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Hereditary Nonpolyposis Colorectal Cancer Gene Detection

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Title: Hereditary Nonpolyposis Colorectal Cancer Gene Detection


1
Hereditary Nonpolyposis Colorectal Cancer Gene
Detection
  • Terdiman JP, et al. 2001. Efficient detection
    of hereditary nonpolyposis colorectal cancer gene
    carriers by screening for tumor microsatellite
    instability before germline genetic testing.
    Gastroenterology 120(1)21-30.
  • Presented by Trina Geiss

2
Goals
  • To find the optimal strategy for the detection of
    Hereditary Nonpolyposis Colorectal Cancer
    (HNPCC).
  • To evaluate the role of microsatellite
    instability (MSI) and MSH2 and MLH1 proteins on
    the detection of HNPCC.

3
Background
  • HNPCC is an autosomal dominant disorder that
    represents 1-3 of all colorectal cancers (CRC).
  • HNPCC is caused by a germline mutation in a gene
    responsible for DNA mismatch repair (MMR).
  • Over 90 of the mutations occur in MSH2 or MLH1,
    which are MMR genes.
  • Present in Incomplete Penetrance.

4
Cancer Sites in HNPCC
  • Colon (proximal)
  • Stomach
  • Uterus
  • Ovary
  • Renal Tract
  • Small Bowel
  • Skin
  • Brain

5
Defects in MMR function
  • Absent or non-functional proteins.
  • Loss of MMR capacity.
  • There is a dominant-negative effect abnormal
    proteins will bind with normal ones.
  • Will no longer be able to separate, destroy, and
    resynthesize mutated DNA.

6
Human MMR Genes
  • MLH1 (3p21)
  • MSH2 (2p16)
  • PMS1 (2q31-33)
  • PMS2 (7p22)
  • MSH3 (5q3)
  • MSH6 (2p16) ( GT Binding Protein)

7
Mismatch Repair Mutations
  • MMR mutations in 70 of families.
  • MLH1 (50), and MSH2 (40).
  • Minor role in the others.
  • Population prevalence 12851 (15-74yrs.)
  • 46 under 45 years.

8
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9
Microsatellite Instability
  • MSI is a simple nucleotide repeat sequence.
  • Length should be stable at any one locus, and any
    alteration will lead to MMR mutation.
  • Poly-A and Poly-CA repeat sequences are the most
    prone to alteration.
  • Also called Replication Error (RER).
  • 90 of CRCs with MSH2 or MLH1 mutations show
    change in length of repeat sequences,
    High-frequency MSI (MSI-H).

10
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11
MI Positive Tumors
  • 90 of HNPCC colorectal cancers.
  • 20 of sporadic colorectal cancers.
  • 30 of sporadic uterine cancers.

12
CRC Prevention
  • Those at risk for HNPCC will decrease their
    chance of death by early and intensive screening
    measures.
  • The tests for detection of HNPCC and mutations of
    MSH2 and MLH1 are now commercially available.
  • Those with the mutation should continue rigorous
    screening.

13
Clinical Criteria for HNPCC
  • Amsterdam
  • Amsterdam II
  • Bethesda

14
History of Amsterdam Criteria
  • Developed in 1991 by the International
    Collaborative Group on HNPCC.
  • Criteria to more accurately identify families
    with HNPCC.
  • 50 of families that meet the criteria have a
    germline mutation of either MSH2 or MLH1.

15
Amsterdam
  • At least 3 relatives with CRC
  • One should be the first-degree relative of the
    other two.
  • Two successive generations affected.
  • 1 CRC diagnosed before the age of 50.
  • Familial adenomatous polyposis should be excluded.

16
Amsterdam II
  • Three relatives with HNPCC associated cancer
    (CRC, cancer of the endometrium, small bowel,
    ureter, or renal pelvis).
  • Same criteria as Amsterdam I.

17
Bethesda
  • Includes the same criteria as Amsterdam II.
  • Includes a broader range of other types of
    familial cancer, and mutated cell types.
  • These criteria will encompass more individuals,
    and therefore more genetic screening.

18
Prevention and Detection Techniques
  • Colonoscopy
  • Gastroscopy
  • Uterine ultrasound and biopsy
  • Colectomy
  • Histerectomy, Oophorectomy

19
Subjects
  • Eligibility of subjects must meet Amsterdam II
    criteria.
  • Total of 109 families were used.
  • Personal and family cancer histories and
    demographic data were obtained.
  • Pedigrees were traced backward and laterally as
    far as possible
  • Tissue sample taken for MSI analysis and
    MSH2/MLH1 immunostaining.

20
Tumor MSI Analysis
  • 5µm thick sections of tissue samples were mounted
    on slides and stained
  • Microsatellite markers were evaluated.
  • PCR was performed and placed on electrophoresis
    gels.
  • MSI-H was diagnosed if 3 or more of any of the
    marker loci had band size shifts.

21
Tumor MSH2 and MLH1 Protein Expression
  • Immunostaining performed using anti-MSH2 and
    MLH1.
  • Overnight incubation with antibodies at 4C
  • Tumor cells were negative for expression if they
    lacked staining in a sample in which normal
    colonocytes and stroma cells were stained.

22
Germline MSH2 and MLH1 Mutation Detection
  • DNA was isolated from peripheral whole blood
    samples.
  • DNA sequences for MSH2 and MLH1 were isolated and
    PCR performed.
  • Exons were placed on gel electrophoresis
  • Changes in electrophoretic mobility and addition
    of bands were seen by staining with ethidium
    bromide.

23
Statistical Analysis
  • Descriptive statistics were calculated to
    characterize the entire data set.
  • The features of the families were compared.
  • Logistic models were made.

24
Probability of detecting an MSI-H tumor at the
earliest age
25
Immunostaining for MSH2 MLH1
  • A normal colon, MLH1 staining present
  • B colon cancer, MLH1 staining absent
  • C colon cancer, MSH2 staining present
  • D normal, MSH2 present
  • E colon cancer, MLH1 present
  • F colon cancer, MSH2 staining absent

26
Results
  • MSH2 or MLH1 mutations in individuals are at high
    risk for CRC.
  • Detection strategies were efficient method of
    detection of HNPCC carriers (MSI analysis and
    MSH2/MLH2 mutation analysis).
  • Amsterdam requirements too strict, so Bethesda is
    better.
  • Continuing research is necessary to better
    predict chance of contracting HNPCC.

27
Summary
  • Inherited mutations of MMR genes lead to high
    relative and absolute risk of cancer.
  • Colorectal and endometrial cancers.
  • Cancer at an early age
  • High risk of further cancers
  • Identification of families and individuals
  • Clinical trials of interventions.
  • Screening is expensive and subject to
    discrimination.

28
References
  • Lynch PM. Clinical challenges in management of
    familial adenomatous polyposis and hereditary
    nonpolyposis colorectal cancer. 1999. Cancer
    86(11)2533-38.
  • Peltomaki P, et al. 1993. Genetic mapping of a
    locus predisposing to human colorectal cancer.
    Science 260810-15.
  • Terdiman JP, et al. 2001. Efficient detection
    of hereditary nonpolyposis colorectal cancer gene
    carriers by screening for tumor microsatellite
    instability before germline genetic testing.
    Gastroenterology 12021-30.
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