The pathology protocol outlines the examination of lymph nodes and tumor specimens from colon cancer patients in a clinical trial, including standard hematoxylin and eosin staining of all lymph nodes as well as further analysis using immunohistochemistry of sentinel lymph nodes from patients with no detected metastases to identify possible micrometastases. The protocol provides definitions for isolated tumor cells and micrometastases and specifies the handling of specimens, identification of sentinel lymph nodes, and pathological assessments that will be performed and documented.

1. PATHOLOGY PROTOCOL

2. INTRODUCTION
• better prognosis colon cancer
• better staging
• basis of the study:
–ex vivo SLNM
–pathology protocol
RATIONALE OF THE STUDY

3. RATIONALE OF THE STUDY
• high disease recurrence in pN0 colon
cancer patients.
• 5-yr disease recurrence 5yr OS
stage I 10% 90%
stage II 15-30% 75%
• ~ 1150 pts yearly in the Netherlands

4. EnRoute⊕ PROTOCOL
CTC
SURGERY
Diagnosis
Colon carcinoma
Informed
Consent
General workup
Colonic cancer workup
SNAQ/dietician
Bloodsampling
RANDOMIZATION
pN0(micro+)
WHO status
ASA classification
FOLLOW UP
FOLLOW UP
CHEMOTHERAPY
CTC CTC
SNAQ/dietician
Bloodsampling Bloodsampling
REGISTRATION
FOLLOW UP
pN0(micro-)
FOLLOW UP
GROUP A
GROUP B
GROUP C
CTC
SURGERY
Diagnosis
Colon carcinoma
Informed
Consent
General workup
Colonic cancer workup
SNAQ/dietician
Bloodsampling
RANDOMIZATION
pN0(micro+)
WHO status
ASA classification
FOLLOW UP
FOLLOW UP
CHEMOTHERAPY
CTC CTC
SNAQ/dietician
Bloodsampling Bloodsampling
REGISTRATION
FOLLOW UP
pN0(micro-)
FOLLOW UP
GROUP A
GROUP B
GROUP C
SURGERY
ex vivo SLNM

5. 1. oncological colonic resection
2. ex vivo sentinel lymph node procedure
3. pathological examination
standard H&E procedure
fine pathological analysis in pN0 patients
EnRoute⊕ PROTOCOL

6. 1. oncological colonic resection
2. ex vivo sentinel lymph node procedure
3. pathological examination
standard H&E procedure
fine pathological analysis in pN0 patients
EnRoute⊕ PROTOCOL

7. RIGHT HEMICOLECTOMY
• suture marked
– a. ileocolica
– a. colica dextra
– right branch
a. colica media
SURGICAL RESECTIONS

8. LEFT HEMICOLECTOMY
• suture marked
– left branch
a. colica media
– a. colica sinistra
SURGICAL RESECTIONS

9. SIGMOID RESECTION
• suture marked
– aa. sigmoidales
– a. rectalis superior
SURGICAL RESECTIONS

10. 1. oncological colonic resection
2. ex vivo sentinel lymph node procedure
3. pathological examination
standard H&E procedure
fine pathological analysis in pN0 patients
EnRoute⊕ PROTOCOL

11. AIM
• more and probably more relevant LN’s
• fine pathology only in SLN’s
(in pN0 patients)
ex vivo SLNM

12. PROCEDURE
• within 15 minutes following resection
• performed by surgeon
• or by pathologist according to local facilities
• opening colon antimesenterial border
• Patent-V blue, 1 ml in toto
• submucosal injection, 5 min massage
• formalin fixation within 15 minutes
ex vivo SLNM

13. ex vivo SLNM preparation

14. ex vivo SLNM opening colon

15. ex vivo SLNM cleaning

16. ex vivo SLNM injection

17. ex vivo SLNM massage

18. ex vivo SLNM detection

19. ex vivo SLNM transportation

20. 1. oncological colonic resection
2. ex vivo sentinel lymph node procedure
3. pathological examination
standard H&E procedure
fine pathological analysis in pN0 patients
EnRoute⊕ PROTOCOL

