1. Using the Kentucky Cancer Registry as a Population-based Virtual Tissue Repository to 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
Examples of studies

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
A population-based sample of cancer case data and tissue are provided to the researcher
Formalin fixed paraffin imbedded tissue samples are collected from the labs by the Registry for a population-based sample of cases

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 white women 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 database to create a research data set.

12. Pre-Invasive Cervical Cancer HPV Genotyping Study: Tissue Procurement (2009-2012)
Table 1: Pre-Invasive Cervical HPV Genotyping Study Data Collection
Non-App White
Non-App Black
App White
AIS
Totals
Recruitment Goal
150
600
Total Specimens Collected
166
130
154
121
571
Insufficient Tissue to test 9 4 12 13 38
Total cases with HPV Genotyping
157
126
142
108
533

13. Pre-Invasive Cervical Cancer HPV Genotyping Study: Research Question 1
Are HPV genotypes different for Appalachian white vs non-Appalachian white females?
There were no significant differences in HPV genotype expression between Appalachian white females diagnosed with CIN-3 vs non-Appalachian white females.
To answer these research questions, a random sample of 200 cases were drawn from four population-based strata: Non-Appalachian white women diagnosed with CIN-3, non-Appalachian black women diagnosed with CIN-3, Appalachian white women diagnosed with CIN-3, and white women diagnosed with AIS. The goal of this study was to collect 150 tissue blocks from cases in each strata. The tissue blocks for cases in the sample are stored in 48 different clinical pathology labs throughout the state. This study is ongoing. However, what is impressive is the efficiency with which the tissues specimens have been collected. In 5 months, more than 80% of the required samples have been obtained. In previous efforts the clinical labs holding tissue samples were asked to stop their clinical production to cut tissue blocks, and prepare H&E slides. This was clearly problematic. Using the MCC Biospecimens and Tissue Procurement Shared Resource Facility, Central Research Lab for all of the processing has made the effort much more effective and avoided all of these issues.

14. Pre-Invasive Cervical Cancer HPV Genotyping Study: Research Question 2
Are HPV genotypes different for non-Appalachian white vs non-Appalachian black females?
HPV Genotypes
RACE
% with genotype
p-value
16 (alone or
White
62.42%
0.0114
with any other HPV)
Black
46.83%
16 alone
0.0074
31.75%
35 (alone or
1.91%
0.0009
11.90%
35 alone
1.27%
0.0035
8.73%
Any oncogenic HPV
29.94%
0.0131
except 16 & 18
44.44%
To answer these research questions, a random sample of 200 cases were drawn from four population-based strata: Non-Appalachian white women diagnosed with CIN-3, non-Appalachian black women diagnosed with CIN-3, Appalachian white women diagnosed with CIN-3, and white women diagnosed with AIS. The goal of this study was to collect 150 tissue blocks from cases in each strata. The tissue blocks for cases in the sample are stored in 48 different clinical pathology labs throughout the state. This study is ongoing. However, what is impressive is the efficiency with which the tissues specimens have been collected. In 5 months, more than 80% of the required samples have been obtained. In previous efforts the clinical labs holding tissue samples were asked to stop their clinical production to cut tissue blocks, and prepare H&E slides. This was clearly problematic. Using the MCC Biospecimens and Tissue Procurement Shared Resource Facility, Central Research Lab for all of the processing has made the effort much more effective and avoided all of these issues.

15. Pre-Invasive Cervical Cancer HPV Genotyping Study: Research Question 3
Are HPV genotypes different for white females with CIN-3 vs white females with AIS?
HPV Genotype
CIN-3 Vs. AIS
% with genotype
p-value
18 (alone or
CIN-3
4.7%
<0.0001
with any other HPV)
AIS
41.7%
18 alone
2.7%
36.1%
31 (alone or
9.7%
0.001
1.0%
31 alone
7.0%
0.0018
0.0%
52 (alone or
0.0126
52 alone
3.7%
0.0418
To answer these research questions, a random sample of 200 cases were drawn from four population-based strata: Non-Appalachian white women diagnosed with CIN-3, non-Appalachian black women diagnosed with CIN-3, Appalachian white women diagnosed with CIN-3, and white women diagnosed with AIS. The goal of this study was to collect 150 tissue blocks from cases in each strata. The tissue blocks for cases in the sample are stored in 48 different clinical pathology labs throughout the state. This study is ongoing. However, what is impressive is the efficiency with which the tissues specimens have been collected. In 5 months, more than 80% of the required samples have been obtained. In previous efforts the clinical labs holding tissue samples were asked to stop their clinical production to cut tissue blocks, and prepare H&E slides. This was clearly problematic. Using the MCC Biospecimens and Tissue Procurement Shared Resource Facility, Central Research Lab for all of the processing has made the effort much more effective and avoided all of these issues.

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 (Necessary but 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
Identify the study Population
Did Not Recur
Recurred
Tissue at the time of recurrence
Using the Registry, 479 female cancer patients treated surgically between 2000 and 2007 for their 1st breast 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
TWIST
SNAIL
PAR4
b-catenin
pCrkL
 Intensity
HER2+ (n=36)
HER2-/ER+/PR+ (n=282) H L
HER2-/ER-/PR+ (n=32)
HER2-/ER-/PR- (n=75)
 Allred Score
HER2+
HER2-/ER+/PR+
HER2-/ER-/PR+
HER2-/ER-/PR-
 Intensity X Percent
H: Significantly high in the primary tumor of recurrent patients
L: Significantly low in the primary tumor of recurrent patients

22. PROTEIN EXPRESSION IN RECURRENT VS. PRIMARY TUMORS
Results
PROTEIN EXPRESSION IN RECURRENT VS. PRIMARY TUMORS
TWIST
SNAIL
PAR4
b-CATENIN
pCrkL
Intensity H
Allred Score
Intensity x Percentage
H: Significantly high at the time of recurrence in the tissue sample of
patient who recurred compared to their primary tumors

23. pCrkL EXPRESSION IN RECURRENT VS. PRIMARY TUMORS
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 you as a registrar?
Examples of studies

27. Examples of Biospecimens for VTR
FFPE tumor blocks
Protein expression
DNA/RNA detection
Formalin-fixed paraffin embedded tumor blocks

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
Example of how a de-identified record with a whole slide image may look

30. Examples of Biospecimens for VTR
Example of how a de-identified record with a whole slide image may look

31. Examples of Biospecimens for VTR
Example of how a de-identified record with a whole slide image may look

32. Examples of Biospecimens for VTR
Example of how a de-identified record with a whole slide image may look

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
Systemic therapy
Radiation treatment
Co-morbidities
Recurrence details
Biomarkers
Not just first course therapy but also subsequent therapies that are not always captured in CPDMS, exact dosages of chemo drugs etc
*same thing on radiation therapy; not just first course but also subsequent therapies
*different studies ask for different things
*specific recurrence details
*specific biomarkers for example CA19-9 for pancreas

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.
*emphasize IRB approval, explain that UK is our governing body for IRB
*emphasize waiver of informed consent
*I will provide documentation for the accrual of research subjects,

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
Examples of categories patients would be eligible under

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
*examples of types of eligible studies that VTR studies may qualify as
*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. *

39. Screenshot from Cancer Answer forum and answer showing that VTR study will count towards accrual for living patients

40. Example of Communication from VTR Staff
Example of communication provided by VTR staff
*note John Doe counts twice-once for imaging study and once for genetic study

41. Mention desire to work with our regional partners as well.

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
Emphasize cancer committee discussion

44. Questions?
Contact Information:
Thomas C. Tucker, PhD, MPH
Rachel Maynard, BHS, CTR