stool dna testing for colon cancer
TRANSCRIPT
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Stool DNA Testing For Colon Cancer
Steven Itzkowitz, MD, FACP, FACG, AGAF
Professor of Medicine
Mount Sinai School of Medicine
New York, NY
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Tests that detect Adenomas and Cancer: (structural)
• Flexible sigmoidoscopy q 5 yrs
• Colonoscopy q 10 yrs
• Barium enema (air contrast) q 5 yrs
• CT Colonography q 5 yrs
CRC Screening Guidelines:
Average-Risk Adults Over Age 50
(ACS, US Multi-Society Task Force, ACR)
Tests that primarily detect Cancer: (stool-based)
• Fecal occult blood test (FOBT) q 1 yr
• Fecal immunochemical test (FIT) q 1 yr
• Stool DNA test (sDNA) interval uncertain
Levin et al. CA-Cancer J Clin 58:130, 2008
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Why Stool DNA Tests?• Colonoscopy is becoming the preferred CRC
screening test.
• However, barriers to colonoscopy include:
– Organizational: access (USA, abroad); capacity
– Patient-associated: discomfort, fear, embarrassment, inconvenience (work absence; patient escort; child care)
• Therefore, non-invasive tests may greatly facilitate CRC screening efforts.
• DNA is theoretically a more specific analyte than blood for stool-based detection.
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Rationale for Stool DNA Testing: Mucocellular Layer
Colon cancer Normal colon
Courtesy: David Ahlquist, MD, Mayo Clinic
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CarcinomaEarly
adenoma
Intermediate
adenoma
Late
adenoma
APC (10)
Normal
mucosa
Molecular Markers of Colon Carcinogenesis
Chromosomal Instability (e.g. FAP)•Aneuploidy
•LOH
•Tumor suppressor gene mutations
Microsatellite Instability (e.g. HNPCC)•Hypermethylation/mutation of DNA MMR genes
•Target gene alterations (TGFbRII; others)
K-ras (3) DCC/18q genes P53 (8)
70-85%
15% Long-DNA (DIA)
BAT26
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Version 1 Stool DNA Test:
Collection Kit (with freezer pacs)
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Stool DNA Testing: Early Studies
Study Sensitivity Specificity
Ahlquist ‘00 91% (20/22) 93% (26/28)
Tagore ‘00 63% (33/52) 98.2% (111/113)
Syngal ‘02,‘03 62% (40/65) --
Brand ‘02 69% (11/16) --
Calistri ‘03 62% (33/53) 97% (37/38)
Syngal ‘06 63% (43/68) --
These studies:
• used the same multi-target DNA panel (Version 1)
• paved the way for a large average-risk pop’n screening study
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sDNA is Better than FOBT in
Average-Risk Individuals2,507 asymptomatic, average-risk subjects over age 50
Fecal DNA assay compared to Hemoccult-II
PreGenPlus Assay:
• 22 Mutations - APC (10), K-ras (3), p53 (8), BAT-26
• DNA integrity assay (DIA)
sDNA Hemoccult-II
Cancer (n=31) 51.6 % 12.9%
Adenomas
• HGD (n=40) 32.5% 15.0%
• Villous (n=133) 18.0% 9.8%
• >1 cm (n=214) 10.7% 10.3%
Normal colon (n=1423) 5.6% 4.8%
Imperiale, Ransohoff, Itzkowitz, et al. NEJM 351:2704, 2004
(p=0.003)
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Patient Preferences
(Based on Imperiale Study)
• Preferred strategy among 4,042 patients in the multicenter study (84% response rate)
• Stool DNA received the same or higher mean ratings than FOBT for prep- and test-related features.
• Stool DNA received higher ratings than colonoscopy for all prep- and test-related features except accuracy.
