Valutazione con metodica
OSNA del linfonodo sentinella
Anna Sapino
Università di Torino
AO-U San Giovanni Battista di
Torino
J Clin Pathol 2004;57:695–701.
Le procedure anatomo-patologiche per la valutazione del LS mancano
di standardizzazione
• riduzione macroscopica
• utilizzo dell’esame al congelatore
• numero di sezioni istologiche da esaminare
• utilizzo di colorazioni ancillari
• stesura del referto
Based on AJCC/UICC TNM, 7th edition
Protocol web posting date: October
2009
(sn):
Only sentinel node(s) evaluated. If 6 or more sentinel nodes and/or
nonsentinel nodes are removed, this modifier should not be used
pN0
No regional lymph node metastasis histologically, no
additional examination for isolated tumor cells
pN0(i–) No regional lymph node metastases
histologically, negative IHC
pN0(i+) Malignant cells in regional lymph node(s) not
greater than 0.2 mm or single tumor cells, or a cluster
of fewer than 200 cells in a single histologic crosssection (detected by H&E or IHC including ITC)
pN1
pN1mi: MICROMETASTASES (greater than 0.2 mm
and/or more than 200 cells, but none greater than 2.0
mm).
pN1 a: METASTASES in 1 to 3 axillary lymph nodes, at
least 1 metastasis greater than 2.0 mm
LS FISSATO IN FORMALINA ED INCLUSO IN PARAFFINA
1 paraffin block
22 (HE+IHC)
(HE+IHC)
150
150µ
22 (HE
(HE ++ IHC)
IHC)
150
150µ
22 (HE
(HE ++ IHC)
IHC)
150
150µ
2
2 (HE
(HE +IHC)
+IHC)
150
150µ
22 (HE
(HE +IHC)
+IHC)
150
150µ
Etc.
Etc.
5
serial (step) sectioning
Mean Number of slides:
Number of sections:
Technical time
(from embedding to final slides with IHC):
Pathologist time:
Reimbursement:
No intraoperative diagnosis
Turn around time to diagnosis:
4-7 days
SLN+
Second operation needed
15
up to 120
1 hours
30 min
250 euros
LS NEL CARCINOMA DELLA MAMMELLA
E STANDARDIZZAZIONE
PROCESSAZIONE
ALLESTIMENTO
LETTURA E PROBLEMI DI INTERPRETAZIONE DIAGNOSTICA
CELLULE TIMORALI ISOLATE
MICROMETASTASI
MACROMETASTASI
Ha maggior peso la quantità di tumore nel
linfonodo di come sono disposte le cellule!
FIG. 2 Lobular carcinoma (test case #56). A dispersed pattern of lobular carcinoma with
fewer cells than the case illustrated in Figure 2 also caused disagreement in classification.
On (A) pre-test, three MDs chose micrometastasis, one chose “other”, and two chose
isolated tumor cells (ITC). On (B) post-test, all six MDs chose ITC [(N0(i)].
392 patients with an invasive lobular carcinoma and positive SN and axillary
lymph node dissection
SNs with multiple single cells and clusters arranged in a discontinuous manner
but dispersed homogeneously in a definable part of the lymph node, classified
as micrometastases according to the EWGBSP interpretations vs. ITC
according to Turner et al.
Frequency and comparison of non-SN involvement according to two different
interpretations of the N staging system.
