Study of aggressiveness prediction of mammary adenocarcinoma by
Raman spectroscopy
Renata Andrade Bitar,1,2 Herculano da Silva Martinho,1 Leandra Náira Zambelli Ramalho,3 Arnaldo
Rodrigues dos Santos Junior,1 Fernando Silva Ramalho,3 Leandro Raniero2 and Airton A. Martin2*
1
Centro de Ciências Naturais e Humanas (CCNH), UFABC, 166, Santa Adélia Street, Santo André,
SP, 09210-170, Brazil
2
Laboratory of Biomedical Vibrational Spectroscopy (LEVB), Institute of Research and
Development, Universidade do Vale do Paraíba, Univap, Avenida Shishima Hifumi, 2911,
Urbanova, CEP 12244-000, São José dos Campos, SP
3
Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto (USP), 3900, Bandeirantes
Avenue, Ribeirão Preto, SP, 14049-000, Brazil
ABSTRACT
Although there are many articles focused on in vivo or ex vivo Raman analysis for cancer diagnosis, to the best of our
knowledge its potential to predict the aggressiveness of tumor has not been fully explored yet. In this work Raman
spectra in the finger print region of ex vivo breast tissues of both healthy mice (normal) and mice with induced mammary
gland tumors (abnormal) were measured and associated to matrix metalloproteinase-19 (MMP-19) immunohistochemical
exam. It was possible to verify that normal breast, benign lesions, and adenocarcinomas spectra, including the subtypes
(cribriform, papillary and solid) could have their aggressiveness diagnosed by vibrational Raman bands. By using MMP19 exam it was possible to classify the samples by malignant graduation in accordance to the classification results of
Principal Component Analysis (PCA). The spectra NM /MH were classified correctly in 100% of cases; CA/CPA group
had 60 % of spectra correctly classified and for PA/AS 54% of the spectra were correctly classified.
Keywords: Raman spectroscopy, Breast cancer detection, Multivariate statistical analysis, Principal
components analysis, MMP-19
1. INTRODUCTION
Raman spectroscopy has emerged as a non-destructive analytical tool for the biochemical characterization of breast
tissues due to several advantages such as sensitivity to small structural changes, non-invasive sample capability, and for
non-requirement sample preparation. Many in vitro studies have been applying Raman spectroscopy to analyze
biological tissue for diagnostic purposes [1-4]. However, in vivo Raman experiments are scarce due to the great difficulty
in differentiate the subtypes of breast cancers, and also, only a few experiments have used animal models for the study of
it clinical applicability [5-6]. Experimental animal models of carcinogenesis seem to be a scientifically acceptable way
to study the carcinogens process in the mammary gland, because they present similar diseases to the human mammary.
The animal model is particularly important in studies of clinical applicability of the optical biopsy in breast cancer
diagnosis. Fournier et al. (2006) have investigated in vivo detection of mammary tumors in a rat model using
autofluorescence imaging in the red and far-red spectral regions. The authors identified intensity variation in the
autofluorescence images of malignant tumors under @670 nm excitation line [7]. It was high the adjacent normal tissue,
whereas intensity of benign tumors was lower compared to normal tissue. In the finger print Raman regions a work of
Moreno et al.(2010) reported the in vivo Raman experiment to evaluate the vibrational modes of malignant and benign
breast tissues with the following diagnosis: fibroadenoma, invasive ductal carcinoma, ductal carcinoma in situ, and
fibrocystic condition. Quadratic discriminate analysis, a multivariate statistical method of analysis, showed 98.5%
separation between normal and altered tissue [8]. Besides the Raman finger print region, the identification of normal and
cancer breast tissue of rats was also investigated by using high-frequency
*amartin@pq.cnpq.br; phone/fax +55 12 39471165
Biomedical Vibrational Spectroscopy V: Advances in Research and Industry,
edited by Anita Mahadevan-Jansen, Wolfgang Petrich, Proc. of SPIE Vol. 8219, 821914
© 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.909159
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FT-Raman spectroscopy with a near-infrared excitation source on in vivo and ex vivo measurements [9].
Aaron M Mohs et al. (2010) report the use of a hand-held spectroscopic device emitting at 785 nm and near-infrared
contrast agents for detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and
surface-enhanced Raman scattering (SERS) signals. In vivo studies using mice breast tumors demonstrate that the tumor
borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated
with tumor bioluminescence [10].
