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

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

Oct 22, 2022 93 likes •363 views

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

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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

steven.itzkowitz@mountsinai.org

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SLIDE 2

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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Carcinoma Early 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 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% 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, 2005 2 Whitney et al. J Mol Diagn 6:386, 2004 3 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 p53

70-85% 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)

10 20 30 40 50 60 70 1 2 3 1 2 3 1 Stool n  Hemoccult HemoccultSensa Stool DNA

* P<0.0001 vs Hemoccult

  • r 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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SLIDE 23

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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SLIDE 24

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