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a r t i c l e i n f o a b s t r a c t
Article history: Raman spectroscopy was employed for the characterization of blood plasma samples from patients at different
Received 27 December 2018 stages of breast cancer. Blood plasma samples taken from clinically diagnosed breast cancer patients were com-
Received in revised form 2 May 2019 pared with healthy controls using multivariate data analysis techniques (principal components analysis – PCA) to
Accepted 26 May 2019
establish Raman spectral features which can be considered spectral markers of breast cancer development. All
Available online 27 May 2019
the stages of the disease can be differentiated from normal samples. It is also found that stage 2 and 3 are bio-
Keywords:
chemically similar, but can be differentiated from each other by PCA. The Raman spectral data of the stage 4 is
Breast cancer found to be biochemically distinct, but very variable between patients. Raman spectral features associated with
Blood plasma DNA and proteins were identified, which are exclusive to patient plasma samples. Moreover, there are several
Raman spectroscopy other spectral features which are strikingly different in the blood plasma samples of different stages of breast can-
Multivariate data analysis cer. In order to further explore the potential of Raman spectroscopy as the basis of a minimally invasive screening
Cancer staging technique for breast cancer diagnosis and staging, PCA-Factorial Discriminant Analysis (FDA) was employed to
classify the Raman spectral datasets of the blood plasma samples of the breast cancer patients, according to dif-
ferent stages of the disease, yielding promisingly high values of sensitivity and specificity for all stages.
© 2019 Elsevier B.V. All rights reserved.
https://doi.org/10.1016/j.saa.2019.117210
1386-1425/© 2019 Elsevier B.V. All rights reserved.
2 H.F. Nargis et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117210
Fig. 3. PCA loadings of pair-wise PCA analysis of Raman spectral data of healthy versus different stages of breast cancer samples (a) PCA scatter plots of; control versus stage-2 (b), control
versus stage-3 (c) and control versus stage-4 (d).
4 H.F. Nargis et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117210
Fig. 4. PCA scatter plot of pair-wise PCA analysis (a) and loading (b) of Raman spectral data of breast cancer samples of stage 2 verses stage 3.
set (3/4 of spectra) and a validation set (1/4 remaining spectra) with different combination of training/validation sets. The overall result ob-
random selection of samples. Additionally, a 100 fold cross validation tained with 10 PC-scores (10 dimensions) is presented under a confu-
has been implemented to evaluated the robustness of the model with sion matrix showing discrimination by mean of specificity and
Fig. 5. PCA scatter plot of pair-wise PCA analysis (a) and loadings (b) of Raman spectral data of breast cancer samples of stage 2 verses stage 4.
H.F. Nargis et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117210 5
Fig. 6. PCA scatter plot of pair-wise PCA analysis (a) and loadings (b) of Raman spectral data of breast cancer samples of stage 3 verses stage 4.
sensitivity of classification. This helps for the better evaluation of the although they are clinically distinguished by the extent of the disease
rate of differentiation obtained based on a data set dictated by the num- progression, they are spectrally, and therefore biochemically similar,
ber of patients. In present study, there are 26 groups of Raman spectra in terms of their manifestation in the blood stream. Notably, the outly-
(8 healthy and 18 female patients with a confirmed clinical and histo- ing clusters of the spectra have been identified as associated with the
pathological diagnosis of breast cancer). samples of stage-4, which are clustered according to individual patients.
They are not significantly more differentiated than the other stages ac-
3. Results and discussion cording to PC1, but are further differentiated according to PC2, indicat-
ing a distinct biochemical manifestation in the blood stream. PCA
Fig. 1 shows the mean Raman spectra with standard deviation of scatter plots for PC2 vs PC3, PC2 vs PC4, PC3 vs PC4, are shown in the
control and breast cancer blood plasma samples (mean Raman spectra supplementary material as S-1, S-2 and S-3 respectively. In all these fig-
of breast cancer stage-2, stage-3 and stage-4), and potentially differen- ures, the differentiation is not good as compared to Fig. 2 of the manu-
tiating features are indicated by vertical lines (Spectra have been offset script (PC1 vs PC2) where PC1 which discriminates Control from
for clarity). Raman spectral features, including those at 689 (nucleotide disease samples and PC2 which discriminates stage 4 from stage-2
conformation), 770 (phosphate), 788 (phosphodiester bands in and stage 3.
