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Time-resolved fluorescence (TRF) and diffuse reflectance spectroscopy (DRS) for margin analysis in breast cancer

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Purpose: One of the major problems in breast cancer surgery is defining surgical margins and establishing complete tumor excision within a single surgical procedure. The goal of this work is to establish instrumentation that can differentiate between tumor and normal breast tissue with the potential to be implemented in vivo during a surgical procedure. Methods: A time-resolved fluorescence and reflectance spectroscopy (tr-FRS) system is used to measure fluorescence intensity and lifetime as well as collect diffuse reflectance (DR) of breast tissue, which can subsequently be used to extract optical properties (absorption and reduced scatter coefficient) of the tissue. The tr-FRS data obtained from patients with Invasive Ductal Carcinoma (IDC) whom have undergone lumpectomy and mastectomy surgeries is presented. A preliminary study was conducted to determine the validity of using banked pre-frozen breast tissue samples to study the fluorescence response and optical properties. Once the validity was established, the tr-FRS system was used on a data-set of 40 pre-frozen matched pair cases to differentiate between tumor and normal breast tissue. All measurements have been conducted on excised normal and tumor breast samples post surgery. Results: Our results showed the process of freezing and thawing did not cause any significant differences between fresh and pre-frozen normal or tumor breast tissue. The tr-FRS optical data obtained from 40 banked matched pairs showed significant differences between normal and tumor breast tissue. Conclusion: The work detailed in the main study showed the tr-FRS system has the potential to differentiate malignant from normal breast tissue in women undergoing surgery for known invasive ductal carcinoma. With further work, this successful outcome may result in the development of an accurate intraoperative real-time margin assessment system. Lasers Surg. Med. 50:236-245, 2018. © 2018 Wiley Periodicals, Inc.
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Lasers in Surgery and Medicine
Time-Resolved Fluorescence (TRF) and Diffuse Reflectance
Spectroscopy (DRS) for Margin Analysis in Breast Cancer
Nourhan Shalaby,
1
Alia Al-Ebraheem,
1
Du Le,
1
Sylvie Cornacchi,
2
Qiyin Fang,
3
Thomas Farrell,
4
Peter Lovrics,
2,5
Gabriela Gohla,
5,6
Susan Reid,
4
Nicole Hodgson,
4
and Michael Farquharson
1
1
School of Interdisciplinary Science, McMaster University, Ontario, Canada
2
Faculty of Health Sciences, Department of Surgery, McMaster University, Hamilton, Ontario, Canada
3
Faculty of Engineering, McMaster University, Hamilton, Ontario, Canada
4
Juravinski Hospital and Cancer Centre, Hamilton, Ontario, Canada
5
St. Joseph’s Healthcare, Hamilton, Ontario, Canada
6
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
Purpose: One of the major problems in breast cancer
surgery is defining surgical margins and establishing
complete tumor excision within a single surgical proce-
dure. The goal of this work is to establish instrumentation
that can differentiate between tumor and normal breast
tissue with the potential to be implemented in vivo during
a surgical procedure.
Methods: A time-resolved fluorescence and reflectance
spectroscopy (tr-FRS) system is used to measure fluores-
cence intensity and lifetime as well as collect diffuse
reflectance (DR) of breast tissue, which can subsequently be
used to extract optical properties (absorption and reduced
scatter coefficient) of the tissue. The tr-FRS data obtained
from patients with Invasive Ductal Carcinoma (IDC) whom
have undergone lumpectomy and mastectomy surgeries is
presented. A preliminary study was conducted to determine
the validity of using banked pre-frozen breast tissue
samples to study the fluorescence response and optical
properties. Once the validity was established, the tr-FRS
system was used on a data-set of 40 pre-frozen matched pair
cases to differentiate between tumor and normal breast
tissue. All measurements have been conducted on excised
normal and tumor breast samples post surgery.
Results: Our results showed the process of freezing and
thawing did not cause any significant differences between
fresh and pre-frozen normal or tumor breast tissue. The tr-
FRS optical data obtained from 40 banked matched pairs
showed significant differences between normal and tumor
breast tissue.
Conclusion: The work detailed in the main study showed
the tr-FRS system has the potential to differentiate
malignant from normal breast tissue in women undergoing
surgery for known invasive ductal carcinoma. With further
work, this successful outcome may result in the develop-
ment of an accurate intraoperative real-time margin
assessment system. Lasers Surg. Med. © 2018 Wiley
Periodicals, Inc.
