Department of Pathology Inflammatory Bowel Disease Neoplasia Mary P. Bronner, MD Division Chief of Anatomic Pathology University of Utah
Department of Pathology Neoplastic Progression in Chronic Inflammatory GI Dz Inflammation Dysplasia Carcinoma
Department of Pathology Chronic Inflammatory GI Disease & Cancer • Barrett’s Esoph Esoph CA • HP Gastritis Gastric CA • Hepatitis B & C HCC • Ch Pancreatitis Panc CA • UC and Crohn’s Intestinal CA
Department of Pathology Ulcerative Colitis: A Paradigm
Department of Pathology
Department of Pathology Managing Cancer Risk in UC • Ignore it •“Prophylactic” colectomy • Colonoscopic surveillance for dysplasia / early carcinoma
Department of Pathology Optimal Colonic Biomarker • Pancolonic distribution • Predate incurable cancer • Objective • Sensitive, Specific, PPV, NPV
Gold Standard Biomarker: Dysplasia
Department of Pathology Dysplasia: Problems • Sampling • Distinction from reactive change • Observer variation • Natural history incompletely understood
Department of Pathology Adequate Bx Sampling Histology Dysplasia Cancer No. Bx’s for 90% confidence 33 34 No. Bx’s for 95% confidence 56 64 From: Rubin CE, et al. Gastroenterology 1992;103:1611
Department of Pathology UC Surveillance Protocol X X X X X X X X X X X X 10 cm X X X X X X X X X X X X X X X X X X X X X X X X X X X X 5 cm X X X X
Department of Pathology Rectosigmoid Predominance of Ulcerative Colitis Cancer Location of Colorectal Carcinoma RS D T A/C 52% 12% 21% 15% Choi PM. Gastroenterology 1993;104:666 Summary of 5 Studies
Department of Pathology Dysplasia: Problems • Sampling • Distinction from reactive change • Observer variation • Natural history incompletely understood
Department of Pathology
Department of Pathology
Department of Pathology Dysplasia: Problems • Sampling • Distinction from reactive change • Observer variation • Natural history incompletely understood
Department of Pathology Outcome of 40 UC LGD Patients • 78% no progression, avg f/u 5y (1-13 y) • 22% HGD, avg f/u 1.5 y (1-3 y) • ≥3 LGD biopsies: 9x progression risk • 2 non-compliant patients developed Dukes’ A cancer Brentnall, Bronner, et al. Prospective study of progression of LGD in UC. Inflamm Bowel Dis 18:2240-6, 2012.
Dysplastic Field: Limited
Department of Pathology Better Biomarkers of Cancer Risk Greatly Needed!
Department of Pathology
Department of Pathology Chromosomal Instability? • FCM Aneuploidy - Detects gross chromosomal instability • CGH - Detects clonal gains and losses of chromosomal regions • FISH - Detects clonal and non-clonal chromosomal abnormalities
Department of Pathology Biopsy Sampling: Flow Cytometry Dysplasia Cancer No. Bx for 90% confidence 20 8 No. Bx for 95% confidence 30 14 Rubin CE, et al. Gastroenterology 1992;103:1611
Morphologic + DNA Ploidy Neoplastic Field: Larger
Department of Pathology Metaphase Comparative Genomic Hybridization in UC 39% (15/38) of diploid bx’s near dysplasia or cancer showed CGH detectable alterations Performed in collaboration with F. Waldman, UCSF
Department of Pathology Array-based Comparative Genomic Hybridization (CGH) Chromosomes replaced by ordered array of targets Karyotyping of metaphase spreads not necessary Greatly increased resolution
Department of Pathology Array CGH in UC • 100% (9/9) UC-progressors extensive chromosomal gains and losses • FISH and PCR targets identified Bronner MP, Mod Pathol 2010;23:1624-33
Department of Pathology Ulcerative Colitis A-CGH PROGRESSORS NON-PROGRESSORS Gain Loss Bronner MP, Mod Pathol 2010;23:1624-33.
Department of Pathology BAC CGH Whole Genome Log2-Ratio Plots of All Chromosomes Normal Non-UC Control UC Non-progressor UC Progressor UC Progressor Bronner MP, Mod Pathol 2010;23:1624-33.
Morphology + DNA Ploidy + CGH Neoplastic Field: Larger Still
Department of Pathology Non-Clonal Change in UC: Wider Field? • DNA Flow & CGH detect clonally expanded abnormalities only • Larger fields of non-clonal instability? Detectable in negative biopsies, even from rectum? • Assessed by Fluorescence In Situ Hybridization (FISH)?
