UNIVERSITY OF PITTSBURGH Institutional Animal Care and Use Committee

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1 UNIVERSITY OF PITTSBURGH Institutional Animal Care and Use Committee Standard Operating Procedure: Tumor Burdon Guidelines: Neoplasia Proposals in Rodents EFFECTIVE ISSUE DATE: 10/1/2001 REVISION DATE(s): 8/1/2004; 9/23/2013; 7/16/2018 Introduction Tumor implantation in research animals is a critically important experimental activity which requires consideration of the effect of the tumor or tumors on the animal. Effective monitoring systems and endpoints should include limits on the tumor burden and severity of tumorassociated disease. The use of altered physiological, biochemical and other biomarkers are suggested as potentially more objective and reproducible endpoints than clinical signs. This document begins with an executive summary related to tumor burden endpoints with expanded information following this summary table related to performing tumor studies in mice and rats. These guidelines limit the tumor burden an animal experiences to that which does not cause excessive pain or distress and are for cumulative tumor burden per animal. If multiple tumors occur, the total tumor burden cannot exceed the parameters noted below without prior IACUC approval. Requirements 1. The University of Pittsburgh s IACUC approves all proposals involving development, induction, or injection of tumors in laboratory animals. To avoid subjecting animals to unnecessary pain or distress, these proposals require periodic follow-up regarding the condition and health of the animals (see Monitoring below). 2. All animal study proposals must address tumor burden effects and endpoints in the applicable sections of the ARO IACUC submission ( Anticipated Effects - Assessment of Animals' Well-Being and Experimental Design and Endpoints ) 3. Although tumor burden measurement and assessment is a complicated issue, The IACUC guidance is to not exceed sizes as follows unless scientifically justified and approved by the IACUC: a. Greatest dimension: measurement should not exceed 2.0 cm in any direction for mice and 4.0 cm in rats. b. Volume measurement: Does not exceed the 2000 mm3 and 5000 mm3 endpoints for mice and rats respectively. 4. All faculty and scientific support staff should be familiar with normal species-specific animal health and behavior, and be able to observe adverse changes in health, behavior,

2 or tumor burden. When adverse cases are identified, and the animal is not immediately euthanized, it is very important that several individuals be notified. They include the principal investigator, investigator support personnel, animal care personnel, facility manager/supervisor, and the Attending Veterinarian or their designee. Clinical signs requiring action are outlined in the box below. 5. In general, the clinical signs shown below are principal indicators of rare but severe symptoms of potential adverse effects, which should be avoided. Where any one sign is present the animal should undergo immediate intervention as approved in the IACUC protocol or be humanely culled and vigilance increased for the remainder of the cohort. Procedures General 1. Transplantable tumors, hybridomas, cell lines, blood products, and other biologic materials can be sources of both murine and human viruses that can contaminate rodents or pose risks to laboratory personnel. These products carry a risk of carrying adventitious rodent pathogens into the specific pathogen free rodent colonies potentially compromising other research models. Therefore, all cell line usage requests must include the required information in the IACUC ARO application section entitled: Procedure Classification - Cells, Tumor Line, Antibody, Serum or Body Fluid Use. Note: the use of immunodeficient or certain genetically modified animals (GMAs) may be more susceptible to illness or more susceptible to shedding human and animal pathogens, when using human tissues and cell lines. (1) This can lead to an increased risk to the health and safety of personnel working with the animals therefore such studies should be done using appropriate Animal Biosafety Level housing and practices as outlined in the EH&S risk assessment attached to each approved IACUC protocol Preclinical cancer studies fall into two broad categories: those using tumor cell transplantation and those in which tumors arise spontaneously or are induced in the host. Particular attention must be paid to the body system most likely to be affected by the tumor type and subsequent metastases (e.g. solid, ascitic, lymphoid, etc.) and organ system (e.g., skin, peritoneum, spleen, lymph node, brain etc.). 3. The duration of the experiment should be as short as possible and the number of animals required for scientific and statistical evaluation of results should be kept to a minimum. 4. The site for injection of solid tumors should be carefully chosen to permit room for tumor growth and to avoid unnecessary distress whenever possible (e.g., subcutaneous flank or peri-midline back are considered to be the least cause for distress). Note: some tumor cells grow SQ, and animals need to be monitored for skin ulceration, necrosis or other problems. Other injection sites (IP, IM, intracranial, intracardiac, prostatic, oropharyngeal, etc.) may have unique site related health concerns such as impaired mobility, breathing, and abdominal distension. Page 2 of 10

