RAPID MOLECULAR ANALYSIS AND COMPARISON OF …...Basalioma 7 Normal lymph node 26 Benign breast...
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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