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INTRODUCTION

• Cancer in our home pets is an emerging issue, and the trend is that the

owners treat their animals

• It is often identified too late, with multiple metastases spread around the

body and leaving the only options, euthanasia or drastic surgery

• Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has been used

for the real-time, in-vivo identification of human cancers

• In animals, there is an option of collecting large masses of tissue from a

primary cancer and multiple metastases even in near in-vivo environment

• The aim of this study was to

Build a pet database and test it for real-time tissue identification

Compare the molecular fingerprint of primary cancers to multiple

metastases

Identify the molecular fingerprints of different canine and feline

cancers and to compare it to adjacent human cancers

RAPID MOLECULAR ANALYSIS AND COMPARISON OF CANINE, FELINE AND HUMAN TUMOUR SAMPLES WITH REIMS; METASTASIS BASED IDENTIFICATION OF PRIMARY TUMOUR

Julia Balog 1,2, Richard Schaffer1, Lajos Balogh3, Julianna Thuroczy3, Steven Pringle4 and Zoltan Takats2 1 Waters Research Center, Budapest, Hungary. 2 Imperial College London, UK. 3 Budafok Allatgyogyaszati Kozpont, Budapest, Hungary 4 Waters Corporation, Wilmslow, UK.

RESULTS

Pet database building

• Tissue is collected from all animals attending the clinic for cancer

surgery euthanasia.

• The current database contains tissue from 31 dogs (Table 2), 7

cats (Table 1) and 1 guinea pig.

• A Principal Component Analysis (PCA) followed by a Linear

Discriminant Analysis (LDA) is used as Classifier (Fig 4).

• The database is validated with 5-fold cross-validation with above

85% correct classification rate.

• For each species it is necessary to build a separate database as

shown with normal breast tissue and breast carcinoma.

• Once a database is built, it will be tested real-time, in-vivo in the

operating theatre of the animal clinic.

CONCLUSION

REIMS method was successfully implemented into

veterinary environment

We have created a cat and dog database from a

diverse range of spontaneous cancers in the pets

We successfully tested our database real-time in-

situ

Primary tumour and its metastases, irrespective of

the localisation, have similar features, thus the

primary cancer can be identified only based on the

molecular fingerprint of the metastasis

There are similar lipid species in human, cat, dog

and guinea pig tissue signal, however invasive

ductal breast carcinoma is different these species,

thus a database has to be built for each species

separately

Comparison of human, dog, cat and guinea pig breast carcinoma

• Human, canine, feline and guinea pig invasive ductal breast carcinoma was

compared (Fig. 9 & 10).

• The signal of the normal tissue and the tumour in different species is different,

the database built on feline spectra cannot be used for canine and the other way

around.

• Specific lipids for the tumour of each species based on the PC loading plots is

shown on Figure 11. Human tissue has more TGs compared to animals,

plasmalogens are more abundant in canine, and there are differences in the PE

levels of feline and guinea pig.

METHODS

REIMS method with monopolar surgical pencil (Fig. 1, 2 & 3)

• Xevo G2-XS Mass spectrometer

• Negative ion mode

• m/z 600-900

• Continous IPA solvent spray with 0.5 ng/ul Leucin-enkephalin

• Samples were collected at Budafok Animal Care Center from

spontaneous cancer pets brought in for surgery or euthanasia

Primary colorectal adenocarcinoma vs

Metastasis in liver and lymph nodes

• Comparison of primary cancer to metastases –

Figure 5 shows that the signal of the primary cancer

and the metastasis (in liver of lymph node) have

similar signal and group together in the PCA space.

The underlying spectra are shown on Figure 7.

• Canine tumour and normal tissue feature similar

species as human tissue, including fatty acids,

phospholipids, diacyl-glycerols, triglycerides and

sphingolipids.

• MSMS signal was recorded using the most

abundant peaks in both normal tissue, primary and

metastatic cancer (Table 4). Most peaks contain

multiple lipid species and the distribution of the

different lipids is different in each tissue type (Fig. 8).

• It is possible to identify the primary tumour based

on the mass spectral fingerprint of the metastasic

tissue.

Figure 10. 3-dimensional linear discriminant plot

of normal breast tissue (green) and breast

carcinomas in dfifferent species

Figure 11. Species specific lipids based on the PCA loading plot.

Figure 9. Mass spectra of breast carcinoma in different species

Figure 7. Mass spectra of normal colon mucosa and liver; primary colorectal adenocarcinoma;

metastases in liver and lymph nodes.

Colon mucosa

Primary colon

adenocarcinoma

Metastasis from colon

adenocc in liver

Normal liver

Metastasis from colon

adenocc in lymph node

Figure 6. 3-dimensional PCA plot of normal colon and liver

tissue vs primary and metastatic adenocarcinoma. The primary

and metastatic cancer group together on the plot.