Although the use of Raman spectroscopy has been widely used for cancer diagnosis, only a few studies have extended
their analysis beyond the diagnostic confirmation. The correlation between the aggressive potential of a lesion and their
nucleic acids and proteins Raman bands, for example, had seldom been discussed in the literature. There are numerous
proteins that can be studied by Raman spectroscopy. However, the metalloproteinases are the ones that are involved with
the maintenance of the collagen extracellular matrix. In normal tissue homeostasis, the interacting network of proteases
and their natural inhibitors maintain a proteolytic balance. During cancer progression, this balance is disturbed by
overexpression of proteases including at least matrix metalloproteases (MMPs) and related families of proteases, the
ADAMs (a disintegrin and metalloproteases) and ADAMTS (ADAM with thrombospondin repeats). This imbalance
alters the non-cellular compartment, which in turn activates downstream molecular effectors leading to the establishment
of a milieu permissive for tumor progression, invasion and dissemination [11-12]. The human MMP-19, recently
identified, has the basic structure characteristic of all MMPs, including: signal sequence on N-terminal, a dominance of
propeptide with residual cystein necessary to maintain the enzymatic latency, the activation sequence that contains a site
of ligation with zinc and a dominance of “hemopexim” terminal [13]. There is a progressive loss of the MMP-19 from
the in situ carcinoma to the mammary invasive cancer, in which there is a nearly complete loss of the expression of the
MMP-19, with only a small amount of tubular carcinomas showing any immunoreactivity. These results can confirm
some finds of this study, where the normal mammary tissue and mammary hyperplasia presented a higher expression of
the MMP-19, followed by the cribriform and lastly by the papillary and solid adenocarcinomas. Thus, these MMPs are
associated with breast cancer development and tumor progression and are proper candidates for further functional
analysis of their role in breast cancer.
Therefore, the purpose of the present study was to investigate the potential of Raman spectra to explain the biochemical
differences between some mammary tumors specimens with dubious and complex histopathology associated with the
MMP-19 immunohistochemical exam to verify the tumor aggressiveness.
2. EXPERIMENTAL DETAILS
2.1 Experimental Mammary Carcinogenesis
This research followed the policies and rules that regulate the researches involving animal and the ethic principles on the
animal experimentation, edited by the Brazilian College of Animal Experimentation (COBEA) and obtained approval
from the Committee of Ethics in Researches of the University of Vale do Paraíba (CEP/UNIVAP).
The experimental group was formed by 20 young virgin Sprague-Dawley female rats. Mammary gland tumors were
induced by a single dose of 50 mg/Kg of DMBA (7,12-dimethylbenz(a)anthracene) diluted in soy oil (1 mL) given
intragastrically by gavage [9]. All of the rats, with an average weight of 185 g, received the chemical carcinogen at the
age of 40 days. The rats were divided in two groups, five of them being the Control Group, where the gavage procedures
were simulated only with soy oil; and the others 15 rats have composed the DMBA Group, where there was application
of the carcinogen itself.
All animals were bred under ideal conditions of temperature, humidity and light, and they were fed with appropriate
rations in pellets and filtered water. We performed physical examinations 3 times a week. The six pairs of mammary
glands were checked by inspection, touching, and palpation. The mammary lesions were found, single or multiple, on
the age of 114 days, approximately 10 weeks after the gavage procedure with DMBA, and the Raman measurements
started. It was noted that all rats submitted to gavage developed malignant tumors in at least one breasts, whilst rats from
the control group did not develop any.
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2.2 Immunohistochemical Evaluation (MMP-19 exam)
Immunohistochemical staining was performed by using an avidin-biotin peroxidase system (Novostain Super ABC kit;
Novocastra Laboratories, Newcastle Upon Tyne, UK). The sections of normal mammary tissue (NM); mammary
hyperplasia (MH); cribriform adenocarcinoma (CA); papillary adenocarcinoma (PA); solid adenocarcinoma (AS); and
cribriform and papillary adenocarcinoma (CPA) were then incubated with anti-matrix metalloproteinase 19 (MMP-19)
(1:100, clone 9F6, Novocastra Laboratories, Newcastle Upon Tyne, UK) as primary antibody, for two hours at room
temperature (25°C) in a humid chamber. After washes in PBS solution, biotinylated universal secondary antibody
(Novocastra Laboratories, Newcastle Upon Tyne, UK) was applied for 30 minutes. The sections were incubated with the
avidin–biotin complex reagent (Novocastra Laboratories, Newcastle Upon Tyne, UK) for 30 minutes and were developed
with 3.3-diaminobenzidine tetrahydrochloride in PBS, pH 7.5, containing 0.036% hydrogen peroxide, for five minutes.
Light Mayer’s hematoxylin was applied as a counterstain. The slides were then dehydrated in a series of ethanols and
mounted with Permount (Fischer, Fairlawn, NJ, USA).