• Preferred test:
– Stool DNA: 45%
– FOBT: 32%
– Colonoscopy: 15%
– No preference: 8%
Schroy et al, Am J Prev Med 28:208, 2005
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sDNA is Better than FOBT in
Average-Risk Individuals
Conclusions:
1. sDNA more sensitive than Hemoccult-II for CRC
2. sDNA similar specificity as Hemoccult-II
3. But, DIA performance lower than expected• DNA degraded in transit, despite use of freezer pacs
and overnight shipping.
Imperiale et al. NEJM 351:2704, 2004
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Improving the Stool DNA Test:
“Version 2”
• Better DNA stabilization
– Adding EDTA-containing buffer to stool
significantly increases the recovery of DNA 1
• Improved DNA extraction method
– Gel-based extraction (instead of beads)
enhances DNA recovery 2
• New markers
– Methylation markers (eg. vimentin) 3
1 Olson et al, Diagn Mol Pathol 14:183, 20052 Whitney et al. J Mol Diagn 6:386, 20043 Chen et al. J Natl Cancer Inst 97:1124, 2005
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Carcinoma
(MSS)
Early
adenoma
Intermediate
adenoma
Late
adenoma
APC
Normal
mucosa
PATHWAYS OF COLON CARCINOGENESIS
Chromosomal Instability (e.g. FAP)•Aneuploid; LOH; Tumor suppressor gene mutations
Microsatellite Instability (e.g. HNPCC)•Mutation/loss of DNA MMR genes; diploid
•Mutations of key target genes (eg, TGFbRII)
K-ras DCC/18q genes p5370-85%
15%
CpG Island Methylation; CIMP (e.g. HPS)•DNA methylation inhibits key gene expression
•BRAF oncogene mutation
Sessile Serrated Polyp
(SSP; SSA)
Carcinoma
(MSI-H)
Carcinoma
(MSI)
15%
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New Stool Collection Kit
(with buffer)
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Results of Version 1 Assay
(MuMu22+DIA)
Version 1(Imperiale, NEJM, „04)
Version 1.1*(with buffer and gel capture)
No. Positive
%Positive
No. Positive
% Positive
Sensitivity:
•All markers 16/31 51.6% 29/40 72.5%
•MuMu22 16/31 51.6% 17/40 42.5%
•DIA 1/31 3.2% 26/40 65.0%****(p<0.0001)
Analyzing the original Version 1 markers, the DNA
stabilizing buffer & gel capture increased sensitivity for CRC
(51.6% -> 72.5%), especially DIA (3.2% -> 65%)
* Itzkowitz et al. Clin Gastroenterol Hepatol 2007, 5:111
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Version 2: Two Markers
Sensitivity (n=40) Specificity (n=122)
No.
Positive
%
(95% C.I.)
No.
Positive
%
(95% C.I.)
DY (DIA) 26 65.0 (49.5-77.9) 9 92.6 (86.6-96.1)
Vimentin 29 72.5 (57.2-83.9) 16 86.9 (79.8-91.8)
Vim + DY 35 87.5 (73.9-94.5) 22 82.0 (74.2-87.8)
Vimentin methylation + DY resulted in optimal sensitivity
(87.5%) & specificity (82.0%)
Itzkowitz et al. Clin Gastroenterol Hepatol, 2007, 5:111
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Sensitivity of Version 2:
by Cancer Stage
No.
