Symposium: Cytological preparation for molecular analysis –

pre-analytical issues for EBUS TBNA specimens

FTA Cards: an additional tool for

NSCLC cells storing Pio Zeppa

Department of Medicine, “Scuola Medica Salernitana” University of Salerno, Italy

Disclosure statement: I have no conflicts of interest to declare

Lung cancer (LC) and FNC

• Roughly half of LC are diagnosed by cytology

• Molecular tests are routinely requested for LC

• Capitalization of cytological material is mandatory

• Cells retrieved for DNA extraction from smears, LBC

and cell-blocks are the most common samples

• EZH2 and CD79B mutations in 13% and 8%, in FL and DLBCL

• Multiplex mutation assay designed to detect point mutations of

EZH2 and CD79B, using MassARRAY spectrometry+ Sanger

sequencing on DNA-FNC of B-NHL stored on FTA cards.

• Mutated and wild types in different samples of the same patients

during follow-up hence mutational status of EZH2 and CD79B

may vary over time.

• individualized therapy should be based on molecular findings at

the time of treatment, rather than on material from previous

specimens.

….FTA cards on lymph node FNC Saieg MA et al.: Cancer Cytopathol. 2013;121:377

FTA CARDS

FTA Cards contain chemicals that lyse cells, denature proteins

and protect nucleic acids from nucleases, oxidation and UV

DNA entrapped within FTA matrix, ready for applications in 30’

DNA stored at room temp. for years before and after utilization

FTA CARDS USAGE

Saieg MA et al., Cancer Cytopathol. 2012; 120: 206.

Peluso AL et al: Use of FTA cards for the storage of breast carcinoma nucleic

acid on fine-needle aspiration samples. Cancer Cytopathol. 2015;123:582-592.

AIMS

• To evaluate whether DNA of NSCLC cells,

obtained by FNC, may be collected, stored,

and preserved on FTA cards

• To assess whether DNA stored on FTA card is

quantitatively and qualitatively suitable for

different molecular techniques

LC-FNC (TC-guided and transbronchial FNC) , material

management, FTA storing and DNA evaluation

TC-guided and trans-bronchial FNC LC on

FTA cards and controls

Cases N. Age, median

(range)

FNA

Diagnosis

Cell block ICC

(CK5/6, TTF1, P63 or P40)

NSCLC 25 57

(35 – 82)

NSCLC 25 NSCLC

Adenocarcinoma 20

Squamous carcinoma 4

NSCLC n.o.s. 1

Controls

Lymph node 56

53

(20-75)

Reactive 40

NHL 13

P63 TTF1

-80 °C

LC

FT

A c

a rd L

C

-80 °C

BR

H

FT

A c

a rd B

RH

0

1 0

2 0

3 0

4 0

5 0

D N A y ie ld

[ng

/l]

p < 0 ,0 0 0 1 p < 0 ,0 0 0 1

NSCLC Controls

-80°C FTA card -80°C FTA card

Median (range) 19,5 (10,6-

45,3)

6,5 (1,3-10,7) 24,9 (8,4-38,7) 10,15 (1,0-

14,4)

t-Student -80°C FTA card

LC vs Controls 0,5234 0,024

NSCLC and BRH DNA yield evaluation (LC in red, controls in blue)

-80 °C

LC

FT

A c

a rd L

C

-80 °C

BR

H

FT

A c

a rd B

RH

1 .6

1 .8

2 .0

2 .2

O D R a tio

R2

60

/28

0

p = 0 ,2 6 7 6 p = 0 ,4 2 0 2

LC Controls

-80°C FTA card -80°C FTA card

Median

(range)

1,94 (1,74-

2,01)

2,00 (1,67-

2,10)

1,85 (1,52-

2,16)

1,89 (1,67-

2,20)

t-Student -80°C FTA card

LC vs LN 0,6825 0,1370

NSCLC and BRH DNA OD evaluation

JAK2 PCR from NSCLC and lymph node

lines 1 and 2: -80°C and FTA cards LC case 1 lines 14 and 15 -80°C and FTA cards BRH case 1

lines 3 and 4: -80°C and FTA cards LC case 2 lines 16 and 17 -80°C and FTA cards BRH case 2

lines 5 and 6: -80°C and FTA cards LC case 3 lines 18 and 19 -80°C and FTA cards BRH case 3

lines 7 and 8: [-80°C and FTA cards LC case 4 lines 20 and 21 -80°C and FTA cards BRH case 4

lines 9 and 10: -80°C and FTA cards LC case 5 line CP -80°C positive control

lines 11 and 12: -80°C and FTA cards LC case 6 line 22 negative PCR control (no DNA in PCR mix)

Line SM molecular weight DNA Molecular weight was 500 base pairs in all samples

1*105

cells/40l

1*106

cells/40l 1*107

cells/40l 1*108

cells/40l -80°C

cells

Median

[DNA] ng/l,

(range)

0,7

(0,5-0,9)

6,9

(3,6 -9,6)

17,2

(12-23)

25,7

(13,1-45)

56

(18,3-91,2)

A: Increasing NSCLC cells concentration (105 a 108) on FTA cards

B: PCR JAK2 of 2 NSCLC FNC at different concentrations.