Samples of normal rat’s mammary tissue were used as positive control for MMP-19. Negative controls for
immunostaining were prepared by omission of the primary antibody. Mammary gland tissues were considered to be
positive when distinct brown cytoplasmic (MMP-19) staining was present homogenously. The number of
immunoreactive cells was assessed semi quantitatively, in 30 fields chosen randomly, at high power (400 times
amplification magnitude): 0 = no stained cells; + = less than 10 % positive cells; ++ = 10–50 % positive cells; and +++ =
more than 50 % positive cells [11].
2.3 Raman experiments and statistical analysis
All Sprague-Dawley rats were sacrificed and the mammary glands, healthy and with tumors, were removed, identified,
placed in cryogenic tubes Nalgene®, and stocked in liquid nitrogen (77K) for ex vivo analysis though FT-Raman
spectroscopy. In order to acquire the FT-Raman spectra, Bruker RFS 100 spectrometer was used. The mammary
samples were defrosted in physiologic solution on 0.9% and sectioned in one or two fractions of 2 mm3 each. To spectral
collection, three different points of each sample were chosen to be measured by using 300 mW of laser power, 300 scans
and 4 cm-1 resolution. Soon after FT-Raman spectra procedure, mammary tissue fragments were stored into 10 %
formaldehyde solution and sent to histopathological exam (Hematoxylin and Eosin stain and MMP-19
immunohistochemical exam).
All Raman spectra were stored and converted to ASCII format in order to be compatible with the Origin®7.0 SR0 and
Minitab®15.1.0.0.l software’s for pre-processing and statistical analysis. For pre-processing procedures, all spectra were
submitted to baseline correction of by automatic subtraction of a fifth degree polynomial followed by vector
normalization. The statistical algorithm suggested for the spectral analysis was composed by Principal Components
Analysis (PCA), which was applied to classify the Raman spectra in the shift region between 500 and 1800 cm-1.
3. RESULTS AND DISCUSSION
Figure 1 show the NM, MH, PA, CA, and the histological sections, in which the MMP-19 expression were indicated by
the arrows. In the NM and MH tissues were observed a larger quantity of stained cells followed by the CA, PA and SA
with a decreasing expression of the MMP-19.
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A
B
C
D
E
Figure 1: MMP-19 expression (X 200 times) indicated by arrows in (a) normal mammary gland (NM); (b) mammary
hyperplasia (MH); (c) cribriform adenocarcinoma (CA); (d) papillary adenocarcinoma (PA); and (e) solid
adenocarcinoma (SA).
Figure 2 shows the Raman spectra box-and-whisker diagrams of normal mammary tissue (NM); (b) mammary
hyperplasia (MH); (c) cribriform adenocarcinoma (CA); (d) papillary adenocarcinoma (PA); (e) solid adenocarcinoma
(AS); and (f) compound cribriform and papillary adenocarcinoma (CPA) at fingerprint region between 500 and 1800 cm1
. The shadows on the median line of each group indicated the degree of dispersion (spread) of those spectra.
Principal Components Analysis (PCA) and Cluster Analysis (CLA), have been employed to develop discrimination
methods, according with tissue types and histological diagnosis. On Figure 3 is shown PCA (which main components
submitted to analysis were PC2, PC3, PC4, and PC5) on the region between 500 and 1800 cm-1. By this analysis, it was
possible to classify these spectra into three distinct clusters. Cluster (1) - 41 MN, 12 MH, 1 PA, and 1 CPA spectra 96.4% of all were classified as “+++” for MMP-19 expression; Cluster (2) - 9 CA, 17 PA, and 9 SA - being referred to
compound adenocarcinomas classified as “+” for MMP-19 expression in 74.3 % of all cases; Cluster (3) - 6 CA, 20 PA,
3 SA, and 14 CPA, identified as “++” for MMP-19 examination in 46.5 % of all spectral. The proportion of correct
classification of assertiveness for each group was CA (66.7%), CPA (86.7%) and PA (31.6%). This result lead us to
conclude that this algorithm and the predictors used for this analysis were able to identify the adenocarcinomas "++",
therefore, the less aggressive ones.
The Principal Components Analysis (PCA) and Linear Discriminant Analysis with cross-validation (LDA), were divided
into three groups according to MMP-19 results: NM/MH +++ (N = 30); CA/CPA ++ (N = 50); and PA/SA + (N = 53).
Predictors used for LDA were PC2, PC3, PC4 and PC5. The spectra NM /MH were classified correctly in 100% of
cases; CA/CPA group had 60 % of spectra correctly classified and for PA/AS 54%spectra were correctly classified. It
was possible to note that that PCA and LDA algorithm have separated normal and hyperplastic mammary from
mammary adenocarcinomas spectra adequately.