Key words: optical spectroscopy; margin assessment;
fluorescence; diffuse reflectance; absorption coefficient;
reduced scatter coefficient
INTRODUCTION
Breast cancer is accountable for 13% of female cancer
deaths in Canada and remains the most frequently
diagnosed cancer in women [1]. Early stage cancers are
managed with breast conserving surgery where surgeons
aim to remove the identified cancer along with a rim of
normal tissue of 1–2 mm to confirm complete cancer
removal. Margin detection between tumor and normal
tissue has proven to be a problematic challenge for
surgeons. Thus, surgical margin assessment is an area of
great relevance and active research in breast cancer. The
presence of a clear surgical margin is the most important
indicator available to ensure completeness of surgical
excision, while a positive surgical margin is a significant
risk factor for predicting local recurrence. Furthermore,
the presence of positive margins, which can be up to
30% [2], generally leads to further surgical resections with
associated morbidity, resource utilization, anxiety, and
delay. Optical biopsies have been recently used to
potentially replace the current assessments where a
pathologist examines the rim of normal tissue around
the cancer to confirm it is clear of any cancer cells as the
current pathological assessment remains time consuming
and labor intensive and is not readily transferable to
routine practice [3–11].
In this study a time-resolved fluorescence and reflec-
tance spectroscopy (tr-FRS) system is utilized to study
samples obtained from patients with breast Invasive
Conflict of Interest Disclosures: All authors have completed
and submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest and none were reported.
Contract grant sponsor: Juravinski Hospital and Cancer
Centre Foundation Fall 2014 Research Grant.
Correspondence to: Nourhan Shalaby, Department of Inter-
disciplinary Science, McMaster University, 1280 Main Street
West, Hamilton, Ontario, L8S 4L8 Canada.
E-mail: shalabyn@mcmaster.ca
Accepted 31 December 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/lsm.22795
ß2018 Wiley Periodicals, Inc.
Ductal Carcinoma (IDC) whom have undergone lumpec-
tomy or mastectomy. By measuring fluorescence intensity
and lifetime as well as collecting diffuse reflectance (DR) of
the tissue, the data can subsequently be used to extract
optical properties (absorption and reduced scatter coeffi-
cient) of the tissue.
Time-resolved fluorescence (TRF) measures the fluores-
cence lifetime and fluorescence intensity response in
endogenous fluorophores present within the tissue. En-
dogenous fluorophores of particular interest in breast
tissue include collagen, reduced nicotinamide adenine
dinucleotide (NADH), and flavin adenine dinucleotide
(FAD). The NADH/FAD red-ox state of the tissue has also
been examined in previous work [12] and has been shown
to be significantly higher in tumor versus normal breast
tissue. The TRF technique has been performed to distin-
guish between glioma specimens from normal tis-
sue [13–15]. Fluorescence lifetime is an intrinsic feature
of fluorophores. Fluorescence lifetime is the time required
by a population of excited fluorophores to decrease
exponentially to N/e or 36.8% of initial population (N)
via the energy loss through fluorescence and other non-
radiative processes. Endogenous fluorophores have life-
times that can vary from picoseconds to nanoseconds [16].
Fluorescence lifetime is independent of biological relevant
fluorophore concentration, absorption by the sample,
fluorescence intensity, photo-bleaching, and/or excitation
intensity.
Diffuse reflectance (DR) uses a broadband light source in
the UV-VIS region providing illumination and the diffuse
reflectance spectra is collected by detection fibers posi-
tioned at various distances from the source fiber. The
diffuse reflectance can interpret how light is either back
scattered or absorbed from tissue. The diffuse reflectance
can display absorption and scattering properties of the
tissue, revealing physiological and morphological changes,
respectively. The absorption coefficient can reveal infor-
mation on major absorbers in the tissue, such as
oxygenated and deoxygenated hemoglobin and beta-caro-
tene, while the reduced scatter coefficient can relay
information on the size and density of scattering centers,
such as nuclei and cells, in the tissue. The optical
properties at 545 and 575 nm will be investigated in this
work as they correspond to the absorption peaks of
oxygenated hemoglobin which has been shown to be
altered in tumor lesion [17].
The preliminary aim of this work is to compare between
fresh and pre-frozen breast tissue to assess the effects of
the freezing and thawing process on the tr-FRS parame-
ters to validate the use of pre-frozen tissue in further work.
