Peroperative Investigations of the Sentinel Node Dr J

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 04 okt. 2008 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: l 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 (2000 -3000 µm) slice of node - Actual tissue viewed is normally only 2 -5% of the node - Will miss 10 -15% of metastases > 0. 2 mm (200 µm) - Requires experienced pathologist but is subjective - 1 -4 day delay = Not intra-operative - sensitivity 83. 4% to 97% ; High Specificity 99 -100% l 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 45 -79 % 0. 2 and <2 mm micromets <0. 2 mm sub-micromets Negative 10 -25 % 7 -15 % ~10 % Degnim, 2003; Van Rijk, 2006; Viale, 2005; and Smeets, 2005

AXILLARY U/S and FNAC (Clinically negative axillas) l Ultrasound alone identified only 34% of positive axillas (Mathijsen; Surgical Oncology, 2006) l U/S plus FNAC identified 21% of positive axillas (Rijk; Annals of Surgical Oncology, 2006) l 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.

Author Method Zuber Sensitivity (%) Specificity (%) Micromet’s (Macro)met’s Frozen section 98 100 95 100 86 100 74 100 Imprint 47, 1 98, 3 Frozen section 88, 2 100 (2008) Leung Frozen section 50 (2007) Hameed imprint (2007) Pugliese imprint 41 (2006) Mori (2006) Pogacnik imprint 37 77 99, 2 Frozen section 27 96 100 Imprint 27 93 99 Imprint 40 78 Frozen section 64 83 (2005) Brogi (2005) Mewes (2003) Creager imprint 53 98 Frozen section 74 99 (2002) Tanis (2001)

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

Frozen Section Histology PRO Moderate sensitivity: 57 -74% l High specificity 99 -100% l Some morphologic information available and rough estimate of size of metastases l 10 -30 minutes turn around time – can be used intra-operatively l CON l l l l 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 l “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% l Specificity 98 -100% l All tissue saved for later permanent section l 10 -30 minutes turn around time – can be used intra-operatively l CON l l l 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 Gene. Xpert Assay l Not Commercially Available – Detects metastases in SLNs – Intraoperative l Test result: Quantitative l Technician operated – Fully automated l Quality Controls – External controls – Internal controls l Markers – TACSTD 1 – PIP l Preliminary Cutoffs determined l Cepheid Gene. Xpert System – Runs 1 to 16 samples Early validation with 90 SLNs complete l Future plans are unknown – last publication was 2006 l (Hughes. Ann Surg 2006; 243)

Molecular: Sysmex OSHA Assay (One Step Nucleic Acid Amplification) l CE Marked - Available in EU – Detects metastases >0. 2 mm in SLNs l Test result + + + = macrometastases + = micrometastases = negative l Technician operated – Part manual, part automated l – No internal control Marker – Cytokeratin 19 (CK 19) - Epithelial RD-100 i – Runs 4 samples plus controls Quality Controls – External controls and calibrators l l and calibrators Analytical determination of cutoffs l Validation with 101 patients l (Clin Cancer Res 2007; 13(16))

Molecular: Veridex Gene. Search™ BLN Assay l FDA approved and CE Marked – Intra-operative or post-operative – Detects metastases >0. 2 mm in SLNs – Allows decisions on ALND l Test result – Positive/Negative l Technician operated – Part manual, part automated l Quality Controls – Positive/negative external controls – Internal control l Markers – Cytokeratin 19 (CK 19) - Epithelial – Mammaglobin (MG) - Breast l Cepheid Smart. Cycler System – Runs 1 -6 samples plus 2 controls – Multiple run capability – Closed tube, real time RTPCR

New Molecular Assay Using Real-time RT-PCR l uses real-time RT-PCR to detect MG (mammaglobin) & CK (cytokeratin) 19 transcripts (m-RNA) l Identifies 0. 2 mm clinically significant metastases >

Real Time RT-PCR Procedure - m. RNA templated converted to c. DNA

Multiple Cycles Allow Amplification of Target Sequences

Polymerase Chain Reaction DNA amplification fluorescence molecules emission

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

Validation Study: Node Sampling Node A 1 2 3. 0 mm Node B 1 2 3 4 5 6 12. 0 mm Nodes parsed into ~2 mm pieces Assay 100% sampling H&E 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