DNA),828 (tyrosine/protein), 848 (single bond stretching vibrations Fig. 3 represents the PCA scatter plots and loadings of the Raman
for the amino acids and valine and polysaccharides), 885(disaccharide spectral data of control/healthy versus each different stage of the breast
(cellobiose), (C-O-C) skeletal mode), 1138 (n(C\\C)-lipids, fatty cancer blood samples. These stages of disease development are well dif-
acids),1173 (cytosine, guanine), 1185 (anti-symmetric phosphate vi- ferentiated in a pair wise comparison and this indicates an important as-
brations), 1268 (d (=C-H) (phospholipids)), 1285 (typical phospho- pect of the PCA as the pair-wise PCA analysis (subsets of the full data
lipids), 1307 (CH3/CH2 twisting, wagging &/or bending mode of set) is expected to better highlight the variability in the data and
collagens & lipids) and 1319 cm−1 (guanine (B,Z-marker)) have higher hence different from the combine PCA analysis of the complete data
intensities in the mean Raman spectra of patient samples. In contrast, set, as indicated in the results presented in this manuscript.
the Raman spectral features at 700 (n (C\\S) trans (amino acid methio- The changes in biological molecules which are responsible for the
nine)), 761 (Tryptophan, d (ring)), 798 (CH out of plane deformation) clustering of healthy and cancerous samples in the PCA scatter plots
and 1410 cm−1 (ns COO2) have higher intensities in the mean Raman
spectra of control/healthy volunteers than in the patient samples. Table 2
Fig. 2 shows the PCA scatter plot of the Raman spectral data of the Results of the PCA-FDA for the Raman spectral data of Normal versus all breast cancer
(stage 2, 3&4) samples.
blood plasma samples of breast cancer patients of all different stages
versus healthy ones. PC1 explains 79% of the variance, and shows rea- Breast cancer Normal Total/group Specificity
sonably good differentiation of the Raman spectral data of healthy and Breast cancer 4358 36 4394 1 × 100
breast cancer patients. Stage 2 and Stage 3 samples are well differenti- =100%
ated from normal controls, but are largely overlapped, although it may Normal 0 2760 2760 Sensitivity
be considered that the Stage 3 samples are more differentiated accord- Total spectra 7154 0.99 × 100
=99%
ing to PC1. The overlap of the Stage 2 and 3 clusters indicates that,
6 H.F. Nargis et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117210
Table 3
Results of the PCA-FDA for the Raman spectral data of breast cancer stage 2 versus stage 3 samples.
are indicated by the loadings of the PC in Fig. 3 (a). In all cases, positive spread. It is still contained to the breast area and is generally very effec-
loadings show the peaks of cancerous samples, while negative loadings tively treated. On the other hand, stage 3 is known to be an advanced
represent the Raman spectral features associated with the healthy sam- stage of breast cancer, at which the cancer has started to invade the sur-
ples, as their respective Raman spectra are clustered in the positive and rounding tissues of the breast. In this regard, Raman spectroscopy can
negative axis of the PC-1. The Raman spectral features, observed in pos- potentially be of benefit in the differentiation of these stages of breast
itive loadings of all stages, include those at 689 (Nucleotide conforma- cancer, hence leading to early diagnosis which could be useful for the ef-
tion), 770 (phosphate), 788 (phosphodiester bands in DNA),828 fective treatment. The technique is shown to do this on the basis of the
(tyrosine/protein), 848 (single bond stretching vibrations for amino identification of the underlying biochemistry, the Raman spectral fea-
acids and polysaccharides), 1138 (n(C\\C)-lipids, fatty acids),1173(cy- tures, as indicated in (Fig. 4 b). It is found that positive loadings of PC2
tosine, guanine), 1185 (anti-symmetric phosphate vibrations), 1268 are associated with the Raman features of stage-3 which have higher in-
(d (=C-H) (phospholipids)), 1285 (typical phospholipids) 1307 (CH3/ tensities than that of stage-2, particularly those of nucleic acids. These
CH2 twisting, wagging &/or bending mode of collagens & lipids) and features associated with DNA are very clear markers of cancer, as circu-
1319 cm−1 (guanine (B,Z-marker)). Notably, these Raman features lating DNA is only present in the blood plasma of the cancer patients.
