# Presentation: 764991

## Star Grant CR-83075701:

-  Children’ Vulnerability to Toxic Substances in the Environment

*Analysis of Genotoxic Biomarkers in Children Associated with a Pediatric Cancer Cluster and Exposure to Two Superfund Sites*
- PI: Barry Finette M.D., Ph.D.
- Co PI’s : J. P. O’Neill and P. Vacek
- Departments of Pediatrics, Microbiology and Molecular Genetics and Medical Biostatistics
- University of Vermont, Burlington VT

- Region 5 STAR Biomarkers Meeting
- July 14, 2004
- Chicago IL

- Slide 1

## RESEARCH PROPOSAL

- Objective
- To Evaluate the utility of specific biomarkers for assessing cancer risk in a pediatric population following environmental exposure to genotoxic chemicals
- 	- Measure biomarkers of effect in children from an exposed population 	with a documented increase in childhood cancer incidence and compare 	them to measurements in unexposed children.

- Slide 2

## RESEARCH PROPOSAL

- 1.  To utilize the *HPRT* biomarker system to investigate the genetic effects of exposure by determining the frequency and mutational spectra in a high risk pediatric cancer population from Tom’s River N.J. who were exposed to contaminated drinking water from two Superfund sites.
- 2.  To compare the frequency of chromosomal aberrations (CA) as a biomarker of effect from exposed and unexposed children to investigate prevalence of genomic damage
- 3.  To determine genotype frequencies for 18 polymorphisms of 11 metabolic genes as biomarkers of susceptibility in exposed children with cancer, their siblings and unexposed children.

- Slide 3

## Current Status of Biomarkers for Assessing Cancer Risk

- -  Somatic mutations are promising biomarkers for cancer risk because they
-    can capture genetic events that are associated with malignant
-    transformation.  To date there are no studies to support a relationship
-    between frequency or specta of somatic mutations and cancer risk.

- -  The association between chromosomal aberrations and neoplastic
-    transformation has been well established.  Two major cohort studies
-    performed in the Nordic countries and Italy have demonstrated that the
-    frequency of CA’s in peripheral MNCs is predictive of cancer risk.

- -  Carcinogenic chemicals can be directly mutagenic or require metabolic activation
-    to gain genotoxic potential.  Metabolic enzymes also exist that detoxify
-    carcinogens *in vivo.  *There is growing evidence that specific polymorphisms in
-    these genes are associated with cancer risk.

- Slide 4

## Genetic Effects of Genotoxic Exposure in Humans

- Slide 5

## Exposure
Food Consumption
Children consume 7 times as much water per pound than an adult during their first 6 months of life.
Children  eat 3 -4 times more food per pound as an adult during there first 5 years of life.

Behavior
Unable to remove themselves from noxious environments
Oral exploration

- Food Consumption
- Children consume 7 times as much water per pound than an adult during their first 6 months of life.
- Children  eat 3 -4 times more food per pound as an adult during there first 5 years of life.
- Behavior
- Unable to remove themselves from noxious environments
- Oral exploration

- Susceptibility of Environmental Exposure in Children

- Slide 6

## Exposure
Physical Location
Developmentally dependent

Breathing Zones
Lower breathing zones that accumulate higher concentrations of heavier chemicals.

Oxygen Consumption
Higher surface to volume ratio resulting in an O2 consumption 2 fold higher than adults.

- Physical Location
- Developmentally dependent
- Breathing Zones
- Lower breathing zones that accumulate higher concentrations of heavier chemicals.
- Oxygen Consumption
- Higher surface to volume ratio resulting in an O2 consumption 2 fold higher than adults.

- Susceptibility of Environmental Exposure in Children

- Slide 7

## Slide 8

## Age Specific Incidence of Cancer

- Slide 8

## Biomarker Monitoring and Assessment

- Slide 9

## Research Program

- Slide 10

**Notes:**
***Over the past 9 years my research program has focused on investigating somatic mutational events in humans from the late stages of fetal development through adolescence using a surrogate biomarker system.
    ***  This approach has allowed us to gain insight into....... 
    -  spontaneous mutational events associated with endogenous mutagens, as well as ....
    -  environmental mutational events associated with transplacental exposure to tobacco smoke, and....
    -  iatrogenic mutational events associated with antineoplastic therapy for the treatment of pediatric malignancies.

