HIDDEN CANCERS AND HUMANE ENDPOINTS Lene Rud, Morten Kobaek Larsen Merel Ritskes-Hoitinga Prof. in Lab. Animal Science Biomedical Laboratory University of Southern Denmark www.felasa.org www.lal.org.uk
Working addresses Biomedical Laboratory Faculty of Health Sciences University of Southern Denmark Odense 1-9-05: Central Animal Laboratory University Medical Center Radboud University Nijmegen The Netherlands
Overview presentation Definitions General guidelines Guidelines cancer research Rat models Literature search Experimental results Future Acknowledgments
Humane Endpoints - definitions OECD (2000): Humane endpoints (HEP) are defined as the earliest possible indicators of distress, severe pain, or impending death, which should be used as measures for termination of the experiment, in order to prevent unnecessary suffering OECD schemes for use in toxicology studies OECD: Organisation for Economic Cooperation and Development
Humane Endpoints - definitions CCAC (1998): The point at which an experimental animal s pain and/or distress is terminated, minimized or reduced, by actions such as killing the animal humanely, terminating a painful procedure, or giving treatment to relieve pain and/or distress CCAC: Canadian Council on Animal Care
Scientific endpoints Always keep in mind: What is the scientific aim of the experiment and when is it reached? Animals are killed when sufficient scientific data have been obtained for the particular aim This can imply that the stage of pain and/or distress is prevented With a little more effort e.g. measure one extra blood parameter- we can often obtain a lot more valuable information (Rose Gaines Das), which can help to define new HEP in future experiments
International harmonization of guidelines on HEP ICLAS wg 04 10 general principles: Animals experience pain and distress in comparable situations as is the case for humans Death or severe pain and distress should be avoided as endpoints The earliest possible endpoint should be used, consistent with the scientific objectives of the study Studies should be designed to minimize any pain or distress The duration of studies involving pain or distress should be kept to a minimum
ICLAS guidelines - continued Pilot studies should be encouraged for determining morbidity, time course of effects and freq. of observations Prospective planning to agree on appropriate endpoints and on persons to be responsible for making the judgments on endpoints Team approach, including scientist, veterinarian, animal care staff and ethics committee to agree on the appropriate endpoint Research and animal care staff must be trained and competent for recognizing behavior and signs of pain, distress and moribundity Animals must be monitored for behavior, physiology and/or clinical signs at an appropriate frequency
High quality education essential LAS courses teach the spirit of the 3R s FELASA accreditation assures high-quality teaching and harmonisation of the courses in the EU www.felasa.org felasa recommendations for the accreditation of laboratory animal science education and training www.felasa.org T. Nevalainen,, Convenor; H.J.M. Blom; ; A. Guaitani; ; P. Hardy; B.R. Howard, P. Vergara
Evaluating pain and distress FELASA wg on pain and distress, Lab. Animals 1994, 28, 97-112 http://www.lal.org.uk/pdffiles/felasapain.pdf An applied approach to the assessment of severity, Jones et al. 1999, Astra Charnwood, UK
Guidelines cancer research United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Workman et al. 1998, Brit. J. Cancer 77, 1-10 Dutch Code of Practice, Dierproeven in het kankeronderzoek 1999 Wallace J. 1999 Proceedings Humane Endpoints in animal experiments for Biomedical Research, Zeist, pp. 79-84 Wallace J. 2000 ILAR Journal 41, 87-93
Rat models at Biomedical Lab. Wish from the clinic to develop an animal model for colorectal cancer (CRC) and its metastases in order to test vaccination therapy PhD study Kobaek-Larsen on development of Colorectal cancer rat model Extensive literature review Use of inbred strains Detailed characterisation Close clinical observations Publications
