Measuring Biomarker Stability in Frozen Tissue · PDF file0 10 20 30 40 50 60 70 Air - 10 Air...

Measuring Biomarker Stability in Frozen Tissue

Post-acquisition variables:• Time at room

temperature• Temperature of room• Type of fixative• Time in fixative• Rate of freezing• Freeze / Thaw• Size of aliquots• Storage temperature• Storage duration• Storage in vacuum• Processing methods

NCI Best Practices for Biospecimen Resources

• Technical Operations Best Practices– Collection, processing, storage, retrieval, use– Collection and managing clinical data– QA/QC– Biosafety– Informatics, inventory control and tracking

• Ethical, Legal and Policy– Custodianship– Informed consent– Privacy protection– Access to biospecimens and data– Intellectual property and resource sharing

Principals of Evidence Based Biospecimen Research

• What practices affect the quality of a specimen?

• Can they be measured?• Do the measurements have meaning to

data acquired in research testing?

Frozen Tissue

• Still the pristine specimen carrying intact non-chemically altered DNA, RNA, and proteins.

• Will be needed for biomarker discover.• May be needed for future clinical

information that can’t be resolved by robust assays.

Sample Packaging and Storage

• Can the quality of frozen specimens be protected with more stringent packaging?

• What is the duration of protection?

Questions• Oxidation - RNA, DNA, &

proteins

• Dehydration• Morphological deformation

• Residual and active enzyme activity

• Storage container, temperature, length of storage

Operational Variables• Sample size• Freezing rates• Packaging analysis

– Sealer– Pouch

• Tissue container

Polymer Laminate Diagrams

Proposed Solution1. Foil laminate (high

barrier packaging)2. Embedding cassettes:

protect tissue from distortion

3. Modified atmosphere packaging: vacuum or inert gas, i.e., argon

30Embedding tray in bag (vacuum)

30Barrier bag (no vacuum)

26Barrier bag (vacuum)

3030 mil clamshell tray in bag (vacuum)

2530 mil clamshell

2320 mil clamshell

70Standard 5 ml tube

50CRE tray in standard bag

Time to Freeze (sec)Container

Based on the exhaustion of LN2 boiling

Vessel Freezing Periods

Sample Dissection for a Single Casemucosal tissue sample

200 mg pieces 200mg pieces 200mg pieces

Baseline ArgonAir (cryovial) Vacuum

(-10C -20C -80C) (-10C -20C -80C)(-10C -20C -80C)

1 2 3 6 12

Months

Months

1 2 3 6 12

1 2 3 6 12

Months

1 2 3 6 12

Months

1 2 3 6 12

Months

1 2 3 6 12

Months

Months

1 2 3 6 12

Months

1 2 3 6 12

Months

1 2 3 6 12

Sample Dissection

100 mg

3 gram tissue sample

200 mg frozen

Total mRNA RIN#

qRT-PCR Biomarkers

Histology100 mg

Immunohistochemistry

Digital Imaging & Analysis

formalin fixation

Biomarkers GAPDH - Glyceraldehyde 3-phosphate dehydrogenase

(message)– implicated in non-metabolic processes, transcription

activation, initiation of apoptosis, and ER to Golgi vesicle shuttling – house keeping gene

CEA – glycoprotein-cell adhesion (protein & message)– CEA is a tumor marker to identify recurrences– In humans, the carcinoembryonic antigen family consists of

29 genes, 18 of which are normally expressed.CDX2 (protein and message)

– CDX2 expression is maintained in the adult small and large intestinal epithelia

– CDX2 expression is induced or up-regulated in pathologic states

CK20 (protein)– cytokeratin, major cellular protein of mature enterocytes

and goblet cells and is specifically expressed in the gastric and intestinal mucosa.

Elongation ratio (Epithelial cells: red; Stromal cells: yellow)

Courtesy of Sanford Barsky, M.D., Ph.D.

