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9/25/2012
Outline
• Lynch syndrome/CRC overview
Colon Cancer –
Lynch Syndrome Tumor
Testing
Dana Zakalik, M.D.
Beaumont Cancer Genetics Program
OUWB Medical School
September 19, 2012
Molecular Pathology Symposium
Colorectal Cancer
– Genetics of LS
• Clinical characteristics/cancer risks
• Management of risk
• Tumor testing
– MSI
– IHC
• Case examples
Epidemiology of Colorectal Cancer
• 3rd most common cancer in the U.S.
– 145,000 new cases per year
• 3rd most common cause of cancer-related
death
• Most common form of hereditary CRC is
Lynch Syndrome (LS)
• Stepwise progression
Sporadic
60%
Familial/
Multifactorial
30%
– Benign mucosa  polyp  cancer
• Effective screening  prevention
Rare Syndromes
~4%
MUTYH
Associated
Polyposis (MAP)
~1%
Familial
Adenomatous
Polyposis (FAP)
~1%
Lynch Syndrome
~2-4%
Cancer 1996;78:1149-67
Am J Med 1999;107:68-77
Gastroenterology 2000;119:837-53
Am J Path 2003;162:1545-8
Lynch Syndrome
(formerly HNPCC: Hereditary Nonpolyposis Colorectal Cancer)
KEY FEATURES:
• CRC (usually dx <50 y)
LYNCH SYNDROME REVIEW
– Usually RT-sided, signet-ring, mucinous, poorly
differentiated, Microsatellite Instability-High
(MSI-H)
• Endometrial cancer <50 y
– Can present prior to CRC dx in women
Other cancers: Ovarian, stomach, urinary tract,
pancreatic, small bowel, bile duct, brain,
sebaceous cysts
• AD inheritance
• 0 to very few polyps
• GENES: MLH1, MSH2, MSH6, PMS2  “mismatch
repair” genes
Slides Courtesy of WBH Cancer Genetics Program
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Lynch Syndrome
Colorectal cancers in LS
• Amsterdam I Criteria, 1991
– Three or more relatives with CRC, which
one case is a first degree relative of the
other two
– At least two successive generations
– One or more cancers diagnosed before
age 50
– FAP excluded
• Distinct histologic features
– Tumor infiltrating lymphocytes
– “Crohn’s-like” lymphocytic infiltrate
– Mucinous histology
• Adenoma to carcinoma – rapid progression
• Metachronous cancer risk – 16% at 10 yrs,
41% at 20 yrs
• Defect in mismatch repair (MMR) system
**Revised in 1999 – Amsterdam II
EPCAM (TACSTD1)
Mismatch Repair Genes
• TACSTD1 lies upstream of MSH2 promoter
– Included in LS testing schema
MSH6 (10%)
MSH2 (40%)
MLH1 (45%)
PMS2
(5%)
• Deletion in the 3’ exons of TACSTD1 gene
leads to hypermethylation of MSH2 promoter
• Leads to silencing of MSH2
– Accounts for 25% of cases in which MSH2 absent
Chr 7
Chr 3
Chr 2
• Absence of MSH2 expression on IHC can be
caused by either: MSH2 mutation or EPCAM
mutation (leading to methylation of MSH2)
Slide courtesy of Monica Marvin, MS; CGC.
