Download Screening for the Lynch Syndrome

Screening for the Lynch Syndrome
(Hereditary Nonpolyposis
Colorectal Cancer)
Heather Hampel, M.S., Wendy L. Frankel, M.D., Edward Martin, M.D., Mark
Arnold, M.D.,Karamjit Khanduja, M.D., Philip Kuebler, M.D., Ph.D., Hidewaki
Nakagawa, M.D., Ph.D., Kaisa Sotamaa, M.D.,Thomas W. Prior, Ph.D., Judith
Westman, M.D., Jenny Panescu, B.S., Dan Fix, B.S., Janet Lockman, B.S.,
Ilene Comeras, R.N., and Albert de la Chapelle, M.D., Ph.D.*
N Engl J Med 2005;352:1851-60.
Background
< Amsterdam Criteria I>
There should be at least 3 relatives with colon cancer and:
1.Large-scale
for germ-line
: increasingly possible
One should screening
be a first degree
relative onmutations
the other two;
2. At least two successive generations should be affected;
3. At least one should be diagnosed before age 50;
 The Lynch syndrome (hereditary nonpolyposis colorectal cancer)
4. Familial adenomatous polyposis (FAP) should be excluded;
: caused
by mutations
DNA mismatch-repair
genes
5. Tumors
should
be verified in
by the
pathological
examination
(MLH1, MSH2, MSH6, PMS2)
: Amsterdam criteria , Bethesda guideline
 The sensitivity of the criteria
: only 40-80 % among person at high risk of cancer
-> The strategies for diagnosing Lynch syndrome need to be improved
-> presence of germ line mutation in mismatch repair gene
< Revised Bethesda Guidelines for testing colorectal tumors for MSI >
1.
Amsterdam I criteria
2.
Indivisuals with 2 HNPCC cancer (including synchronous/
metachronous colorectal cancer)
3.
Indivisuals with colorectal cancer and a first degree relative with
colorectal cancer or HNPCC extracolonic cancer or colorectal
adenoma (<50yr)
4.
Colorectal or endometrial cancer (<50yr)
5.
Rt sided colorectal cancer with undifferentiated pattern on histology
(<50yr)
6. Signet cell type colorectal cancer (<50yr)
7. colorectal adenoma (<40yr)
 Searching of mutation in four mismatch-repair genes
: difficult and expensive
-> molecular prescreening for microsatellite instability
 Microsatellite instability (MSI)
: expansion or contraction of short repeated DNA sequences
that are caused by the insertion or deletion of repeated unit
: the presence of MSI -> defect in a DNA mismatch repair gene
: hallmark of the Lynch syndrome (>90 % of tumors)
-> prescreening for the syndrome
: more favorable prognosis, do not benefit from chemotherapy
with fluorouracil
-> important because of prognosis, therapeutic implication
 Aims of this study
: frequency of Lynch syndrome in pts with colorectal cancer
: examine strategies for molecular screening to identify the
Lynch syndrome
: compare effectiveness two techniques to prescreen for
mismatch repair deficiency
genotyping for MSI <-> immunohistochemical analysis
Methods
<Patients>
 1066 patients with new diagnosis of colorectal carcinoma
at the major hospitals in metropolitan Columbus, Ohio
 Primary screening : genotyping of tumor for MSI
 Positive for MSI
: search for germ-line mutations in the MLH1, MSH2, MSH6,
PMS2 genes with the use of immunohistochemical staining for
mismatch-repair proteins, genomic sequencing, deletion studies
 Family members of carriers of the mutation were counseled
: those at risk -> mutation testing
<Microsatellite instability>
 To determine microsatellite instability of the tumor
: ascertain genotypes using polymorphic markers
(BAT25, BAT26, D2S123,D5S346, and D18S69 or D17S250)
 High-frequency MSI : two or more marker
Low-frequency MSI : only one marker
Negative : none of the markers
<Immunohistochemical staining for mismatch repair protein>
 Immunoperoxidase staining on formalin-fixed tissue
 Primary antibodies : MLH1, MSH2, MSH6 or GTBP, PMS2
<Detection of mutations>
 To search for germ-line mutations, DNA was directly
sequenced with the use of primers
<Methylation of the promoter region of MLH1>
 A region in the 5' part of the promoter region
:was studied with the use of the methylation-specific PCR
 Another region closer to exon 1
:was studied with use of combined bisulfite restriction analysis
Results
Figure 1. Analytic Strategy
of the Study and Numbers
of Patients for Each
Analysis. IHC denotes
immunohistochemical, MSI
microsatellite instability, and
MLPA multiplex ligationdependent probe amplification.
3pts
15pts
* Of the 119 relatives who received counseling and were offered testing,
2 chose not to undergo testing.
† Of these 52 relatives, 14 had previously had a cancer related to the
Lynch syndrome and 38 were unaffected at the time of testing. None of
the 52 relatives had previously received a diagnosis of the Lynch syndrome.
* Only 20% of pts need molecular testing
 Considerable savings of time, effort
and cost over the method
Figure 2. Proposed
Strategy for Screening
Patients with Colorectal
Cancer for Lynch
Syndrome Mutations and
Mismatch-Repair
Deficiency.
Figure 3. Immunohistochemical Staining for Mismatch-Repair Proteins in
Colorectal Adenocarcinoma.
Panel
Panel
Panel
Panel
A shows positive staining for MLH1, Panel B negative staining for MLH1,
C positive staining for MSH2, Panel D negative staining for MSH2,
E positive staining for MSH6, Panel F negative staining for MSH6,
G positive staining for PMS2, and Panel H negative staining for PMS2.
Conclusions
 Routine molecular screening of patients with colorectal
adenocarcinoma for the Lynch syndrome identified mutations
in patients and their family members that otherwise would not
have been detected.
 These data suggest that the effectiveness of screening
with immunohistochemical analysis of the mismatch-repair
proteins would be similar to that of the more complex strategy
of genotyping for microsatellite instability.