Colorectal Cancer Molecular Diagnostics
Transcript of Colorectal Cancer Molecular Diagnostics
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Mary P. Bronner, M.D.
Division Chief of Anatomic and Molecular Oncologic Pathology
Colorectal Cancer
Molecular Diagnostics
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• MSI, MMR IHC
• KRAS
• BRAF
• PIK3CA
• PTEN
• APC, SMAD4, BMPRIA,STK11
• Septin 9
Molecular CRC Testing
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MSI Testing
• MSI-H Sporadic (15%)
• MSI-H Lynch (2-3%)
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• Colorectal CA 80%
• Endometrial CA 50%
• Pancreatic, gastric, small bowel, sebaceous
skin, ovarian, genitourinary, GBM cancers
• Screening: Age 25 or 10 years < than youngest
Annual colonoscopy & endometrial bx,
periodic EGD, EUS of pancreas, pelvic exam,
brain scans, urine cytology
HUGE & LIFELONG IMPACT ON
LYNCH PATIENTS: DX IS CRITICAL
Lynch Syndrome Cancers
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Microsatellites: Short, repetitive DNA sequences prone
to error during replication
Normally repaired by MMR gene proofreading
complex
Microsatellite instability (MSI)
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Microsatellite instability (MSI) Mutations in MMR genes lead to accumulation of
altered length microsatellites (MSI)
MLH-1, MSH-2, MSH-6, PMS-2 alterations cause
MSI-H cancer
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MSI-High Colon Cancer • Sporadic: ~100% MLH1
(methylated)
• Lynch: 60% MLH1, 35% MSH2,
5% PMS2, MSH6, other
• Familial & sporadic path: identical
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Right-sided location
Age < 50 years (Lynch)
Poor differentiation
Absence of dirty necrosis
>2 tumor infiltr lymphs/hpf
Mucinous change
Crohn’s-like lymphoid reaction
MSI-H CRC: Clinicopathologic Features
Greenson JK, et al. Am J Surg Pathol 27:563-570, 2003.
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Consider FAP and Lynch Syndrome
Duodenal or Gastric Adenoma
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• Hereditary and syndromic
components of Lynch
• Prognosis
• Therapy
Reasons to Diagnose MSI-H CRC
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Tumor screening assays (90% sens)
Detect affected patients with tumor
MSI by PCR (paraffin works well)
MMR Immunohistochemistry:
MLH-1, MSH-2, MSH-6, PMS2
Blood germline mutation analysis
Detect affected family members
without tumor
Lynch Testing
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• Non-colon tumor tissue
• Endometrial neoplasia
• Other syndromic tumors
• Fresh or fixed (formalin)
MSI/IHC: Other Tumors
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• Also need constitutional
(normal) DNA
• Non-tumor paraffin tissue
• Blood
• Buccal swab
MSI Requirements
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Normal
Carcinoma
Unstable Unstable Unstable Unstable Unstable
Microsatellite-High (MSI-H) Instability at > 2 loci
Microsatellite-Low (Indeterminate) Instability at 1 locus
Microsatellite stable (MSS) None of 5 loci unstable
MSI Electropherogram Results
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IHC in Lynch Syndrome
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Family history
MSH2, MSH6, PMS2 IHC loss
Adenoma involvement
MLH1 promoter methylation
BRAF point mutation (V600E)
Germline MMR gene mutation
Sporadic vs. Lynch CRC
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MSI-H in randomized stage-
matched sporadic tumors predicts:
• Longer survival
• Chemotherapy
MSI: Prognosis & Adjuvant Rx
Zaanan A, et al. Clin Cancer Res 17:7470;2011
Ribic CM, et al. NEJM 349:247;2003
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Probably not Lynch syndrome
Consider Lynch syndrome
Negative for MSI Positive for MSI
Abnormal MLH1 staining
Normal MLH1 staining and abnormal for MSH2, MSH6, or PMS2
Alteration absent (wild-type) Alteration present
BRAF V600E mutation,
MLH1 methylation Seq&Del/Dup MSH2, MSH6, PMS2
Probable sporadic colorectal cancer
Seq &Del/Dup MLH1
MSI by: Immunohistochemical stains or PCR
Lynch Syndrome Testing Algorithm
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KRAS Testing
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• 50-60% CRC patients present with or
develop metastases
• 5-yr survival
Stage I + II (N0) 91%
Stage III (N1,2) 70%
Stage IV (M1) 11%
The Metastasis Problem
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5 FU/Leucovorin mainstay for decades
After 2000 new therapies
Oxaliplatin (FOLFOX)
Irinotecan (FOLFIRI)
Anti-VEGF (bevacizumab)
Anti-EGFR (cetuximab, panitumumab)
Search for alternate Rx’s
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• <1% response rate to anti-EGFR Rx with
codon 12 or 13 or 61 mutations (~40% of
CRC)
• ~40% response rate with KRAS WT (~60%
of CRC)
• But…. ~ 60% KRAS WT will not respond
• Other markers play a role
KRAS mutation
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• ~30,000 new metastatic CRC annually
• KRAS testing = $13 million ($452/pt)
• Cetuximab Rx= $2.1 billion ($71,120/pt)
• Mutated KRAS (~40%) excluded from
cetuximab
• Cost savings: ~$750 million annually
• High toxicity; ~2 month added survival
Cost (savings)
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No need for normal tissue
for KRAS testing
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KRAS Methods
• All methods applicable to formalin-
fixed paraffin-embedded tissue
• Tumor microdissection
• Sequencing, Sequenom, allele
specific PCR, and melt curve
analysis
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• Impact of specific KRAS 12/13 mutations?