MMR System and LS
• DNA MMR system maintains genomic
integrity by correcting DNA errors during
replication
• Recognizes base-pair mismatches and
repairs them
• Failure to repair DNA mismatches leads to
genomic instability
• Occurs in regions of repetitive nucleotide
sequences - microsatellites
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Red Flags for Lynch Syndrome
• Lynch Syndrome
– Colon/ uterine cancer under age 50
– Presence of > one LS-associated cancer
in same individual
– Specific pathological features
– 3-2-1-0 Amsterdam Criteria
CLINICAL PRESENTATION
•
•
•
•
3- 3 relatives with a LS associated tumor
2- Two successive generations affected
1- At least one diagnosed under age 50
0- Polyposis syndrome ruled out
*
LS cancers: colorectal, endometrial, gastric, ovarian, ureter/renal pelvis, biliary tract,
small bowel, pancreas, brain, sebaceous adenoma
Lynch Syndrome- Cancer Risks
Lynch Syndrome- Cancer Risks
• Cancer Risks
Avg age dx = 42-61
EC Avg age dx = 47-55
Lynch Syndrome- Cancer Risks
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Lynch Syndrome: Management
Colon Cancer
Surveillance
Colonoscopy – every 1-2 y
starting at age 20-25 y
Surgery
Prophylactic Colectomy
Consider if:
• Cancer diagnosis
• Large polyp burden
• Pt unwilling to undergo
surveillance
Endometrial
/Ovarian Cancer
Surveillance
(no clear evidence to support)
• Transvaginal U/S
• Endometrial sampling
• Starting at age 30-35 y
Surgery
• Hysterectomy
• RRSO
Lynch Syndrome: Management
• Gastric and Small Bowel cancer (?)
– No clear evidence supporting screening but may consider:
– EGD with extended duodenoscopy
– Capsule endoscopy for small bowel cancer
– At 2-3 y intervals starting at age 30-35 y
• Uroepithelial cancer
– Consider annual urinalysis starting at 25-30 y
• CNS cancer
– Annual physical examination staring at 25-30 y
• Pancreatic cancer
– No recommendation, limited data
Chemoprevention of LS
(Burn at al Lancet 10/12)
•
•
•
•
•
•
•
•
CAPP2 Trial – largest LS intervention trial
861 pts with LS (UK)
ASA 600 mg vs placebo
> 4 y f/u (mean 56 mos)
63% reduction in CRC (HR 0.41, p=0.02)
Also decrease in non-CRC LS cancers
No protection if < 2 y of intervention
No increase in ulcers, GI bleed, anemia
Lynch Syndrome Evaluation
• Germline Testing
– Molecular genetic testing of the germline genes
MLH1, MSH2, MSH6, PMS2 for a deleterious
mutation
• Tumor screening
– Microsatellite Instability (MSI)
– Immunohistochemistry (IHC)
LYNCH SYNDROME
TUMOR TESTING
Lynch Syndrome Testing
• Germline Testing
– Molecular genetic testing of the germline genes
MLH1, MSH2, MSH6, PMS2 for a deleterious
mutation
• Tumor screening
– Microsatellite Instability (MSI)
– Immunohistochemistry (IHC)
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Germline Testing
Mismatch Repair Genes
• MLH1 and MSH2 (EPCAM)
~90%
• MSH6 ~7-10%
• PMS2 <5%
• Tumor screening (MSI/IHC)
allows for targeted germline
testing
Lynch Syndrome Testing
• Germline Testing
– Molecular genetic testing of the germline genes
MLH1, MSH2, MSH6, PMS2 for a deleterious
mutation
• Tumor screening
– Microsatellite Instability (MSI)
– Immunohistochemistry (IHC)
Microsatellite Instability (MSI)
Tumor Tests to Screen for Lynch
Syndrome
• Microsatellite Instability (MSI) testing
– Performed on DNA extracted from tumor and normal tissue
– 15% of sporadic CRC cases are MSI-H
• Epigenetic silencing of MLH1 due to hypermethylation
– Test is positive (MSI-H) in 77-89% of LS cases
– 5-10% false negative rate
• Immunohistochemistry staining
– Abnormal (absence of MMR protein) in 10-15% of sporadic
CRC
– 5-10% false negative rate
MSI testing in LS
• MSI-H is associated with Lynch syndrome
• MSI-stable and MSI-low is not
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Lynch Syndrome
••Revised Bethesda Guidelines, 2004
Bethesda Criteria, 2004
Tumors from individuals should be tested for MSI in the following Tumors from individuals should be tested for MSI in the following situations: situations: 1. Colorectal cancer diagnosed in a patient who is less than 50 years of age. < 50 y.