were also observed in Fig. 1 as elevated in the mean Raman spectra of Moreover, this also indicates the progression from stage 2 to stage 3,
breast cancer blood plasma samples, and therefore these positive load- as indicated by the dominant presence of these Raman features in the
ings can be associated with the presence of disease. Similarly, in the Raman spectra of the patients of stage-3 patients.
negative loadings, the features at 700 (n (C\\S) trans (amino acid me- Fig. 5 (a) shows the PCA scatter plot of breast cancer stage-2 and
thionine)), 761 (Tryptophan, d (ring)), 798 (CH out of plane deforma- stage-4 and it can be seen that Raman spectral data of the stage-2
tion) and 1410 cm−1 (ns COO2) have higher intensities in mean (blue dots) is differentiated from that of the stage-4 (red dots) by clus-
Raman spectra of healthy volunteers than patient samples. Other tering in the negative and positive axis of the PC-1 respectively. In Fig. 5
Raman spectral features observed include those at 1100 cm−1 (C\\C vi- (b), the differences observed as positive loadings have higher intensities
bration mode of the gauche-bonded chain) as positive loadings and in the Raman spectral data of stage-4 than stage-3 patients, including
1440 (CH2 deformation in normal breast tissue) and 1663 cm−1 those at 689 cm−1 (nucleotides), 1185 cm−1 (anti-symmetric phos-
(DNA) as negative loadings. Moreover, Raman spectral features, includ- phate vibrations), 1285 cm−1 (phospholipids) and 1319 cm−1 (guanine
ing those at 689, 1138, 1173, 1185, 1268, 1319, 1440 and 1663 cm−1, (B,Z-marker)).
are elevated in breast cancer stage 4, and hence can be considered as a In Fig. 6 (a) PCA scatter plot of stage-3 and stage-4 of the breast can-
marker associated with the progression of disease. cer is shown and separation between these two stages is observed ac-
In Fig. 4 (a), the PCA scatter plot shows the comparison of two differ- cording to PC-1, by clustering in the negative and positive axis
ent breast cancer stages, stage-2 and stage-3. The spectra of the different respectively. In Fig. 6 (b), which shows the loadings of the PC1, the pos-
stages are differentiated according to PC2, indicating that Raman spec- itive loadings represent the Raman spectral features those have higher
troscopy of the blood serum can differentiate these two stages of the intensities in stage-4 as compared the stage-3. Notably, these Raman
disease. The positive loadings of PC2 is associated with the Raman fea- spectral features, including those at 689 cm−1 (nucleotides),
tures of stage-3, which have higher intensities than that of stage-2, in- 1185 cm−1 (anti-symmetric phosphate vibrations), 1285 cm−1 (phos-
cluding those at 625 cm−1, 689 cm−1 (nucleotides), 770 (phosphate), pholipids) and 1319 cm−1 (guanine), have also been observed (Fig 5)
788 (phosphodiester bands in DNA), 828 (tyrosine/protein), 1285 as the features associated with stage-4 and in Fig. 4 these features
(phospholipids) 1307 (CH3/CH2 twisting, wagging and/or bending have higher intensities in stage-3 as compared with stage-2. Hence,
mode of collagens & lipids) and 1319 cm−1 (Guanine). these changes observed in the stage wise comparison for the breast can-
Therefore, although stage 2 and stage 3 are not well distinguished in cer indicate the biochemical changes taking place during the develop-
the overall comparison of all patient samples to normal controls (Fig. 2), ment and progression of breast cancer.
the two stages of disease development are well differentiated in a pair In stage 4, the disease has spread to the surrounding tissues of the
wise comparison (Fig. 4 a). breast and it is considered the most advanced stage of breast cancer. It
After the diagnosis of breast cancer, it is important to determine the is very difficult to treat effectively at this stage of the cancer. The differ-
staging of the cancer, because this will help to know that how far the entiation between stage 2/3 and stage 4 is important because this can
disease has progressed and also to determine the best way to contain lead to early diagnosis which could be useful for the effective treatment.
and eliminate the breast cancer. Stage 2 breast cancer is still in the ear- Notably, stage 2 is difficult to diagnose by histopathology unless a num-
lier stages, but there is evidence that the cancer has begun to grow or ber of biopsies are taken. Moreover, this process is time consuming and
Table 4
Results of the PCA-FDA for the Raman spectral data of breast cancer stage 3 versus stage 4 samples.
