1. Molecular markers to aid in early diagnosis of pancreatic cancer Michael Goggins, MD Professor of Pathology, Medicine and Oncology Johns Hopkins Medical Institutions, Baltimore, MD “7th Annual Symposium on Gastrointestinal Cancers " St. Louis, Mo, 9/20/08

2. Disclosure Dr. Goggins has licensing agreements with Oncomethylome sciences for several DNA methylation markers

3. Early detection of asymptomatic disease Early diagnosis of symptomatic disease

4. positive margin negative margin poor or undifferentiated well or moderate no positive nodes positive nodes < 3 cm ≥ 3 cm p<0.0001 Survival for Ductal Pancreas Adenocarcinoma Tumor Diameter Margin Status Lymph Node Status Histologic Grade

5. Early detection of small asymptomatic neoplasms may result in cure Best outcome: 79 patients wth small, asymptomatic, < 1 cm cancers 5- year survival 100% after surgery if PC limited to duct epithelium (CIS?) Ariyama 1997

6. What is the natural history of patients who present with symptoms and are ultimately diagnosed with pancreatic cancer? We do not have much quantitative information on the obstacles to diagnosis for patients with pancreatic cancer

7. When there are delays in diagnosis, what are their causes?

8. Chari et al, AJR 2004;182:897 Pancreatic CT abnormalities up to 18 months prior to a diagnosis of pancreatic cancer

9. How many patients would have a significantly improved outcome if they were optimally diagnosed as soon as symptoms presented?

10. Markers of Pancreatic Neoplasia What performance characteristics are needed for a(serum) molecular marker for the Diagnosis of pancreatic cancer

11. The performance characteristics of a marker often vary by population (disease stage, heterogeneity): Ca19-9 PC AmpCA CP islet ca controls JHU unpublished Sensitivity for resectable PC =65% (Sensitivity for unresectable PC=80%)

12. 900 (TP)200 (FP) 100 (FN)800 (TN) DiseaseNo Disease Positive Negative Test Sensitivity: 90% Specificity: 80% (for cancer) PPV: 900/1100=81% NPV: 800/900=91% 1,000 1,100 900 Disease prevalence 50% (e.g. pancreatic mass)

13. 182 (TP)360 (FP) 18 (FN)1540 (TN) DiseaseNo Disease Positive Negative Sensitivity: 90% Specificity: 80% (for resectable cancer) PPV: 182/542=34% NPV: 1540/1558=98% 1,800 200 542 1558 Disease prevalence ~10%, e.g. mid-back pain, wt loss

14. What performance characteristics are needed for a molecular marker for Screening for pancreatic cancer?

15. General Population: Age 65: 5 year Risk PPV NPV Surveillance Epidemiology and End Result

16. 18 (TP)396 (FP) 2 (FN)1584 (TN) DiseaseNo Disease Positive Negative Sensitivity:90% Specificity: 80% PPV: 18/414=4.3% NPV: 1584/81856=99.8% 1,98020 1586 414 Disease prevalence, 1%

17. New Onset Diabetics: 3 yr PC risk PPV NPV Chari et al. Gasto 2005

18. Family Hx PC 3 1 st -degree relatives: 3-yr PC risk PPV NPV Klein et al. Cancer Research 2004

19. PPV NPV Klein et al. Cancer Research 2004 Family Hx PC 2 1 st -degree relatives: 3-yr PC risk

20. Is this the right question to ask of our markers? What performance characteristics are needed for a molecular marker to Screen for pancreatic cancer?

21. What about the performance characteristics for a molecular marker of neoplastic precursors?

22. 9510 590 DiseaseNo Disease Positive Negative Test: Sensitivity: 95% Specificity: =90% (EUS or MRI) PPV: 95/105=91% NPV: 90/95=95% 100 105 95 Strong Family Hx of PC, prevalence: 10% (IPMN)

23. Cancer of the Pancreas Screening (CAPS) clinical trials Who are we screening? Asymptomatic High risk individuals with a strong family history of pancreatic cancer and certain germline mutation carriers CAPS1:1999 CAPS2: 2001 CAPS3: 2006 CAPS4: 2008

