Valutazione con metodica OSNA del linfonodo sentinella. Anna Sapino Università di Torino AO-U San Giovanni Battista di Torino

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1 Valutazione con metodica OSNA del linfonodo sentinella Anna Sapino Università di Torino AOU San Giovanni Battista di Torino

2 J Clin Pathol 2004;57: Le procedure anatomopatologiche 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 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

4 LS FISSATO IN FORMALINA ED INCLUSO IN PARAFFINA

5 1 paraffin block 5 2 (HE+IHC) 150µ 2 (HE + IHC) 150µ 2 (HE + IHC) 150µ 2 (HE +IHC) 150µ 2 (HE +IHC) 150µ Etc. serial (step) sectioning

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: 47 days SLN+ Second operation needed

7 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!

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) pretest, three MDs chose micrometastasis, one chose other, and two chose isolated tumor cells (ITC). On (B) posttest, 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 nonsn involvement according to two different Frequency and comparison of non interpretations of the N staging system. SN classification Number of patients with nonsn involvement (%; 95% CI)) Difference % (95% CI) EWGBSP Turner ITC 3/27 (11%; ) 11/71 (15%; ) 4% ( ) Micrometastases 22/107 (21%; ) 29.2) 28/96 (29%; ) 9% ( ) Macrometastases 158/258 (61%; ) 144/225 (64%; ) 3% ( )

11 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 OSNA diagnosis Size of metastasis CK19 mrna MacroMetastasis MicroMetastasis ITC / background copies mrna/µl CK19 (1.0 x 10 8) copies mrna/µl CK19 (5.0 x 10 6 )

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 METODICHE A CONFRONTO: ISTOLOGIA E OSNA Grado di concordanza molecolareistologico: % Sensibilità: % Specificità dal %

15 Quality of the assay Detection of pyrophospate Determination of Rise Time Determination of RNA amount Magnesium pyrophosphate Daily calibration

16 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

17 Time of execution Workflow of the OSNAassay fat tissue clearing Weight Lymph nodes are simply homogenised in a special homogenising reagent. The liquid phase is taken and inserted in the RD100i 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

18 Molinette utilizzo dell intero linfonodo

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20 PATHOLOGICAL PARAMETERS OSNA 110 (%) NON OSNA 169 (%) Pvalue Age yr Median (range) < > (3882) 5 (5) 30 (27) 32 (29) 43 (39) 61.2 (2386) 17 (10) 35 (21) 45 (26) 72 (43) Ns Tumor Size (mm) < > (30) 19 (17) 58 (53) 41 (24) 45 (27) 83 (49) Ns Histological Grade (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 010% >10% 10 (9) 100 (91) 14 (8) 155 (92) Ns Progesterone Receptor 010% >10% 22 (20) 88 (80) 38 (22) 131 (77) Ns HER2 negative positive 108 (98) 2 (2) 144 (85) 25 (14) Ns Ki67 010% >10% 35 (32) 75 (68) 55 (32) 114 (67) Ns

21 OSNA 110 casi Metodo Tradizionale 169 casi Macrometastasi 11% 20 % Micrometastasi 18% 8% P<0.01 ITC / 7 % Negativo 71% 66%

22 Macrometastasi Cavo ascellare positivo Micrometastasi Cavo ascellare positivo OSNA 42% OSNA 22% Metodo Tradizionale 48% Metodo Tradizionale 22%

23 RISULTATI OSNA 2010 Cytology (HE/IHC) OSNA Assay Macrometastases (++) Micrometastases ( +) Negative Positive % Negative%

24 Cases Imprint Cytology OSNA CK19 Copy number/µl Result CK19 Cutoff 31.5 Ct SYBRGreen RTPCR SPDEF Cutoff 31.6 Ct Result ALN (positive LN/Total LN) L2 4.6x Borderline Yes (0/20) L32 4.9x Positive Yes (0/8) L12 4.7x Positive No L26 6.6x Borderline No L13 2.0x Positive Yes (0/23) L38 3.4x Borderline No L35 1.4x Positive Yes (0/13) L28 2.7x Positive Yes (2/18) L75 2.9x Borderline No L35 b 6.9x Positive Yes (0/13) L50 2.8x Borderline No L31 3.4x (I) Positive No L6 4.1x (I) Borderline No L26 4.9x (I) Borderline No L2b 3.3x (I) Positive Yes (0/19) L31b 1.0x (I) Borderline No L33 1.3x (I) Positive No L3 1.6x Positive Yes (0/19) L52 2.3x Positive Yes (0/14) L71 + <250 (L) Positive Yes (0/12)

25 Breast Unit San Giovanni Hospital 200 SLN/years 1 sn/pts Patients (number) Time for technician (hours) Time for pathologist (hours) Histology Negative (150) RIDUZIONE TEMPO TECNICO DEDICATO Histology False negative (28) Histology Positive (32) RIDUZIONE TEMPO MEDICO DEDICATO Total working hours 50% 60% 210 OSNA negative (150) OSNA positive (50) Total working hours

26 Costi OSNA caso per 200 pazienti / anno 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

27 GRAZIE PER L ATTENZIONEL

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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 Oct;76(10):