Post on 09-Jul-2020
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