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