1 Regulatory Implications of Neoplastic Cell Substrate Tumorigenicity Andrew M. Lewis Jr. M.D. DVP, OVRR, CBER, FDA

2 Outline Review regulatory concerns - define tumorigenicity Review tumorigenicity testing: 1. How tumorigenicity is evaluated 2. Evaluating the aggressiveness of tumorigenic cell substrates 3. Regulatory implications associated with highly tumorigenic cells 4. Aspects of enhanced tumorigenicity testing relevant to evaluation of tumorigenic cell substrates Review mechanisms of neoplastic development and their implications for neoplastic cell-substrate evaluation

3 Regulatory Concerns Associated with the Tumorigenicity of Cell Substrates Induction of tumor allografts Example: Reports of humans being engrafted with cells from human tumors (Southam, Science 125: 158, 1957). Transfer of known or unknown viruses 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. Transfer of oncogenic agents 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) Transfer of cell components that might initiate neoplastic processes 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.

4 Regulatory Concerns Associated with Highly Tumorigenic Neoplastic Cell Substrates General perception that the more aggressive (tumorigenic) the neoplastic cell substrate, the greater the risk of its components inducing neoplastic processes Factors that contribute to the highly aggressive phenotype require further explanation (Liotta and Kohn, Nature 411:375, 2001). No attempts to correlate the oncogenic activity of cell substrate DNA with the aggressiveness of their tumorigenic phenotype. Fewer the cells required to produce a tumor, the smaller the safety factor that can be attributed to the transfer of factors that might induce neoplastic activity by first-order (1-hit) kinetics.

5 Tumorigenicity 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

6 How is the tumorigenic phenotype expressed by neoplastic cell substrates evaluated?

7 Evaluating the Tumorigenicity of Neoplastic Cells: In Vivo Models Inoculation of athymic (nude) mice or nude rats. Inoculation of mice or newborn rats treated with irradiation or anti-thymocyte globulin (ATG). Inoculation into immunologically privileged sites such as the brain, cheek pouch (hamster) or the eye. For cell lines from inbred mice, rats, and hamsters inoculation of syngeneic animals. 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.

8 Cell-Substrate History and Its Implications for Tumorigenicity Testing Concerns about using neoplastic cells as vaccine substrates were voiced in 1954 by the Armed Forces Epidemiology Board with a recommendation that only “normal” cells be used Prior to 2000, only cells that were shown to be non-tumorigenic were used in the manufacture of viral vaccines

9 Tumorigenicity Testing of Cell Substrates Prior to 2000 (1) Single-Dose Assays: ` 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)

10 Tumorigenicity Testing of Cell Substrates Prior to 2000 (2) Single-Dose Assays: ` 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.

11 Tumorigenicity Data: Endpoints and Interpretation 1. Tumor incidence - No. animals with progressively growing tumors/No. animals surviving (Almost always used) 2. Tumor latency - time to tumor appearance (days, weeks, months) (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

12 Why are Single-Dose Assays Inadequate? Dose-response and time to response are basic parameters of most types of bioassays Single-dose assays provide only 1 data point in the dose response that is inherent to the process of tumor formation in animals Single-dose, short term assays can give data that are unreliable on the ability of neoplastic cells to form tumors in vivo Data on the kinetics of tumor formation by neoplastic cell substrates provide additional data that contribute to vaccine safety

13 Problems with the Validity of Tumor Incidence and Tumor Latency Endpoints in Single-Dose, Short- Term Tumorigenicity Assays 1 Cell Lines 2 No. Exps. Tumor incidence after indicated No. of weeks (Inoculum/host cells/ adult nude mouse) SV40ME120/88/8 SV40 ME220/8 4/86/7 1.Data from Lewis et al. Cancer Lett 93:179, Independent clones of BALB/c mouse embryo cells transformed in tissue culture by SV inoculated into 6-8 week old nude mice 10 6 cells/mouse.

14 Rationale for Expanded Tumorigenicity Testing of Neoplastic Cell Substrates Introduction of highly tumorigenic cell substrates in the manufacture of viral vaccines sets new precedents Presence of unknown agents/factors in highly tumorigenic cell substrates represents their greatest risk Testing algorithm for evaluating highly tumorigenic cell substrates, designed to be state-of-the-art, has limited track record as use of tumorigenic cell substrates is a recent event Every practical technique needs to be used to minimize the risk of transferring infectious/oncogenic agents/factors by vaccines Detection of unknown agents/factors can be enhanced by expanding tumorigenicity testing methods and evaluating the data available from such assays

15 Recommendations for Tumorigenicity Testing of Neoplastic Cell Substrates Evaluate the kinetics of tumor formation by determining the tumor incidence at doses of 10e7, 10e5, 10e3, and 10e1 cells/adult nude mouse Record incidence of palpable tumors at weekly intervals over 4-5 month interval Determine species of origin of cells in tumors across the range of tumor-forming doses, with particular attention to tumors at limiting cell doses Necropsy all mice at end of the study and obtain histopathology on tumors/injection sites Evaluate any spontaneous tumors that develop for evidence of genomic DNA from the cell substrate

16 Expanded Tumorigenicity Testing Enhances the Regulatory Management of Neoplastic Cell Substrates Defines the tumorigenic phenotype of the cell substrate Assess the level of neoplastic aggressiveness expressed by the tumorigenic phenotype –Different levels of aggressiveness influence the level of concern and the evaluation of the substrate - the more aggressive, the higher the level of concern. More will be presented later in the talk. Tests for unrecognized oncogenic agents by identifying the species of the cells that grow into tumors (Brooks et al. Lab Invest 58:518, 1988) May indicate the presence of unrecognized oncogenic agents by looking for aberrations in the kinetics by which tumors are formed by the cell substrate (Reid et al. J Gen Virol 42:609, 1979)