Validation Study – Results 78 cases Permanent Section H&E & IHC + RT – PCR molecular Assay - + 12 2 - 1* 63 13 65 + for permanent section H&E or IHC must be >0. 2 mm *Only sample positive by IHC alone

Assay False Negative/ IHC Positive Histology Result Picture Goes here! micrometastasis of 0. 25 mm. IHC + Micromet

Validation Results Country/ Location Belgium Institute Jules Bordet U. S. A. 14 sites No. Histology Positives 13/78 16. 7% 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 %

Clinical Use: Standard Sampling Node A 1 2 3. 0 mm Node B 1 2 3 4 5 6 12. 0 mm Cutting Scheme same as in the Validation Study Assay 100% sampling Histology H&E IHC • Initially one SLN was tested, now all SLNs are being tested 2 mm • Histological sampling is different in Clinical Use

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

Clinical Use: Performance of the Assay 300 cases RT – PCR molecular Assay Permanent Section H&E & IHC + + 45 13* - 6** 236 51 249 + for permanent section H&E or IHC must be >0. 2 mm *In one of the 45 patients, histology was positive in a different SLN than the one tested in the assay **4 were (MI)<1 mm (size 0. 3 mm to 0. 75 mm) And all by IHC only 1 case: micrometastases between 1 and 2 mm

BLN Assay Performance Jules Bordet Institute Validation Study N=78 Jules Bordet Institute Post-Market Data N=300 Blumencranz et al. 2007 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 * Metastases size (mm) Sensitivity 0. 2 -1 70% (7/10) 1. 1 -2 84% (5/6) 2. 1 -4 90% (9/10) >4. 1 100% (19/19) * Overall BLN Assay Sensitivity vs. Histology 88. 2% (45/51) Overall Histology Sensitivity vs. BLN Assay 77. 6% (45/58) SLN Status BLN Assay ALND Status # Performed # (%) Positive 236 48 1 (2%) Micro (0. 2 mm – 2. 0 mm) 4 3 0 Macro (> 2. 0 mm) 1 1 0 Negative 13 13 2 (15%) ** Micro (0. 2 mm – 2. 0 mm) 12 12 2 (17%) Macro (> 2. 0 mm) Positive N Negative Histology 28 28 11 (39%) • *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 Frozen Section BLN Assay N Sensitivity % (95% CI) Specificity % (95% CI) 77 (61 – 89) (96 – 100) 95 93 (83 – 99) 22 3 99 (89 – 97) PPV % NPV Agreement % % 94 95 95 76 99 94 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) 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

CONCLUSION RT-PCR l Intraoperative Sensitivity > Frozen Section and Imprint l Performance is comparable to the standard of care = permanent H&E – Better? : Increased node tissue sampling l The BLN Assay identifies clinically relevant (>0. 2 mm) metastatic cancer l Detects metastases with challenging histology (lobular Ca) l Standardized and validated – Eliminates intra- and inter-laboratory variability l Objective and reproducible – Simple enough to be performed by a histo. technician or med. technician

Future l Will continue intra-operatively using the BLN Assay at the Institut Jules Bordet – Effectively being used intra-operatively and Performance is as expected l Will continue with current histology cutting – But are hoping that the assay once become the standard ; no need of histology anymore? l Research : l 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% Specificity 99 -100% 98 -100% 93 -94% Standardized No No Yes Labor required* Pathologist Cytologist Technologist Ease of evaluation Moderate Difficult Automated Nodal sampling* Limited 50% Sensitivity across cancer types Moderate High Morphologic Info Yes No No Turn around time 10 -30 minutes 30 -40 minutes

Conclusions l Current intraoperative histopathology/cytopathology on SLNs: – has high specificity but lower sensitivity – requires high level professional experience but still subjective – nodal sampling is limited l 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 l Technique and interpretation is pathologist and institution dependent l Non-standard procedure l Only 5 -10 % off the tissue analysed l Labor intensive / time consuming l Low sensitivity (micromet’s) l Evaluation challenging in some cases l Even for experienced pathologists (e. g. , Lobular Cancer) l Not being used at the Institut Jules Bordet