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

Outline Define tumorigenicity and oncogenicity Review regulatory concerns associated with tumorigenic cell substrates, especially cell substrates that are highly tumorigenic Review tumorigenicity testing: 1. How tumorigenicity is evaluated 2. How highly tumorigenic cells can be identified 3. Uses of expanded models of tumorigenicity testing and their contributions to cell substrate evaluation Review mechanisms of neoplastic development and their implications for neoplastic cell-substrate evaluation

Definitions, Explanations, Implications Tumorigenicity The process by which neoplastic cells growing in tissue culture form tumors when inoculated into animal Tumorigenicity vs. Oncogenicity During tumorigenicity, the inoculated cells grow into tumors During oncogenicity, oncogenic agents transform the cells of the injected species into neoplastic cells that grow into tumors

Regulatory Concerns Associated with the Tumorigenicity of Cell Substrates Induction of tumor allografts Example: Reports of humans being engrafted with cells from human tumors Transfer of known or unknown viruses Examples: Unrecognized agent (LCMV) in cells from a human breast carcinoma; Variety of agents (herpesviruses, retroviruses, polyomaviruses, papillomaviruses) in human tumors. 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 DNA Transfer of cell components that might initiate neoplastic processes Example: Cellular oncogene H-ras, present in the DNA of neoplastic cells, induces tumors in Swiss mice

Highly Tumorigenic Cell Substrates: Regulatory Concerns General perception that the more tumorigenic (aggressive) the neoplastic cell substrate, the greater the risk of its components inducing neoplastic processes Factors that contribute to the highly tumorigenic phenotype require further explanation 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

Estimated Risk Factor Levels Cell substrate components Perceptions of the Risks Posed by Tumorigenic Neoplastic Cell Substrates Cell Substrate Type (Example) Estimated Risk Factor Levels Perceived Risk Level Advent Agents from: Cell substrate components Host Laboratory exposure DNA oncogen. DNA infect Other Adventitious agents Oncogenic Primary ++++ + ++ Diploid - Immortal non-tumorig (VERO) Immortal. weakly tumorig 1 (293) highly tumorig 2 (HeLa) +++ 1.Cell lines are usually laboratory generated. High cell doses (10e6 to 10e7 cells/mouse) needed for tumor formation in immunosuppressed host. No reports of recovery of dominant cellular oncogenes. With defective adenovirus vectors, replication competent viruses can be recovered. 2. Capacity to form tumors at 10e1 to 10e4 cells/animal. Oncogenic viruses and dominant activated cellular oncogenes can be recovered from tumor cells. Reports of adventitious agent contamination.

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

Evaluating the Tumorigenicity of Neoplastic Cells: In Vivo Models Currently Used Inoculation of athymic (nude) mice or nude rats Inoculation of newborn mice or newborn rats treated with either irradiation or anti-thymocyte globulin (ATG

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, excepting 2 experimental, highly-purified, protein vaccines, only cells that were shown to be non-tumorigenic were used in the manufacture of virus vaccines

Tumorigenicity Testing of Cell Substrates Prior to 2000 Single-Dose Assay: CBER assay: (Points To Consider in the Characterization of Cell Lines Used to Produce Biologicals 1993) Inoculum - 107 cell/animal Possible Host - 10 nude mice; 10 NB rats, NB mice, or NB hamsters immunosuppressed with antithymocyte globulin (ATG); 10 mice thymectomized, irradiated and reconstituted with bone marrow from healthy 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 Endpoint - Tumor incidence (i.e.. No. with tumors/No. surviving)

Limitations of Single-Dose Assays Single dose assays are appropriate for documenting the lack of tumor forming capacity but provide only 1 data point Single dose assays become less useful when cells possess a capacity to form tumors Single-dose, short term assays can give data that are unreliable on the ability of some neoplastic cells to form tumors

Problems with the Validity of Tumor Incidence and Tumor Latency Endpoints in Single-Dose, Short- Term Tumorigenicity Assays1 Cell Lines2 No. Expts. Tumor incidence after indicated No. of weeks (Inoculum/host - 106 cells/ adult nude mouse) 5 10 15 20 25 SV40ME1 2 0/8 8/8 SV40ME2 4/8 6/7 Data from Lewis et al. Cancer Lett 93:179, 1995. Independent lines of BALB/c mouse embryo cells transformed in tissue culture by SV40 777 inoculated into 6-8 week old nude mice 106 cells/mouse.

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 Detection of unknown agents/factors that could transfer oncogenic activity can be enhanced by expanding tumorigenicity testing methods and evaluating the data available from such assays Every practical technique needs to be used to minimize the risk of transferring infectious/oncogenic agents/factors by vaccines

Expanded Tumorigenicity Testing Enhance the Regulatory Management of Neoplastic Cell Substrates The tumorigenic phenotype of the cell substrate can be defined by evaluating the kinetics of tumor formation at doses of 107, 105, 103, and 101 cells/adult nude mouse Determining the tumor forming capacity establishes the level of tumorigenicity (aggressiveness) expressed by the tumorigenic phenotype Unrecognized oncogenic agents can be detected by identifying the species of the cells that grow into tumors across the range of tumor-forming doses and evaluating any spontaneous tumors that appear for evidence of DNA from the cell substrate Unrecognized oncogenic agents can also be detected by looking for aberrations in the kinetics by which tumors are formed by the cell substrate

Identifying Neoplastic Cell Substrates that Exhibit Highly Tumorigenic Phenotypes Determining the dose-response dynamics of tumor formation, expressed as TPD50 values (tumor-producing doses at the 50% endpoint), provides useful data on: No. of cells required for tumor development - the fewer the cells required, the more aggressive the phenotype Time of tumor appearance (tumor latency) - the more rapidly the tumors appear, the more aggressive the phenotype Capacity of the tumors that form to metastasize also contributes to assessing the aggressiveness of the phenotype