• Other predictors of anti-EGFR response
• Other KRAS mutations: codon 61, others?
• BRAF
• EGFR copy number (FISH, CISH, PCR), specific
mutations
• PTEN
• PIK3CA
Future
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• KRAS mutations in 30-40% CRC’s
• Highly predictive of lack of response to
anti-EGFR Rx
• Laboratory plays key role determining
proper and most cost-effective Rx in
stage III-IV CRC
• BRAF, PIK3CA, PTEN downstream markers
may be useful in KRAS wild type tumors
• Additional biomarkers expected
KRAS Summary
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BRAF Testing
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• ~ 10% colorectal cancers have BRAF mutations
• Predicts anti-EGFR non-response in KRAS WT
BRAF
Study Pts
BRAF
Mutation
%
BRAF
Wild Type
Response
BRAF
Mutated
Response
Cappuzzo ’08 79 5% 17% 0%
De Roock ’08 113 6% 27% 17%
Di Nicolantonio ’08 79 14% 32% 0%
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BRAF mutation
T to A transversion
Valine to glutamate at codon 600 (BRAF V600E)
BRAF: Downstream from KRAS
BRAF and KRAS mutations:
Mutually exclusive
KRAS-WT/BRAF-MUT
Anti-EGFR therapy non-responders
KRAS
BRAF
MEK
MAPK
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Chemoradiation Rx: Does it affect
GI cancer molecular testing?
• Neoadjuvant Rx common in rectal &
esophageal adenocarcinomas
• Ondrejka SL, et al. Am J Surg Pathol
35:1327,2011
• Pre and Post neoadjuvant Rx
• No change in 18 patients for MSI PCR
• No change in 18 patients for KRAS mutations
by Sanger sequencing
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Gastrointestinal Polyposes Predisposing to CRC
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Case 1
• 18 yo boy currently asymptomatic but strong family history of colon cancer
• Colonoscopy reveals hundreds of colonic polyps
• Colectomy is performed
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Familial Adenomatous Polyposis
• Sequencing reveals APC knockout mutation
• Risk of colorectal cancer 100%, average age onset 39 years
• Extra-colonic intestinal manifestations: duodenal & jejunal adenomas/ carcinomas, gastric polyps (?cancer)
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Case 2
• 45 yo man with 85 adenomas
• No APC mutation in germline
• Family history of colon cancer, recessive inheritance
• MUTYH gene is sequenced; compound Y179C & G396D germline mutation detected
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MYH-assc polyposis (MAP)
• Colon polyps usually like attenuated FAP
• Extra-colonic: duodenal ad/ca, cancer of ovary, bladder, skin, sebaceous glands
• Most homozy or compound heterozy
of Y179C and G396D mutations
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Case 3 • 35 yo woman with breast & thyroid
cancer
• Sister with ovarian cancer in her 20’s, thyroid nodule at 32, & childhood colon polyps
• FH of endometrial cancer
• Endoscopy reveals colonic, gastric & esophageal polyps
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Glycogenic acanthosis in esophagus
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Cowden’s syndrome
• Personal medical and FH history implicating Cowden’s syndrome
• PTEN mutation testing to reveal pathogenic germline change (R335X) of Cowden’s
• Emerging literature on colon cancer risk, plus previously known breast, thyroid, endometrial CA risks
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Case 4
• 15 yo girl presents with rectal bleeding & small bowel obstruction
• Jejunal intussusception & polyposis
• Bowel resected
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Peutz-Jeghers • Hamartomatous polyps, mostly small
bowel (jejunum), also stomach and colon
• Mucocutaneous hyperpigmentation
• Autosomal dominant mutation in STK11 (LKB1) gene (50-90 %)
• Multi-organ cancer syndrome: breast, colon, pancreas, stomach, lung, gyn, testes: 93% lifetime cancer risk
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Case 5
• 57 yo man with upper GI bleeding
• Upper GI endoscopy revealed 5x6 cm gastric mass & multiple smaller polyps throughout stomach (biopsied as hyperplastic polyps)
• Mass & few smaller polyps resected
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Hyperplastic Polyp
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Juvenile Polyposis
• 28% SMAD4, 24% BMPR1A
• SMAD4 also causes hereditary hemorrhagic telangiectasia (HHT)
• SMAD4 mutations may also have severe gastric polyposis & ↑ gastric cancer risk
• JP at high risk for colon (20-70%) & gastric cancer (mostly SMAD4 for gastric)
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Summary: CRC Molecular Dx: Current Impact on Practice: 15 Genes
• Metastatic CRC for Anti-EGFR Rx (50-60%): KRAS
• KRAS WT for Anti-EGFR Rx (40%): BRAF, PIK3CA,
PTEN
• Sporadic MSI-H CRC (15%): MMR IHC, MSI, MLH1
Methylation, BRAF
• Lynch MSI-H (2-3%): MMR IHC, MSI, MLH1
Methylation, BRAF
• FAP (<1%): APC
• Juvenile Polyposis, Peutz-Jeghers, Cowden’s
(<1%): SMAD4, BMPRIA, STK11, PTEN
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