Department of Pathology UC FISH Hypothesis: UC progressors differ from UC non-progressors using non-clonal genomic instability biomarkers on single negative rectal biopsies
Department of Pathology FISH • Interphase nuclear suspensions placed on glass slide • Locus specific probes (Chrom 8, 11, 17, 18) & centromeres (green and red) • Red and green FISH spots counted per 100 nuclei
Normal Cells Abnormal Cells
Department of Pathology Control Normal Colon FISH Chrom11 Probe Set 90 100 % of Nuclei 80 60 40 20 1 0 2 0 1 2 0 2 2 0 0r2g 1r1g 1r2g 1r3g 2r1g 2r2g 2r3g 2r4g 3r2g 3r3g Red and Green Signal Counts
Department of Pathology Diploid Neg Rectal Bx UC Progressor Chrom11 Probe Set 100 74 80 % of Nuclei 60 40 11 8 20 4 2 0 0 0 1 1 0 0r2g 1r1g 1r2g 1r3g 2r1g 2r2g 2r3g 2r4g 3r2g 3r3g Red and Green Signal Counts
Department of Pathology FISH in Ulcerative Colitis p 0.001 Non-UC controls N=10 p 0.001 UC non-progressors N=18 10 UC progressors N=12 % cells with FISH 8 p 0.001 abnormalities p=0.001 6 4 2 0 Arm Centromere Arm Centromere loss gain loss gain Bronner MP, et al. Am J Pathol 2008;173:1853.
Department of Pathology ROC Analysis of FISH Biomarkers 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 8q: c-myc 11q: CyclinD1 Sensitivity Sensitivity 0.0 0.2 0.4 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Specificity 1-Specificity 1-Specificity 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Sensitivity 18q: DCC Sensitivty 17p: p53 1.0 0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 1.0 Specificity 1-Specificity 1-Specificiy Bronner MP, et al. Am J Pathol 173:1853-1860, 2008
Department of Pathology ROC Analysis of FISH Biomarkers All 4 chromosomes combined 1.0 0.8 Sensitivity 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1-Specificity Bronner MP, et al. Am J Pathol 2008;173:1853.
Department of Pathology Consequences of Shortened Telomeres • Sticky chromosomal ends • Bridge-breakage-fusion cycles • Chromosomal arm losses/gains and dicentrics Studied by peptide nucleic acid (PNA) probe ISH or RT PCR
Department of Pathology
Department of Pathology Telomere Shortening in UC Epithelial: Stromal Telomere Ratio p=0.001 1.4 p=0.08 1.2 p=0.02 1.0 0.8 0.6 0.4 0.2 0 Non-UC Non- Progressors control progressors O’Sullivan J, et al. Nat Genet 2002;32:280-284.
Anaphase Bridges in UC p=0.0002 0.03 p=0.011 % Anaphase Bridges 0.025 0.02 0.015 0.01 0.005 0 Non- Non-UC Progressors progressors control Bronner MP, et al. Am J Pathol 173:1853-1860, 2008
Department of Pathology NGS miRNA bioclassifier of UC patients at increased risk of colon cancer • Why miRNAs? – Small size (~21nt) more stable, less ribonuclease degradation – Readily detectable in FFPE and stained slides – Important roles in immune regulation
miRNAs misregulation in UC-P, UC-NP Department of Pathology UC-NP vs. nl (26 UC-P vs. nl (29 • Linear discriminant miRNAs) miRNAs) analysis to predict UC-P vs. UC-NP • Robust candidate panel 15 18 11 selected for RT-PCR & additional cohort validation UC_NP UC_P miRNA Normalized Read count Panel 1000 UC-NP UC-P 9/10 10/10 100 mir UC_NP+UC_P
Histology + DNA Ploidy + CGH +FISH +Telomeres +Ana Bridges +miRNA Neoplastic Field: Entire Colon
Department of Pathology
Department of Pathology UC Polypoid Dysplasia You’re dalmed if you do, and dalmed if you don’t Teri Brentnall,MD
Department of Pathology
Department of Pathology Dysplasia in UC vs Adenoma • No clinical features • No endoscopic features • No pathologic features • No molecular tests
Department of Pathology HOWEVER • If the lesion can be demonstrably completely removed endoscopically • Has only Low-Grade Dysplasia • There is no other dysplasia on adequate sampling • Then, careful follow-up may be considered
Department of Pathology UC Dysplasia Management Continue Surveillance with adequate sampling: – Single site LGD while in surveillance – Indefinite of negative for dysplasia
Department of Pathology UC Dysplasia Management Consider Colectomy: – Multiple LGD sites – LGD on more than one endoscopy – LGD at initial colonoscopy – Excessive inflammatory polyps
Department of Pathology Inflammatory Polyps
Department of Pathology UC Dysplasia Management Colectomy Indicated: – HGD – Endoscopically unresectable dysplastic lesion
Department of Pathology Conclusions • Molecular alterations are widespread in UC, CD, CP, HP, HCV • Single non-dysplastic bx alterations show promise for reducing sampling error • Paradigm for cancer in chronic inflammatory disease
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