***Our goal is to investigate these gene-environment interactions during human development to gain insight into the.... 

    ***pathogenesis, diagnosis and treatment of multifactorial human diseases, in particular cancer.

4 ANIMATIONS

## Slide 12

## The HPRT Biomarker System

-  *HPRT* gene is located on the X-chromosome at Xq26
- 	– Therefore this locus is functionally heterozygous in which a single mutation can result in
-         mutant phenotype.
- 	– Mutations in this gene can be used as a biomarker of effect, reflective of genome wide
-         mutational events.
-  Selection of *HPRT* mutant clones in peripheral T-cells
- 	– Lymphocytes that have a mutation resulting in altered *HPRT* activity will grow in the
-        presence of the purine analog 6-thioguanine.
- 	– This allows for the selection of rare mutational events.

- The *HPRT* Biomarker System

- Slide 13

## The HPRT Biomarker System

- 	– This allows for an accurate determination of somatic mutant frequency.
-  Molecular Analysis
- 	– Mutant isolates can be expanded in vitro.
- 	– *HPRT* mutant clones can be characterized at the molecular level to gain insight into
-        mutational mechanisms and mutational spectra.
-  Determination of T cell clonality
- 	– *HPRT* mutants can be specifically identified by their T cell receptor (TCR) CDR3 region.
- 	– Allows for the identification of in vivo sequential mutational events
-       in independent single mutant isolates or those associated with a
-       proliferating clonal population.

- The *HPRT* Biomarker System

- Slide 14

## Longitudinal Genotoxic Effects of Treatment for ALL

- Rice et al. 2004. Cancer Research

- Slide 15

## Distribution of Aberrant V(D)J Recombinase mediated HPRT Deletions in Newborns Following Transplacental Exposure to Environmental Tobacco Smoke

- χ

- 2

- = 5.11     p = 0.023

- Unexposed

- Exposed

- Finette et al, 1998. Nature Medicine

- Slide 16

## Frequency of in vitro V(D)J recombinase events following exposure to alkylating agents (EMS and MMS) and radiation

- Slide 17

**Notes:**
The alkylating agents EMS and MMS yield a dose as well as time dependent increase in V(D)J mediated recombination of the plasmid substrate.
Cytotoxicity analysis was performed in the Reh cell line to control for the effective dose used in the experiments

## Dover Township resides in Ocean County N.J. off the Atlantic seacoast and was established in 1767.  Within Dover township resides Toms River Village

- Slide 18

## Background for Tom’s River N.J. Pediatric Cancer Cluster

- Children residing in Dover Township, New Jersey between 1979-1995 are an ideal population for evaluating biomarkers of the health effects of exposure because their elevated risk of childhood cancer is well-documented and has been linked to their exposure to industrially contaminated groundwater.

- Extensive  epidemiological studies by the New Jersey Department of Health and Senior Services and the U.S. Agency for Toxic Substances and Disease Registry found that the incidence of childhood cancer in Dover Township during 1979-1995 was 34% higher than the statewide rate.

- Slide 19

## Background for Tom’s River N.J. Pediatric Cancer Cluster

- Chemical contaminants came from two industrial sources that have been designated Superfund Sites by the U.S. Environmental Protection Agency, and included aniline- and benzidine-based dyes, epichlorohydrin, trichloropropane substituted anthraquinones and styrene-acrylonitrile trimer, as well as 150 additional chemicals including a particles radionucleotides decay contaminants such as 226Ra, 224Ra, 222Rn and 220Rn

- In the Toms River, a section of Dover Township defined by four census tracks, the incidence was 70% higher than the statewide rate.  In particular female children with ALL under 5 years of age, as well as brain and other CNS tumors.  A case-control study indicated that the cancers were associated with exposure to contaminated drinking water, either *in utero* or during early childhood  and extensive toxicologic and hydro-geologic studies demonstrated that chemicals and radionucleotides have contaminated public groundwater in some areas of Dover Township for the past 50 years.

- Slide 20

## Specific Aim 1

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Gender |  |  |  |  |  |  |  |  |  |  |
| Male | 30 |  | 61.2% |  |  | 23 | 53.5% |  |  | 0.454 |
| Female | 19 |  | 38.8% |  |  | 20 | 46.5% |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |
| Age (yrs) |  | 17.5 |  | 7.9 |  | 17.4 | 8.2 |  |  | 0.956 |
| CE |  | 0.55 |  | 0.17 |  | 0.45 | 0.15 |  |  | 0.005 |
| Mf x 10-6 |  | 3.9 |  | 2.85 |  | 5.06 | 4.54 |  |  |  |
| lnMf x 10-6 |  | 1.12 |  | 0.7 |  | 1.35 | 0.73 |  |  | 0.135 |
| Residual lnMf |  | -0.15 |  | 0.47 |  | -0.07 | 0.6 |  |  | 428 |

- Exposed

- Controls

- n

- %

- n

- %

- p-value

- Mean

- S.D.