The primary aim is to evaluate whether the tr-FRS system
is capable of differentiating between tumor and normal
breast tissue. These aims serve towards the ultimate goal
of establishing credible technology that can be used in a
device with the potential of providing substantially higher
confidence of accurate and complete tumor removal within
a single surgical procedure in vivo in real time.
METHODS
Samples
Matched pair breast samples were collected from St.
Joseph’s Healthcare and the Juravinski Hospital and
Cancer Centre directly after surgery and subsequently
measured using the tr-FRS system. A small piece of excised
tumor tissue was provided from surgery, with a minimum
size requirement of 4 32 mm. A further small piece of
normal tissue, from the same patient, with equivalent size
was excised at a distance from the tumor area to avoid any
boundary effects. Furthermore, histological analysis was
performed to confirm normal specimens were free of any
tumor content. Measurements were made on post-surgical
excised samples at three different locations as illustrated
in Figure 1 and each measurement was repeated three
times. The data presented in this work represents the
average of all measurements made on each sample. The
protocol and study processes were approved and performed
in accordance with the recommendations and regulations
of “Hamilton Integrated Research Ethics Board.”
Preliminary study-sample set. Ten matched pair IDC
specimens were collected and directly measured with the
tr-FRS system within 30 minutes after surgery to extract
Fig. 1. (A) A breast specimen that has undergone lumpectomy showing the locations of excision
with a distance of 3–5cm between tumor and normal. (B) An image of an excised tumor breast
specimen. Blue dots represent the approximate measurement locations, where each location was
measured three times.
2 SHALABY ET AL.
the optical parameters. After data collection, the fresh
tissues were frozen from room temperature to a freezer
with subzero temperature of 808C for a period of time
between 4 and 6 weeks. After this time, data collection was
repeated subsequent to complete thawing of the tissue
samples. This temperature was chosen to avoid mechanical
changes proven to cause Collagen damage with use of snap
freezing using liquid nitrogen (1968C) [18]. Exposing
tissue to room temperature for a few minutes allowed the
thawing process to be completed without any external
interference or further processing. Comparison was
carried out to investigate if there were differences in the
data from samples when collected from the fresh tissues
and after the same tissue had been frozen.
Main study-sample set. Forty matched pair IDC pre-
frozen banked specimens were stored in a freezer with
subzero temperature of 808C until time of measurement.
Methodology
An integrated, dual-modality tr-FRS instrument was
used to collect intrinsic fluorescence and reflectance data
from the specimens. The TRF spectroscopy measures
fluorescence intensity and lifetime of fluorophores, provid-
ing information on biological composition of tis-
sue [16,19,20]. DRS reveal optical properties that relay
information on absorber concentration as well as scatter-
ing size, structure, and density of cells [17,21,22]. Although
both techniques have been used separately to classify
tissue types, the integration of both modalities allows the
yield of higher sensitivity and specificity than each
modality alone. Figure 2 shows the integration of the
two subsystems. The TRF module uses a UV pulsed laser
source at 355 nm and pulse width of 0.3 ns (PNV-001525-
140, Teem Photonics, Meylan, France) to excite endoge-
nous tissue fluorophores and collect the spectral
fluorescence intensity and lifetime response of breast
tissue with a range 350–550 nm. An acousto-optic tunable
filter (AOTF, TEAF5-0.36–0.52-S, Brimrose, MD) is used
to apply rapid wavelength switch while a photomultiplier
tube and a high-speed digitizer are used to retrieve decay
at each wavelength in real time [19,23]. In the DRS
subsystem, a broadband light source (Dolan-Jenner MI-
150, Edmund Optics, NJ) is used to illuminate the sample
and diffuse reflectance from 300 to 800 nm at various
source-detector distances (0.23, 0.59, and 1.67 mm) are
collected. A fiber optic probe of 2 mm diameter (illustrated
in Fig. 3) is used to integrate both excitation sources and
collect the output signal from the TRF and DRS in a hand-
held device. For more information on the system, please
refer to the methods section described by Nie et al. [15].
All statistical analysis was performed using IBM SPSS
Statistics Version 22. The P-value has been reported as
significant for each variable as P<0.05. The Shapiro-test
was used for each category to test if the data set was
consistent with a Gaussian distribution function. Wilcoxon
Signed Ranks Test was used for comparing the difference
of the parameters for non-normally distributed data while
Paired-Sample T Test was used for normally distributed
data.
RESULTS
Preliminary Study-Results
TRF results.
