Translational
Molecular Genetics
Harold Frucht, M.D.
Division of Digestive and Liver Diseases
Columbia University
April 14, 2005
Cases with Familial
Risk (~25%)
Hereditary NonPolyposis Colorectal
Cancer (3-5%)
Familial
Adenomatous
Polyposis (<1%)
Hamartomatous
Polyposis Syndromes
(<1%)
Sporadic Cases
(~70%)
Disease-Associated Mutations Alter
Protein Function
Gene Mutations
Somatic Mutation - Sporadic Cancer
Germ Line Mutation - Inherited Syndrome
Mechanisms of Cancer Gene Action
M (mitosis)
G1
(cell growth)
Oncogenes:
promote cell growth
G2
S (synthesis)
G0 (resting)
Mismatch repair
genes: correct
replication errors
Modifier genes:
influence cell function
Suppressor genes:
inhibit cell cycle; promote apoptosis
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Somatic Mutation (Sporadic Disease)
2 normal copies
of the gene
in every cell
One copy
mutated
in cell
(1st hit is acquired)
Second copy
mutated
in cell
(2nd hit is also
acquired)
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Germline Mutation (Inherited Disease)
One copy
mutated
in every cell
(1st hit is inherited)
Second copy
mutated
in cell
(2nd hit is acquired)
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Chung, DC. Gastroenterology 2000; 119: 854-865
CIMP = CpG Island Methylator Phenotype
• epigenetic phenomenom
• hypermethylation of the promoter region of
the hMLH1 gene
• responsible for MSI in 15% of sporadic
colon cancers
Polymorphism
APC Gene
I1307K Mutation
Germline Mutation of Codon 1307
T-A Transversion
(Leucine - Isoleucine Substitution)
Causes hypermutability in adjacent sequences resulting
in somatic alterations which predispose to colon cancer
Incidence in Ashkenzai Jews
6.1 %
Lifetime risk of colon cancer in people with mutation 18-30 %
Non-Jewish
population
All Ashkenazi Jews
0%
6%
Unselected
Ashkenazi Jews w/
CRC
10%
Ashkenazi Jews w/
CRC and family
history of CRC
0%
28%
10%
20%
30%
% with mutation
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Inherited Syndromes Predisposing to
Colon Cancer
Gene
Lifetime
Risk of CRC
apc
~100%
Hereditary Non-Polyposis Colon Cancer
MMR
>80%
Peutz-Jeghers Syndrome
STK11
2-13%
Juvenile Polyposis
SMAD4
~<50%
Cowden Syndrome
PTEN
small
Familial Adenomatous Polyposis
Correlations between the APC Genotype
and the Clinical Phenotype
NEJM 2003; 349:1750-1760
Clinical Criteria for Hereditary Non-Polyposis Colorectal Cancer
Amsterdam criteria
At least three relatives with colon cancer and all of the following:
•One should be the first-degree relative of the other two
•Two successive generations should be affected
•At least one colon cancer should be diagnosed before the age of 50
•FAP should be excluded
Modified Amsterdam
criteria
As for the Amsterdam criteria except that the cnacers need to be an HNPCCassociated cancer (colon, endometrium, small bowel, ureter, renal pelvis)
instead of specifically colon cancer.
Bethesda criteria
Families meeting the Amsterdam criteria
Individuals with 2 HNPCC-associated cancers, including synchronous or
metachronous cancers
Individuals with colon cancer and a first-degree relative with an HNPCCassociated cancer and/or colonic adenoma; 1 cancer diagnosed at age <
45 years and the adenoma diagnosed at age < 40 years
Individuals with colon or endometrial cancer diagnosed at < 45 years
Individuals with right-sided colon cancer having an undifferentiated pattern
(solid/cribiform) or signet cell histopathology diagnosed at <45 years
Individuals with adenomas diagnosed at < 40 years
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Autosomal Dominant Inheritance
• Each child has 50%
chance of inheriting
the mutation
• No “skipped
generations”
• Equally transmitted
by men and women
Normal
Affected
HNPCC: Direct Mutation Testing
hMLH1
hMSH2
hMSH3
hPMS1
hMSH6
hPMS2
HEREDITARY COLON CANCER - Germline Mutation
SPORADIC COLON CANCER -
Somatic Mutation
FAMILIAL COLON CANCER -
Germline Mutation Causing
Hypermutability and
Subsequent Somatic
Mutation
Germline Mutations - Inherited Disease
APC - Familial Polyposis Coli
MMR - HNPCC (Lynch Syndrome)
MYH- Familial Polyposis Coli
Somatic Mutations - Sporadic Disease
Oncogenes: myc, ras, src
Tumor Suppressors: p53, DCC, APC, MCC
Mismatch Repair Genes: MSH2, MSH3, MSH6,
MLH1, PMS1, PMS2
“Genetic Polymorphisms” - Familial Disease
APC - Familial Colon Cancer
APC Gene
Germline Mutation - Familial Polyposis Coli
Somatic Mutation - Sporadic Colon Cancer
I1307K Germline Mutation - Familial Colon Cancer
GENETIC COUNSELING
GENETIC TESTING
Failure to Diagnose Hereditary Colorectal
Cancer and Its Medicolegal Implications
A Hereditary Nonpolyposis Colorectal Cancer Case
Henry T. Lynch, M.D.,* Jane Paulson, J.D.,† Matthew Severin, J.D., Ph.D.,*
Jane Lynch, B.S.N.,* Patrick Lynch, J.D., M.D.‡
From the *Department of Preventive Medicine, Creighton University School of
Medicine, Omaha, Nebraska, †Paulson & Baisch, Portland, Oregon, and
‡Department of GI Oncology/Digestive Diseases, MD Anderson Cancer Center,
Houston, Texas
Diseases of the Colon & Rectum 1999: Jan 42(1); 31-35
The Use and Interpretation of Commercial APC Gene Testing for
Familial Adenomatous Polyposis
Francis M. Giardiello, M.D., Jill D. Brensinger, M.S., Gloria M. Petersen, Ph.D., Michael C. Luce, Ph.D., Linda M.
