Presented by: Thomas C. Tucker, PhD, MPH Associate Director Kentucky Cancer Registry and

1. Using the Kentucky Cancer Registry as a Population-based Virtual Tissue Repositoryto Advance the Science of Cancer Research Presented by: Thomas C. Tucker, PhD, MPH Associate Director Kentucky Cancer Registry and Rachel Maynard, BHS, CTR VTR Project Manager

2. Topics to be discussed • How can central cancer registries improve the science of cancer research? • What is a population-based Virtual Tissue Repository? • Example 1: Using the Kentucky Cancer Registry as a virtual Tissue Repository • Example 2: Using the Kentucky Cancer Registry as a virtual Tissue Repository • Current research using the Kentucky Cancer Registry as a virtual Tissue Repository • How this can be helpful to you

3. Two important concepts • Internal validity • External validity

4. Internal Validity • When differences between the experimental (exposed) group and the control group are completely accounted for, the study is said to have internal validity and causal inferences can be made. • In other words, it is possible to determine whether the exposure causes some outcome (disease, etc.). • Many have argued that “randomization” was the most important scientific advance of the 20th century. • Why is it that the findings from randomized clinical trials with internal validity almost never have the same effect when they are applied to general populations?

5. External Validity • When the findings from a research project or study can be generalized to some defined population, they are said to have external validity. • Population-based cancer registries provide the tools to explore external validity and many argue that moving from studies with strong internal validity to studies with strong external validity is the next step in advancing our scientific understanding. • The continuum from research with strong internal validity to studies with strong external validity is also part of “Translational Research”.

6. Population-based cancer registries can enhance cancer research by providing tools that lead to studies with strong “external validity”.

7. Using the Registry as a Population-Based Sample Frame Geographic Area Covered by the Population-Based Cancer Registry Cancer cases occurring each year Cancer research with strong “external validity” (the ability to generalize the findings to the underlying population) Information gathered on each cancer case The Population-Based Cancer Registry A scientific sample of data from cancer patients representing the population covered by the registry is provided to the researcher

8. Using the Registry as a Population-Based Virtual Tissue Repository Geographic Area Covered by the Population-Based Cancer Registry Cancer research with strong “external validity” (the ability to generalize the findings to the underlying population) Pathology Labs The Population-Based Cancer Registry Formalin fixed paraffin imbedded tissue samples are collected from the labs by the Registry for a population-based sample of cases A population-based sample of cancer case data and tissue are provided to the researcher

9. Pre-Invasive Cervical Cancer HPV Genotyping Study: Research Questions • Are the HPV genotypes different for non-Appalachian white women diagnosed with pre-invasive cervical cancer (CIN-3) compared to Appalachian white women? • Are the HPV genotypes different for non-Appalachian white women diagnosed with pre-invasive cervical cancer (CIN-3) compared to non-Appalachian black women? • Are the HPV genotypes different for white women diagnosed with CIN-3 compared to whitewomen diagnosed with AIS?

10. Study Population • All Kentucky women age 18 or older diagnosed with CIN-3 or AIS between January 1, 2009 and December 31, 2012 were available for this study. • The cases were divided into four strata (non-Appalachian white women, non-Appalachian black women, Appalachian white women and AIS cases). • A total of 4903 pre-invasive cervical cancer cases fell into one of these strata. • 3099 were non-Appalachian white women diagnosed with CIN-3. • 290 were non-Appalachian black women diagnosed with CIN-3. • 1360 were Appalachian white women diagnosed with CIN-3. • 154 were white women diagnosed with AIS. • The recruitment goal was to obtain tissue specimens from a random sample of 150 cases in each strata (for a total of 600 cases). A random sample of 200 cases was drawn from each strata except AIS to allow replacement for cases for which tissue could not be obtained.

11. Study Procedures • The Kentucky Cancer Registry (KCR) served as the “Honest Broker” for this study. • KCR solicited the required tissue blocks from each of 48 pathology labs where the tissue was stored. • Tissue blocks were sent directly to KCR, the blocks were anonymized and given a unique code. • The coded blocks were cut by the Markey Cancer Center, Biospecimens and Tissue Procurement Research Lab and the tissue specimens sent to CDC for genotyping. • The original blocks were returned to the contributing lab by KCR. • All of the HPV genotyping was done at CDC. • The HPV genotype or types for each specimen and the associated unique code were then returned to KCR and KCR linked HPV genotype information with the pre-invasive cervical cancer databaseto create a research data set.

12. Pre-Invasive Cervical Cancer HPV Genotyping Study: Tissue Procurement (2009-2012)

13. Pre-Invasive Cervical Cancer HPV Genotyping Study:Research Question 1

14. Pre-Invasive Cervical Cancer HPV Genotyping Study:Research Question 2

15. Pre-Invasive Cervical Cancer HPV Genotyping Study:Research Question 3

16. Conclusions Appalachian women compared to non-Appalachian women • There are no significant differences between non-Appalachian white women diagnosed with CIN-3 compared to Appalachian white women African American women compared to white women • HPV 16 alone or with any other HPV type was significantly more common among white women diagnosed with CIN-3 compared to black women. • In contrast, HPV 35 alone or with any other HPV type was significantly more common among black women diagnosed with CIN-3 compared to white women. Women diagnosed with CIN-3 compared to women diagnosed with AIN. • HPV 18 was significantly more common among women diagnosed with AIN compared to women diagnosed with CIN-3. • In contrast, HPV 31 was significantly more common among women diagnosed with CIN-3 compared to women diagnosed with AIN.