Fluorophore lifetime. Table 1 compares fresh with pre-
frozen in both normal and tumor breast tissue at 400, 460,
and 515 nm, corresponding to the emission wavelength of
the endogenous fluorophores Collagen, NADH, and FAD
respectively. No significant difference was observed
between the fresh and pre-frozen samples in normal and
tumor samples. This suggests that the freezing and
Fig. 2. An illustration of the integration of the DRS subsystem (left) and the TRF subsystem (right)
with data collection occurring through a probe and a central control unit (computer).
TRF AND DRS FOR MARGIN ANALYSIS IN BREAST CANCER 3
thawing process did not yield significant changes on the
fluorescence lifetime.
Fluorophore intensity. Figure 4 compares fluorescence
intensity spectra for (A) fresh to pre-frozen normal samples
and (B) fresh to pre-frozen tumor samples. All spectra are
normalized to the applied voltage and are represented as
arbitrary units with the maximum value as the NADH
peak value at 460 nm. The spectra were analyzed using
Peak Fit software (PeakFit TM v.4.12, Seasolve Software
Inc.) where the emission peaks of fluorophores (collagen at
400 nm, NADH at 460 nm, and FAD at 515 nm) were
smoothed and treated as Gaussian distributions to deter-
mine the amplitude, FWHM and integral area of each
fluorophore. For each peak, the integral area was deter-
mined by PeakFit using the following FWHM and
amplitude equation:
y¼aexp ln 2ðÞxx0
dx

2

where as the amplitude represented in arbitrary units,
dxs the half width at half maximum (HWHM) and x
0
is the
maximum position.
Collagen. Table 2 shows collagen integral area in freshly
excised and pre-frozen between normal and tumor breast
tissue (n¼10).
FAD. Table 3 shows FAD integral area in freshly excised
and pre-frozen between normal and tumor breast tissue
(n¼10).
NADH/FAD. Table 4 shows NADH/FAD integral area in
freshly excised and pre-frozen between normal and tumor
breast tissue (n¼10).
Tables 2–4 show no significant difference between fresh
and pre-frozen collagen, FAD and NADH/FAD integral
area in both normal and tumor breast tissue.
DR results.
Optical properties. Table 5 compares the absorption
coefficient between fresh and pre-frozen normal and tumor
breast tissue at 545 and 575 nm. No significant difference
was observed for both the normal and tumor subset.
Reduced scatter coefficient. Table 6 compares the
reduced scatter coefficient between fresh and pre-frozen
in both normal and tumor breast tissue at 545 and 575 nm.
No significant differences in absorption coefficient and
reduced scatter coefficient were observed for both the
normal and tumor subset in Tables 5 and 6. This implies
that the freezing and thawing process did not contribute to
significant differences in optical properties. In both
Tables 5 and 6 power analysis has been performed to
determine the strength of the sample size on the signifi-
cance level and yielded in low power which can be
contributed to the small sample size used in this sub-study.
Main Study-Results
TRF results.
Fluorophore lifetime. Table 7 shows the lifetime values
at 400, 460, and 515 nm, the wavelength corresponding to
collagen, NADH and FAD emissions, respectively.
Fluorophore intensity. Normalization and fitting of the
fluorescence intensity was performed as mentioned above.
Figure 5 compares the fluorescence intensity of 40 matched
pair cases of normal and tumor breast samples.
Fig. 3. Transverse view of fiber optic probe. All fibers are 0.23 mm,
unless indicated otherwise.
TABLE 1. Lifetime at 400, 460, 515 nm in Freshly Excised and Pre-Frozen Normal and Tumor Breast Samples
(n¼10)
Normal Tumor
Mean (ns) Standard error P-value Mean (ns) Standard error P-value
Fresh lifetime 400 nm 3.126 0.194 P¼0.303
a
4.381 0.179 P¼0.055
a
Pre-frozen lifetime 400 nm 3.391 0.247 4.921 0.228
Fresh lifetime 460 nm 6.605 0.266 P¼0.664
a
5.334 0.341 P¼0.878
b
Pre-frozen lifetime 460 nm 6.711 0.311 5.495 0.323
Fresh lifetime 515 nm 8.255 0.451 P¼0.249
a
5.818 0.508 P¼0.721
b
Pre-frozen lifetime 515 nm 7.830 0.491 5.841 0.510
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Significance tested at P<0.05.
4 SHALABY ET AL.
Collagen. Table 8 shows the collagen integral area
between normal and tumor breast samples.