Hylind, B.S., R.N., Judith A. Bacon, B.S., Susan V. Booker, B.A., Rodger D. Parker, Ph.D., and Stanley R. Hamilton,
M.D.
From the Departments of Medicine (F.M.G., J.D.B., L.M.H., J.A.B., S.V.B.) and Pathology (S.R.H.) and the Oncology Center (F.M.G.,
G.M.P., S.R.H.), John Hopkins University School of Medicine, Baltimore; the Departments of Epidemiology (G.M.P.) and Health Policy
and Management (R.D.P.), John Hopkins University School of Hygiene and Public Health, Baltimore; and the Department of Molecular
Biology, LabCorp, Research Triangle Park, N.C. (M.C.L.). Address reprint requests to Dr. Giardiello at Blalock 935, Johns Hopkins
Hospital, 600 N. Wofe St., Baltimore, MD 21287-4461.
Background The use of commercially available tests for genes linked to familial cancer has aroused concern about the impact of these
tests on patients. Familial adenomatous polyposis is an autosomal dominant disease caused by a germ-line mutation of the adenomatous
polyposis coli (APC ) gene that causes colorectal cancer if prophylactic colectomy is not performed. We evaluated the clinical use of
commercial APC gene testing.
Methods We assessed indications for APC gene testing, whether informed consent was obtained and genetic counseling was offered
before testing, and the interpretation of the results through telephone interviews with physicians and genetic counselors in a nationwide
sample of 177 patients from 125 families who underwent testing during 1995.
Results Of the 177 patients tested, 83.0 percent had clinical features of familial adenomatous polyposis or were at risk for the disease —
both valid indications for being tested. The appropriate strategy for presymptomatic testing was used in 79.4 percent (50 of 63 patients).
Only 18.6 percent (33 of 177) received genetic counseling before the test, and only 16.9 percent (28 of 166) provided written informed
consent. In 31.6 percent of the cases the physicians misinterpreted the test results. Among the patients with unconventional indications
for testing, the rate of positive results was only 2.3 percent (1 of 44).
Conclusions Patients who underwent genetic tests for familial adenomatous polyposis often received inadequate counseling and would
have been given incorrectly interpreted results. Physicians should be prepared to offer genetic counseling if they order genetic tests.
NEJM 1997; 336:823-27
Microsatellite Instability (MSI)
• 10% - 15% of sporadic tumors have MSI
• 95% of HNPCC tumors have MSI
• Routine MSI assays soon available
Normal
Electrophoresis gel
MSI tumor
Genetic Testing Lab Methods
Normal
DNA
Mutated
Linkage Analysis: Probability of Inheritance.
MSI Assays: Highly predictive for MMR mutation.
Gene Sequencing: Approaches 100%.
DGGE: Highly sensitive (>90%).
SSCP: Detects 60%-95% of mutations.
mRNA
Protein
Protein Truncation: Point of mutation dependent.
Immunohistochemistry: Antibody dependent.
Gel
Calvert & Frucht, Ann Int Med, 2002;137:603-613
History Suggestive of Inherited Colon Cancer
Probable FAP
Probable HNPCC
APC genetic test of an
affected individual
negative
positive
annual endoscopy APC gene testing
of family members
for all family
members
positive negative
no
adenomas
annual
endoscopy
Genetic test of an
affected individual
annual
endoscopy
adenomas
no
adenomas
prophylactic
colectomy
Colon Cancer
screening as
recommended for the
general population
positive
negative
HNPCC genetic
testing of family
members
continued high
risk colon cancer
screening of the
individual and all
family members
negative
positive
positive
for colon
cancer
negative
colectomy
continued survellance for rectal
adenomas and extra-colonic tumors.
consider chemoprevention.