SN classification
Number of patients with non-SN involvement (%;
95% CI))
Difference
% (95% CI)
EWGBSP
Turner
ITC
3/27 (11%;3.9-28.1)
11/71 (15%; 8.9 -25.7)
4% (-13.8-16.9)
Micrometastases
22/107 (21%;14.0-29.2)
28/96 (29%; 21.0-38.9)
9% (-3.3-20.4)
Macrometastases
158/258 (61%;55.267.0)
144/225 (64%;57.570.0)
3% (-5.9-11.3)
OSNA
• PROCEDURA AUTOMATIZZATA DI
AMPLIFICAZIONE DEGLI ACIDI NUCLEICI
• VERIFICA LA PRESENZA DEL GENE DELLA
CK19 NEL TESSUTO LINFONODALE
• CONSENTE UNA DIAGNOSI MOLECOLARE DEL
LS INTRAOPERATORIA
• FORNISCE UN RISULTATO DI NEGATIVO,
MICROMETASTASI O MACROMETASTASI
OSNA diagnosis
Size of metastasis
CK19 mRNA
Macro-Metastasis
Macro-Metastasis
++
++
5.000 copies
mRNA/µL – CK19
(1.0 x 108)
Micro-Metastasis
Micro-Metastasis
++
250 copies
mRNA/µL – CK19
ITC
ITC // background
background
(5.0
-- x 106)
osna and sentinel lymph node metastases
Tsujimoto et al 2007 - Clinical Cancer Research
Visser et al 2008 - Int J Cancer
Schem et al 2009 - Virchows Arch
Tamaki et al 2009 – Clinical Cancer Research
METODICHE A CONFRONTO: ISTOLOGIA E OSNA
Grado di concordanza molecolare-istologico: 92- 98.2%
Sensibilità: 95- 98.1%
Specificità dal 94.7-100%
Quality of the assay
Detection of
pyrophospate
Determination of
Rise Time
Magnesium
pyrophosphate
Daily calibration
Determination of
RNA amount
Quality of the assay
Undesired amplification false positive results. of genomic DNA
is avoided due to:
• 6 different primers which have been specifically designed to
avoid the amplification of CK19 pseudogenes or their
transcripts,
•precipitation of DNA at low pH during sample preparation
and the isothermal reaction temperature of 65°C.
False negative?
CK19 negative
Time of execution
Workflow of the OSNA-assay
fat tissue clearing
Weight
Lymph nodes are simply homogenised in a
special homogenising reagent.
The liquid phase is taken and inserted in the
RD-100i which automatically performs pipetting,
amplification, and detection.
The total time required starting from the
preparation of the lymph node until results
are displayed is about 30 minutes for one
lymph node and about 40 minutes for four
lymph nodes.
Clin Cancer Res 2007;13(16) August15, 2007
Molinette utilizzo dell’intero
linfonodo
PATHOLOGICAL
PARAMETERS
OSNA
110 (%)
NON OSNA
169 (%)
66.7(38-82)
5 (5)
30 (27)
32 (29)
43 (39)
61.2 (23-86)
17 (10)
35 (21)
45 (26)
72 (43)
Ns
Tumor Size (mm)
<10
1.1-1.5
>1.5
33 (30)
19 (17)
58 (53)
41 (24)
45 (27)
83 (49)
Ns
Histological Grade
1
2
3
46 (42)
48 (44)
16 (14)
66 (39)
78 (46)
25 (15)
Ns
Histological Type
Ductal
Lobular
Special Type
81 (74)
16 (14)
13 (12)
109 (64)
29 (17)
31 (18)
Ns
Vascular invasion
Absent
Present
80 (73)
30 (27)
118 (70)
51 (30)
Ns
Estrogen Receptor
0-10%
>10%
10 (9)
100 (91)
14 (8)
155 (92)
Ns
Progesterone
Receptor
0-10%
>10%
22 (20)
88 (80)
38 (22)
131 (77)
Ns
HER2
negative
positive
108 (98)
2 (2)
144 (85)
25 (14)
Ns
Ki67
0-10%
>10%
35 (32)
75 (68)
55 (32)