1. CRC dx
2. Presence of multiple colorectal, or other Lynch syndrome‐associated 2. Presence of
synchronous or metachronous LS‐associated tumors, tumors,* regardless of age.
regardless of age.
3. Colorectal cancer with the MSI‐H histology diagnosed in a patient 3. CRC with MSI‐H histology in pt < 60 y. who is less than 60 years of age. 4. CRC in
pt with one or more first‐degree relatives with a LS‐related 4. Colorectal cancer diagnosed in one or more first‐degree relatives tumor, with one of the cancers
< 50 y
with an Lynch syndrome‐related tumor, and with one of the cancers 5. CRC dx
in pt with two or more first‐ or second‐degree relatives with a being diagnosed under age 50 years. LS‐related cancers, regardless of age. 5. Colorectal cancer diagnosed in two or more first‐ or second‐degree relatives, regardless of age. * Lynch syndrome (HNPCC)-related tumors include colorectal, endometrial, stomach, ovarian, pancreas, ureter and
renal pelvis, biliary tract, and brain (usually glioblastoma as seen in Turcot syndrome) tumors, sebaceous gland
adenomas and keratoacanthomas in Muir–Torre syndrome, and carcinoma of the small bowel.
Patient #1
Patient #2
IHC results – Interpretation
Immunohistochemistry
(IHC)
• Normal : “staining present” or “positive
staining of MMR protein”
• Patient 1: Absent MLH1/PMS2
• Patient 2: Absent MSH2/MSH6
• Abnormal : “Staining absent” or “negative
staining for MMR protein”
www.springerimages.com
Five Possible Results of IHC test:
2. Abnormal – MLH1 & PMS2 Absent
1. Normal – All 4 Stains Present
• 80% of the time will get this result
• May need genetics evaluation if suspect LS,
patient dx <45, patient has had multiple CRC
primaries (meets Amsterdam II criteria)
• 80% acquired
methylation of
MLH1
• BRAF testing
• 20% will be LS
MLH1
MSH6
MSH2
PMS2
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4. Abnormal – MSH6 Absent
3. Abnormal – MSH2 & MSH6 Absent
• Most likely LS due to
either MSH2 or MSH6
gene mutation
•
Posible EPCAM
mutation
• Most likely LS due to
an MSH6 gene
mutation
MLH1
MSH2
MSH6
PMS2
MLH-1
MSH-6
5. Abnormal – PMS2 Absent
MLH1
MSH6
PMS-2
LS is under-recognized
• Most likely LS due to
an PMS2 gene
mutation
• Older age of cancer
MSH-2
MSH2
PMS2
• Amsterdam criteria too restrictive
• ~25 - 50% of individuals with LS do not meet
Amsterdam or Bethesda criteria
• Accuracy of family history poor
• Estimated 1.2% of all individuals with LS are
aware of their LS diagnosis (Hampel et al 2011)
• Solution – Universal Tumor Screening for LS
“The Search for Unaffected Individuals with LS: Do
the Ends Justify the Means?”