1-2: -80°C case 1 [1] e

2 [2];

3-5: 1*105cells/40L

case 1 [3] e case 2 [5];

4-6: 1*106cell/40L case

1 [4] e case 2 [6];

7-8: 1*107cell/40L case

1 [7] e case 2 [l8];

9-10: 1*108cell/40L

case 1 [9] e case 2 [10].

Absorbance capacity of NSCLC cells on FTA cards

LC cells DNA on whole FTA card

FT

A c

ard

OD

-80°C

OD

1 .4

1 .6

1 .8

2 .0

2 .2

O D R a tio o f 1 0 L C c a s e s

R2

60

/28

0

FT

A c

ard

Yie

ld

-80

°C Y

ield

2 0

4 0

6 0

8 0

1 0 0

D N A y ie ld o f 1 0 L C c a s e s

ng

/l

Yield (ng/l) OD Ratio

FTA card -80°C FTA card -80°C

Median 39,21 40,00 1,81 1,74

Min 19,10 17,60 1,60 1,58

Max 99,80 89,20 2,01 1,89

T-Student p=0,5203 p=0,4928

STUDENT-T TEST

Yield

(ng/l)

OD

Ratio

Paired data

-80°C vs FTA card p=0,0154 p=0,234

Cases

-80°C LC cells FTA Card LC

cells JAK2 PCR

Yield

(ng/l)

OD

Ratio

Yield

(ng/l)

OD

Ratio -80°C

FTA

card

1 35,10 1,65 31,30 1,89 Yes Yes

2 35,20 1,83 24,90 1,72 Yes Yes

3 43,30 2,01 48,70 1,73 Yes Yes

4 74,50 1,85 62,80 1,86 Yes Yes

5 65,40 1,69 52,80 1,72 Yes Yes

6 19,10 1,63 17,60 1,58 Yes Yes

7 99,80 1,60 89,20 1,74 Yes Yes

8 31,30 1,89 28,40 1,77 Yes Yes

9 26,00 1,80 27,10 1,75 Yes Yes

10 79,70 1,93 54,40 1,76 Yes Yes

LC DNA maximum from FTA card

Yield (ng/l) OD Ratio

FTA

card -80°C

FTA

card -80°C

Median 39,21 40,00 1,81 1,74

Min 19,10 17,60 1,60 1,58

Max 99,80 89,20 2,01 1,89

Lung Cancer Cells bio-banking

Bio-banking Advantags Limitations

Formalin-fixed

paraffin-

embedded

blocks

• Long time tissue storage

• Morphology preservation

• ICC classification

• DNA fragmentation

• DNA/RNA lower quality

• Lower accuracy

Frozen cells • Long time tissues and cells storing

• Cells storing (FNC, others)

• DNA/RNA sufficient yield, high quality

• high-throughput analysis

• Costs

• Standardization?

• Needs for specific

infrastructure

FTA cards • Cheep and handily

• No specific infrastructure requested

• Room temp. storing for long time

• Cell storing (FNC, others)

• High quality of nucleic acids

• Lower yield

• No suitable for

proteomic studies

• RNA?

Why LC-FNC is an ideal tool for molecular purposes

needs for molecular procedures,

FNC and cell harvesting

• The amount of cells, obtainable by FNC, is not

constant depending on the needles gauge, the

technical procedures and the nature of the lesions

• Cells harvesting by 1 pass FNC with 23G needles:

4.00 X 106 from lymphoma, 2.50 X 106 from

breast carcinoma, 1.65 X 106 from thyroid

carcinoma

• Forty DNA/RNA ng should be sufficient for any

molecular test and can be obtained from 4.00 X 106

cells; one or two additional FNC from LN should be

sufficient to obtain genetic material sufficient for any

procedure

cells harvesting by 1 pass FNC with 23G needles:

40.00 X 106 from lymphoma, 2.50 X 106 cells from

breast ca, 1.65 X 106 cells from thyroid ca. 40.00 X 106 cells from Lymph node NHL

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry.

Peluso et al. Acta Cytol 2016 in press

Conclusions

• FTA cards can be used to store DNA of NSCLC cells

obtained by FNC for molecular investigations

providing a reliable alternative to traditional methods

• The relative little amount of stored genetic material

on the FTA cards is fully counterbalanced by the

easy, fast, inexpensive, and operative-friendly

procedure and the good quality of stored DNA

Thank you for your attention