OPTIMARK project preliminary results on antigenicity and integrity in

non-tumor tissue samples

Cristina Villena Pulmonary Biobank Consortium CIBERES. Scientific coordinator

Spanish National Biobank Network – ISCIII. Coordinator

Spanish National Biobank Network

0001 CIBERES- H. U. SON ESPASES 0002 IDIS- C.H.U SANTIAGO 0004 CNIO 0006 FJD 0007 NAVARRABIOMED 0008 MD ANDERSON 0010 HOSPITAL RAMON Y CAJAL 0011 IMIM 0013 H12O 0014 F-CIEN 0016 IDIBAPS 0017 FISABIO 0018 CLINICA UNIVERSIDAD DE NAVARRA 0019 IDIVAL 0020 HOSPITAL UNIV. PUERTA DE HIERRO 0021 BIOBANCO VASCO 0023 ISPA 0024 IDIBELL 0025 IDIPAZ 0027 IRBLleida 0029 INSTITUTO INV. S. PERE VIRGILI 0032 INSTITUTO INV. BIOMED. LA CORUÑA (INIBIC) 0033 INSTITUTO INV. BIOMED. GIRONA DR.JOSEP TRUETA 0034 INSTITUTO INV. SANITARIA GALICIA SUR 0036 HOSPITAL SANT JOAN DE DEU 0037 H. U. FUNDACION ALCORCON 0038 IMIB

0039 INSTITUTO ARAGONES CIENCIAS SALUD 0040 INSTITUTO INV. SANIT. H. CLINICO SAN CARLOS 0041 BBSPA 0042 INSTITUTO INV. SANITARIA GREGORIO MARAÑON (IiSGM) 0043 H LA FE 0044 U Salamanca 0045 HUGTiP 0047 VHIR 0049 INCLIVA 0050 HOSPITAL DE LA PRINCESA 0051 FIVO XXXX ISCIII -BioNER

OPTIMARK project

1. Identify the pre-analytical variables more relevant in tissues, which could affect the clinical biomarkers development.

OBJECTIVES

2. Develop and validate a common procedure to clarify the potential use of the samples in research, especially those without pre-analytical control.

3. To allow collections to be available to the scientific and biotechnological community, with a very good control of their pre-analytical variables, so that they can evaluate the sensitivity of their developing biomarkers to these factors.

Retrospective phase: sample conservation time

If we are able to find a sensitivity biomarker with the sample age, probably we could use it to evaluate the impact of other pre-analytical factors.

Colon (n=104) Brain (57)

Lung (76) Breast (85)

Stomach (17) Endometrium (35)

374 patients (30-65 y.o.; in brain up to 75 y.o. without vascular pathology)

Healthy tissue (non-tumor)

Flash-frozen and FFPE paired samples.

Checking non other relevant pathologies in the organ selected.

B: extraction time between 5 to 10 years

control: extraction time <1 year

A: extraction time between 1 to 5 years

C: extraction time between 10 to 20 years

D: extraction time >20 years

Groups

Experimental design

Biobanks sample suppliers (10)*

Colon (5) Brain (3) Lung (3) Breast (4) Endometrium (1) Stomach (1)

RNA Integrity analysis (9) Antigenicity analysis

Flash frozen Slides FFPE

Biobanks stainers IHQ Vimentin (2)

Biobanks stainers IHQ CD31 (2)

Biobanks stainers IHQ Ki67 (2)

Scanning center (system 1)

Scanning center (system 2)

Scanning center (system 3)

(a) (b)

Nuclear Cytoplasm Membrane

Ki67 Vimentin CD31

TTF-1 SDHB Beta-catherin

FOS BCL-2

A A

Antigenicity Colon Ki67 (nucleus)

CONTROL CONTROL

Intensity: +++ red, ++ orange, + yellow, 0 blue

B B

C C

D

Intensity: +++ red, ++ orange, + yellow, 0 blue

Antigenicity Colon Ki67 (nucleus)

Analysis Antigenicity Colon Ki67 (nucleus)

Intensity: +++ red, ++ orange, + yellow, 0 blue

Antigenicity Endometrium CD31 (membrane)

CONTROL A

B C

Analysis Antigenicity CD31 (membrane)

0

20

40

60

80

100

120

140

160

180

Brain Lung Colon Breast Endometrium Stomach

Control

A

B

C

D

CD

31

Ave

rage

Po

siti

ve In

ten

sity

Intensity: +++ red, ++ orange, + yellow, 0 blue

Antigenicity Lung TTF-1 (nucleus)

CONTROL A

B C

Analysis Antigenicity Lung TTF-1 (nucleus)

Regarding... RNA Integrity Number (RIN)

Tissue Biobank N

(%)

Patient age

(years)*

Obtention Warm Ischemia

Time (min)*

Cold Ischemia

Time (min)*

Lung

1, 2 41

(21,1)

53 ± 10

Surgery

22 ± 10

22 ± 10

Brain

3, 4 27

(13,9)

67 ± 11

Post mortem

NA**

NA

Colon

4, 5, 6, 7 49

(25,3)

58 ± 9

Surgery

17 ± 2

16 ± 5

Breast

4, 5, 7, 8 34

(17,5)

50 ± 8

Surgery

NA

18 ± 6

Endometrium

9 26

(13,4)

53 ± 7

Surgery

NA

61 ± 38

Stomach

10 17

(8,8)

47 ± 12

Surgery

NA

229 ± 285

*Average ± SD, **Non available

194 paired tissue samples

RNA Integrity Number (RIN)

Endom. Lung Brain Colon Breast Stomach

Error barr: +/- 1 SD

less than 1 year old

1-5 years old

5-10 years old

10-20 years old

RIN

ave

rage

+/-

SD

Acknowledgments

Villena C, Almenara I, Artiga MJ, Bahamonde O, Bermudo R, Castro E, De la Puente R, Escámez T, Esteva-Socias M, Fraga M, Jauregui-Mosquera L, Martin-Arruti M, Morente M, Peiró-Chova L,

Pons DG, Rejón JD, Ruiz-Miró M, Vieiro P, Zazo S, Villar V y Rábano A on behalf of R&D working group of Spanish Biobank Platform