114 (67)
Ns
Age yr
Median (range)
<45
46-55
56-65
>65
P-value
OSNA
110 casi
Metodo
Tradizionale
169 casi
Macrometastasi
11%
20 %
Micrometastasi
18%
8%
/
7%
71%
66%
ITC
Negativo
P<0.01
Macrometastasi
Cavo ascellare
positivo
Micrometastasi
Cavo ascellare
positivo
OSNA
42%
OSNA
22%
Metodo
Tradizionale
48%
Metodo
Tradizionale
22%
RISULTATI OSNA 2010
Cytology (HE/IHC)
Positive %
Negative%
Macrometastases (++)
83
17
Micrometastases ( +)
23
77
Negative
1
99
OSNA Assay
OSNA
SYBR-Green RT-PCR
Cases
Imprint
Cytology
CK19
Copy
number/µl
Result
CK19
Cut-off 31.5
Ct
SPDEF
Cut-off 31.6
Ct
Result
ALN
(positive LN/Total LN)
L2
-
4.6x 103
+
32
32.6
Borderline
Yes (0/20)
L32
-
4.9x 102
+
27.1
27.5
Positive
Yes (0/8)
L12
-
4.7x 103
+
25.8
25.7
Positive
No
L26
-
6.6x 102
+
32.8
32.9
Borderline
No
L13
-
2.0x 103
+
28.1
27.7
Positive
Yes (0/23)
L38
-
3.4x 103
+
30
32.3
Borderline
No
L35
-
1.4x 103
+
25.4
24.7
Positive
Yes (0/13)
L28
-
2.7x 102
+
26.4
25.4
Positive
Yes (2/18)
L75
-
2.9x 102
+
31.8
32
Borderline
No
L35 b
-
6.9x 102
+
26.3
26.1
Positive
Yes (0/13)
L50
-
2.8x 102
+
31.3
32.9
Borderline
No
L31
-
3.4x 102
+(I)
21
21.6
Positive
No
L6
-
4.1x 103
+(I)
33.2
34
Borderline
No
L26
-
4.9x 102
+(I)
32
34
Borderline
No
L2b
-
3.3x 102
+(I)
25.1
24.8
Positive
Yes (0/19)
L31b
-
1.0x 103
+(I)
30.8
31.5
Borderline
No
L33
-
1.3x 103
+(I)
31.1
25.1
Positive
No
L3
-
1.6x 104
++
23.3
25.0
Positive
Yes (0/19)
L52
-
2.3x 104
++
25.1
25.4
Positive
Yes (0/14)
L71
+
<250
-(L)
27.3
28.9
Positive
Yes (0/12)
Breast Unit San Giovanni Hospital
200 SLN/years– 1 sn/pts
Patients
(number)
Time for technician
(hours)
Time for
pathologist
(hours)
Histology Negative (150)
150
75
RIDUZIONE TEMPO TECNICO
DEDICATO
Histology False negative (28)
28
14
-50%
Histology Positive (32)
32
RIDUZIONE TEMPO MEDICO
DEDICATO16
Total working hours
105
-60% 210
OSNA negative (150)
100
25
OSNA positive (50)
33
9
133
34
Total working hours
Costi OSNA
caso per 200 pazienti / anno
z
z
z
z
z
z
z
z
z
200 pazienti con biopsia del linfonodo sentinella / anno
Media di 1 LN / Paziente
Costo medio OSNA: circa € 350 / paziente inclusi:
– Noleggio strumentazione automatica e accessori
– Full risk
– Reagenti e consumabili dedicati per eseguire 200 pazienti o linfonodi
La variabilità dei costi dipende da:
Numero pazienti con biopsia del LS / anno
Numero di linfonodi /paziente
Numero di giornate OSNA /settimana
Numero di settimane lavorative /anno
Numero anni di contratto
GRAZIE PER L’ATTENZIONE
Risk: UP STAGING OF MICROMETASTASES
WATCH AND SEE
Multidisciplinary discussion taking into account
•the histology of tumor (dimension, grade, vascular invasion)
•the patient clinical feature
Axillary recurrence is low in patients with breast cancer who do not undergo
completion axillary lymph node dissection for micrometastases in sentinel lymph
nodes.
Rayhanabad J, Yegiyants S, Putchakayala K, Haig P, Romero L, Difronzo LA.
Am Surg. 2010 Oct;76(10):1088-91.