adverse effects on the animals may be greater and a number of different scientific assays and humane endpoints may need to be employed. Whenever possible, scientific and humane endpoints should utilize criteria or assays that avoid significant animal distress or death. Studies of tumor growth or therapy frequently utilize either in situ or excision assays to establish changes in the pattern of tumor growth or cell survival. Assays conducted in situ involve either various methods of determining changes in the pattern of growth of the tumors in the host or extension of the survival time of the host. Excision assays normally require removal of the tumor at the end of the experiment for additional laboratory evaluation. Tumor growth or excision assays should replace “survival” endpoints. Tumor growth delay, tumor doubling time (Td 1 ), tumor regression, and clonogenic survival of tumor cells are alternative assays for assessing the growth of solid tumors. When tumor growth delay, tumor regression, or Td is the scientific endpoint, the final size of the tumor(s) in both treated and untreated control animals should be the smallest practicable (e.g, 2 to 3 Tds of the size) at the beginning of treatment, rather than any fixed final tumor volume or weight. The potential problems with in situ assays are that (1) tumors may become too large, (2) tumor-associated disease may appear in the host, or (3) animals may die as a result of tumor development. Excision assays assess what fraction of the tumor cells are still clonogenic and can form colonies in vivo, in other animals, or in vitro ( Courtney and Mills 1978 ). Where either ascitic tumors or leukemias are involved, ex vivo clonogenic assays or the onset of clinical signs should replace moribundity or death of the host as an experimental endpoint. Whenever possible, the potential consequences (in terms of pain, distress, or lasting harm) of the experimental challenge to the animals should be identified and controlled or eliminated at the outset ( Wallace et al. 1990 ). A review of the literature relevant to the use of animal models in cancer research ( Clarke 1997 ; Gart et al. 1986 ; Hanfelt 1997 ; Kallman 1987 ; UKCCR 1988 ; Welch 1997 ) will illustrate the practical cal and ethical issues involved in the design and conduct of cancer studies involving animals. Animal welfare considerations have both qualitative and quantitative constituents. Experimental protocols should be subjected to statistical review to ensure that they employ refined experimental technique and use the minimum number of animals to produce valid scientific results ( Gart et al.; 1986 ; Hanfelt 1997 ). Management of the experimental cancer program and animal care must also be considered ( Schiffer 1997 ). Pilot experiments, dose range finding studies, or a staged experimental approach using a small number of animals can help determine the response to an unknown or novel experimental challenge and provide data to develop scientific and humane endpoints for later studies using larger groups of animals. These approaches are particularly encouraged when there is the potential for significant animal distress. Detailed knowledge of the growth characteristics and biology of the proposed tumor model and the onset and nature of any adverse effects on the animals is crucial to the establishment of both scientific and humane endpoints ( Siemann 1987 ). Tumors in laboratory animals may be induced or transplanted or may occur spontaneously. Established methods of inducing experimental cancer in animals include exposure to carcinogenic substances, local or whole body irradiation, inoculation with viruses or malignant cells, genetic mutation, or (increasingly) the introduction of oncogenes by genetic modification of animals or transfection of cells. Experimentally induced tumors may develop at a predictable site and within a specific time frame, or their onset may be difficult to predict. Tumors may be solid and localized or may be solid and metastasizing to produce secondary disease in other tissues. Solid tumors may develop either in the superficial tissues or internally. Certain types of malignancies may arise in the blood, bone marrow, or lymphatic system. In contrast to the development of clinical cancer, experimental tumor systems in animals are frequently characterized by a relatively short latent period and rapid tumor growth. The incidence, site of origin, and growth rate of an experimental tumor and the onset and nature of the adverse effects on the host will vary with (1) methods used to induce the tumors, (2) the biology of the tumor, (3) the site of development and the issues involved, (4) any associated experimental challenge, and (5) the response of the host. The ideal experimental tumor model system would be stable and exhibit reproducible growth, behavior, morphology, and sensitivity to the potential experimental challenge. Immunogenicity, cell kinetics, and heterogeneity should be known. It should be possible to predict the incidence, origin, and time of onset of tumor development and any associated disease. However, experimental tumor cell lines are frequently genetically unstable and subject to constant selection processes at the cellular level in the host and through laboratory processing. Continuous serial transplantation in vivo or transfer into a host of a different genotype may transform the growth characteristics of a tumor line ( Rockwell 1987 ). Typical changes in tumor biology associated with serial transplantation include a reduced latent period, an increase in tumor incidence, reduced tumor doubling time, increased malignancy or ability to metastasize ( Visonneau et al. 1998 ), and changes in the sensitivity to therapeutic agents ( Clarke 1997 ). If cancer studies are to yield reliable and reproducible scientific results, then care must be exercised to minimize experimental