CEA

CK20 CDX2

0

10

20

30

40

50

60

70

Air -10

Air -20

Air -80

Arg -10

Arg -20

Arg -80

Vac -10

Vac -20

Vac -80

CDX2 % PosCDX 2 Intensity

0

10

20

30

40

50

60

Air -10

Air -20

Air -80

Arg -10

Arg -20

Arg -80

Vac -10

Vac -20

Vac -80

CEA % PosCEA Intensity

0

10

20

30

40

50

60

Air -10

Air -20

Air -80

Arg -10

Arg -20

Arg -80

Vac -10

Vac -20

Vac -80

CK20 % PosCK20 Intensity

Degree of change between different packaging methods versus storage temperature for CDX2, CEA, and CK20

CDX2 CEA

CK20

qRT-PCR Baselines

10

15

20

25

30

1 2 3 4 5 6 7 8 9

Subjects

Cyc

le T

hres

hold

CDX2

CEA

GAPDH

# Cycles / Time point of storage - Baseline # cycles

Measurement of Change in mRNA

RIN Degradation Varying Temperature at Month 3

0

12

34

5

67

89

10

T0 T3 -10 T3 -20 T3 -80

Temperature

RN

A I

nteg

raty

Num

ber

A IR

A RGON

V a cu u m

Storage [-10C] : Packaging [Air]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyl

ce C

hang

e

Month 1 Month 2 Month 3

Storage [-20C] : Packaging [Air]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyc

le C

hang

es

Month 1 Month 2 Month 3

Storage [-80C] : Packaging [Air]

-6.00-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyc

le C

hang

e

Month 1 Month 2 Month 3

Changes in Biomarker qRT-PCR

Storage [-20C] : Packaging [Air]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2qRT-

PCR

Cyc

le C

hang

Month 1

Month 2Month 3

Storage [-20C] : Packaging [Argon]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyl

ce C

han

Month 1Month 2

Month 3

Storage [-20C] : Packaging [Vacuum]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2qRT-

PCR

Cyc

le C

hang

e

Month 1Month 2Month 3

Storage [-80] : Packaging [Air]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2qRT-

PCR

Cyc

le C

hang

e

Month 1Month 2Month 3Month 6

Storage [-80C] : Packaging [Argon]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyc

le C

han

Month 1Month 2

Month 3

Month 6

Storage [-80C] : Packaging [Vacuum]

-4.00-2.000.002.004.006.008.00

10.0012.0014.0016.00

GAPDH CEA CDX2

qRT-

PCR

Cyc

le C

han

Month 1

Month 2

Month 3

Month 6

Biomarker Measurement Varying Temperature at Month Three

15

20

25

T0 -10 -20 -80Temperature (°C)

Cyc

le T

hres

hold

AIR GAPDH

ARGONGAPDH

VacuumGAPDH

AIR CEA

ARGON CEA

VacuumCEA

GAP-DH Cycle Threshold in Air

y = 17.345e0.054x

R2 = 0.845

y = 16.462e0.018x

R2 = 0.402

y = 16.667e0.102x

R2 = 0.868

15

16

17

18

19

20

21

22

23

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Timepoint

Cyc

le T

hres

hold

Predicted -10 Observed -10 Predicted -20 Observed -20Predicted -80 Observed -80

Air (cryovial) Packaging(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Perc

ent C

hang

e

GAPDH CEA CDX2

Argon Packaging(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Perc

ent C

hang

e

GAPDH CEA CDX2

Vacuum Packaging(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Perc

ent C

hang

e

GAPDH CEA CDX2

GAPDH(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Perc

ent C

hang

e

Air Argon Vacuum

CDX2(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Per

cent

Cha

nge

Air Argon Vacuum

CEA(-80C)

-10

-5

0

5

10

15

1 2 3 6 12

Perc

ent C

hang

e

Air Argon Vacuum

Conclusions / Future Studies• Degradation occurs at about 5% in the first 12

months according to percent change in the original quantity of message

• No difference in packaging seen at this time • Continue to measure the duration of time and

packaging for affects in tissue quality• Varying types of biomarkers need to be tested• Compare fixation methods to the frozen tissue

AcknowledgmentsDepartment of Pathology• Dr. Yufang Tang • Dr. Sanford Barsky• Dr. Kurtis Yearsley• Dan Rohrer • Susie Jones• Mary Marin • Susan Long • Nehad Mohamed

Department of Agriculture Engineering

• Dr. Gönül Kaletunç• Jason Baker• Wes Halderman*• Sara Kallio• Lily Meyer

Thank You!