17 How to Evaluate Neoplastic Cell Substrates that Exhibit Highly Tumorigenic Phenotypes 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

18 TPD 50 Endpoint Explained TPD 50 = Tumor producing dose at the 50% endpoint (i.e. No. of cells required for tumor formation) TPD 50 values change (evolve) as the tumor incidence changes during the observation period until it reaches the endpoint of the capacity of the cells to form tumors TPD 50 values are best determined by the Spearman- Karber estimator of 50% endpoints (Miller, Biometrica 60: 535, 1973) (Example presented in next slide)

19 Tumor Formation by HeLa Cells in Nude Mice Time (weeks) Tumor incidence at dose of cells inoculated (log10) TPD 50 (log10) /80/ /813/134/130/ /813/137/130/ /813/137/131/130/ /813/137/131/130/ /813/137/131/130/ /813/1310/131/130/ /813/1310/131/130/ /813/1310/131/130/ Estimated by Spearman-Karber 2. Data from 2 independent assays. 3. Data from 3 independent assays.

20 Graph of the TPD 50 Data at Weekly Intervals in Nude Mice Injected with HeLa Cells (TPD 50 Evolution Curve) TPD 50 (log10) Time (weeks)

21 TPD 50 Evolution Curves Represent survival curves of average tumor latency Can be converted to survival curves Conversion to survival functions simplifies statistical analyses

22 Tumor Formation Dynamics of Weakly Tumorigenic and Highly Tumorigenic Cell Substrates TPD 50 (log10) Time (weeks)

23 TPD 50 Cell Line Comparison of TPD 50 Values for Cell Lines of Human, Mouse, and Hamster Origin Human MouseHamster

24 Ability of the Adult Nude Mouse Model to Detect Tumorigenic Phenotypes Cell Line/Tumor Category Number Forming Tumors/Number Tested Stiles et al. (Can Res 36, 1353, 1976) Gershwin et al. (JNCI 58: 1455, 1977) Giovanella et al. (Cancer 42: 2269, 1978) Sordat et al. WNM Cell lines from human tumors 3/35/8 2 96/965/7 (7/7) 3 Cell lines from rodent tumors 2/2NT Cell lines from spontaneously transformed normal tissue 1/5 4 NT Cell lines from normal tissue transformed in culture by viruses 7/13NT Individual Totals (%) Grand total 119/134 (89%) 13/23 (56)5/8 (63)96/96 (100)5/7 (71) 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 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.

25 Newborn Nude Mice are More Sensitive than Adult Nude Mice in Detecting Tumor Formation by High Passage (252) ATCC VERO cells Weeks after injection Mice Without Tumor (%) Newborn-HP Adult-HP 0/18 Adult-LP 0/14 16/

26 Factors Known to Modify the Tumor-Forming Capacity of Neoplastic Cells Growing in Tissue Culture 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)

27 Biological and Molecular Mechanisms Involved in Neoplastic Development and Tumor Formation (1) Multi-Step Models of Carcinogenesis 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)

28 Biological and Molecular Mechanisms Involved in Neoplastic Development and Tumor Formation (2) 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

29 Mechanisms of Neoplastic Development and the Regulatory Management of Tumorigenic Cell Substrates Tumor development is a multi-step process requiring independent genetic alterations involving different genetic loci Every mutation above one decreases the possibility of transferring neoplastic activity by the power of the mutation number Tumor development represents the end stage of neoplastic development that begins with an initiating event Transfer of viral oncogene or dominant activated oncogene activity that is capable of inducing neoplastic activity resulting in tumor formation can be detected in animals models The sensitivity of some testing models to detect this activity is low (Drs Khan and Peden will have more to say on this point) Initiating events represent single, 1st order genetic/epigenetic processes Initiating events are not reversible and may or may not evolve along the path of neoplastic development during the life of an individual Currently there is no way to test cell substrate components for neoplastic initiation

30 Summary Cell substrate tumorigenicity testing can provide data on: 1. Tumorigenic phenotype 2. Relative aggressiveness 3. Transfer of oncogenic viruses 4. Presence of adventitious agents Tumorigenicity testing in adult nude mice can detect tumor- forming capacity in 9/10 cell lines tested (Newborn nude mice possibly provide a more sensitive alternative) Tumor formation represents an end-stage of neoplastic progression With exception of neoplastic initiation events, the multi-step process of neoplastic development makes it unlikely that neoplastic activity can be transferred by cell components other than oncogenic viruses Currently, there are no assays for evaluating biological products for their potential to induce neoplastic initiation

31 OVRR Recommendations for Tumorigenicity Testing of Neoplastic Cell Substrates Evaluate the kinetics of tumor formation by determining the tumor incidence at doses of 10e7, 10e5, 10e3, and 10e1 cells in adult nude mice Record incidence of palpable tumors at weekly intervals over 4-5 month interval. Determine species of origin of cells in tumors across the range of tumor-forming doses, with particular attention to tumors at limiting cell doses Necropsy all mice at end of the study and obtain histopathology on tumors/injection sites, Evaluate any spontaneous tumors that develop for evidence of genomic DNA from the cell substrate