Valutazione con metodica OSNA del linfonodo sentinella

Anna SapinoUniversità di TorinoAO-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

pN1

Based on AJCC/UICC TNM, 7th editionProtocol 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

pN0No regional lymph node metastasis histologically, no additional examination for isolated tumor cellspN0(i–) No regional lymph node metastases histologically, negative IHCpN0(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)

LS FISSATO IN FORMALINA ED INCLUSO IN PARAFFINA

5

2 (HE+IHC)2 (HE+IHC)150µ150µ

150µ150µ

150µ150µ

Etc.Etc.

150µ150µ

150µ150µ

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

Mean Number of slides: 15 Number of sections: up to 120Technical time (from embedding to final slides with IHC): 1 hoursPathologist time: 30 minReimbursement: 250 euros

No intraoperative diagnosisTurn around time to diagnosis: 4-7 daysSLN+Second operation needed

PROCESSAZIONE

ALLESTIMENTO

LETTURA E PROBLEMI DI INTERPRETAZIONE DIAGNOSTICA

LS NEL CARCINOMA DELLA MAMMELLA E STANDARDIZZAZIONE

CELLULE TIMORALI ISOLATEMICROMETASTASIMACROMETASTASI

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 withfewer 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 choseisolated 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. ITCaccording to Turner et al.

Frequency and comparison of nonnon--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%;3.93/27 (11%;3.9--28.1)28.1) 11/71 (15%; 8.9 -25.7) 4% (-13.8-16.9)

Micrometastases 22/107 (21%;14.022/107 (21%;14.0--29.2)29.2) 28/96 (29%; 21.0-38.9) 9% (-3.3-20.4)

Macrometastases 158/258 (61%;55.2158/258 (61%;55.2--67.0)67.0)

144/225 (64%;57.5-70.0)

3% (-5.9-11.3)

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

++

+

-

++

+

-

Macro-Metastasis

Micro-Metastasis

ITC / background

Macro-Metastasis

Micro-Metastasis

ITC / background

CK19 mRNACK19 mRNASize of metastasis Size of metastasis

250 copies mRNA/µL – CK19

(5.0 x 106)

5.000 copies mRNA/µL – CK19

(1.0 x 108)

OSNA diagnosis

osna and sentinel lymph node metastases

Tsujimoto et al 2007 - Clinical Cancer ResearchVisser 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

QualityQuality of the of the assayassay

Detection of pyrophospate

Determination of RNA amount

Determination of Rise Time

Magnesium pyrophosphate

Daily calibration

Undesired amplification false positive results. of genomic DNA is avoided due to:

• 6 different primers which have been specifically designed toavoid the amplification of CK19 pseudogenes or theirtranscripts,

•precipitation of DNA at low pH during sample preparationand the isothermal reaction temperature of 65°C.

False negative?CK19 negative

QualityQuality of the of the assayassay

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 resultsare displayed is about 30 minutes for one lymph node and about 40 minutes for fourlymph nodes.

Workflow of the OSNA-assay

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

Time of executionTime of execution

fat tissue clearing

Weight

Molinette utilizzo dell’intero linfonodo

Ns55 (32)114 (67)

35 (32)75 (68)

Ki670-10%>10%

Ns144 (85)25 (14)

108 (98)2 (2)

HER2negativepositive

Ns38 (22)131 (77)

22 (20)88 (80)

Progesterone Receptor0-10%>10%

Ns14 (8)155 (92)

10 (9)100 (91)

Estrogen Receptor0-10% >10%

Ns118 (70)51 (30)

80 (73)30 (27)

Vascular invasionAbsentPresent

Ns109 (64)29 (17)31 (18)

81 (74)16 (14)13 (12)

Histological TypeDuctalLobularSpecial Type

Ns66 (39)78 (46)25 (15)

46 (42)48 (44)16 (14)

Histological Grade1 2 3

Ns41 (24)45 (27)83 (49)

33 (30)19 (17)58 (53)

Tumor Size (mm)<101.1-1.5>1.5

Ns61.2 (23-86)17 (10)35 (21)45 (26)72 (43)

66.7(38-82)5 (5)

30 (27)32 (29)43 (39)

Age yrMedian (range)<4546-5556-65>65

P-valueNON OSNA169 (%)

OSNA110 (%)

PATHOLOGICAL PARAMETERS

66%71%Negativo7 %/ITC8%18%Micrometastasi

20 %11%Macrometastasi

Metodo Tradizionale

169 casi

OSNA 110 casi

P<0.01

48%42%Cavo ascellare positivo

Metodo TradizionaleOSNA Macrometastasi

22%22%Cavo ascellare positivo

Metodo TradizionaleOSNA Micrometastasi

991Negative7723Micrometastases ( +)1783Macrometastases (++)

Negative%Positive %OSNA Assay

Cytology (HE/IHC)

RISULTATI OSNA 2010

Yes (0/12)Positive28.927.3-(L)<250 +L71

Yes (0/14)Positive25.425.1++2.3x 104-L52

Yes (0/19)Positive25.023.3++1.6x 104-L3

NoPositive25.131.1+(I)1.3x 103-L33

NoBorderline31.530.8+(I)1.0x 103-L31b

Yes (0/19)Positive24.825.1+(I)3.3x 102-L2b

NoBorderline3432+(I)4.9x 102-L26

NoBorderline3433.2+(I)4.1x 103-L6

NoPositive21.621+(I)3.4x 102-L31

NoBorderline32.931.3+2.8x 102-L50

Yes (0/13)Positive26.126.3+6.9x 102-L35 b

NoBorderline3231.8+2.9x 102-L75

Yes (2/18)Positive25.426.4+2.7x 102-L28

Yes (0/13)Positive24.725.4+1.4x 103-L35

NoBorderline32.330+3.4x 103-L38

Yes (0/23)Positive27.728.1+2.0x 103-L13

NoBorderline32.932.8+6.6x 102-L26

NoPositive25.725.8+4.7x 103-L12

Yes (0/8)Positive27.527.1+4.9x 102-L32

Yes (0/20)Borderline32.632+4.6x 103-L2

ResultSPDEFCut-off 31.6

Ct

CK19Cut-off 31.5

Ct

ResultCK19Copy

number/µl

ALN(positive LN/Total LN)

SYBR-Green RT-PCROSNAImprintCytologyCases

Breast Unit San Giovanni Hospital200 SLN/years– 1 sn/pts

933OSNA positive (50)25100OSNA negative (150)

105210Total working hours

1428Histology False negative (28)

34133Total working hours

1632Histology Positive (32)

75150Histology Negative (150)

Time forpathologist

(hours)

Time for technician(hours)

Patients(number)

RIDUZIONE TEMPO TECNICO DEDICATO-50%

RIDUZIONE TEMPO MEDICO DEDICATO -60%

Costi OSNA caso per 200 pazienti / anno

200 pazienti con biopsia del linfonodo sentinella / annoMedia di 1 LN / PazienteCosto 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 / annoNumero di linfonodi /pazienteNumero di giornate OSNA /settimanaNumero di settimane lavorative /annoNumero anni di contratto

GRAZIE PER LGRAZIE PER L’’ATTENZIONEATTENZIONE

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 undergocompletion 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.