variables by scrupulously following experimental tumor propagation protocols ( Welch 1997 ). Changes in the immunogenicity or antigenicity of tumor lines may alter the growth characteristics of the tumor and modify responses to experimental therapy. The strain, sex, age, health status, and genetic background of the host animal are additional variables that can influence tumor development. The genetic and microbiologic status of the tumor cell lines and the animal recipients should be ascertained to avoid experimental artifacts ( McKisic et al. 1996 ; Schiffer 1997 ). Transplantable tumors may be induced orthotopically, in the tissue or site of origin, or ectopically, in the or by the inoculation site may the growth characteristics of the tumors ( et al. 1988 ; and 1990 ). of the origin, incidence, and time of onset of developing tumors in the host animal is if experiments or animal welfare are not to be To promote the of experimental tumor it may be to modify the or physiologic whole body or agents are frequently used to further the response of immunodeficient rodents inoculation with human tumor of the and may be to in the establishment of tumors. Experimental challenges may include novel or anticancer treatments or the of potential Experimental protocols should the are to be at potentially techniques may be to induce experimental cancer, an experimental challenge, or an response an experiment. These may a greater challenge to an animal than that of a developing Humane endpoints should the of the experimental challenges on the animals. or tumor development can result in animal distress or Studies on experimental tumors frequently be until a tumor of the size is the experimental the development of the tumor and condition of the animal should be of data on tumor growth the study may yield results than of the final tumor will also in tumor development and possible adverse effects on the animal. should be tumor development or tumor-associated disease causes death or a significant in the host. on tumor development will be by the experimental growth characteristics of the tumor, site of tumor and clinical condition of the animals. must be in to the animals for tumor with limits placed on the tumor or severity of tumor-associated disease. of will be by the growth characteristics of the tumor and the onset of in the experimental or development of the Animals with transplanted tumors may require observation the of tumor however, animals in the of tumor-associated disease or toxicity may require a The incidence and growth rate of tumors, induced tumors, or in animals may be difficult to predict. When as tumors as the mouse ( et al. ) and the cell tumor ( et al. ) are when growth is Care must be to the distress of animals potentially tumors, particularly used as untreated and not therapy. When experiments involve the use of potentially tumors, the experimental should be the onset of the period of or death. the development of solid tumors in the superficial tissues and the condition of the animals is relatively can large, tumors any adverse effects or on their However, it is to limits on tumor with the objectives of the scientific The UKCCCR ( UKCCCR 1997 ) solid tumors to a of of the body weight. However, the of a tumor in to the of the animal can be particularly if the tumor is the host is weight. the tumor with is a reliable of tumor growth. either tumors of a fixed size or treatment a fixed time period may particularly if tumors or or the tissues. superficial tumors may to their or Some protocols may require tumors into the number of tumors an animal can be to is and specific is it is to use animals with tumors or a greater number of animals with tumor must be a between and animal The use of tumor may into question the of the scientific data with to possible tumor tumors may and the however, the use of tumor in a single animal has been to and experimental data ( Hanfelt 1997 ; et al. ). or of the the developing tumor may can occur when tumors either develop or are into the of the host. causes of can include (1) in tumors develop on the and are subjected to constant and (2) certain tumor types and (3) cell lines that are to may occur when the tumors are relatively When is of the tumor line, the should be to the experiment in the latent period as a tumor has the growth pattern will may be for terminating the experiment. or tissue may result in a continuous of body and/or When it is to an animal with an tumor, both the status of the tissues and the condition must be Tumors or tumor-associated metastatic disease may be difficult to ( Welch 1997 ). development of tumors in may cause distress or death. There is increasing in experimental tumors orthotopically, for example, in the tissue or site of as in or ( 1999 ; Kerbel et al. ; et al. 1999 ; et al. ), in to or inoculation into may increase the of the distress the growth and of tumors may be difficult to Tumor development in for tumor is the or may cause distress and require specific and in the and other tissues may occur as a result of either inoculation of tumor cells into the system, or cells from a primary tumor, or from cancers of the or lymphatic The removal of a primary tumor may the development of latent metastatic disease The onset of clinical signs or endpoint assays conducted at are used to the of or tumor methods to or tumors in animals may include serial of animals, investigative medical or for either or tumor development. of tumor as of body ( et al. ) and or may also be used as to either clinical signs or death. The increasing of as ( et al. ) or ( et al. ; 1999 ) to tumor cells can provide an to the of tumor metastatic cells with or can single cell in tissues ( Welch 1997 ). techniques can in animals ( and 1999 ). The of human or tumor can be used to tumor or cancer cells in an animal engrafted with human tumors. can be in the living animal by for the of