Peroperative Investigations of the Sentinel Node Dr. J.C. Schobbens President of Belgian Society of Senology Institut Jules Bordet, Brussels, Belgium Dr. I Veys, Dr. D Noterman, Dr. D Herten, Dr. P. Deneubourg Dr. V. Durbecq, Dr. P. Bourgois,Dr.JM Nogaret, Dr. D. Larsimont Dr. V. Durbecq, Dr. P. Bourgois,Dr.JM Nogaret, Dr. D. Larsimont 04 okt Diegem

SLN = Golden Standard In recent years SLN procedure has become the standard for small breast cancer lesions. Sentinel Lymph Node (SLN) accurately reflect the presence of metastases in axillary LN (ALN) Goal = avoiding axilla dissection in node negative patients

Standard Care SLN: post-operative H&E permanent sections ( Yared et al, Am J Surg Pathol 2002, ASCO 2005 ) - 3 levels (5 µm each) from each 2-3 mm ( µm) slice of node - Actual tissue viewed is normally only 2-5% of the node - Will miss 10-15% of metastases > 0.2mm (200 µm) - Requires experienced pathologist but is subjective day delay = Not intra-operative - sensitivity 83.4% to 97% ; High Specificity % immunohistochemistry (IHC) if H&E negative

Incidence of Further Axillary Metastasis Predicted by Size of the SLN Metastasis Size of SLN metastases Incidence of further axillary metastases >2 mm macromets % 0.2 and <2 mm micromets % <0.2 mm sub-micromets 7-15 % Negative~10 % Degnim, 2003; Van Rijk, 2006; Viale, 2005; and Smeets, 2005

AXILLARY U/S and FNAC (Clinically negative axillas) Ultrasound alone identified only 34% of positive axillas ( Mathijsen; Surgical Oncology, 2006) U/S plus FNAC identified 21% of positive axillas ( Rijk; Annals of Surgical Oncology, 2006) 33% of node + diagnosted with FNAC 11,6 % SLN avoided 8 % cost saving (Genta; world J. Surg, 2006)

One of the most important issues is whether accurate diagnosis of sentinel node metastases can be done intraoperatively.

AuthorMethodSensitivity (%) Micromet’s Sensitivity (%) (Macro)met’s Specificity (%) Zuber (2008) Frozen section98100 Leung (2007) Frozen section Hameed (2007) imprint86100 Pugliese (2006) imprint Mori (2006) Imprint Frozen section 47,1 88,2 98,3 100 Pogacnik (2005) imprint377799,2 Brogi (2005) Frozen section Imprint Mewes (2003) Imprint Frozen section Creager (2002) imprint5398 Tanis (2001) Frozen section7499

2-Dimensional Slices of Complex 3-Dimensional Tumors Make Accurate Detection Difficult No Met Micromet Macromet Lymph Node Cancer

Frozen Section Histology PRO Moderate sensitivity: 57-74% High specificity % Some morphologic information available and rough estimate of size of metastases minutes turn around time – can be used intra-operatively CON No standard methods Lack of higher sensitivity: Impractical to sample node more thoroughly Less distinct staining: More difficult to interpret Subjective evaluation Limited ability to identify lobular cancer Loss of tissue when cutting Freezing node can make later permanent section histology less distinct

Technical Issues Intraoperative alternatives “Exhaustive” frozen section (EIO Milan) – immediate FS of the entire SLN (35 of 60 sections), with H&E and quick-IHC technique – pro: 100% sensitivity – con: effort, time, cost, consumes the node Viale et al ; Cancer 1999

Touch Preparation / Imprint Cytology PRO Moderate sensitivity: 53-56% Specificity % All tissue saved for later permanent section minutes turn around time – can be used intra-operatively CON No standard methods Lack of higher sensitivity: Impractical to sample node more thoroughly Difficult to interpret – requires expert cytologist Subjective evaluation No size estimation