(Hampel ’11)
• Population incidence of LS: 1 in 370
• LS: 2.8% of all newly dx CRC
• Estimated 829,747 of the 307,006,550
people in U.S. could have LS
• < 10,000 LS cases currently diagnosed in US
• 1.2% (10,000/829,747) of all individuals with
LS are aware of their diagnosis
Universal Tumor Screening
•
Reduce the morbidity and mortality from LS-related
cancer in at-risk affected and unaffected relatives
•
Sufficient evidence to recommend offering Lynch
syndrome tumor screening to all individuals with
newly diagnosed colorectal cancer
– Justified from a national health care system
perspective
– ICER is $22,522 (anything below $50K
considered cost-effective)
– The Evaluation of Genomic Applications and
Prevention Practice (EGAPP) Guidelines, 2009
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EGAPP Guidelines
Lynch Syndrome Testing
• Endorsed tumor testing of all patients with
CRC (cost-effective)
• Many (71%) NCI designated Cancer Centers
have adopted reflex IHC/MSI to screen for
LS
• Community hospitals less often (15%)
• LSSN – Lynch syndrome screening network
– Beaumont a member
The most cost effective strategy involves
IHC testing first and subsequent targeted
gene testing (Mvundura et al 2010)
• MLH1, MSH2, MSH6, and PMS2 analysis = $4000‐4500
• MSI & IHC = $900‐1000
• IHC alone = $300‐400
• Single Gene Analysis = $1000‐1400
• Beamer et.al. JCO 4/1/12
Cost-effectiveness data
• Universal tumor testing of all CRC
• Preferred approach:
– IHC followed by BRAF mutation testing
• Incremental cost-effectiveness ratio (ICER)
Universal Tumor Screening for LS:
The Beaumont Experience
– $36,200 per life-year gained
• Requires participation of at-risk relatives (3 or
more)
Myundura et.al. 2010
The Beaumont Experience
IHC for the four Lynch associated proteins
initiated January 1, 2012 for ALL newly
diagnosed colorectal cancer resections ONLY
– Biopsies may not provide enough normal
tissue for internal control
– Infrastructure in place to handle screening
results and insure communication and
proper follow-up (Cancer Genetics)
The Beaumont Testing Schematic
IHC Normal
CRC > 50 yo
&
no family hx
no personal hx
IHC Abnormal
Absent Staining
CRC ≤ 50 or
FDR w/ CRC
or multiple
primaries
MSH2 & MSH6,
or PMS2
absent
MLH1& PMS2
absent
Somatic
tumor testing
STOP
Refer to Genetics
STOP
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Beaumont Data 1/1/12 – 6/1/12
Beaumont Data 1/1/12 – 6/1/12
• Reflex IHC for MMR proteins
• Abnormal results:
(MLH1,MSH2,MSH6,PMS2)
– 22 showed absent MLH1 and PMS2 (22%)
– 109 IHC performed on colorectal cancer
resections
– 10 currently pending
• Expected 15%
• 20% of those expected to have LS
• 80% Sporadic etiology (somatic hypermethylation
MLH1/BRAF mutation)
• 75 of 99 IHCs were normal (76%)
– Expected 80-85%
– 2 showed absent MSH6 and MSH2 (2%)
• Expected 3%
• Most will likely have LS
• 24 / 99 IHCs were abnormal (24%)
– Expected 15-20%
– 0 MSH6 or PMS2 only absent
• Expected 2%
IHC Results
Expected
Observation
CASE EXAMPLES
Normal
MLH1/PMS2
Absent
MSH2/MSH6
Absent
MSH6 Absent
PMS2 Absent
Case Example 1
n
• 21-year-old male presented with a bowel
obstruction due to carcinomatosis
• Found to have adenocarcinoma of the colon
with liver metastases
– Treatment included a left hemicolectomy and
palliative chemotherapy
No
Info
n
d. 70s
Ca Type
Unknown
60s
d. 65
80s
d. 80s
Lymphoma Breast
dx 60
No
Info
60
No
CScope
d. 40
Uterine
dx 37
Colon
dx 40
• Patient was referred to cancer genetics due
to his early age of diagnosis of CRC
21
Colon
dx 21
24
d. 56
d. 38
Ovarian
dx 27
Ovarian
dx 37
d.
d.70
Lung
Uterine
(Smoker)
dx 30s
Pancreatic
dx ?
49
Colon
dx 37
53
Polyps?
d. late 70s
Lymphoma
dx 60-70s
60
26
Maternal Ancestry: Polish
Paternal Ancestry: Irish
AJ: No
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n
n
No
Info
d. 70s
Ca Type
Unknown
60s
n
80s
d. 80s
Lymphoma Breast
dx 60
No
Info
d. 65
60
No
CScope
21
Colon
dx 21
d. 56
d. 40
Uterine
dx 37
Colon
dx 40
24
d.70
d.
Uterine
Lung
dx 30s
(Smoker)
Pancreatic
dx ?