cancer cells or changes in the cellular of the blood. in tumor cells or changes in can the onset of clinical When human cells are engrafted into immunodeficient the number of human cancer cells is less of either the onset or severity of clinical In the of reliable laboratory-based animals with or should be for clinical signs as of condition or and of the and Scientific endpoints should clinical signs as clinical or death. solid tumor and of condition are associated with the development of When the mouse is as it is The period is associated with and the the animals should be and for each Tumor cell survival assays may be used as endpoints in of clinical signs or death of the host. tumors can arise in and their development the clinical tumors are characterized by a variety of and cell if they may be The onset of is frequently and cancer arise in or at the time to provide for experimental The of tumor, incidence, and clinical onset of tumor-associated disease will vary with the strain, age, and of the host. or other variables may also influence tumor incidence and time of of the background incidence and biology of tumors in the host animal is essential if their development is not to with experimental cancer studies or cause animal distress or Some of laboratory as and have a predictable incidence of tumors, may their use in scientific studies. Tumor development may be associated with significant of the physiologic or metabolic of the serious and is characterized by an to of and death. not the may be associated with the use of specific tumor lines as the mouse ( et al. 1987 ) or cell tumor ( et al. however, animals should be for signs of The onset of induced in animals may be A significant and of may be is The scientific objective should be to the experimental as as possible the onset of the of clinical Humane endpoints for animals should the of of less than starting body and as by condition and animal models with a genetically to develop cancer are used in cancer research ( Amundadottir et al. 1996 ; et al. ; Lipkin 1997 ; Rovigatti et al. 1998 ; Rosenberg and Bortner 1999 ). Cancer development is a and there is the that animal models will provide on the of specific in and in the development of novel anticancer The mouse is to develop a variety of different relatively in ( et al. ). The mouse ( et al. 1990 ) is an the cancer in The is in the will develop tumors of the small that result in and A major welfare with the genetic of animals is the of the adverse particularly cancer, of animals. When or are changes may act increasing the incidence and of onset of experimental cancer and other effects ( Amundadottir et al. 1996 ; Rosenberg and Bortner 1999 ). may be by in the genetic background of the genetically modified animals ( and ). Continuous of genetically modified animals for tumor development or may be from to the condition of the animals and to any in their condition is an essential of scientific and humane endpoints. with the use and care of the animals should be with the systems and how to will be by the status and of the experimental challenge. systems may include a of of and behavior, clinical signs of pain or distress, and specific to changes from the scientific challenge ( ). Experimental protocols should include a of the proposed scientific challenge and a of any tumor models The objective of pain or distress in animals as a result of scientific is with A number of systems have specific and clinical signs of pain and distress in laboratory animals ( Montgomery 1990 ; and ; 1989 ; Wallace et al. 1990 ). adverse signs and a to each of the increasing severity of the and the on the animal ( and ). clinical signs for animals are in the UKCCCR ( UKCCCR 1997 ). distress systems for cancer studies on the condition and can lead to of humane end However, systems can animals that are they not provide a reliable of either survival or time of death. adverse clinical signs are equally of the of an body and that an animal is in a body or that an animal is not not the of the When cancer studies are involved, may be a reliable of a serious condition than of body weight. There is that terminating an experiment on the of a of clinical lead to a of essential data and a of animals. between the changes in the condition, the and the potential should be in experimental and humane endpoints may require of distress systems and of alternative The physiologic and health system is a severity of disease system used by for human ( et al. ). The system a of health and and to assess the condition of human and provide a of the and is using a from to of the condition can then be using a of specific physiologic less A approach using specific of tests be for use in laboratory animals a variety of or may the onset of clinical or other of may provide objective and reproducible experimental or humane endpoints than clinical ( and 1999 ). cell and can toxicity ( Clarke 1997 ), and other and provide a endpoint in of clinical In to tissue clinical and experimental cancer can produce or specific and metabolic in the host. is increased in many malignancies, particularly ( and 1997 ). The of malignancies and response to treatment can be by the of specific tumor as for cancer and in tissue or associated with tumor development can also be from or Laboratory assays small of body or tissues and it possible to metabolic or physiologic in small animals. The is a human that ( et al. 1999 ). and 2 , and 2 can be from a single small to This and systems may have a in the physiologic status of small laboratory animals. in molecular and to physiologic and metabolic changes in the experimental animal. These with an approach of experimental animals as can yield scientific data from animals and lead to refined scientific and humane endpoints. The use of objective of in animals will also in the of developing experimental and humane endpoints in cancer studies.