# Presentation: 263065

## 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
- 	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

## 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.

## 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.

## 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

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

## 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.

## 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

## 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)

## 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.

## 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

## 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

## Problems with the Validity of Tumor Incidence and Tumor Latency Endpoints in Single-Dose, Short- Term Tumorigenicity Assays1  

| Cell Lines2 | No. Exps. | 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 | 8/8 | 8/8 | 8/8 |
| SV40 ME2 | 2 | 0/8 | 0/8 | 4/8 | 6/7 | 6/7 |

- 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.

## 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

## 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

## 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)

## 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

## 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 it reaches the endpoint of the capacity of the cells to form tumors
- TPD50 values are best determined by the Spearman-Karber estimator of 50% endpoints
- 	(Miller, Biometrica 60: 535, 1973)
- 	(Example presented in next slide)

## Tumor Formation by HeLa Cells in Nude Mice

| Time
(weeks) | Tumor incidence at dose of cells inoculated (log10) |  |  |  |  |  | TPD50
(log10)1 |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | 72 | 63 | 5 | 4 | 4 | 4 |  |
| 1 | 8/8 | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 6.50 |
| 2 | 8/8 | 13/13 | 4/13 | 0/13 | 0/13 | 0/13 | 5.19 |
| 3 | 8/8 | 13/13 | 7/13 | 0/13 | 0/13 | 0/13 | 4.96 |
| 4 | 8/8 | 13/13 | 7/13 | 1/13 | 0/13 | 0/13 | 4.88 |
| 5 | 8/8 | 13/13 | 7/13 | 1/13 | 0/13 | 0/13 | 4.88 |
| 6 | 8/8 | 13/13 | 7/13 | 1/13 | 0/13 | 0/13 | 4.88 |
| 7 | 8/8 | 13/13 | 10/13 | 1/13 | 0/13 | 0/13 | 4.75 |
| 8 | 8/8 | 13/13 | 10/13 | 1/13 | 0/13 | 0/13 | 4.75 |
| 9-12 | 8/8 | 13/13 | 10/13 | 1/13 | 0/13 | 0/13 | 4.75 |

- 1. Estimated by Spearman-Karber
- 2. Data from 2 independent assays.
- 3. Data from 3 independent assays.

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

**TPD****50**** (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

**TPD****50**** (log10)**

**Time (weeks)**

## Comparison of TPD50 Values for Cell Lines

- TPD**50**

- Cell Line

**Comparison of TPD****50**** Values for Cell Lines**
**of Human, Mouse, and Hamster Origin**

- Human

- Mouse

- Hamster

## 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 315 19771  |
| Cell lines from human tumors | 3/3 | 5/82 | 96/96 | 5/7 (7/7)3 |
| Cell lines from rodent tumors | 2/2 | NT | NT | NT |
| Cell lines from spontaneously transformed normal tissue | 1/54 | NT | NT | NT |
| Cell lines from normal tissue transformed in culture by viruses | 7/13 | NT | NT | NT |
| 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 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.

## Newborn Nude Mice are More Sensitive than Adult Nude Mice in Detecting Tumor Formation by High Passage (252) ATCC 10-87 VERO cells

## 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)

## Biological and Molecular Mechanisms Involved in Neoplastic Development and Tumor Formation (1)

**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)

## 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

## 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 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

## 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

## 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