Table 5
Results of the PCA-FDA for the Raman spectral data of breast cancer stage 2 versus stage 4 samples.
histology protocols are not necessarily reliable. In this regard, Raman carried out. The results are shown in Table 2, which indicate that the de-
spectroscopic analysis of blood samples, a much more rapid analysis, velopment of the breast cancer can be identified by employing blood
is shown to be helpful in the detection of the early stage and differenti- serum samples with a sensitivity of 99% and a specificity of 100%. Fur-
ation of the later stages of breast cancer, predominantly on the basis of thermore, PCA-FDA shows sensitivity of 95% and specificity of 94% for
increasing DNA contents during the progression of the breast cancer Raman spectral data of breast cancer of stage 2 versus stage 3
from stage 2 to stage 4. The Raman spectral features, including those (Table 3), sensitivity of 95% and specificity of 81% for stage 3 versus
at 689 cm−1 (nucleotides), 1185 cm−1 (Anti-symmetric phosphate vi- stage 4 (Table 4) and sensitivity of 95% and specificity of 100% for breast
brations), 1285 cm−1 (phospholipids) and 1319 cm−1 (Guanine), are cancer of stage 2 versus stage 4 (Table 5).
observed (Fig. 5) as the features associated with stage-4 and in Fig. 4
these features have higher intensities in stage-4 as compared with
4. Conclusions
stage-2/3. Hence, these changes observed in the stage wise comparison
for the breast cancer indicate the biochemical changes taking place dur-
Raman spectroscopy along PCA has shown its potential to be used
ing the development and progression of breast cancer. Notably, all the
for the diagnosis of breast cancer at an early stage, by employing
patients of the stage 4 are found very much distinct from each other, in-
blood plasma samples and healthy controls taken from clinically diag-
dicated by the clustering pattern of the Raman spectral data of this stage
nosed breast cancer patients. Among several, Raman spectral features
in the PCA scatter plots (Fig. 5a and Fig. 6a) which may be due to the in-
associated with DNA and proteins are identified, which are solely ob-
crease in the degree of the severity of the breast cancer, and the diver-
served in the blood plasma samples of the breast cancer patients. More-
sity of routes towards metastasis. The source of this variability has
over, there are several other spectral features which are strikingly
been shown below in S-4 as mean Raman spectra of each sample of
different in the blood plasma samples of different stages of breast can-
stage-4. The variability in these samples is due to the difference in
cer. Raman spectral data of different stages of the disease is compared
peak intensities as indicated by vertical lines including 640 (C\\S
by employing PCA. PCA scatter plots have shown clear differentiation
stretching & C\\C twisting of proteins), 675 (Amide I, b-sheet), 689
between healthy and disease as well as different stages of breast cancer.
(Nucleotide conformation), 700 (aminoacid methionine), 752 (Gua-
The differentiation between Stage 2/3 and stage 4 is important because
nine), 770 (Phosphate group), 788 (phosphodiester bands in DNA),
this can lead to early diagnosis which could be useful for the effective
828 (tyrosine/protein), 901(C-O-C) skeletal str. and 932 cm-1 (C\\C
treatment.
stretching).
This study advances previously published work related to the use of
Raman spectroscopy for the characterization/diagnosis of breast cancer Acknowledgements
by using blood plasma/serum samples from clinically diagnosed pa-
tients, as the spectral markers of 689 (Nucleotide conformation), 788 Higher Education Commission (HEC), Islamabad, Pakistan. Project
(phosphodiester bands in DNA), 828 (tyrosine/protein), 848 (most no.6400/Punjab/NRPU/R&D/HEC/2016.
probably due to single bond stretching vibrations for amino acids and
polysaccharides) and 1663 cm−1 (DNA) have not previously been iden- Appendix A. Supplementary data
tified. In these reports, Bilal et al. applied Partial least squares (PLS) re-
gression analysis to the Raman spectral data acquired from blood Supplementary data to this article can be found online at https://doi.
serum of breast cancer and healthy individuals and coefficients of re- org/10.1016/j.saa.2019.117210.
gression were determined with sensitivity of 90% and specificity 75%
which are associated with the changes at the bio-molecular level [22],
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