3 5. For tumor regression studies, careful attention should be paid to any animal exhibiting an ulcerated and/or necrotic tumor. To deter cannibalization or adverse welfare concerns, any animal exhibiting an ulcerated or necrotic tumor should be euthanized or medically managed as approved in the IACUC protocol. Affected animals should be separated immediately and singly housed until tumor regression is complete. Recordkeeping for such cohorts is essential. Use of a blue procedural card or pink copy of the Illness report form are required and should be placed on each individual cage containing a rodent with an open tumor, recording the date of the tumor opening on the card. The principal investigator is responsible for ensuring adherence to: Monitoring: a. IACUC-approved regression timelines b. Approved pain management/analgesic plan c. Endpoints as described in the animal study proposal The IACUC Guidelines for Endpoints in Animal Study Proposals (i.e., euthanizing the mouse if the tumor becomes infected, interferes with ambulation/eating/drinking, or the mouse becomes otherwise debilitated) 3. In circumstances involving declining health status, morbidity, or unrelieved pain and discomfort, every attempt will be made to reach consensus with the principal investigator bearing experimental endpoints approved in the IACUC protocol in mind. However, the final analysis and discharging of the University of Pittsburgh s animal care and use regulatory responsibility rests with the Attending Veterinarian or their designee. If determined necessary in the judgment of the attending veterinarian, animals will be euthanized. 1. Monitoring procedures must be closely adhered to. The research technical staff must be aware of the parameters of the study, such as tumor growth potential and whether a tumor is likely to become ulcerated. The Investigator must clearly define study parameters and endpoints in their IACUC Study Proposal Application (ARO) and must provide guidance to the technical staff on all study matters. Failure to adequately monitor animals or to abide by the conditions stated in the approved IACUC proposal will result in disciplinary action by the IACUC. 2. A plan for monitoring the animals both before and after a change in clinical status, providing care if appropriate, and increasing the level of monitoring as necessary, should be in place. 3. Monitoring or clinical care on weekends and holidays may require involvement of the investigative staff to supplement that standardly provided by the animal care and veterinary staff. Unless otherwise specified in the IACUC proposal, monitoring and Page 3 of 10

4 taking the appropriate actions is the responsibility of the investigative staff, including on weekends and holidays. 4. The monitoring plan should identify the personnel responsible for evaluation, record keeping, notification of the investigator and/or veterinarian and persons responsible for euthanasia. Checklists or score sheets may be helpful in ensuring appropriate observations are made, consistently interpreted, and properly documented. 5. Clinical observations and/or palpation may be necessary to monitor for deterioration of clinical condition. Special examination techniques may be required for specific sites (e.g. respiratory rate for lung involvement, neurological disturbance for brain neoplasms, and blood cell counts for leukemia) Clinical observation parameters used in cancer research and toxicological studies have been described and typically include changes in general appearance, skin and hair, eyes, nose, mouth and head, respiration, urine, feces and locomotion Body condition scoring can be applied to most species, BC 1 through 5 6 & 7. a. BC 1 - Emaciated skeletal structure extremely prominent, little or no flesh/muscle mass, vertebrae distinctly segmented. b. BC 2 - Underconditioned segmentation of vertebrate column evident, dorsal pelvic bones readily palpable, reduced muscle mass. c. BC 3 - Well conditioned vertebrae and dorsal pelvis are not prominent/visible, palpable with slight pressure. d. BC 4 - Over conditioned spine is a continuous column, vertebrae palpable only with firm pressure. e. BC 5 - Obese, contour is smooth and bulky, bone structure disappears under flesh and subcutaneous fat. 8. Schedule a. Mice and rats with developing tumors are to be observed no less than three times weekly until a palpable tumor nodule is present (5-7.5mm in greatest diameter), followed by daily monitoring (including weekends and holidays). Deviations from this monitoring schedule must be discussed and justified in the protocol. b. If tumors are located in a location that is not palpable, a monitoring schedule should be established based on pilot studies. Pilot studies can be used to familiarize the animal researcher to possible adverse effects and to define the critical time scale of adverse effects. Features to consider include tumor site, growth rate, invasion, distension, ulceration, metastasis, and production of cachectic factors. Page 4 of 10

