1. Susceptibility Markers

3. Susceptibility Markers
Susceptibility markers represent a group of biological markers, which may make an individual susceptible to cancer.
These markers may be genetically inherited or determined or acquired.
They are independent of environmental exposures.

4. Biomarker of Genetic Susceptibility
High risk genes
Low risk genes

5. Genetic Susceptibility to Cancer
Mutations with strong influence on risk
Variations with weak functional effect
Rare in the population (<1%)
Low to high frequency in the population (1-50%)
Results in familial clustering
Limited familial clustering
Can be studied in families
Can be studied in populations
e.g. BRCA germline mutations
010205

6. Frequency Distribution of Breast Cancer
Hereditary
Familial
10%
20%
70%
Unexplained by Family History
or Inherited Predisposition

7. BRCA1 and BRCA2 Mutations in the Ashkenazi Jewish Population
An estimated 1 in 40 Ashkenazi Jews carries a BRCA1 or BRCA2 mutation
BRCA1
185delAG
Prevalence = ~1%
5382insC
Prevalence = ~0.15%
Slide 3: Genes Are the Units of Inheritance
People inherit traits from their parents through genes. It is believed that humans have about 30,000 different genes. Genes are organized into 23 different chromosomes and are stored in the nucleus of each cell. Thus, chromosome contain approximately 1000 to 2000 genes each.
Genes consist of long segments of a special molecule called DNA that is designed to store important biological information. Every cell in the body contains approximately 3 feet of DNA! Information stored in genes is retained whenever cells divide, so that all of the cells in the body contain the same genes. Not all cells use the information in every gene, though. In fact, differences in the ways that cells use genetic information, referred to as “gene expression,” account for differences between cells. Thus, the cells in the breast use a different set of genes than the cells that comprise the bone, heart or other tissues.
Each gene contains the instructions for making a protein, which does the work of the cell. Abnormalities in the genes, called “mutations,” result in changes in the protein. Some mutations completely prevent proteins from working correctly.
Core slide for a community presentation
BRCA2
6174delT
Prevalence = ~1.5% 2 2

8. Hereditary Breast and Ovarian Cancer
Other genes (16%)
BRCA1 (52%)
BRCA2 (32%)
7-10%
Hereditary
Slide 5: Hereditary Breast and Ovarian Cancer
All cancer is genetic, because it results from mutations in genes that normally control cell division. Most such mutations are acquired during a person's lifetime, but in a minority of people mutations in critical genes are inherited.
Approximately 7% of breast cancer and 10% of ovarian cancer results from such genetic mutations passed down from either the father or mother1. The majority (approximately 84%) of hereditary breast cancer results from inherited mutations in two genes called BRCA1 and BRCA22. Although sometimes referred to as the “breast cancer genes,” BRCA1 and BRCA2 are also associated with the majority of hereditary cancers of the ovary.
Although the risk of breast or ovarian cancer may sometimes be increased in other hereditary cancer syndromes, there do not appear to be other genes (such as a so-called "BRCA3") that are responsible for a significant proportion of hereditary breast and ovarian cancer. Recent studies have in fact indicated that “if there are additional genes, they are of minor importance, compared with BRCA1 and BRCA2, in families with breast and ovarian cancer3."
It is estimated that millions of people, worldwide, carry mutations in BRCA1 or BRCA2.
References:
1. Claus EB, Schildkraut JM, Thompson WD, Risch NJ: The genetic attributable risk of breast and ovarian cancer. Cancer 1996;77:
2. Ford D, Easton DF, Stratton M, et al: Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. American Journal of Human Genetics 1998;62:
3. Gayther SA, Russell P, Harrington P et al: The contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer: No evidence for other ovarian cancer-susceptibility genes. American Journal of Human Genetics 1999;65:
Core slide for a health care professional presentation
Core slide for a community presentation
Predisposing factor in 15-45%
of hereditary breast cancer
Sporadic
Am J Hum Genet 1998; 62:676-89