21. STANDARD H&E PROCEDURE
• all lymph nodes (LN vs. SLN)
• all lymph nodes in marked cassettes
• blue vs non-blue
• non-SLN bivalve
• one H&E slide per level
PATHOLOGY PROTOCOL

22. FINE PATHOLOGY
• only in SLN, only in pN0 patients
• definition and number of sentinel nodes
(SLN)?
– the first three blue nodes nearest to the tumour
(defined by pathologist)
PATHOLOGY PROTOCOL

23. FINE PATHOLOGY
• one SLN per cassette (marked)
• inclusion according to size
– <5mm in toto
– 5-10 bivalve
– >10 4 sections
PATHOLOGY PROTOCOL

24. PATHOLOGY PROTOCOL
FINE PATHOLOGY
• pN0: all SLN’s studied at 3 levels
• slides from each level HE and IHC
• section interval 250 μm
• IHC: CK AE1/AE3,
CAM5.2, LU5

25. PATHOLOGY PROTOCOL
STEP 1 resection - fixation
STEP 2 fixation – conv. H&E staining
STEP 3 conv. H&E staining – IHC

26. PATHOLOGY PROTOCOL
STEP 1
FRESH SPECIMEN
digital picture
specimen variables
- length colon segment
- length tumour-base radix
- total weight specimen
- width mesocolon
- size of the tumor
- number of blue nodes
- tumor specimen
fixation protocol for
at least 24h
fixation protocol for
at least 24h
FIXED SPECIMEN
ex vivo SLNM
#
# according to
local facilities
#

27. PATHOLOGY PROTOCOL
STEP 1
digital picture
specimen variables
- length colon segment
- length tumour-base radix
- total weight specimen
- width mesocolon
- size of the tumor
- number of blue nodes
- tumor specimen
#
# according to
local facilities

28. PATHOLOGY PROTOCOL
STEP 2 fixation protocol for
at least 24h
identification LNN
(≥ 10 LNN)
first 3 blue SL nodes
nearest to the tumour
other blue/non-blue
LNN
marked cassettes 1-3 marked cassettes >3
< 0.5 cm: in toto
0.5 < 1cm: bivalve
> 1 cm: lamellate
bivalve
conventional H&E staining

29. PATHOLOGY PROTOCOL
STEP 3
a.
b.
conventional H&E staining
(all sections LNN 1 – 10)
macrometastasis
(pN+)
no macrometastasis
(pN0)
NO IHC staining H&E and IHC staining
of all SLNs (max 3)
detection of
micrometastasis
EXCLUSION CRITERIA
< 10 LNN
T4
angioinvasion
colon perforation at presentation
obstruction at presentation

30. PATHOLOGY PROTOCOL
MICROMETASTATIC DISEASE
• definitions used in EnRoute⊕
- isolated tumor cells (ITC)
- micrometastasis (MM)

31. PATHOLOGY PROTOCOL
DEFINITIONS USED IN EnROUTE⊕
Definitions used in the En Route⊕ study
Isolated Tumor Cells
(ITCs)
Micrometastases
(MMs)
size < 0.2 mm 0.2 – 2 mm
localization no defined no defined
detection method HES and/or IHC HES and/or IHC
formal designation (present vs
absent)
pN0i+ vs pN0i- pN0mi+ vs pN0mi-
EnRoute⊕ if present pN0micro+ pN0micro+

32. PATHOLOGY PROTOCOL
• pathology flow diagram available
• pathology (e)CRF / PALGA
• financial compensation
– € 100,- per patient (all registered patients)
• protocol discussion “Pathologendagen”
PATHOLOGY PROTOCOL

33. ACKNOWLEDGEMENTS
Steering pathologists
– H. J. (Hans) van de Linden (JBZ)
– P (Peet) Nooijen (JBZ)
– V.T.H.B.M. (Vincent) Smit (LUMC)
Educational Grant:
PATHOLOGY PROTOCOL