Colon mucosa

Primary colon

adenocarcinoma

Normal colon serosa Normal liver

Metastasis from colon

adenoccarcinoma

Histology Diagnosis - Feline # of DB

entries

Breast carcinoma 12

Metastasis from breast

carcinoma

26

Lymphoma in kidneys 27

Leiomyoma 15

Capsule of leiomyoma 4

Squamous cell carcinoma 9

Metastasis in liver from mouth

squamous cell carcinoma

26

Sarcoma 18

Figure 4. Pseudo 3 dimensional LDA plot of different

feline cancers and adjacent normal tissue.

Sampling (cutting line)

Scan 19

Scan 24

Testing the canine/feline database

real-time, in-situ

Figure 5. Real-time classification of feline

mouth carinoma metastasis in liver. In every

second, the software will display a tissue

identification result . The metastasis was

identified correctly in scans 22-24.

Histology Diagnosis –

Canine

# of DB

entries

Histology Diagnosis # of DB

entries

Basalioma 7 Normal lymph node 26

Benign breast adenoma 19 Normal breast 35

Breast carcinoma 21 Normal colon mucosa 10

Colon adenocarcinoma 10 Normal colon serosa 10

Metastasis from hemangiosarcoma 37 Normal liver 44

Metastasis in liver from pancreatic

carcinoma

10 Normal lung tissue 19

Mastocytoma grade II-III 45 Normal spleen 16

Melanoma 5 Normal vagina 6

Metastasis from colon adenocarc. 21 Normal pancreas 15

Metastasis from head squamous cell

carinoma

14 Normal skin 9

Metastasis from mastocytoma 20 Normal testicle 35

Metastasis in spleen 13 Normal epididymis 11

Pancreatic cancer 15

Perianal tumor 5 Sertoli cell tumor 17

Pododermal carcinoma 13 Squamous cell carcinoma 20

Prostate cancer 12 Vagina leiomyoma 33

Sarcoma 9

Number of spectra

Number of correct

classifications

Number of incorrect

classifications

Number of outliers

Correct Classification

Rate (Exluding outliers)

Correct Classification

Rate (Including outliers)

Total – Canine 599 537 4 58 99.26% 89.65%

Total – Feline 150 130 0 20 100% 86.67%

Figure 8. Xevo G2-XS Mass Spectrometer with REIMS source

Metastasis from colon

adenocarcinoma in liver

Primary colon

adenocarcinoma

Normal colon

mucosa

MSMS of peak

687.527

m/z Exact

mass Lipid Identification Ion

Cancer

vs

Normal

657.518 657.5230 DG(20:1/16:0) M+Cl

657.5230 DG(18:1/18:0) M+Cl

673.483

673.4814 PA(16:0/18:1) M-H

673.4815 PA(16:1/18:0) M-H

673.4814 PE(16:0/16:0) M-NH3-H

687.527 687.5335 SM(d18:1/16:0) M-H

687.5335 SM(d16:0/18:1 M-H

697.482 697.4814 PA(18:2/18:1) M-H

697.4814 PE(18:2/16:0) M-NH3-H

718.540 718.5392 PE(16:0/18:0) M-H

718.5392 PE-NMe2(16:0/16:0) M-H

723.506 723.497 PA(20:3/18:1) M-H

723.497 PE(18:1/18:0) M-NH3-H

725.514 725.5127 PE(18:2/18:0) M-NH3-H

725.5127 PE(18:1/18:1) M-NH3-H

742.541 742.5392 PE(18:0/18:2) M-H

742.5392 PE(18:1/18:1) M-H

744.557 744.5549 PE(18:1/18:0) M-H

744.5549 PE-NMe2(18:1/16:0) M-H

749.512

749.5127 PE(18:1/20:3) M-NH3-H

749.5127 PA(18:1/22:4) M-H

749.5127 PA(18:0/22:5) M-H

749.5127 PA(18:2/22:3) M-H

750.541 750.5443 PE(P-18:0/20:4) M-H

750.5443 PE(P-16:0/22:4) M-H

766.540 766.5392 PE(18:0/20:4) M-H

766.5159 PC(16:1/16:0) M+Cl

770.571

770.5705 PE(18:0/20:2) M-H

770.5705 PE-NMe2(18:1/18:1) M-H

770.5705 PE-NMe2(18:0/18:2) M-H

794.565 794.5705 PE(22:4/18:0) M-H

794.5472 PC(18:1/16:0) M+Cl

Figure 2. Schematics of REIMS source

Figure 1. REIMS source

Table 1. Felline database

Table 3. 5-fold cross-validation results on feline and canine database

Table 2. Canine database

Table 4. Abundant lipids in normal colon

mucosa and adenocarcinoma. Top candidates

of possible lipid IDs.

Sampling –

Spectrum acquisition Pre-processing

• lockmass correction

• background subtraction

• TIC normalisation, bining to 0.1

Multivariate statistics

• PCA

• LDA

1 2 3

Figure 3. Data analysis overview