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Regulatory Implications of Neoplastic Cell Substrate Tumorigenicity. Andrew M. Lewis Jr. M.D. DVP, OVRR, CBER, FDA. Outline. Review regulatory concerns - define tumorigenicity Review tumorigenicity testing: 1. How tumorigenicity is evaluated
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Andrew M. Lewis Jr. M.D.
DVP, OVRR, CBER, FDA
1. How tumorigenicity is evaluated
2. Evaluating the aggressiveness of tumorigenic cell
3. Regulatory implications associated with highly
4. Aspects of enhanced tumorigenicity testing relevant to
evaluation of tumorigenic cell substrates
Example: Reports of humans being engrafted with cells from human tumors (Southam, Science 125: 158, 1957).
Examples: Unrecognized agent (LCM) in cells from a human breast carcinoma (Zavada et al. J. Gen Virol 24:327, 1974); Variety of agents (herpesviruses, retroviruses, polyomaviruses) in human tumors. More details will be presented by Dr. Khan .
Example: Virus-free SV40-transformed human meningioma cells inoculated into nude mice induced mouse host-cell fibrosarcomas and lymphomas that contained SV40 (Brooks et al. Lab Inv, 58: 518, 1988)
Example: Cellular oncogene H-ras induces tumors in Swiss mice
(Burns et al. Oncogene 6:1973, 1991)
More details to be presented by Dr. Peden.
(Liotta and Kohn, Nature 411:375, 2001).
The process by which neoplastic cells growing in
tissue culture form tumors when inoculated into animals
Tumorigenicity vs. Oncogenicity
During tumorigenicity, the cells that were inoculated grow into tumors
During oncogenicity, oncogenic agents transform the cells of the species injected into neoplastic cells that grow into tumors
Note - Of these models, only the athymic mouse model and the
ATG-treated newborn rat model are currently used for the
regulatory assessment of cell-substrate tumorigenicity.
1. WHO assay (WHO Tech Rept Series 673: 72, 1982):
Inoculum - 10e6 cells/animal
Host -20 ATG-treated newborn(NB) rats, NB mice or NB hamsters; bone marrow-reconstituted, thymectomized and irradiated mice; chick embryo skin organ cultures
Observation period - minimum of 3 weeks with necropsy-histopathology of injection site, tumors, lymph nodes-organs for metastases.
(Revised in 1998 to 10 mice, 5 observed for 3 weeks and 5 observed for 12 weeks)
2. CBER assay: (CBER PTC in the Characterization of Cell Lines Used to Produce Biologicals 1993)
Inoculum - 10e7 cell/animal
Host - 10 nude mice or ATG-treated NB rats, NB mice, or NB hamsters or bone marrow-reconstituted, thymectomized and irradiated mice;
Observation period - 3 weeks for half and 12 weeks for half, unless tumor growth intervenes, with necropsy/histopathology of injection site, tumors, lymph nodes-organs for metastases.
1. Tumor incidence - No. animals with progressively growing
tumors/No. animals surviving
(Almost always used)
2. Tumor latency - time to tumor appearance (days, weeks,
(Almost always used)
3. Tumor size, weight, mass
(Used occasionally) - the faster (larger in size/wt./volume)
the tumor grows, the more aggressive the phenotype
4. For regulatory purposes, assays are considered valid if 9/10
positive controls develop progressively growing tumors
Data from Lewis et al. Cancer Lett 93:179, 1995.
Independent clones of BALB/c mouse embryo cells transformed in tissue culture by SV40 777 inoculated into 6-8 week old nude mice 106 cells/mouse.
(Brooks et al. Lab Invest 58:518, 1988)
(Reid et al. J Gen Virol 42:609, 1979)
Kinetics of tumor formation provides data on:
1- No. of cells required for tumor development - the fewer the cells required, the more aggressive the phenotype
2. Time of tumor appearance (tumor latency) - the more rapidly the tumors appear, the more aggressive the phenotype
3. Capacity of the tumors that form to metastasize may also contribute to assessing the aggressiveness of the phenotype
(Miller, Biometrica 60: 535, 1973)
(Example presented in next slide)
1. Estimated by Spearman-Karber
2. Data from 2 independent assays.
3. Data from 3 independent assays.
of Human, Mouse, and Hamster Origin
1. Proceedings of the second international workshop on nude mice, U. Tokyo Press, 1977, Gustav Fischer Verlag, Stuttgart
2. Cell lines from adenocarcinomas of the pancreas, breast, brain (glioma) failed to form tumors at 10 6 -7 cells/mouse after 28 wks.
3. Cells from lymphomas and leukemias failed to form tumors in adult nude mice but formed tumors in newborn nudes.
4. Of the cell lines from rats, mice, and dogs (MDCK); only cells from mice were tumorigenic.
Mice Without Tumor (%)
16 32 48
Weeks after injection
Newborn Nude Mice are More Sensitive than Adult Nude Mice in Detecting Tumor Formation by High Passage (252) ATCC 10-87 VERO cells
1. Contamination of cell substrate with viruses or bacteria (Reid et al. J Gen Virol 42:609, 1979; Baldwin and Pimm, Br J Ca 28: 281, 1973).
2. Infection of rodent hosts used in tumorigenicity testing (Lipman and Perkins in Laboratory Animal Medicine, Fox, Anderson, Loew, and Quimby, Eds, p1143, 2002)
3. Level of immunocompetence of rodent host (adults > newborns ≥ adult nude mice > newborn nude mice)
Multi-Step Models of Carcinogenesis
4 - 6 somatic mutation model for progression of colon carcinoma
(Vogelstein et al. NEJM, 319:525, 1988)
3-gene model of the neoplastic transformation of human cells in culture to cells that can form tumors in nude mice
(Hahn et al. Nature 400: 464, 1999)
3-Stage Model of Neoplastic Development
1. Initiation - 1st, apparently irreversible, stage of neoplastic development - Initiating event represents a single genetic/epigenetic DNA change
2. Promotion - 2nd, apparently reversible, stage of neoplastic development - Promotional events represent 1 or more additional genetic/epigenetic changes (oncogene activation/ tumor suppressor gene deactivation)
3. Progression - (See above) final genetic/epigenetic events resulting in tumor formation, invasion, and metastases
(Drs Khan and Peden will have more to say on this point)
1. Tumorigenic phenotype
2. Relative aggressiveness
3. Transfer of oncogenic viruses
4. Presence of adventitious agents