Peroperative Investigations of the Sentinel Node Dr. J.C. Schobbens

1. PeroperativeInvestigations 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.JMNogaret, Dr. D. Larsimont 04 okt. 2008 Diegem

2. 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

3. Standard Care SLN: • post-operative H&E permanent sections ( Yared et al, Am J SurgPathol 2002, ASCO 2005) • 3 levels (5 µm each) from each 2-3 mm (2000-3000 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 • 1-4 day delay = Not intra-operative • sensitivity 83.4% to 97% ; High Specificity 99-100% • immunohistochemistry (IHC) if H&E negative

4. Incidence of Further Axillary Metastasis Predicted by Size of the SLN Metastasis Degnim, 2003; Van Rijk, 2006; Viale, 2005; and Smeets, 2005

5. 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)

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

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

13. Frozen Section Histology PRO • Moderate sensitivity: 57-74% • High specificity 99-100% • Some morphologic information available and rough estimate of size of metastases • 10-30 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

14. Technical IssuesIntraoperative 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

15. Touch Preparation / Imprint Cytology PRO • Moderate sensitivity: 53-56% • Specificity 98-100% • All tissue saved for later permanent section • 10-30 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

16. Molecular Intra-operative Options

17. 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)

18. 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))

19. Molecular: VeridexGeneSearch™ 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

20. New MolecularAssayUsing 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

21. Real TimeRT-PCR Procedure - mRNA templated converted to cDNA

22. Multiple Cycles Allow Amplification of Target Sequences

23. Polymerase Chain Reaction DNA amplification fluorescence molecules emission

24. Qualitative Interpretation of CK 19CYCLE THRESHOLD VALUES = CTs Cut-off Negative Positive Level of Fluorescence Negative Ct Value (Threshold) Positive Negative 15 0 5 10 20 25 30 35 40 Number of Amplification Cycles

25. Validation Study: Node Sampling Node A Node B 1 2 1 2 3 4 5 6 3.0 mm 12.0 mm Nodes parsed into ~2mm pieces H&E Assay100% sampling Histology IHC 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) 2 mm

26. Validation Study – Results • + for permanent section H&E or IHC must be >0.2mm *Only sample positive by IHC alone

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

28. Validation Results

29. Clinical Use: Standard Sampling Node A Node B 6 1 2 1 2 3 4 5 3.0 mm 12.0 mm Cutting Scheme same as in the Validation Study Assay100% sampling Histology H&E • Initially only one SLN was tested, now all SLNs are being tested • Histological sampling is different in Clinical Use IHC 2 mm

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

31. Clinical Use: Performance of the Assay • + for permanent section H&E or IHC must be >0.2mm *In one of the 45 patients, histology was positive in a different SLN than the one tested in the assay **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

32. BLN Assay Performance

33. BLN AssayAccording to Metastases Size * * • *Note: the 6 patients with positive histology and unknown size are not included in the above tables • ** USA : 25%

34. Performance of BLN Assay vs. Frozen All comparisons to permanent section H&E Clinical Use of the BLN Assay at Morton Plant Dr. Blumencranz ; ASCO Breast 2008

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

36. 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

37. 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

38. Comparison of Currently Available Intra-operative Tests

39. 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

40. Thankyoufor the attention! St.-Agatha Catania 255

41. Current Used Techniques Have Limitations • Technique and interpretation is pathologist and institution dependent • Non-standard procedure • Only 5 -10 % 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