Positive
%
(95% CI)
Total: 35/40 87.5 (73.9-94.5)
• Stage I 6/8 75.0 (40.9-92.8)
• Stage II 9/10 90.0 (59.6-98.2)
• Stage III 16/17 94.1 (73.0-99.0)
• Stage IV 4/5 80.0 (37.6-96.4)
• DY+Vim detected the vast majority of CRC regardless of
tumor stage
Itzkowitz et al. Clin Gastroenterol Hepatol, 2007, 5:111
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Version 2 Detects CRC
Regardless of Location
PV1 DY Vim DY + Vim
Right (n=11) 54.5% 36.4% 72.7% 90.9%
Left (n=29) 79.3% 75.9% 72.4% 86.2%
P value NS 0.03 NS NS
• DY preferentially detected distal CRC
• Vim detected CRC regardless of location
• Therefore, DY+Vim detected the majority of CRC’s
regardless of location
Itzkowitz et al. Clin Gastroenterol Hepatol 5:111,2007
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Version 2:
Patient Satisfaction Survey
Percent
Male 41%
Age >60 yrs 40%
Perform the test; easy/very easy 97%
Open the preservative bottle; easy/very easy 96%
Add the preservative to specimen; easy/very easy 100%
Very comfortable performing the test 93%
Would repeat test if doctor recommended it 84%
Itzkowitz et al. Clin Gastroenterol Hepatol 5:111, 2007
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Stool DNA Test - Version 2
CRC NL Sensitivity Specificity
Phase 1a 40 122 88% (74-95) 82% (74-88)
Phase 1b 42 241 86% (72-93) 73% (67-78)
TOTAL 82 363 83% (73-90) 82% (77-85)
Note: 6/7 (86%) adenomas with HGD/CIS were also positive
Itzkowitz et al, Am J Gastroenterol 103:2862, 2008
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2nd MultiCenter sDNA Study
sDNA
Positive
(%)
Hemoccult-II
Positive (%)
HOSensa
Positive (%)
P value
SDT-1 20 (14-26) 11 (6-16) 21 (15-27) NS
SDT-2 46 (38-55) 16 (10-22) 24 (17-31) <0.001
Cancer 58 (36-80) 47 (25-70) 63 (41-85) NS
Adenoma >1 cm 46 (35-54) 10 (4-15) 17 (9-24) <0.001
Normal 16 (8-24) 4 (1-11) 5 (1-13) 0.03
• 3,764 asymptomatic, average-risk subjects over age 50; 22 centers
• Stool DNA assay compared to Hemoccult-II & HemoccultSensa
Stool DNA test:
• SDT-1: MuMu22+DIA
• SDT-2: K-ras, APC scan, methyl-vimentin (better adenoma markers)
Ahlquist et al. Ann Intern Med 149:441, 2008
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Stool DNA Test Sensitivity for
Screen-Relevant Neoplasia (n=142)
0
10
20
30
40
50
60
70
1 2 3 1 2 3 1Stool n
Hemoccult HemoccultSensa Stool DNA
* P<0.0001 vs Hemoccult or HemoccultSensa *
%
Ahlquist et al, Ann Int Med 149:441, 2008
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New Stool DNA Methylation Markers
Marker Sensitivity Specificity
Cancer Adenoma
SFRP2 63-94% 12-62% 77-100%
SFRP1 84% 100% 86%
NDRG4 53-61% -- 93-100%
TFPI2 76% 21% 79-93%
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New Stool DNA Assay:
Digital Melt Curve Assay
DMC Exact V. 1.1 Hemoccult-II Hemoccult-Sensa
Sensitivity (AAP)* 59% 26% 7% 15%
Specificity 92% 100% 92% 92%
• Analyzed 27 advanced adenomas with k-ras mutation
Zou et al. Gastroenterology 136:459, 2009
• Adenomas >2 cm: 8/10 (80%)
• Adenomas with HGD: 5/5 (100%)
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Stool DNA: Cost Effectiveness
With Perfect Adherence Reduction in
CRC Incidence
Reduction in
CRC Mortality
No screening -- --
FOBT 49% 66%
sDNA test (V 2.0) q 3 yrs 43% 63%
FIT 66% 78%
Colonoscopy 73% 80%
• FOBT ($15), FIT ($22), Stool DNA ($300), C’scopy ($920)
• FIT dominated other stool tests.
• sDNA V2 (with 100% adherence) more effective when per-
cycle FIT adherence fell below 50%
Parekh et al. Aliment Pharmacol Ther 27:697, 2008
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Conclusions
• Newer stool DNA tests:
• Are much less complex
• Are less expensive
• Can theoretically be run by local laboratories
• Are showing promise for detecting important
adenomas
• The future:
• newer assays/markers under development
• reducing cost