Calvert & Frucht, Ann Int Med, 2002:137;603-613
FAMILY JW-39
11-17-94
60
heart
disease
70’s
BR, 70
70’s
BR, 70
88
48
heart
disease
32
85
60’s
LU, 60
84
72
90
52
uBR, 54
CO, 72
53
50
EN, 45
29
EN, 42
27
25
CO, 26
ascending,
Dukes, C
Legend:
= male;
= female;
= deceased;
= proband
Solid figures = cancer; BR = breast cancer; CO = colon cancer; LU = lung cancer;
EN = endometrial cancer; number refers to age diagnosis
Calvert & Frucht, Ann Int Med, 2002:137;603-613
FAMILY JW-37
9-7-94
70
77
75
CO, 59
EN, 76
68
RE, 68
67
73
73
65
CO, 39
BL, 66
BO, 73
CO, 39
CO, 64
46
49
CO, 37
27
21
90
65
60’s, 70’s
EN, 65
47
12
Legend:
= male;
= female;
= deceased;
= proband
Solid figures = cancer; CO = colon cancer; EN = Endometrial cancer; BL = Bladder cancer;
BO = Bone cancer; RE = Rectal cancer; number refers to age diagnosis
Calvert & Frucht, Ann Int Med, 2002:137;603-613
Incidence of Pancreatic Cancer by
Number of Affected First Degree Relatives
•
10% of patients with pancreatic cancer have a familial aggregation
or an inherited predisposition
Number of FDRs with
Pancreatic Cancer
Incidence (per 100,000)
in the U.S. Population
General U.S. (reference)
9
1
41
2
58
3 or more
288
Klein AP, et al., Cancer Research 2004; 64: 2634-2638
END OF PRESENTATION
Clinical Features of Inherited Cancer Syndromes
Feature
FAP
HNPCC
Age of onset
Early
Early
No. of adenomas
> 100
< 10
Adenoma distribution
Total
Mainly right side
Cancer distribution
Random
Mainly right side
Other cancers
Periampullary
Endometrial, other
Lynch HT et al, Clinical Risk Factors for Colorectal Cancer
Cancer Family Syndromes
Colon
Endometrium
Gastric
Biliopancreatic
Genitourinary
Ovary
Breast
Sarcomas
Skin
Small Bowel
Lung
Other
(63 %)
(8/28 %)
(6 %)
(4 %)
(2 %)
(1/3 %)
(2/6 %)
(2 %)
(2 %)
(1 %)
(1 %)
(2 %)
GENETIC TESTING FOR FAP
1. Linkage Analysis
2. In vitro truncated protein testing (transcription translation method)
3. Mutation Testing
GENETIC TESTING FOR HNPCC
1. Linkage Analysis
2. Truncated Protein Testing
3. Mutation Testing
4. Microsatellite Instability Testing
Clincial Cancer Screening Recommendations*
Colon Cancer Screening Recommendations
RISK
SCREENING
MODALITY
AGE AT WHICH
TO BEGIN
FREQUENCY
Average
FOBT
Sigmoidoscopy
FOBT and Sigmoidoscopy
DCBE
Colonoscopy
50
50
50
50
50
annually
every 5 years
every 5 years
every 5-10 years
every 10 years
First-degree relative with colon
cancer or adenomatous polyp
at age  60 years
Same as for average risk
individuals
40
same as for average risk
individuals
Two or more first-degree
relatives with colon cancer or
adenomatous polyp at age < 60
years
Colonoscopy preferred
FAP
Sigmoidoscopy
HNPCC
Colonoscopy
40 or 10 years younger than
the earlier diagnosis
10-12 years
20-25 years or 10 years
younger than the earliest colon
cancer diagnosis
every 3-5 years
annually
every 1-2 years
Extracolonic Cancer Screening Recommendations
FAP:
Duodenal cancer
EGD
20-25 years
every 1-3 years
HNPCC:
Endometrial and
ovarian cancer
Pelvic exam
Trans vaginal ultrasound
25-35 years
every 1-2 years
Gastric cancer
EGD
30-35 years
every 1-2 yeas
*DCBE = double contrast barium enema; EGD = esophagogastroduodenoscopy; FOBT = fecal occult blood test.
Columbia Colon Cancer Prevention
Program (C3P2)
History and Physical
Risk Assessment
Screening Guidelines
Genetic Counseling and Testing
Chemoprevention