5 c. If tumor growth is rapid in the days before termination, twice daily monitoring may be necessary. 9. Variables: Some tumors can cause significant changes in animal health. In particular it is recommended, animals should be observed regularly as specified in the protocol for any indication of the following: Visual weight loss Changes in feces/urine Restlessness Vocalization Cranial deformity/neurological signs Rough/unkempt hair coat Skin pathology Jaw deformity/malocclusion Ascites and/or abdominal distension Decreased food/water intake Lethargic/depressed activity Respiratory difficulty Perianal soiling Hunched posture Restricted mobility Hypothermia Clinical Signs Necessitating Immediate Intervention 1. Failure to eat or drink over 24 to 48-H period resulting in emaciation or dehydration. 2. Consistent or Rapid body weight loss reaching 20% at any time or 15% maintained for 72h compared with the pre-treatment weight of adult mice or age-matched, vehicletreated controls. With some tumors body weight is a very poor indicator and muscle atrophy or emaciation is more useful. Body condition scoring provides a very useful indication of muscle loss (Ullman-Cullere and Foltz, 1999). 3. Persistent Hypothermia. 4. Bloodstained or mucopurulent discharge from any orifice. 5. Labored respiration, particularly if accompanied by nasal discharge and/or cyanosis. 6. Enlarged lymph nodes or spleen. 7. Hind-limb paralysis or weakness. 8. Anemia as indicated by symptoms such as pale feet, or hematological measures. 9. Significant abdominal distension or where ascites burden exceeds 10% of the bodyweight of age-matched controls. Accurate determination is difficult but body girth is useful and 20% increase should be the maximum normally allowed; similar to the appearance of a pregnant mouse. 10. Incontinence or diarrhea over a 48-h period. Page 5 of 10

6 11. Tumors that interfere with locomotion or cause abnormal vocalization, animal behavior or function. Endpoints 1. There are several examples in the literature that describe endpoint criteria 7, 8, 9, 10. For most species they include: body weight, physical appearance, measurable clinical signs, unprovoked behavior and response to external stimuli. The overall well-being of the animal takes priority over precise tumor measurements in decisions regarding euthanasia or other interventions. Tumors induced in body cavities (cranium, orbit, abdomen, or thorax) may have additional limitations as to the maximum acceptable size. These animals must be monitored very closely for any severe impairment in physiological or neurological function and be euthanized as soon as such signs become apparent. 2. All animal experiments must provide for a humane endpoint. As a general guideline, animals used in experimental procedures involving tumor development must be considered for euthanasia if any of the following conditions occur: 13,14,15, and 17. a. Primary Tumor Size: i. Two primary methods are acceptable. Greatest dimension: measurement should not exceed 2.0 cm in any direction for mice and 4.0 cm in rats. ii. Volume measurement: Does not exceed the 2000 mm3 and 5000 mm3 endpoints for mice and rats respectively. Note- Where two tumors per animal are grown, for example, in contralateral flanks, the size should be correspondingly less and should cumulatively not exceed the maximum burden of a single tumor. Justification to exceed these size or volume restrictions must be approved by the University of Pittsburgh s IACUC in advance. Tumor Volume Measurement: Estimation of tumor size and burden is an important consideration in determining endpoints. Assessment of the size of superficial tumors using calipers (usually of two diameters at right angles) is an easy and definable method. Measurement variations can be minimized by ensuring that the same welltrained technician is involved for the duration of the study. The most accurate volume calculations are obtained using the formula V = (W2 L)/2 for caliper measurements and the formula V = (4/3) π (L/2) (L/2) (D/2) for ultrasonography, CT, or other imaging measurements, where V is tumor volume, W is tumor width, L is tumor length and D is tumor depth 16. b. Tumor interferes with normal behavior and condition of the animal (e.g., locomotion, exploration, grooming). Page 6 of 10

