Transcript 0.2 mm

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:

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
Predicted by Size of the SLN Metastasis
Size of SLN metastases
>2 mm
macromets
0.2 and <2 mm
micromets
<0.2 mm
sub-micromets
Negative
Incidence of further axillary
metastases
45-79 %
10-25 %
7-15 %
~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.
Author
Method
Zuber
Frozen section
Sensitivity (%) Sensitivity (%) Specificity (%)
Micromet’s
(Macro)met’s
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%
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 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 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
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

– PIP
Preliminary Cutoffs determined
Cepheid GeneXpert System
– Runs 1 to 16 samples

Markers
– TACSTD1



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

– No internal control
Marker
– Cytokeratin 19 (CK19) - Epithelial
RD-100i
– Runs 4 samples plus
Quality Controls
– External controls and calibrators


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 RTPCR
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
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 ~2mm 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
RT –
PCR
molecular
Assay
Permanent Section H&E & IHC
+
-
+
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
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 only 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
Clinical Use
H&E
Sections
are 150
µm apart
IHC
H&E
Sections
are 100
µm apart
IHC
2 mm
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 >0.2mm
*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
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
Negative
Positive
ALND Status
Histology
N
# Performed
# (%) Positive
Negative
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)
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
N
Frozen
Section
BLN
Assay
223
Sensitivity
%
(95% CI)
Specificity
%
(95% CI)
77
99
(61 – 89)
(96 – 100)
95
93
(83 – 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)
Turn Around Time
70
Current average turn around time:
65
1 node: 30 min
60
> 1 node: 35 min
50
45
40
35
30
Patient Number
Turn Around Time = time from node removal to time BLN Assay result reported
97
93
89
85
81
77
73
69
65
61
57
53
49
45
41
37
33
29
25
21
17
13
9
5
25
1
Minutes
55
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

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%
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
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 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