peroperative investigations of the sentinel node dr. j.c. schobbens
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Peroperative Investigations of
the Sentinel Node
Dr. J.C. Schobbens
President of Belgian Society of Senology
Institut Jules Bordet, Brussels, BelgiumInstitut Jules Bordet, Brussels, BelgiumDr. I Veys, Dr. D Noterman, Dr. D Herten, Dr. P. DeneubourgDr. I Veys, Dr. D Noterman, Dr. D Herten, Dr. P. Deneubourg Dr. V. Durbecq, Dr. P. Bourgois,Dr.JM Nogaret, Dr. D. LarsimontDr. V. Durbecq, Dr. P. Bourgois,Dr.JM Nogaret, Dr. D. Larsimont
04 okt. 2008 Diegem04 okt. 2008 Diegem
SLN = Golden StandardSLN = 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: 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 (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
Incidence of Further Axillary Metastasis Incidence of Further Axillary Metastasis Predicted by Size of the SLN MetastasisPredicted by Size of the SLN Metastasis
Size of SLN metastasesIncidence of further axillary
metastases
>2 mm
macromets45-79 %
0.2 and <2 mm
micromets10-25 %
<0.2 mm
sub-micromets7-15 %
Negative ~10 %
Degnim, 2003; Van Rijk, 2006; Viale, 2005; and Smeets, 2005
AXILLARY U/S and FNACAXILLARY U/S and FNAC(Clinically negative axillas)(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 FNAC11,6 % SLN avoided8 % 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 Sensitivity (%)Micromet’s
Sensitivity (%)(Macro)met’s
Specificity (%)
Zuber(2008)
Frozen section 98 100
Leung (2007)
Frozen section 50 95 100
Hameed (2007)
imprint 86 100
Pugliese (2006)
imprint 41 74 100
Mori
(2006)
Imprint
Frozen section
47,1
88,2
98,3
100
Pogacnik (2005)
imprint 37 77 99,2
Brogi
(2005)
Frozen section
Imprint
27
27
96
93
100
99
Mewes
(2003)
Imprint
Frozen section
40
64
78
83
Creager (2002)
imprint 53 98
Tanis(2001)
Frozen section 74 99
2-Dimensional Slices of Complex 3-Dimensional Tumors Make Accurate Detection Difficult
No Met
Micromet
Macromet
Lymph Node
Cancer
Frozen Section HistologyFrozen Section HistologyPRO 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
Technical IssuesTechnical IssuesIntraoperative alternativesIntraoperative 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 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
Molecular Intra-operative Molecular Intra-operative OptionsOptions
Molecular: QRT-PCR GeneXpert AssayMolecular: 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 AssayMolecular: Sysmex OSHA Assay(One Step Nucleic Acid Amplification)(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 GeneSearchMolecular: Veridex GeneSearch™™ BLN BLN
AssayAssay 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 New Molecular Assay Using Real-time RT-PCRRT-PCR
uses real-time RT-PCR to detect MG (mammaglobin) & CK (cytokeratin) 19 transcripts (m-RNA)
Identifies clinically significant metastases > 0.2 mm
Real TimeReal TimeRT-PCR ProcedureRT-PCR Procedure
- mRNA templated converted to cDNA
Multiple Cycles Allow Amplification of Target Sequences
Polymerase Polymerase Chain ReactionChain Reaction
DNA amplification
fluorescence
molecules emission
Level of
Level of
Flu
ore
scence
Flu
ore
scence
Qualitative Interpretation of CK 19Qualitative Interpretation of CK 19
CYCLE THRESHOLD VALUES = CTsCYCLE THRESHOLD VALUES = CTs
3015 20 25 35 401050
Positive Negative
Ct Value (Threshold)
Cut-off
Negative Positive
Negative
Number of Amplification CyclesNumber of Amplification Cycles
Validation Study: Node Validation Study: Node SamplingSampling
3.0 mm
6
12.0 mm
Node A Node B
1 2 1 2 3 4 5
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)
Assay100% sampling
HistologyH&E
IHC
Validation Study – Results
78 78 casescases Permanent Section H&E & IHC
RT – PCR
molecular Assay
+
+ -
12 2
- 1* 63
13 65
+ for permanent section H&E or IHC must be >0.2mm
*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
13/78
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 %
Validation Results
Clinical Use: Standard Clinical Use: Standard SamplingSampling
• Initially only one SLN was tested, now all SLNs are being tested
• Histological sampling is different in Clinical Use
Assay100% sampling
Histology
3.0 mm
6
12.0 mm
Node A Node B
1 2 1 2 3 4 5
Cutting Scheme same as in the Validation Study
2 mm
H&E
IHC
Validation Study vs. Clinical UseValidation 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
300300
casescasesPermanent Section H&E &
IHC
RT – PCR
molecular Assay
+
+ -
45 13*
- 6** 236
51 249
+ 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 only1 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 PerformanceBLN Assay Performance
Jules Bordet InstituteValidation Study
N=78
Jules Bordet InstitutePost-Market Data
N=300
Blumencranz et al. 2007 Am J SurgUS Clinical Study
N= 407
OverallAgreement 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 BLN Assay According to Metastases SizeMetastases Size
SLN Status ALND Status
BLN Assay Histology N # Performed # (%) Positive
Negative
Negative 236 48 1 (2%)
Micro (0.2 mm – 2.0 mm) 4 3 0
Macro (> 2.0 mm) 1 1 0
Positive
Negative 13 13 2 (15%) **
Micro (0.2 mm – 2.0 mm) 12 12 2 (17%)
Macro (> 2.0 mm) 28 28 11 (39%)
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)
•*Note: the 6 patients with positive histology and unknown size are not included in the above tables
•** USA : 25%
*
*
Performance of BLN Assay vs. FrozenPerformance of BLN Assay vs. Frozen
Test Method
N Sensitivity
% (95% CI)
Specificity %
(95% CI)
PPV
%
NPV
%
Agreement
%
Frozen Section
223
77 (61 – 89)
99(96 – 100)
94 95 95
BLN Assay
95 (83 – 99)
93 (89 – 97)
76 99 94
All comparisons to permanent section H&E
Clinical Use of the BLN Assay at Morton PlantDr. Blumencranz ; ASCO Breast 2008
Clinical Use: Timing (first 100)Clinical Use: Timing (first 100)Turn Around Time
25
30
35
40
45
50
55
60
65
70
1 5 9
13
17
21
25
29
33
37
41
45
49
53
57
61
65
69
73
77
81
85
89
93
97
Patient Number
Min
ute
s
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-PCRCONCLUSION 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
FutureFuture 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 Comparison of Currently Available Intra-operative TestsIntra-operative Tests
Frozen SectionTouch Prep
CytologyMolecular
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 Limited 50%
Sensitivity across cancer types
Moderate Moderate High
Morphologic Info Yes No No
Turn around time 10-30 minutes 10-30 minutes 30-40 minutes
ConclusionsConclusions 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!Thank you for the attention!
St.-Agatha Catania 255
Current Used Techniques Have Current Used Techniques Have LimitationsLimitations
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
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