9. BRCA1-2 Mutations Increase the Risk of Early-Onset Breast Cancer
By age 40
By age 50
By age 70
Slide 10: BRCA1-2 Mutations Increase the Risk of Early-Onset Breast Cancer
Without intervention, the majority of women with inherited mutations in BRCA1 and BRCA2 will develop breast and/or ovarian cancer. The range of risks of breast and ovarian cancer associated with mutations in these genes has been characterized through numerous studies. The lower estimates of risk are derived from analysis of mutations in an unselected general population of individuals, whereas higher estimates of mutation-associated cancer risk are believed to be more appropriate for individuals with a strong family history of cancer.
Generally, mutations in BRCA1 and BRCA2 are associated with a 56% to 87% risk of breast cancer by age 701,2. Most importantly, hereditary breast cancer occurs at a far earlier age than the nonhereditary (sporadic) form. Women in the general population have only a 2% chance of developing breast cancer before age 50. As shown in the middle panel, however, a woman with a mutation in BRCA1 or BRCA2 has a 33% to 50% likelihood of developing breast cancer before reaching 50 years of age2,3.
References:
1. Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE, Breast Cancer Linkage Consortium: Risks of cancer in BRCA1-mutation carriers. Lancet 1994;343:
2. Struewing JP, Hartge P, Wacholder S, et al: The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. New England Journal of Medicine 1997;336:
3. Easton DF, Ford D, Bishop DT, Breast Cancer Linkage Consortium: Breast and ovarian cancer incidence in BRCA1-mutation carriers. American Journal of Human Genetics 1995;56:
Core slide for a health care professional presentation
Core slide for a community presentation
Population Risk 9% 2%
0.5%
Hereditary Risk
10%-20%
33%-50%
45%-87%

10. BRCA1-2 Mutations Increase the Risk of Ovarian and Related Cancer
By age 70
Slide 11: BRCA1-2 Mutations Increase the Risk of Ovarian Cancer
Women with mutations in BRCA1 and BRCA2 have a greatly elevated risk of ovarian cancer as well as breast cancer. In fact, the risk of ovarian cancer in a woman with a mutation in BRCA1 and BRCA2 is far greater than the lifetime risk of lung cancer in an individual who smokes four packs of cigarettes every day for 30 years.
The risk of ovarian cancer due to inherited BRCA1 mutations is 28%1 to 44%2,3 by age 70, compared to the general population risk of 1%. Mutations in BRCA2 confer an increased risk of ovarian cancer of approximately 27% by age 704, which represents a 15-fold increase compared to the general population. Although it was originally proposed that the risk of ovarian cancer was higher for mutations in certain regions of the BRCA1 and BRCA2 genes, other studies have not confirmed a relationship between the location of a mutation and the likelihood of developing ovarian cancer5.
The age of onset of hereditary ovarian cancer is typically later than that of hereditary breast cancer. In particular, most ovarian cancers associated with mutations in BRCA2 occur after age 504.
References:
1. Whittemore AS, Gong G, Itnyre J: Prevalence and contribution of BRCA1 mutations in breast cancer and ovarian cancer: results from three U.S. population-based case-control studies of ovarian cancer. American Journal of Human Genetics 1997;60:
2. Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE, Breast Cancer Linkage Consortium: Risks of cancer in BRCA1-mutation carriers. Lancet 1994;343:
3. Easton DF, Ford D, Bishop DT, Breast Cancer Linkage Consortium: Breast and ovarian cancer incidence in BRCA1-mutation carriers. American Journal of Human Genetics 1995;56:
4. Ford D, Easton DF, Stratton M, et al: Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. American Journal of Human Genetics 1998;62:
5. Frank TS, Manley SA, Olopade OI, et al: Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk. Journal of Clinical Oncology 1998;16:
Core slide for a health care professional presentation
Core slide for a community presentation
1 - 2%
Population risk
% (BRCA1)
% (BRCA2)
Hereditary risk

11. G1 S G2 M
P53
Cyclin D1
P16
Environmental Carcinogens / Procarcinogens Exposures
PAHs, Xenobiotics, Arene, Alkine, etc
Active carcinogens
Detoxified carcinogens
DNA Damage
Normal cell
Carcinogenesis
Programmed cell death
Tobacco consumption
Occupational Exposures
Environmental Exposure
CYP1A1
GSTP1
mEH
NQO1
XRCC1
GSTM1
2-1. Background: Theoretical model of gene-gene/environmental interaction pathway
Ile105Val 
Ala114Val
Tyr113His
His139Arg
Pro187Ser
MspI
Ile462Val 
Arg194Trp, Arg399Gln, Arg280His
Null 
Ala146Thr
Arg72Pro
G870A
DNA damage repaired
Defected DNA repair gene
If DNA damage not repaired G0
If loose cell cycle control

12. Susceptibility Markers: Metabolic Genes
Tumor susceptibility markers such as P450s, GSTs, and NATs, act in enzymatic pathways related to metabolizing and eliminating carcinogens.

13. Phase I Enzymes
The phase I enzymes such as p450 enzyme superfamily metabolize exogenous or endogenous agents or carcinogens to intermediates, which can result in DNA damages and act as risk factors for cancer.

14. Phase I Genes
The phase I genes encode detoxifying enzymes that recognize a large variety of substrates.
Many drugs, poisons, and other exogenous chemicals, as well as a number of natural endogenous compounds are metabolized by these enzymes.