Colorectal cancer rat model Refinements Improve scientific and HEP Kobaek-Larsen et al. 2000: Literature review for obtaining a good overview Selection of 3 inbred strains on the basis of syngeneic cell lines available Kobaek-Larsen et al. 2002: 4 AOM-injections in 3 inbred strains Aim: selection most sensitive and reproducible model, comparable proces to human 4 AOM inj. sufficient: CRC freq. in BDIX rats 75-100% (literature: up to 27 weekly injections!) Frequent BW measurements: after the 2nd. AOM injection a drop in BW, so a one week break was introduced Close observations of BW, appearance, abdominal palpation, faecal blood led to euthanasia in time: no signs of pain/distress, intestinal obstruction, spontaneous deaths or body weight loss occurred
CRC rat model Refinements (2) Good indicators for the presence of CRC tumors: Fresh blood on the faeces NB. Haemoccult test faeces is always positive! Abdominal palpation skilled technicians can register thesizeofthetumors Kobaek-Larsen et al. 2004: prolonged latency period (52 wks) will intensify AOM side-effects Latency period of maximum 32 wks advisable By using inbred instead of outbred strains, tumors in theexternalearcanalareavoided
Rat model for liver metastases By intraportal transplantation of cells from the CC531 colon cancer cell line in the syngeneic WAG/Rij rat strain, liver metastases can be induced (Thomas et al. 1993, Hagenaars et al. 1998) Well-characterised and reproducible model (Kobaek-Larsen et al. 2000) MSc thesis Rud (2004) - 2 experiments were performed: To establish the correct number of cells for transplantation to induce liver metastases reproducibly in a controllable number over a reasonable period of time To determine which parameters could be used as (biomarkers for) HEP s
Literature search MSc thesis Rud 2004:Many articles about Liver metastases models in rats None have primary focus on animal welfare Body weight is the most often used parameter registered to evaluate animal welfare, which is often unreliable for the animal s condition and welfare in cancer studies Most studies use a fixed endpoint, e.g. 4 weeks after injecting tumour cells Often no humane endpoints are identified in the experimental protocol
General HEP for cancer UKCCCR: The tumour burden must not exceed 10% of the host animals normal body weight Consistent or rapid body weight loss of 20% within 72 hours There is not always a scientific basis for the described HEP And these HEP are not relevant for all studies Relevant humane endpoints should be identified, described, and incorporated into the experimental protocol, during the experimental planning UKCCCR The United Kingdom Coordinating Committee on Cancer Research
Liver metastasis model Intraportal injection of colon adeno-carcinoma cell line aggregates (CC531) into syngeneic WAG rats Previous pilot studies showed that this model gave reproducible results Two studies were performed: Expt. 1: 100.000 cells, lasted max. 6 wks Expt. 2: 6 different amounts of cells (25.000-100.000), lasted max. 9 wks Rats were euthanised when tumour size exceeded 3 cm in diameter
Experiment 1 Previous pilot studies: no sex difference in response 10 4 cells: virtually no liver metastases 10 6 cells: >50 small liver tumors Latency period 4-6 wks 20 male WAG rats of 250 g 10 test rats 10 5 CC531 cells 10 controls physiological saline, sham operated Laparosopy at wks 2, 3, 4 and 6 HEP: Total tumor mass max. 3 cm in diameter.
Measurements expt. 1 Body weight Clinical observations Laparoscopy Blood parameters Albumen Bilirubin GGT ALP ALT AST Autopsy: size and location of the tumors
Clinical Examination Daily inspection (routine) Detailed clinical examination of the animals was carried out twice a week after a modified OECD observation scheme Observation in cage in rest and when handling: (In)activity Hunched back Piloerection Discharge orifices Respiration rate / type Form of / Palpation abdomen Dehydration /oedema Icterus Rotte nr : Observation af rotten før den tages op af buret (1) : Dato : Uge nr : Pirrelig/ nervøs/aggresiv Inaktiv Hunched back (2) Kløen /krassen Piloerektion Respiration Observation af dyret på tæt hold Dato Uge nr Vægt Abdominal stivhed, ømhed, oppustet Vokalisering /resp.stop ved palpation Blødning fra endetarm Blod/sekret omk. Øjne/næse Usorineret/unormal udseende Dehydration (4) Fikseret el. sunkne øjne Icterus Ødemer Bemærkninger :
Laparoscopy Follow development of hidden tumours Laparoscopy: Stryker Ancleoscope, diameter 2.7 mm, view angle 30 degrees Kobaek-Larsen et al., Lab. Animals 2004, 38, 162
Laparoscopy Relatively simple technique The major part of liver surface and peritoneum can be examined Direct information number and size metastases Correlation between metastases development and clinical parameters can be established
