Iban Aldecoa Ansorregui

Hospital Clínic de Barcelona

Universitat de Barcelona

ialdecoa@clinic.cat

Tools for improving LN analysis

• Specimen tattooing:

o Greater LN yields

o LNS most prone to harbour metastases?

• Pooling:

o Higher feasibility

o Acceptable results?

Colonoscopy

Williams, J. G. et al. Color. Dis. 15, 1–38 (2013)

Dawson, K., et al. Arch. Surg. 145, 826–830 (2010)

Bartels, S. A. L., et al. Gastrointest. Endosc. 76, 793–800 (2012)

Dawson, K., et al. Arch. Surg. 145, 826–830 (2010)

Bartels, S. A. L., et al. Gastrointest. Endosc. 76, 793–800 (2012)

Feo, C. V et al. J. Negat. Results Biomed. 14, 9 (2015)

• Bartels et al 95 tattooed patients; 210 non-tattooed as controls

• Median LN tattooed 15 (IQR 10-20) vs. non tattooed 12 (9-16) (P=0,014)

• Multivariante analysis: tattoo, specimen location, pT and pN stage (P<0,05)

Endoscopic tattooing for LN retrieval

To determine the usefulness of preoperative tattooing

• Colonic lesions (colon carcinoma and preinvasive lesions)

• Correlation with OSNA LN staging

Aim

• Colon carcinomas / adenomas May'12-Dec-'13

• Inclusion criteria:

+ 18 years old IHC CK19 +

• pT1 polyps with risk factors

• Unresectable adenomas

• pT1-2 colon carcinoma

• Exclusion criteria:

pT3-4 Rectal T. Appendicular Ca

Synchronic t. CC in IBD Other malignant tumours

Formalin Stent Adipose tissue involvement

Sample

• Screening (FIT) + → Colonoscopy

• Tattoo (median 63 days):

1. Polyps ≥ 2 cm

2. Suspicious lesions (excluding cecum/rectal)

• Unresectable polyps

• Suspected invasive submucosal carcinoma

• Partially resected advanced adenomas (> 10 mm,

villous, high grade dysplasia)

Endoscopic evaluation and tattooing

Results

(...)

15.3 % (44/286) tattooed LN

7.3 % (28/386) non-tattoed LN

OR 3.1

OSNA +

Results

• OSNA + 42/71 (59,2%) → cut-off 100 c/μL

• TTL median: 1.350 c/μL

• pN0 cases: 1.275 c/μL

• Two pN+ cases: 560.000 y 41.160 c/μL

• OSNA result associated to:

• Tumour size (P=0,02)

• High grade (P<0,01)

OSNA Results

• Median 984 days

• 2 pN+ cases → chemotherapy

• 2 cases → metastases at 4 and 21 months f/u

• pT2N0, Right c., low grade, 25 y 30 mm

1. No tattooed OSNA assessed 14/18 LN TTL 47,760 c/μL

2. Tattooed OSNA assessed 10/20 LN TTL 0

OSNA Results

Conclusions

• Colonoscopic tattooing highly efficient LN procurement

• Tattooing helps:

1. Endoscopist and the surgeon localize the tumour

2. Pathology tool to:

o Harvest a higher amount of LN

o Find those LNs which might shelter tumour

• Expansion of presurgical endoscopic tattooing

o Benefit patient’s diagnosis and therapeutic management

HE section

Neoplastic foci

Tissue allocation bias (TAB)

2-5 µm

Primary tumor5 year survival (%)

N0 / N1 / N2

T1 97.4 / 87.6 / 68.7

T2 96.8 / 87.7 / 76.6

T3 87.5 / 68.7 / 47.3

T4 71.5 / 50.5 / 27.1

Gunderson et al., JCO 28: 264-271, 2010

LN status

Micromtx

ITC

Sloothaak, D. A. M. et al. Eur. J. Surg. Oncol. 40, 263–269 (2014)

Occult disease in LN

But,

How much tumor does a LN have?

How do we quantify it?

H&E (conventional method) OSNA

(molecular method)

OSNA in breast SLN

Predictors of additional axillary metastases

Peg, V. et al. Breast Cancer Res. Treat. 139, 87–93 (2013)

Espinosa-Bravo, M. et al. Eur. J. Surg. Oncol. 39, 766–773 (2013)

Feasibility

Aim

To propose a new economical and effective

method of molecular lymph node analysis

in colorectal cancer for routine practice

H&E

≤600 mg

OSNA

Lymph node Pooling

Samples

• Colon carcinomas / adenomas

• Inclusion criteria:

+ 18 years old IHC CK19 +

CC Adenoma

• Exclusion criteria:

Rectal T. Metastatic disease

CC in IBD FAP Other malignant tumours

Formalin Stent Adipose tissue involvement

Neoadjuvant CT

– 86 CC / 1.757 LN OSNA

– Prospective (Feb 2015 - Dec 2015)

Cohorts: Pooling

– 102 CC cases/1.461 LN OSNA

– Retrospective (June 2012 - Dec 2013). Validation set

– *Excl. criteria cN1, gross metastatic LN

Cohorts: Individual

Clinical parameter n (%) / median (IQR) p-value

OSNA pooling

cohort

OSNA individual

cohort

Grade <0.001

High 49 (57.0) 26 (25.5)

Low 37 (43.0) 76 (74.5)

Vascular invasion 0.045

No 65 (75.6) 91 (89.2)

Yes 21 (24.4) 11 (10.8)

Perineural invasion 0.013

No 76 (88.4) 98 (96.1)

Yes 10 (11.6) 4 (3.9)

pT 0.004

pT0 6 (7.0) 8 (7.8)

pTis 6 (7.0) 17 (16.7)

pT1 9 (10.4) 26 (25.6)

pT2 16 (18.6) 19 (18.6)

pT3 32 (37.2) 24 (23.5)

pT4a 17 (19.8) 8 (7.8)

Results

Results

• Time spared: 2-3 hours/case

Results: Concordance with HE

Pooling

cohort (%)

Individual

Cohort (%)

Sens (pN+ and OSNA+) 88,9 100

Spec (pN0 and OSNA-) 79,2 44,6

PPV 33,3 16,4

NPV 98,4 100

Concordance 80,2 50

Internal validation test

• 42 cases LN collected in 2–7 tubes

• Sens 83.3%; spec 93.3%; agreement of 90.5% (k = 0.756)

• Discordant cases

↓

low TTL values

Conclusions Pooling Study

• Pooling allows analyzing a high number of LN with

few molecular determinations per patient

• This approach grants a cost-effective means of

introducing LN molecular analysis of CRC into

pathology departments

• Warrants a more accurate LN pathological staging

• The OSNA in CC must overcome different challenges:

– Feasibility

– Submitting the whole LN

– Characterization: tumor and patient

– Correlation of different prognostic factors

Perspectives

Citology (CK19) HE

Touch-prep. Five LNs/slide

Thank you

• RT-LAMP with/without Indian ink at 1:100

Indian ink – OSNA interference test