OSNA del linfonodo sentinella Anna Sapino

(1)

Valutazione con metodica

OSNA del linfonodo sentinella Anna Sapino

Università di Torino

AO-U San Giovanni Battista di

Torino

(2)

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

(3)

pN1

Based on AJCC/UICC TNM, 7th edition Protocol web posting date:

October 2009

pN1 a: METASTASES in 1 to 3 axillary lymph nodes, at least 1 metastasis greater than 2.0 mm

pN1mi: MICROMETASTASES (greater than 0.2 mm and/or more than 200 cells, but none greater than 2.0 mm).

(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 cross-

section (detected by H&E or IHC including ITC)

(4)

LS FISSATO IN FORMALINA ED INCLUSO IN PARAFFINA

(5)

5

2 (HE+IHC) 2 (HE+IHC)

150

µ

150

µ

150

µ

150

µ

150

µ

150

µ

Etc.Etc.

150

µ

150

µ

150

µ

150

µ

2 (HE + IHC) 2 (HE + IHC)

2 (HE +IHC) 2 (HE +IHC)

2 (HE +IHC) 2 (HE +IHC) 2 (HE + IHC) 2 (HE + IHC)

serial (step) sectioning

1 paraffin block

(6)

Mean Number of slides: 15

Number of sections: up to 120 Technical time

(from embedding to final slides with IHC): 1 hours

Pathologist time: 30 min

Reimbursement: 250 euros

No intraoperative diagnosis

Turn around time to diagnosis:

4-7 days SLN+

Second operation needed

(7)

PROCESSAZIONE ALLESTIMENTO

LETTURA E PROBLEMI DI INTERPRETAZIONE DIAGNOSTICA

LS NEL CARCINOMA DELLA MAMMELLA E STANDARDIZZAZIONE

CELLULE TIMORALI ISOLATE MICROMETASTASI

MACROMETASTASI

Ha maggior peso la quantità di tumore nel

linfonodo di come sono disposte le cellule!

(8)

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)].

(9)

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.

(10)

Frequency and comparison of

non-

non

-SN involvement 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

EWGBSP Turner

ITC 3/27 (11%;3.9-3/27 (11%;3.9-28.1)28.1) 11/71 (15%; 8.9 -25.7) 4% (-13.8-16.9)

Micrometastases 22/107 (21%;14.0-22/107 (21%;14.0-29.2)29.2) 28/96 (29%; 21.0-38.9) 9% (-3.3-20.4)

Macrometastases 158/258 (61%;55.2-158/258 (61%;55.2- 67.0)

67.0)

144/225 (64%;57.5- 70.0)

3% (-5.9-11.3)

(11)

OSNA 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

(12)

++

+

-

++

+

-

Macro-Metastasis

Micro-Metastasis

ITC / background

Macro-Metastasis

Micro-Metastasis

ITC / background

CK19 mRNA

CK19 mRNA

Size of metastasis

Size of metastasis

250 copies mRNA/µL – CK19

(5.0 x 10⁶)

5.000copies mRNA/µL – CK19

(1.0 x 10⁸⁾

OSNA diagnosis

(13)

osna and sentinel lymph node metastases

(14)

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

Grado di concordanza molecolare-istologico: 92- 98.2%

Sensibilità: 95- 98.1%

Specificità dal 94.7-100%

METODICHE A CONFRONTO: ISTOLOGIA E OSNA

(15)

Quality

Quality of the of the assay assay

Detection of pyrophospate

Determination of RNA amount Determination of

Rise Time

Magnesium pyrophosphate

Daily calibration

(16)

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

Quality

Quality of the of the assay assay

(17)

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.

Workflow of the OSNA-assay

Clin Cancer Res 2007;13(16) August15, 2007

Time of execution Time of execution

fat tissue clearing

Weight

(18)

Molinette utilizzo dell’intero

linfonodo

(19)

(20)

Ns 55 (32)

114 (67) 35 (32)

75 (68) Ki67

0-10%

>10%

Ns 144 (85)

25 (14) 108 (98)

2 (2) HER2

negative positive

Ns 38 (22)

131 (77) 22 (20)

88 (80) Progesterone

Receptor 0-10%

>10%

Ns 14 (8)

155 (92) 10 (9)

100 (91) Estrogen Receptor

0-10%

>10%

Ns 118 (70)

51 (30) 80 (73)

30 (27) Vascular invasion

Absent Present

Ns 109 (64)

29 (17) 31 (18) 81 (74)

16 (14) 13 (12) Histological Type

Ductal Lobular Special Type

Ns 66 (39)