24. Familial PC Screening Programs PopnScreening Tests Diagnostic Yield (IPMN/PC) Canto, CGH 2004,n=36 FPC (> 3) PJS EUS 5.8% Canto, CGH 2006,n=78 FPC (> 3) PJS EUS + CT10.2% Kurtz, DDW 2007,n=66 FPCCT/MRI +/- EUS 7.6% Poley, DDW 2007,n=46 FPC, PJS, FAMM, BRCA1/2 EUS 23.9% Other studies ongoing, eg. Brentnall et al (Seattle), Europac, U Pitt, Zubarik et al, (Vt)

25. Cancer of the Pancreas Screening Study (CAPS) 3 CAPS 3 – 1 st national American multicenter screening study  EUS/CT/MRI + biomarkers (juice)  Hopkins, Mayo, MDACC, Dana Farber, UCLA  www.clinicaltrials.gov www.clinicaltrials.gov  Email: caps3@jhmi.edu  lizst.onc.jhmi.edu/caps3

26. CAPS 4 Single center, long-term screening and surveillance (V foundation) Evaluate expanded eligibility –1 FDR, 1 SDR (eg. parent and grandparent) –BRCA2 mutation carriers Biomarker discovery

27. 3 months 10 mm 12 months 18 mm baseline

28. Lobular CP like changes on EUS

29. Extensive PanIN  Lobulocentric atrophy Brune et al, AJSP, 2006

30. Is the identification of IPMNs and PanINs in asymptomatic individuals justified when we have not proven that surveillance of these lesions leads to improved outcome?

31. What molecular markers are on the horizon and can they help identify cancer or advanced neoplasia?

32. Molecular alterations in pancreatic cancer DNA: Somatic mutations Aberrant DNA methylation Chromosomal losses/gains RNAs: RNAs and microRNAs Proteins Peptides, glycopeptides Other eg. Autoantibodies Infiltrating pancreatic ca

33. Normal Duct PanIN 1APanIN 1B PanIN 2PanIN 3 Invasive AdenoCa Intraductal Papillary Mucinous Neoplasm (IPMN) Mucinous Cystic Neoplasm (MCN) Cystic Lesion AdenomaCarcinoma in situ Invasive AdenoCa

34. Research Activity Time 1985199019952000 2005 2010 2015 2020 Candidate Markers Pancreatic cancer research: Era of systematic discovery Translational Evaluation of markers 100% 0% Systematic Discovery (“Omics”)

35. The Pancreatic Cancer Genome

36. Data figure on the pc genome Number of somatic mutations in pancreatic cancer

37. Walter et al, Cancer Biol Ther, 2008 Gains Losses Pancreatic cancer chromosomal gains + losses

38. Quantifying mutant KRAS in Pancreatic juice: LigAMP Shi et al, Cancer Biol Ther, 2008 PC CP

39. Discovering the cancer methylome Methylome = genome wide DNA methylation patterns

40. Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 Chr11 Chr12 Log ratio

41. Hong et al, Modern Path in press Aberrant DNA methylation in IPMNs %

42. Quantitative Methylation analysis of ERCP Brushings of common bile duct strictures

44. QMSP ≥1 gene + 3 gene panel, >3% conc.n Sens (CI)Spec (CI)Accuracy (CI) Pancreatic adenocarcinoma4173.2 (58-84) a 86.4 (7-24) 81 (73-88) i Biliary tract cancer1080 (49-94) b 86.4 (7-24)86 (76-92) Cytology Pancreatic adenocarcinoma4119.5 (14-34) c 100 (95-100) 69 (60-77) Biliary tract cancer1030 (11-60) d 100 (95-100)92 (84-96) Cytology + QMSP Pancreatic adenocarcinoma4176 (61-76) e 86.4 (7-24)82 (74-88) h Biliary tract cancer1090 (60-96) f 86.4 (7-24)88 (79-94) QMSP of biliary and Pancreatic duct Brushings performed to diagnose strictures Parsi et al, Clin Gastro Hep, 2008

45. Pancreas juice sampling for markers

46. Quantifying pancreatic juice DNA methylation alterations 5 gene panel, quantified by QMSP, CAPS2 study population Cancer Res 2006:66:1208

47. Marker Discovering Pancreatic cancer expression patterns

48. Clin Cancer Res 2006;12:442 Candidate pancreatic cancer markers: Serum Macrophage inhibitory cytokine-1 (MIC-1) PC=pancreatic cancer CP=chronic pancreatitis Nl=normal ELISA

50. MicroRNA alterations in pancreatic cancer Hahn et al, Oncogene 2007;264442