Stool DNA Testing For Colon Cancer Steven Itzkowitz, MD, FACP, FACG, - PowerPoint PPT Presentation

Stool DNA Testing For Colon Cancer Steven Itzkowitz, MD, FACP, FACG, AGAF Professor of Medicine Mount Sinai School of Medicine New York, NY steven.itzkowitz@mountsinai.org

CRC Screening Guidelines: Average-Risk Adults Over Age 50 (ACS, US Multi-Society Task Force, ACR) 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 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

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.

Rationale for Stool DNA Testing: Mucocellular Layer Colon cancer Normal colon Courtesy: David Ahlquist, MD, Mayo Clinic

Molecular Markers of Colon Carcinogenesis Chromosomal Instability (e.g. FAP) • Aneuploidy • LOH • Tumor suppressor gene mutations 70-85% K-ras (3) DCC/18q genes P53 (8) APC (10) Normal Early Intermediate Late Carcinoma mucosa adenoma adenoma adenoma Long-DNA (DIA) 15% Microsatellite Instability (e.g. HNPCC) • Hypermethylation/mutation of DNA MMR genes • Target gene alterations (TGF b RII; others) BAT26

Version 1 Stool DNA Test: Collection Kit (with freezer pacs)

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

sDNA is Better than FOBT in Average-Risk Individuals 2,507 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% (p=0.003) 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

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

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

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, 2005 2 Whitney et al. J Mol Diagn 6:386, 2004 3 Chen et al. J Natl Cancer Inst 97:1124, 2005

PATHWAYS OF COLON CARCINOGENESIS Chromosomal Instability (e.g. FAP) • Aneuploid; LOH; Tumor suppressor gene mutations 70-85% K-ras DCC/18q genes p53 APC Normal Early Intermediate Late Carcinoma mucosa adenoma adenoma adenoma (MSS) 15% Microsatellite Instability (e.g. HNPCC) • Mutation/loss of DNA MMR genes; diploid Carcinoma • Mutations of key target genes (eg, TGF b RII) (MSI) 15% CpG Island Methylation; CIMP (e.g. HPS) • DNA methylation inhibits key gene expression • BRAF oncogene mutation Carcinoma Sessile Serrated Polyp (MSI-H) (SSP; SSA)

New Stool Collection Kit (with buffer)

Results of Version 1 Assay (MuMu22+DIA) Version 1 Version 1.1* (Imperiale, NEJM , „04) (with buffer and gel capture) No. % No. % Positive Positive 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

Version 2: Two Markers Sensitivity (n=40) Specificity (n=122) No. % No. % Positive Positive (95% C.I.) (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

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

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

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

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

2nd MultiCenter sDNA Study • 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) sDNA Hemoccult-II HOSensa P value Positive Positive (%) Positive (%) (%) 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 Ahlquist et al. Ann Intern Med 149:441, 2008

Stool DNA Test Sensitivity for Screen-Relevant Neoplasia (n=142) 70 60 * P<0.0001 vs Hemoccult * or HemoccultSensa 50 40 % 30 20 10 0 Stool n  1 2 3 1 2 3 1 HemoccultSensa Stool DNA Hemoccult Ahlquist et al, Ann Int Med 149:441, 2008

New Stool DNA Methylation Markers Marker Sensitivity Specificity Cancer Adenoma 63-94% 12-62% 77-100% SFRP2 SFRP1 84% 100% 86% 53-61% -- 93-100% NDRG4 76% 21% 79-93% TFPI2

New Stool DNA Assay: Digital Melt Curve Assay • Analyzed 27 advanced adenomas with k-ras mutation DMC Exact V. 1.1 Hemoccult-II Hemoccult-Sensa Sensitivity (AAP)* 59% 26% 7% 15% Specificity 92% 100% 92% 92% • Adenomas >2 cm: 8/10 (80%) • Adenomas with HGD: 5/5 (100%) Zou et al. Gastroenterology 136:459, 2009

Stool DNA: Cost Effectiveness Reduction in Reduction in With Perfect Adherence CRC Incidence 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

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