15. Phase II Genes The major function is to detoxify carcinogens
Including GSTs, NATs

17. PCR P450 2E1 after Using Pst1 RFLP
PCR P450 2E1 after Using Pst1 RFLP

18. Figure. GSTP1 polymorphism
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8
ile/val ile/val ile/ile val/val ile/val ile/ile ile/val ile/ile
Figure. GSTP1 polymorphism

19. Figure. P53 polymorphism at codon 72 from buccal cell DNA.
Case 1 Case 2 Case 3 Case Case 5
Arg/Arg Arg/Arg Pro/Pro Arg/Arg Arg/Pro
Figure. P53 polymorphism at codon 72 from buccal cell DNA.

20. DNA Repair Genes

21. Polymorphism of DNA repair genes
DNA repair system maintains the intergrity of the genome by:
Reducing the mutation frequency of cancer-related genes,
minimizing replication errors,
removing DNA damage
Minimizing deleterious rearrangement arising via aberrant recombination
Four pathways for repairing DNA damage
Base excision repair (BER)
Excision of a damaged region  fill-in repair synthesis using opposite strand as template
Nucleotide excisions repair (NER)
Remove photoproducts from UV radiation and bulky adducts
Recombination repair
When both strands are damaged  acts on double-strand breaks and inter-strand links
Mismatch repair
On base mismatches that arise during replication by misincoporation or slippage on the template strand

22. Genetic Variation in Repair Genes
DNA Repair
Genetic Variation in Repair Genes ?
DNA Repair Capacity
ORs
Cancer Occurrence

23. Base Excision Repair
hOGG1
LIG1

24. hOGG1 Often deleted in a variety of cancers
Somatically mutated in some cancer cells
Ser326Cys, exon 7: highly polymorphic
0.2 Caucasian— Chinese
Ser326 protein 7-fold greater repair activity
ORs: Lung , Orolaryngeal

25. LIG1 Encodes DNA ligase I, a DNA joining enzyme
Participates in BER, NER, MMR, and HRR
Exon 6, codon 170 polymorphism
0.5 allele frequency in Caucasians
No studies in esophageal, stomach, liver cancers
No studies in Chinese

26. Nucleotide Excision Repair
Removes bulky adducts caused by environmental agents
UV radiation
Chemical carcinogens

27. XPD/ERCC2
ERCC1
LIG1

28. XPD/ERCC2 Involved in DNA unwinding during NER
Helps repair genetic damage induced by tobacco and other carcinogens
Exon 10, codon 312
Exon 23, codon 751
Most studies of both polymorphisms have been done in lung cancer (most ORs )

29. Homologous Recombinational Repair
One mechanism for repairing double-strand breaks in DNA
Accurately replaces sequence information by physically exchanging a segment from an homologous intact DNA molecule

30. XRCC3
LIG1

31. Adjusted Odds Ratios of Selected DNA Repair Genes for Stomach Cancer
Adjusted ORs
95% CIs
XRCC1-399 (AA GG vs. GA)
1.38
XRCC3 (TT TM vs. MM)
0.57
XPD10 (GG GA vs. AA)
0.77
XPD23 (LL LG vs. GG)
0.82
HOGG1 (SS SC vs. CC
1.20
LIG1 (AA AC vs. CC)
0.71

34. Interactions between smoking and GST M1 (odds ratios
Interactions between smoking and GST M1 (odds ratios* and 95% confidence intervals)
5.29
(1.81, 15.4)
2.79
(0.97, 7.99)
1.13
(0.32, 3.95)
1.00
*Adjusted for age, sex, race, and level of education
More than multiplicative interaction

35. Polymorphism of DNA repair genes
DNA repair system maintains the intergrity of the genome by:
Reducing the mutation frequency of cancer-related genes,
minimizing replication errors,
removing DNA damage
Minimizing deleterious rearrangement arising via aberrant recombination
Four pathways for repairing DNA damage
Base excision repair (BER)
Excision of a damaged region  fill-in repair synthesis using opposite strand as template
Nucleotide excisions repair (NER)
Remove photoproducts from UV radiation and bulky adducts
Recombination repair
When both strands are damaged  acts on double-strand breaks and inter-strand links
Mismatch repair
On base mismatches that arise during replication by misincoporation or slippage on the template strand

36. Lung Cancer Study

37. Associations between Cigarette Smoking, Selected Susceptibility Genes and Lung Cancer
Adjusted ORs
95% CIs
Smoking (Yes/No)
3.70
P53 (P/P vs. A/A or A/P)
1.00
GSTP1 (Val/Val +Val/Ile vs. Ile/Ile)
1.01
Adjusted for age(continuous),sex and race(white/non-white)