17. A retrospective cohort study using the registry as a virtual tissue repository Purpose of the Study To explore 5 distinct proteins associated with breast cancer recurrence and to determine whether female breast cancer patients who were disease-free following surgery but subsequently had a recurrence of their breast cancer within five years had altered levels or activity of one or any combination of five proteins at the time of their first surgery compared to women who were disease-free and did not have a recurrence.

18. Causal Relationships A D (Necessary & Sufficient) A B D (Necessarybut not Sufficient) C & &

19. The Five Proteins Study • Par-4: pro-apoptotic tumor suppressor protein downregulated during breast cancer recurrence • Wnt/b-catenin: signaling pathway induces epithelial-mesenchymal transition (EMT) and promotes metastasis • SNAIL, TWIST: transcription factors, promote EMT promote cancer progression (metastasis) elevated in metastatic and recurrent tumors • c-Abl, Arg: tyrosine kinases, oncoproteins promote survival, proliferation, and metastasis activity levels measured by pCrkL activity

20. A Retrospective Cohort Study 2000-2007 2012 Using the Registry, 479female cancer patients treated surgically between 2000 and 2007 for their 1stbreast Ca. and determined to be disease free were identified. Tissue blocks from the initial surgery were obtained for all of these patients, TMAs were constructed and stained to determine activity levels for each protein. This cohort was then traced forward in time to determine which patients recurred and which did not. Comparisons were made between activity levels of each protein among the primary tumors of recurrent patients (62) and the non-recurrent patients (417). For patients that recurred, comparisons were made between the tumor tissue at first surgery (the primary tumor) and the tissue taken at the time of recurrence. Tissue at the time of recurrence was only available for 22 patients.

21. Results PROTEIN EXPRESSION IN PRIMARY TUMORS FROM RECURRENT VS. NON-RECURRENT PATIENTS H: Significantly high in the primary tumor of recurrent patients L: Significantly low in the primary tumor of recurrent patients

22. Results PROTEIN EXPRESSION IN RECURRENT VS. PRIMARY TUMORS H: Significantly high at the time of recurrence in the tissue sample of patient who recurred compared to their primary tumors

23. Results pCrkL EXPRESSION IN RECURRENT VS. PRIMARY TUMORS

24. Conclusions • Elevated levels of Twist, and low or not activated c-Abl/Arg (pCrkL) in HER2-/ER+/PR+ breast tumors may potentially serve as a novel biomarker for the recurrence of breast cancer. • c-Abl/Arg was activated and over expressed in the tumors of patients at the time of recurrence. Inhibiting c-Abl/Arg activation may potentially prevent recurrence. • It is difficult to imagine doing this study without using a population-based approach.

25. Current and Pending VTR Studies • NCI Pancreatic Cancer Short and Long Term Survivors Study • NCI Breast Cancer Short and Long Term Survivors Study • Activity Levels on ABL/ARG among recurrent Melanoma Patients • Genomic Sequencing of Pediatric Brain Cancers

26. How does the VTR impact youas a registrar?

27. Examples of Biospecimens for VTR • FFPE tumor blocks • Protein expression • DNA/RNA detection

28. Examples of Biospecimens for VTR • Histology slides • Pathology Review • Immunohistochemistry • Digital Images • Quality control • Digital pathology research (WSI-Whole Slide Imaging)

29. Examples of Biospecimens for VTR

30. Examples of Biospecimens for VTR

31. Examples of Biospecimens for VTR

32. Examples of Biospecimens for VTR

33. What you can expect working with the Virtual Tissue Repository (hint: not more work!)

34. Custom Clinical Annotation • Not an audit • In addition to what has already been collected

35. Custom Clinical Annotation • Systemictherapy • Radiationtreatment • Co-morbidities • Recurrence details • Biomarkers

36. Standard 1.9 Clinical Research Accrual • All eligible, cancer-related clinical research studies involving human subjects must be approved by an internal or external Institutional Review Board (IRB) that is responsible for the oversight and review of the research study. • Subjects participating in clinical research studies must give their informed, written consent unless the requirement for written consent has been waived by the overseeing IRB. • The program is responsible for documenting enrollment and accrual of subjects to cancer-related clinical research studies within their program or once a patient is referred and enrolled onto an external research study.

37. Standard 1.9 Clinical Research Accrual • Patients eligible to meet this standard are: • Diagnosed and/or treated, and enrolled in a cancer-related clinical research study within the program or facility. • Diagnosed and/or treated, and enrolled in a cancer-related clinical research study within a staff physician’s office of the program or facility • Diagnosed and/or treated at the program or facility, then referred by your facility for enrollment onto a cancer-related clinical research study through another program or facility • Referred to your facility for enrollment onto a cancer-related clinical research study through another program or facility

38. Standard 1.9 Clinical Research Accrual Cancer-related research studies eligible for accrual under Standard 1.9 include: • Cancer-specific biorepositories or tissue banks • Prevention trials • Screening trials • Diagnostic trials • Treatment trials • Economics of care related to cancer care • Quality-of-life or supportive care trials • Genetic studies • Patient registries with an underlying cancer research focus *Subject accrual must be monitored and reported to the cancer committee each year by the Clinical Research Coordinator. The review of the report is documented in cancer committee minutes. *

40. Example of Communication from VTR Staff

42. How can you help? • Discuss the VTR Project in your Cancer Committee Meetings • Discuss the VTR Project with your pathologists • Pass along contact information for any questions your management, pathology, or laboratory staff may have

44. Questions? Contact Information: Thomas C. Tucker, PhD, MPH tct@kcr.uky.edu Rachel Maynard, BHS, CTR rmaynard@kcr.uky.edu 859-323-3553