78 (46) 25 (15) 46 (42)

48 (44) 16 (14) Histological Grade

1 2 3

Ns 41 (24)

45 (27) 83 (49) 33 (30)

19 (17) 58 (53) Tumor Size (mm)

<10 1.1-1.5

>1.5

Ns 61.2 (23-86)

17 (10) 35 (21) 45 (26) 72 (43) 66.7(38-82)

5 (5) 30 (27) 32 (29) 43 (39) Age yr

Median (range)

<45 46-55 56-65

>65

P-value NON OSNA

169 (%) OSNA

110 (%) PATHOLOGICAL

PARAMETERS

(21)

66%

71%

Negativo

7 % /

ITC

8%

18%

Micrometastasi

20 % 11%

Macrometastasi

Metodo Tradizionale

169 casi OSNA

110 casi

P<0.01

(22)

48%

Cavo ascellare 42%

positivo

Metodo

Tradizionale OSNA

Macrometastasi

22%

Cavo ascellare 22%

positivo

Metodo

Tradizionale OSNA

Micrometastasi

(23)

99 1

Negative

77 23

Micrometastases ( +)

17 83

Macrometastases (++)

Negative

%

Positive

%

OSNA Assay

Cytology (HE/IHC)

RISULTATI OSNA 2010

(24)

Yes (0/12) Positive

28.9 27.3

-(L)

<250 +

L71

Yes (0/14) Positive

25.4 25.1

++

2.3x 10⁴ -

L52

Yes (0/19) Positive

25.0 23.3

++

1.6x 10⁴ -

L3

No Positive

25.1 31.1

+(I) 1.3x 10³

- L33

No Borderline

31.5 30.8

+(I) 1.0x 10³

- L31b

Yes (0/19) Positive

24.8 25.1

+(I) 3.3x 10²

- L2b

No Borderline

34 32

+(I) 4.9x 10²

- L26

No Borderline

34 33.2

+(I) 4.1x 10³

- L6

No Positive

21.6 21

+(I) 3.4x 10²

- L31

No Borderline

32.9 31.3

+ 2.8x 10²

- L50

Yes (0/13) Positive

26.1 26.3

+ 6.9x 10²

- L35 b

No Borderline

32 31.8

+ 2.9x 10²

- L75

Yes (2/18) Positive

25.4 26.4

+ 2.7x 10²

- L28

Yes (0/13) Positive

24.7 25.4

+ 1.4x 10³

- L35

No Borderline

32.3 30

+ 3.4x 10³

- L38

Yes (0/23) Positive

27.7 28.1

+ 2.0x 10³

- L13

No Borderline

32.9 32.8

+ 6.6x 10²

- L26

No Positive

25.7 25.8

+ 4.7x 10³

- L12

Yes (0/8) Positive

27.5 27.1

+ 4.9x 10²

- L32

Yes (0/20) Borderline

32.6 32

+ 4.6x 10³

- L2

Result SPDEF

Cut-off 31.6 Ct CK19

Cut-off 31.5 Ct Result

CK19 Copy number/µl

ALN

(positive LN/Total LN) SYBR-Green RT-PCR

OSNA Imprint

Cytology Cases

(25)

Breast Unit San Giovanni Hospital 200 SLN/years– 1 sn/pts

9 33

OSNA positive (50)

25 100

OSNA negative (150)

105

210

Total working hours

14 28

Histology False negative (28)

34 133

Total working hours

16 32

Histology Positive (32)

75 150

Histology Negative (150)

Time for pathologist

(hours) Time for technician

(hours) Patients

(number)

RIDUZIONE TEMPO TECNICO DEDICATO -50%

RIDUZIONE TEMPO MEDICO DEDICATO

-60%

(26)

Costi OSNA

caso per 200 pazienti / anno

z 200 pazienti con biopsia del linfonodo sentinella / anno

z Media di 1 LN / Paziente

z Costo medio OSNA: circa € 350 / paziente inclusi:

– Noleggio strumentazione automatica e accessori

– Full risk

– Reagenti e consumabili dedicati per eseguire 200 pazienti o linfonodi

z La variabilità dei costi dipende da:

z Numero pazienti con biopsia del LS / anno

z Numero di linfonodi /paziente

z Numero di giornate OSNA /settimana

z Numero di settimane lavorative /anno

z Numero anni di contratto

(27)

GRAZIE PER L

GRAZIE PER L ’ ’ ATTENZIONE ATTENZIONE

(28)

(29)

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

(30)

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.