49
Colon
dx 37
d. 38
Ovarian
dx 27
Ovarian
dx 37
d. late 70s
Lymphoma
dx 60-70s
No
Info
n
d. 70s
Ca Type
Unknown
60s
60
53
Polyps?
60
No
CScope
26
IHC
Loss of
MSH2
80s
d. 80s
Lymphoma Breast
dx 60
No
Info
d. 65
d. 40
Uterine
dx 37
Colon
dx 40
24
d. 22
Colon
dx 21
MSH2
IVS4+1G>T
Maternal Ancestry: Polish
Paternal Ancestry: Irish
d. 56
d.70
d.
Uterine
Lung
dx 30s
(Smoker)
Pancreatic
dx ?
49
Colon
dx 37
d. 38
Ovarian
dx 27
Ovarian
dx 37
d. late 70s
Lymphoma
dx 60-70s
60
53
Polyps?
26
Maternal Ancestry: Polish
Paternal Ancestry: Irish
AJ: No
AJ: No
Case Example 2
d. 37
• 51-year-old male presented for a routine
screening colonoscopy
d. 89
d. 68
d. 54
Possible
Cervical
– Polyp in right colon
• Pathology revealed a high grade
adenocarcinoma
• Tumor screening performed following colon
resection:
d. 70s
d. 86
d. 70s
d. 77
CHF
CScope
d. 72
Bladder
2
d. 16
d. 71
3
68
61
52
48
51
No
CScope
51
Colon
dx 51
50
d. 52
– IHC loss of MSH6 expression
– MSI-H 3/6 markers
2
Maternal Ancestry: Irish
• Patient presented to cancer genetics
22
24
Paternal Ancestry: Polish
AJ: No
d. 37
d. 89
d. 68
d. 70s
d. 54
Possible
Cervical
d. 86
d. 70s
d. 77
CHF
CScope
d. 72
Bladder
2
d. 16
d. 37
d. 71
d. 89
Maternal Ancestry: Irish
61
52
48
51
No
CScope
51
Colon
dx 51
MSH6+
p.R1005X
50
2
d. 54
Possible
Cervical
d. 52
d. 16
d. 77
CHF
CScope
d. 72
Bladder
d. 71
3
68
Maternal Ancestry: Irish
22
d. 86
Gastric
Tumor
dx 68
d. 70s
2
3
68
d. 68
d. 70s
24
61
52
48
51
No
CScope
51
Colon
dx 51
MSH6+
p.R1005X
Paternal Ancestry: Polish
AJ: No
AJ: No
d. 52
2
22
Paternal Ancestry: Polish
50
24
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Maternal ancestry: Irish / German / English
Paternal ancestry: German
Summary
d. 80
d. “old age”
Colon dx ?
Prostate dx ?
d. ?
d. 80s
Cancer
Colon
Type
dx 60s
d. 70
d. 72
Colon
Stomach
dx ?
dx ?
d. 80s
d. 67
“old age”
heart
d. 58
No info
93
Lung
dx?
unknown
68 – MLH1+
Colon dx 40 / 68
61
Renal dx 64
Key:
Bladder dx 59
Colon cancer
Stomach cancer
Other cancer
Renal cancer
42
46 – polyps
Colon
MLH1 neg
dx 34
• Lynch Syndrome is the most common
inherited colorectal cancer syndrome
• Intense cancer surveillance and/or
prophylactic surgery decreases morbidity
and mortality from LS-related cancers
• Family history and testing criteria alone are
insufficient in identifying individuals at risk
• Tumor screening is a cost-effective approach
to increasing the detection rate of Lynch
Syndrome
25 - Preca
Bladder cancer
9
16
21
Appendix cells
Beaumont Cancer Genetics
Program
Dana Zakalik, MD
Genetic Counselors:
Lindsay Dohany, MS, CGC
Heidi Dreyfuss, MS
Jennifer Fulbright, MS, CGC
Ashley Reeves, MS
248-551-3388
http://cancer.beaumont.edu/genetics
[email protected]
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