7 c. Tumor interferes with the animal s ability to eat and/or drink. d. 20% weight loss (emaciated appearance, cachexia); rapid weight loss over two to four days; or progressive weight loss over a few weeks). e. Body Condition Scoring (BCS) < 2; Muscle atrophy or emaciation. f. Tumor becomes ulcerated, infected, or necrotic with break of overlying skin. Note: Ulceration may be acceptable for certain skin tumors or regression studies. This condition and the treatment must be discussed in the protocol with specific attention to pain and wound medical management plans. g. Palpation of tumor elicits a pain response. h. Animals become moribund, weak, comatose, unresponsive, or death appears imminent. i. Animal showing signs of respiratory difficulty. j. Animal showing signs of hypothermia (i.e., cold to the touch, pale extremities). k. Bloodstained or mucopurulent discharge from any orifice (unresolved). l. Labored respiration particularly if accompanied by nasal discharge or cyanosis. m. Significant abdominal distension (appearance of a full term gravid mouse/rat). n. Incontinence, inappetence or prolonged diarrhea (unresolved ). 3. Death as an endpoint in the experimental design is not routinely acceptable for cancer studies. Extenuating circumstances for which death as an endpoint is expected requires scientific justification, a risk/benefit-based justification, and prior approval by the IACUC. 11 & Refining humane endpoints: Pilot studies and optimization 8, 10, 12 & 13. There are ethical, scientific and legal reasons for ensuring that adverse effects are minimized. Choice of appropriate humane endpoints provides significant opportunities for refinement and should be developed in tandem with the requirements for a valid scientific outcome. Early endpoints reduce non-specific systemic effects and so may increase the precision of the results obtained. Pilot studies, including autopsy to determine the full extent of tumor growth, will facilitate the definition of robust and refined endpoints. Endpoints for particular models must also take account of the known pathogenesis of the particular tumor model in question and should be regularly reviewed in the light of experience. Page 7 of 10

8 Determining the tumor burden of internal orthotropic cancers, systemic lymphoreticular tumors or metastatic disease is challenging. Pilot experiments using small numbers of animals are important to allow characterization of the kinetics and patterns of spread, to predict clinical signs and to define humane endpoints. Biomarkers or circulating cancer cells may be used as surrogates for assessing the burden of lymphomas and leukemia, and real-time imaging is a valuable adjunct. Pilot tumor growth studies using small numbers of animals (5 10) are recommended to establish that patterns of local and metastatic growth are reproducible. They also show any adverse effects associated with tumor progression and enable humane endpoints to be identified. The data derived should feed into group numbers used for definitive studies (e.g., therapy experiments) in order for experimental time frames and statistically significant endpoints to be established. Use of a relevant positive control treatment may be useful at this stage to ensure that tumor growth/responsiveness is as expected. This can be dictated by a variety of factors, including the site of growth. Subcutaneous tumors may grow rapidly and some are prone to developing hemorrhagic areas, which can cause rapid expansion and ulceration (e.g., human A2780 ovarian carcinoma and AR42J pancreatic carcinoma xenografts). For tumors growing as a suspension in the peritoneal cavity, it is important to establish clear criteria to ensure that studies are terminated before animal welfare is compromised. This site is only appropriate for models where ascites is a feature of the natural progression of the human cancer (e.g., ovarian carcinoma, peritoneal mesothelioma, gastrointestinal tumor carcinomatosis). Similar criteria apply to other sensitive specialized sites such as muscle or brain. For metastatic models, pilot experiments should define the extent and time course of dissemination to internal organs. Pilot studies should include sequential analysis of animals to determine the time course required to achieve scientific goals. Termination of studies at the earliest possible point will minimize adverse effects on the animal. Where possible, use of biomarkers (e.g., serum levels of prostate-specific antigen, PSA) and real-time imaging are highly recommended. It is also possible to measure circulating tumor cells using fluorescence and PCR-based techniques. In genetically modified animals, particular care is needed to ensure detection of unexpected sites of tumor development. As with all internal tumor sites, this includes clinical examination, measurement of body weight, abdominal palpation and loss of condition. Humane endpoints, specialist care and interventions should reflect best practice and be discussed and agreed between researchers, veterinarians and animal care staff before commencement of the experiment. Development and publication of appropriate experimental analyses (e.g., pharmacodynamic determinations, functional imaging) to capture detailed phenotypic information assists rational determination of endpoints. 5. Imaging for tumor endpoints: a. General Considerations Page 8 of 10