Molecular Intra-operative Options

Molecular: QRT-PCR GeneXpert Assay Not Commercially Available – Detects metastases in SLNs – Intraoperative Test result: Quantitative Technician operated – Fully automated Quality Controls – External controls – Internal controls Markers – TACSTD1 – PIP Preliminary Cutoffs determined Cepheid GeneXpert System – Runs 1 to 16 samples Early validation with 90 SLNs complete Future plans are unknown – last publication was 2006 (Hughes. Ann Surg 2006;243)

Molecular: Sysmex OSHA Assay (One Step Nucleic Acid Amplification) CE Marked - Available in EU – Detects metastases >0.2mm in SLNs Test result = macrometastases + = micrometastases - = negative Technician operated – Part manual, part automated Quality Controls – External controls and calibrators – No internal control Marker – Cytokeratin 19 (CK19) - Epithelial RD-100i – Runs 4 samples plus controls and calibrators Analytical determination of cutoffs Validation with 101 patients (Clin Cancer Res 2007;13(16))

Molecular: Veridex GeneSearch™ BLN Assay FDA approved and CE Marked – Intra-operative or post-operative – Detects metastases >0.2mm in SLNs – Allows decisions on ALND Test result – Positive/Negative Technician operated – Part manual, part automated Quality Controls – Positive/negative external controls – Internal control Markers – Cytokeratin 19 (CK19) - Epithelial – Mammaglobin (MG) - Breast Cepheid SmartCycler System – Runs 1-6 samples plus 2 controls – Multiple run capability – Closed tube, real time RT- PCR

New Molecular Assay Using Real-time RT-PCR uses real-time RT-PCR to detect MG (mammaglobin) & CK (cytokeratin) 19 transcripts (m-RNA) Identifies clinically significant metastases > 0.2 mm

Real Time RT-PCR Procedure - mRNA templated converted to cDNA

Multiple Cycles Allow Amplification of Target Sequences

Polymerase Chain Reaction DNA amplification fluorescence molecules emission

Level of Fluorescence Qualitative Interpretation of CK 19 CYCLE THRESHOLD VALUES = CTs Positive Negative Ct Value (Threshold) Cut-off Negative Positive Negative Number of Amplification Cycles

Validation Study: Node Sampling 3.0 mm mm Node ANode B Nodes parsed into ~2mm pieces 2 mm Histological sampling was more extensive than standard of care for the site : alternating levels 150 µm apart per piece H&E and IHC Slides reviewed by 4 pathologists (2 juniors/2 seniors) Assay 100% sampling Histology H&E IHC

Validation Study – Results 78 cases Permanent Section H&E & IHC RT – PCR molecular Assay * for permanent section H&E or IHC must be >0.2mm *Only sample positive by IHC alone

Assay False Negative/ IHC Positive Histology Result micrometastasis of 0.25 mm. IHC + Micromet Picture Goes here!

Country/ Location Belgium Institute Jules Bordet U.S.A. 14 sites No. Histology Positives 13/ % 121/416 29% Sensitivity (%)92.3%87.6% Specificity (%)96.9%94.2% PPV (%)85.7%86.2% NPV (%)98.4%94.9% Overall Agreement (%) 96.2%92.3 % Validation Results

Clinical Use: Standard Sampling Initially only one SLN was tested, now all SLNs are being tested Histological sampling is different in Clinical Use Assay 100% sampling Histology 3.0 mm mm Node ANode B Cutting Scheme same as in the Validation Study 2 mm H&E IHC

Validation Study vs. Clinical Use 2 mm H&E IHC 2 mm H&E IHC Validation Study Clinical Use Histological sampling is different in Clinical Use Sections are 150 µm apart Sections are 100 µm apart

Clinical Use: Performance of the Assay 300cases Permanent Section H&E & IHC RT – PCR molecular Assay * -6** for permanent section H&E or IHC must be >0.2mm **4 were (MI)<1mm (size 0.3 mm to 0.75 mm) And all by IHC only 1 case: micrometastases between 1 and 2 mm *In one of the 45 patients, histology was positive in a different SLN than the one tested in the assay