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Normal Mammary Gland (41 spectra)
6,0
4,5
3,0
1,5
0,0
Mammary Hyperplasia (12 spectra)
6,0
4,5
3,0
1,5
0,0
Cribriform Adenocarcinoma (15 spectra)
6,0
4,5
3,0
1,5
0,0
Papillary Adenocarcinoma (38 spectra)
6,0
4,5
3,0
1,5
0,0
Solid Adenocarcinoma (12 spectra)
6,0
4,5
3,0
1,5
0,0
Cribriform and Papillary Adenocarcinoma (15 spectra)
6,0
4,5
3,0
1,5
0,0
500
595
690
790
885
980
1075
1175
1270
1365
1465
1560
1655
1750
Raman Shift (cm-1)
Figure 2. Raman spectra box-and-whisker diagrams of (a) normal mammary tissue (NM); (b) mammary hyperplasia
(MH); (c) cribriform adenocarcinoma (CA); (d) papillary adenocarcinoma (PA); (e) solid adenocarcinoma (AS); and (f)
compound cribriform and papillary adenocarcinoma (CPA) at fingerprint region.
(a)
Dendrogram
Average Linkage; Correlation Coefficient Distance
54,69
Cluster 3
Cluster 2
77,35
AP 3
A P3 1
A P3 8
A CP 7
A CP 5
AC P 1 1
AP 2
A CP 1
A P3 7
A S1 0
A CP 3
A CP 9
AC P 1 3
AP 1
A S1 1
A P3 2
A CP 6
A P3 6
A CP 4
A P1 6
A P3 4
A P2 7
A CP 2
AC P 1 5
AC P 1 2
AP 5
A CP 8
A P2 9
A P3 0
A P3 3
AP 4
AP 6
A C1 5
A S1 2
A C1 2
A C1 4
A P3 5
A C1 3
A C1 1
A P2 8
AC P 1 4
AS 7
AS 8
A P1 8
AC5
A C1 0
A P2 5
A P1 9
A P2 6
AS 9
AS 3
A P2 4
A P1 2
A P1 0
AC4
AS 1
AC8
A P1 1
AP 9
AP 8
AP 7
A P1 4
A P2 2
A P2 0
AC9
AC7
AS 4
AS 2
A P2 1
AS 6
AC2
A P2 3
A P1 3
N2
AS 5
AC6
A P1 5
N9
AC3
AC1
A P1 7
N3 0
AC P 1 0
N4 0
M
H5
N4 1
M
H4
M
H1
N2 3
N3 9
N3 8
M
M
M
M
H8
N3 7
N3 2
N1 1
N2 7
M
N2 6
M
M
M
M
M
M
N6
N1 6
N1 7
M
N1 9
M
M
M
N1 0
M
M
N1 2
M
M
N3 3
M
N8
N1 3
M
H1 2
M
H2
N2 2
M
M
N3 1
H9
N2 1
H7
N1 5
H6
H1 1
N2 0
M
H1 0
M
M
M
M
M
M
N3 5
N4
N2 8
M
M
N5
M
N3
M
H3
M
N1 8
M
M
N1 4
N7
N2 5
N1
N2 9
N3 6
N2 4
N3 4
M
M
M
M
M
M
M
M
M
M
M
100,00
Cluster 1
M
Similarity (%)
32,04
Variables (PC2, PC3, PC4 and PC5)
(b)
PC1
40
20
0
(c)
PC2
10
0
-10
(d)
PC3
2
0
-2
PC4
1
(e)
0
-1
0,7
PC5
(f)
0,0
-0,7
550
660
770
880
990
1100
1210
1320
1430
1540
1650
1760
Raman Shift (cm-1)
Figure 3. Dendrogram of the Cluster Analysis (CLA) results applied to PC2, PC3, PC4 e PC5 of the Raman data of the
normal mammary tissue (NM), mammary hyperplasia (MH), cribriform adenocarcinoma (CA), papillary adenocarcinoma
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(PA), solid adenocarcinoma (SA), and compound cribriform and papillary adenocarcinoma (CPA) at fingerprint region
between 500 and 1800 cm-1
4. CONCLUSIONS
The results of the FT-Raman spectroscopy and MMP-19 study reveal themselves as a highly relevant medical finding.
Here we were able to demonstrate that Raman spectroscopy may assist in the screening of breast cancer, because it brings
sufficient information to classify the breast tissue through its biochemical phenotype. The spectra NM /MH were
classified correctly in 100% of cases; CA/CPA group had 60 % of spectra correctly classified and for PA/AS 54% of the
spectra were correctly classified. The search for the biochemical phenotype has been the greatest challenge of recent
years in the immunohistochemical research.
ACKNOWLEDGMENTS
This work was supported by the grant of the FAPESP (01/14384-8 and 05/58565-7) and CNPQ (Project 301066/2009-4).
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