- Mean

- S.D.

- Analysis of the frequency of *HPRT* mutations (Mf) of unexposed children as compared to children exposed to contaminated groundwater from two superfund sites in Toms River N.J.

- Slide 21

## Comparative analysis of HPRT Mf in  Exposed and Control Subjects

- Exposed
- Unexposed

- The relationship between mutant frequency and age in expose siblings of children with cancer who are from a population with an elevated incidence of childhood cancer (●) and unexposed children from neighboring communities with no increase in cancer incidence (○).  lnMf values have been adjusted to the average unselected CE of 50% for all study subjects: adjusted lnMf  = lnMf + 1.676(CE – 0.50).

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | -0.992 | 0.542 |  | -1.003 | 0.645 |  | -0.956 | 0.411 |  | -0.074 | 0.103 |
|  | -1.518 | 0.0389 |  | -0.903 | 0.634 |  | -1.388 | 0.389 |  | -1.486 | 0.209 |
|  | 1.040 | 0.166 |  | 0.977 | -0.214 |  | 1.108 | 0.132 |  |  |  |
|  |  |  |  |  |  |  |  |  |  | 0.765 | 0.023 |

- Exposed

- Controls

- Both
- Groups

- Vermont
- Data

- B

- S.E.

- B

- S.E.

- B

- S.E.

- B

- S.E.

- Intercept

- CE

- ln(Age+1)

- Comparative analysis of *HPRT *Mf in  Exposed and Control Subjects

- Linear Regression Analysis

- Slide 22

## Specific Aim 2

- -  We have a repository of 661 *HPRT* mutant clones from 49 exposed children
-    and 647 *HPRT* mutant clones from 43 unexposed children.

- -  Our double blind approach is to initially perform mutational analysis (RT/
-    PCR, genomic PCR and DNA sequencing) on a total of 460 mutant clones (5
-    mutant isolates from each subject).  This is being done in groups of 10
-    subjects (5 exposed and 5 controls).

- -  To date, we have completed the mutational analysis over 150 mutants from
-    30 subjects.

- To investigate the *HPRT* mutation spectrum of exposed and unexposed groups to determine if genetic differences exist as a biomarker of effect following genotoxic exposure

- Slide 23

## Specific Aim 3

- To perform a comparative analysis between the frequency of chromosomal aberrations in peripheral MNC’s from exposed children and unexposed controls to investigate the prevalence of widespread genomic damage and its association between genotoxic exposure and cancer risk

- -  We have a successful approach for making high quality metaphase spreads with 500 - 700 metaphases per slide.

- -  We are completing our testing of a 2 dye, 6 chromosome for determining the frequency of chromosomal aberrations

- -  To date, we have made metaphase spreads from 60 of the 92 subjects that will be included in this double blind analysis.

- Slide 24

## Specific Aim 4

- To determine genotype frequencies for 18 metabolic polymorphisms of 11 genes in the exposed children with cancer, their siblings and unexposed controls to determine correlations of genotype, *HPRT* Mf  and mutational spectra, and CA’s

- -  The first part of this study is the recruitment of peripheral blood samples for MNC analysis from the affected siblings of those exposed subjects previously enrolled for specific aims 1-3.  We have recruited 15 of the 40 blood samples for this study.

- Other

- We have also developed an new questionnaire for obtaining information about all residences and schools attended by the study participants, to enable us to determine potential exposure to contaminated drinking water, as well questions regarding other potential exposures.  This information will be used in conjunction with the reconstruction models developed by ATSDR for water-distribution system serving Dover Township.

- Slide 25

## Summary

- Children residing in Dover Township, NJ between 1979-1995 are an ideal population for evaluating biomarkers of effect because their elevated risk of childhood cancer has been well documented and linked to their exposure to contaminated ground water from two EPA designated superfund sites.

- Information on the relationships between exposure, genetic effects and susceptibility obtained from a study of biomarkers would provide valuable biologic insight for interpretation of the epidemiologic findings and for studying the utility of biomarkers for assessing pediatric cancer risk.

- Slide 26