TPD50 Endpoint Explained TPD50 = Tumor producing dose at the 50% endpoint (i.e., No. of cells required for tumor formation) TPD50 values change (evolve) as the tumor incidence changes during the observation period until they reach the limit of the capacity of the cells to form tumors TPD50 values are best determined by the Spearman-Karber estimator of 50% endpoints

Tumor Formation by HeLa Cells in Nude Mice Time (weeks) Tumor incidence at dose of cells inoculated (log10) Mean TPD50 (log10) 7 6 5 4 3 2 1 8/8 0/13 6.50 13/13 4/13 5.19 7/13 4.96 1/13 4.88 10/13 4.75 8 9-12

Graph of the TPD50 Data at Weekly Intervals in Nude Mice Injected with HeLa Cells (TPD50 Evolution Curve) TPD50 (log10) Time (weeks)

TPD50 Evolution Curves Represent survival curves of average tumor latency Can be converted to survival curves Conversion to survival functions simplifies statistical analyses

Tumor Formation Dynamics of Weakly Tumorigenic and Highly Tumorigenic Cell Substrates

Comparison of TPD50 Values for Cell Lines of Human, Mouse, and Hamster Origin Human Mouse Hamster TPD50 Cell Line

Ability of the Adult Nude Mouse Model to Detect Tumorigenic Phenotypes Data from 4 different studies found that 119/134 cell lines tested, at 106 - 107 cells/mouse, had the capacity to form tumors in adult nude mice Cell lines established from carcinomas of the pancreas, breast, and gliomas as well a lymphomas and leukemias from humans have failed to form tumors in adult nude mice Newborn nude mice have been shown to detect many of those human tumors that are not tumorigenic in adults

Factors Known to Modify the Tumor-Forming Capacity of Neoplastic Cells Growing in Tissue Culture 1. Contamination of cell substrate with viruses or bacteria 2. Infection of rodent hosts used in tumorigenicity testing 3. Level of immunocompetence of rodent host (adults > newborns ≥ adult nude mice > newborn nude mice)

Impact of Viral Contamination (Cells) and Viral Infection (Host) on Neoplastic Cell Tumorigenicity in Nude Mice Cell Line 106 to 107 Cells/mouse Incidence of Progressively Growing Tumors in Nude Mice Cells/Mice Infected with:1 Mice infected with mouse hepatitis virus2 Uninfected VSV Mumps Flu/NWS Measles BHK-21 14/14 0/22 0/3 0/19 NT HeLa 5/5 0/12 0/4 SH-Me (human melanoma) 21/21 5/8 1. Reid et al. J Gen Virol 42:609, 1979 2. Akimaru et al. J Surg Oncology 17: 309, 1981

3-Stage Model of Neoplastic Development Mechanisms Involved in Neoplastic Development and Tumor Formation in Experimental Animals 3-Stage Model of Neoplastic Development 1. Initiation - first and apparently irreversible, stage of neoplastic development; initiating event can represent a single genetic change 2. Promotion - apparently reversible, stage of neoplastic development; promotional events can represent 1 or more additional genetic changes (oncogene activation/ tumor suppressor gene deactivation) 3. Progression - final genetic events (further genetic changes) resulting in tumor formation, invasion, and metastases

Multi-Step Models of Carcinogenesis Mechanisms Involved in Neoplastic Development and Tumor Formation in Humans 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)

Mechanisms of Neoplastic Development and the Regulatory Management of Tumorigenic Cell Substrates Tumor development is a multi-step process requiring 4 - 6 independent genetic alterations involving different genetic loci Every “neoplastic 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 animal models to detect oncogenic activity is low Initiating events can represent single genetic processes, do not appear to be 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

OVRR Recommendations for Expanded Tumorigenicity Testing of Tumorigenic Neoplastic Cell Substrates Evaluate and analyze for aberrations the dynamics of tumor formation by determining the tumor incidence at doses of 107, 105, 103, and 101 cells in adult nude mice Record incidence of visible/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/organs, Evaluate any spontaneous tumors that develop for evidence of DNA from the cell substrate

Data of Regulatory Significance Provided by Expanded Tumorigenicity Testing of Neoplastic Cell Substrates Data on tumor formation kinetics reveals weakly/highly tumorigenic phenotypes, which influences the level of concern over adventitious agent contamination and oncogenic/infectivity activity of cell-substrate DNA Data on aberrations in tumor formation, especially at high cell doses, may be indicative of cell-substrate contamination with known/unknown agents Data on the species of origin of the cells that form tumors at the injection site or at distant sites (possibly spontaneous tumors) determine whether oncogenic activity can be transferred from the neoplastic cell substrate to the host Histopathology on injection sites, tumors, and organs establishes/confirms the identity and possible aggressiveness of the neoplastic cell substrate

Summary Cell substrate tumorigenicity testing can provide data on: 1. Tumorigenic phenotype - whether the substrate cells are weakly or highly tumorigenic 2. Transfer of oncogenic viruses 3. Presence of adventitious agents Tumorigenicity testing in adult nude mice can detect tumor-forming capacity in 9/10 cell lines tested (newborn nude mice may provide a more sensitive alternative) Tumor formation represents the end-stage of the multi-step process (initiation, promotion, progression) of neoplastic development With exception of neoplastic initiation events, which can not be evaluated, the multi-step process of neoplastic development makes it unlikely that neoplastic activity can be transferred by cell components other than oncogenic viruses