9 References Imaging techniques now have a principal role in translational cancer research, enabling sequential analysis of biological endpoints in the same animal, with obvious welfare benefits. The main utility of small-animal imaging is for monitoring deep-seated tumors and metastases with or without treatment. Applications include studies of basic biological processes and of tissue pharmacokinetics and pharmacodynamic responses to treatment. However, animal numbers may not be reduced if, for example, full endpoint analysis requires surgical intervention such as cannulation of blood vessels or when contrast agents have a long half-life. Here, sequential imaging may not be possible and alternative techniques involving tissue excision may provide more information (usually at higher spatial resolution) from the same number of animals. There is an increasing clinical need for pharmacodynamic imaging with molecularly targeted cancer therapeutics. However, interpretation of imaging signals is often difficult and animal models have an important role in rigorous validation of new techniques. This needs to be accompanied by consideration of unique animal welfare issues. Use of external imaging techniques on small animals is not completely noninvasive as some form of anesthesia or physical restraint is necessary and surgery or administration of contrast agents may be required 8. b. Imagine Techniques 8 The applications, advantages and disadvantages of commonly used imaging technologies have also been reviewed recently 8. Whole-body optical imaging is relatively simple and cost-effective. Tumor cells are genetically modified to constitutively or inducibly express a fluorescent protein (e.g., egfp, dsred) or an enzyme that activates an exogenously administered substrate to a bioluminescent molecule (usually luciferase for activation of a luciferin). The whole animal is imaged using sensitive optical detectors, which may or may not incorporate a tomographic facility. The potential influences of genetic modification and/or substrate administration on immunogenicity and response to treatment, as well as animal welfare, must be considered 8 & Guide for the Care and Use of Laboratory Animals 8th ed pg BMBL 5th ed 3. See IACUC SOP Pain Management in Laboratory Animals Montgomery CA (1990), Oncological and toxicological research: Alleviation and control of pain and distress in laboratory animals. Cancer Bulletin 42: Ullman-Culleré M and Foltz C. Body condition scoring: a rapid and accurate method for assessing health status in mice. Laboratory Animal Science 49 (3): Toth (1997) The moribund state as an experimental endpoint. Contemp Top Lab Anim Sc 36: Workman P, et al; Guidelines for the welfare and use of animals in cancer research. British Journal of Cancer (2010) 102, Page 9 of 10

10 9. UKCCCR [United Kingdom Coordinating Committee on Cancer Research] Guidelines for the Welfare of Animals in Experimental Neoplasia, 2nd ed. London: UKCCCR. Wallace J, Humane endpoints in cancer research. ILAR 41: 79-84, Guide for the Care and Use of Laboratory Animals 8th ed ref Review of Cost-benefit Assessment in the Use of Animals in Research Animal Procedures Committee. London. Available at accessed January 24, Guide for the Care and Use of Laboratory Animals 8th ed pgs Recognition and Alleviation of Pain in Laboratory Animals; NRC Roughan JV, Flecknell PA, Davies BR (2004) Behavioural assessment of the effects of tumour growth in rats and the influence of the analgesics carprofen and meloxicam. Lab Anim 38: Guidelines for Pain and Distress in Laboratory Animals: Responsibilities, Recognition and Alleviation, Revised 11/14/12. (PDF Document, 7 pages) 16. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements Ana Faustino-Rocha, Paula A. Oliveira, Jacinta Pinho-Oliveira, MSc1, Catarina Teixeira-Guedes, MSc1, Ruben Soares-Maia, MSc1, Rui Gil da Costa, MSc, DVM, PhD3, 4, Bruno Colaço, MSc, DVM, PhD2, Maria João Pires, DVM, PhD2, Jorge Colaço, DVM, PhD2, Rita Ferreira, PhD5 & Mário Ginja, DVM, PhD6 Ref Lab Animal - 42, (2013) Published online: 21 May 2013; doi: /laban Eden V Paster, Kimberly A Villines, and Debra L Hickman, Endpoints for Mouse Abdominal Tumor Models: Refinement of Current Criteria, Comparative Medicine, Vol 48, No 3, June 2009, Pages Page 10 of 10