BLN Assay Performance Jules Bordet Institute Validation Study N=78 Jules Bordet Institute Post-Market Data N=300 Blumencranz et al Am J Surg US Clinical Study N= 407 Overall Agreement 96.1% 93.7% 94% Specificity 96.9% (63/65) 94.8% (236/249) 93% Sensitivity 92.3% (12/13) 88.2% (45/51) 92% PPV 85.7% (12/14) 77.6% (45/58) 76% NPV 98.4% (63/64) 97.5% (236/242) 99%

BLN Assay According to Metastases Size SLN StatusALND Status BLN AssayHistologyN# Performed# (%) Positive Negative (2%) Micro (0.2 mm – 2.0 mm)430 Macro (> 2.0 mm)110 Positive Negative13 2 (15%) ** Micro (0.2 mm – 2.0 mm)12 2 (17%) Macro (> 2.0 mm)28 11 (39%) Metastases size (mm)Sensitivity % (7/10) % (5/6) % (9/10) >4.1100% (19/19) Overall BLN Assay Sensitivity vs. Histology 88.2% (45/51) Overall Histology Sensitivity vs. BLN Assay 77.6% (45/58) *Note: the 6 patients with positive histology and unknown size are not included in the above tables ** USA : 25% * *

Performance of BLN Assay vs. Frozen Test Method N Sensitivity % (95% CI) Specificity % (95% CI) PPV % NPV % Agreement % Frozen Section (61 – 89) 99 (96 – 100) 9495 BLN Assay 95 (83 – 99) 93 (89 – 97) All comparisons to permanent section H&E Clinical Use of the BLN Assay at Morton Plant Dr. Blumencranz ; ASCO Breast 2008

Clinical Use: Timing (first 100) Turn Around Time = time from node removal to time BLN Assay result reported Current average turn around time: 1 node: 30 min > 1 node: 35 min

CONCLUSION RT-PCR Intraoperative Sensitivity > Frozen Section and Imprint Performance is comparable to the standard of care = permanent H&E – Better? : Increased node tissue sampling The BLN Assay identifies clinically relevant (>0.2 mm) metastatic cancer Detects metastases with challenging histology (lobular Ca) Standardized and validated – Eliminates intra- and inter-laboratory variability Objective and reproducible – Simple enough to be performed by a histo. technician or med. technician

Future Future Will continue intra-operatively using the BLN Assay at the Institut Jules Bordet – Effectively being used intra-operatively and Performance is as expected Will continue with current histology cutting – But are hoping that the assay once become the standard ; no need of histology anymore? Research : Possible Correlation of Cts with metastases size and its clinical significance

Comparison of Currently Available Intra-operative Tests Frozen Section Touch Prep Cytology Molecular BLN Assay Sensitivity *57-77%53-56%88-95% Specificity99-100%98-100%93-94% StandardizedNo Yes Labor required*PathologistCytologistTechnologist Ease of evaluationModerateDifficultAutomated Nodal sampling*Limited 50% Sensitivity across cancer types Moderate High Morphologic InfoYesNo Turn around time10-30 minutes minutes

Conclusions Current intraoperative histopathology/cytopathology on SLNs: – has high specificity but lower sensitivity – requires high level professional experience but still subjective – nodal sampling is limited Molecular assay: – has higher sensitivity – is reproducible with less labor – provides more thorough node sampling – may reduce second surgeries for ALND

Thank you for the attention! St.-Agatha Catania 255

Current Used Techniques Have Limitations Technique and interpretation is pathologist and institution dependent Non-standard procedure Only % off the tissue analysed Labor intensive / time consuming Low sensitivity (micromet’s) Evaluation challenging in some cases Even for experienced pathologists (e.g., Lobular Cancer) Not being used at the Institut Jules Bordet