38. P53 Codon 72 Polymorphism, Smoking, and Lung Cancer
Smoke
P53
Cases
Controls
OR(95%CI)
adjusted
Never
Arg/Arg 28
125
1.0
Arg/Pro
Pro/Pro 38
139
1.13 ( )
Yes
133
132
4.32 ( )
P/P
170
4.03 ( )

39. GSTP1 Polymorphism, Smoking and Lung Cancer
Smoke
GSTP1
Cases
Controls
OR(95%CI)
adjusted
Never
Ile/Ile 31
111
1.0
Any Val 28
140
0.69 ( )
Yes
125
135
3.16 ( )
164
152
3.64 ( )
OR int =1.66 ( )

40. PCR primers and enzymes
Genes
Primers 5’ to 3’
Enzyme
ALDH2
Sense
CAA ATT ACA GGG TCA AGG GCT
Mbo II
Anti-sense
CCA CAC TCA CAG TTT TCT CTT
ADH2
GAAGGGGGGTCACCAGGTTG
Mae III
AATCTTTTCTGAATCTGAACAG
ADH3
AAT AAT TAT TTT TCA GGC TTT AAG AGT AAA TAT TCT GT
Ssa I
AAT CTA CCT CTT TCC AGA GC
CYP 2E1
TTCATTCTGTCTTCTAACTGG
Rsa I
CCAGTCGAGTC GAGTCTACATTG TCA
NQO1
TAT CAG AGT GTC TTA CTG AGA
AAT GCT ATA TGT CAG TTG AGG
GTG GCT TCC AAG TCT TAG AAT
TTT CTA GCT TTG ATC TGG TTG
XRCC1 399
CCC CAA GTA CAG CCA GGT C
Msp I
TGT CCC GCT CCT CTC AGT AG
XRCC1 194
GTT CCG TGT GAA GGA GGA GGA
Pvu II
CGA GTC TAG GTC TCA ACC CTA CTC ACT
P53 Intron 3
TGGGACTGACTTTCTGCTCTT
TCAAATCATCCATTGCTTGG
P53 Intron 6
TGGCCATCTACAAGCAGTCA
TTGCACATCTCATGGGGTTA

41. Stomach Cancer Study

43. Adjusted Odds Ratios of Selected Metabolic and Cell-Cycle Genes for Stomach Cancer
Adjusted ORs
95% CIs
GSTM1 (null vs. non-null)
1.15
GSTT1 (null vs. non-null)
0.93
GSTP1 (Val/Val +Val/Ile vs. Ile/Ile)
1.01
ALDH2 (AG/AA vs. GG)
1.00
CYP2E1 (c1c1 & c1c2 vs. c2c2)
0.82
NQO1 (CC/CT vs. TT)
1.5
MTHFR (CC/CT vs. TT)
2.0
p53 (AA/AP vs. PP)
1.2

44. Polymorphism of Susceptibility Genes: Methylenetetrahydrofolate Reductase (MTHFR)
MTHFR gene deals with folate metabolism.
Folate metabolism may play an important role in carcinogenesis through its involvement in both DNA methylation and nucleotide synthesis
A common Ala(222)/Val variant in the methylenetetrahydrofolate reductase (MTHFR) gene leads to a disturbed folate metabolism and is associated with decreased genomic DNA methylation.
Heijmans et al. Cancer Res Mar 15;63(6):

45. Effects of MTHFR and Green Tea Drinking on the Risk of Stomach Cancer
2.94
( )
1.48
( )
2.27
( )
MTHFR CC CT/TT CC CT/TT
Green tea Drinker Drinker Non-Drinker Non-Drinker
OR for interaction: 0.88 ( )

46. 2-3 Results. Gene-Gene interactions
APEX is involved in the restoration phase of BER: it removes the abasic site after DNA cleavage by OGG1
Small number
Biologically plausible

47. 2-3. Results: Multigenetic analyses by pathway
Metabolic genes trend p=0.50
DNA repair trend p=0.83

48. Genes with suggestive effects trend p=0.02
2-3. Results: Multigenetic analyses for all genes, and genes with suggestive evidence of effect
All genes trend p=0.01
Genes with suggestive effects trend p=0.02
(Genes with suggestive effects: NQO1 Pro187Ser, XRCC1 Arg194Trp, OGG1 Ser326Cys, CCND1 G870A)
Suggests that genetic susceptibility depends on multiple loci
Considering variants in different pathways might give a more complete picture of carcinogenesis

49. 2-3 Results: multigenetic factors by smoking status and packyear ( all genes)** ** *

50. 2-3 Results: multigenetic factors by smoking status and packyear ( genes with suggestive effects)** * ** ** **

51. 2-3. Results: Interaction with ETS among never smokers