Laparoscopy Tumour size was estimated semiquantitatively Good coherence estimation and autopsy measurements Digital photography during laparscopy will improve quantification of growing tumours over time
Results expt. 1 All test rats developed metastases Large individual variation No indicators of pain/distress found Levels below detection level: Bilirubin Gamma Glutamyl Transferase (GGT) No significant differences: Albumin ALT Significant results: ALP at wk 6 AST at wk 3, 4, 6
Body weight expt. 1 300 Weigth 280 260 Metastases Controls 240 0 10 20 30 40 50 Days after the operation
Experiment 2 Define optimal nr of cells for transplantation in V. Porta 7 groups of 4 animals each, one as a non-operated control grp Grp 0 (sham-op.) Grp 1 Grp 2 Grp 3 Grp 4 Grp 5 0 cells 100.000 (10 5 ) 75.000 50.000 25.000 7.500 Laparoscopy in wks 2, 4, 6 and 9
Measurements expt. 2 Body weight Laparoscopy Clinical Examination Blood analyses: AST ALT ALP Autopsy measurements
Expt. 2 Tumor development Number and Size of Liver Metastases in Experiment 2. Group Number of cells 2 week 4 week 6 week 9 week Autopsy (9 weeks) Control 0 I 7,500 II 25,000 III 50,000 IV 75,000 V 100,000 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -? A 1 A 1 A 1 - A 2 A 3 A 4 A 4 - - - - - - - - - - - - - - - - - - - - - - - - - - A 1 A 1 A 1 A 1? - - - -? A 2 A 4 A 4 A 4 - - A 4 A 5 A 5 -? A 1 A 2 A 2? - C 1 C 1 C 1? A 1? - - - - - - - -? C 4 C 5 C 4 - - - 6 6 Nr. of tumors: A=1 B=2-5 C >5 Size of tumors: 1 < 1 mm 2 1-5 mm 3 5-10 mm 4 1-2 cm 5 2-3 cm 6 <3 cm
Body Weight expt. 2 320 Weight 300 280 Metastases N=9 No Metastases N=15 260 0 10 20 30 40 50 60 Days after the operation
BW expt. 2 320 Weight 300 280 Metastases No Metastases No surgery 260 0 10 20 30 40 50 60 Days after the operation
Expt. 2: clin. exam. and liver enzymes Clinical examination resulted in no measurable differences between animals with and without metastases ALP gave contrasting results in the 2 experiments, which may be related to the degree of liver damage, or the stadium (degeneration and/or regeneration) ALT was significantly higher in expt. 2 only, at wks 6 and 9 AST was significantly higher in the animals with metastases in expt. 1 at wks 3, 4 and 6, and in expt. 2 at wks. 4 and 6
ALP Expt. 1 Expt. 2 ALP U/L 200 180 160 140 200 * * ALP U/L 180 160 140 * 120 120 100 Week 0 Week 2 Week 3 Week 4 Week 6 100 Week 0 Week 2 Week 4 Week 6 Week 9 Controls Test group No Metastases n=15 Metastases n=9
ALT Expt. 1 Expt. 2 ALT U/L 130 110 90 ALT U/L 130 110 90 * * 70 70 50 Week 0 Week 2 Week 3 Week 4 Week 6 50 Week 0 Week 2 Week 4 Week 6 Week 9 Controls Test group No metastases (n=15) Metastases (n=9)
AST Expt. 1 Expt. 2 180 * 130 AST U/L 130 80 * * AST U/L 80 * * 30 Week 0 Week 2 Week 3 Week 4 Week 6 30 Week 0 Week 2 Week 4 Week 6 Week 9 Controls Test group No metastases n=15 Metastaser n=9
Conclusions HEP liver metastases 75.000 cells resulted in metastases after 6-9 wks Laparoscopy provides a direct view on hidden liver tumours, but conflict Reduction versus Refinement Body weight is not a reliable parameter for HEP Clinical examination has not revealed indications of pain and/or distress AST and ALT mean group values rise, however, there is not a clear correlation between individual metastases size and values A combination of parameters is necessary
Clinical Examination It was not possible to find an indication from the clinical examinations that any of the rats experienced any pain or distress, even though total tumour diameter could be over 3 cm in diameter in some rats There were no differences seen between the rats with and without metastases Explanation: The rats did not experience any pain or distress The rats do not show the pain We do not look well enough We do not know what to look for
Future: additional measurements Telemetry Body temperature Heart rate Blood pressure Physical activity Video Recording behaviour Blood Analysis Tumour markers Cytokeratin 18 In combination with laparoscopy and AST and ALT measurements
Acknowledgments Support Medical Faculty, University of Southern Denmark Special thanks to co-supervisors of this project for valuable discussions and contributions: Peter Sandoe, Rene Remie, Coenraad Hendriksen ECLAM and ESLAV Foundation, one of the very few European funds for this type of research