DR Results:. Figure 6 displays the average diffuse
reflectance spectra from fiber 1 between normal and tumor
breast samples from the 420 to 670 nm range.
In Table 9 below, the average diffuse reflectance from
fiber 1 at 545 and 575 nm, the wavelengths corresponding
to the absorption of hemoglobin, is shown.
Optical properties.
Absorption coefficient. Figure 7 below displays the
average absorption coefficient from 480 to 620 nm between
normal and tumor breast samples.
Table 10 represents the average absorption coefficient in
normal and tumor breast samples at 545 and 575 nm.
Reduced scatter coefficient. Figure 8 displays the average
reduced scatter coefficient from 480 to 620 nm between
normal and tumor breast samples.
Table 11 presents the reduced scatter coefficient in
normal and tumor breast samples at 545 and 575 nm.
DISCUSSION
Preliminary Study
Several groups have used steady-state fluorescence and
diffused reflectance to investigate the cryogenic effects on
soft tissue. In one study it was reported that there was no
significant deviation in ex vivo fluorescence intensity upon
freezing and thawing of rodent cheek pouch tissue but
rather a significant change in the diffuse reflectance
measurements can be observed [24]. The study attributed
this to changes in hemoglobin and oxygen content in the
pre-frozen samples. Other work [25] compared optical
properties between intact soft tissue and defrosted tissue
paste of calf aorta, rat jejunum, and rabbit sciatic nerve
and found no significant differences between the optical
properties. An earlier study investigated the effects of
freezing and thawing on the optical properties of human
aorta and reported a significant decrease in absorption
coefficient over the 300–800 nm range and reduced scatter
coefficient in a smaller range from 300 to 335 nm [26].
Fig. 4. A direct comparison between freshly excised and pre-
frozen normal breast tissue is demonstrated in Figure 4A followed
by freshly excised and pre-frozen tumor breast tissue in Figure 4B.
Errors are SE of the mean.
TABLE 2. Collagen Integral Area in Freshly Excised
and Pre-Frozen Normal and Tumor Breast Tissue
(n¼10)
Mean
Std.
error P-value
Fresh normal collagen area 10.227 1.807 P¼0.988
a
Pre-frozen normal collagen
area
10.199 1.702
Fresh tumor collagen area 20.035 2.862 P¼0.280
b
Pre-frozen tumor collagen
area
23.528 3.038
a
Paired-Samples t-test.
b
Significance tested at P<0.05.
TABLE 3. FAD Integral Area in Freshly Excised and
Pre-Frozen Normal and Tumor Breast Tissue (n¼10)
Mean Std. error P-value
Fresh normal FAD area 16.777 0.882 P¼0.463
a
Pre-frozen normal FAD area 17.655 1.379
Fresh tumor FAD area 15.208 1.356 P¼0.386
b
Pre-frozen tumor FAD area 13.671 1.829
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Significance tested at P<0.05.
TABLE 4. NADH/FAD Integral Area in Freshly Excised
and Pre-Frozen Normal and Tumor Breast Tissue
(n¼10)
Mean
Std.
error P-value
Fresh normal NADH to FADH
area
3.483 0.187 P¼0.878
a
Pre-frozen normal NADH to
FADH area
3.432 0.372
Fresh tumor NADH to FAD
area
3.906 0.401 P¼0.213
a
Pre-frozen tumor NADH to FAD
area
4.687 0.497
a
Paired-Samples t-test.
Significance tested at P<0.05.
TRF AND DRS FOR MARGIN ANALYSIS IN BREAST CANCER 5
Our results have indicated that the process of freezing
and thawing did not cause any significant differences
between fresh and pre-frozen breast tissue in both normal
and tumor breast tissue for fluorescence lifetime at 400,
460, and 515 nm as well as collagen peak area, FAD peak
area, NADH/FAD peak area, absorption coefficient at 545
and 575 nm as well as the reduced scatter coefficient at 545
and 575 nm. This justifies the suitability of using banked
frozen tissue bank specimens in our work. The fact that our
study used different tissue types and followed a different
method of sample preparation involving no grinding or
physical changes to the tissue, would explain the discrep-
ancies between other studies and does not allow for direct
comparison as no significant differences in absorption were
observed in our study.
Main Study
Fluorophore lifetime. There has been a range of
values for collagen fluorescence and lifetime in the
literature [27,28]. However, the complexity of collagen
fluorescence makes it harder to differentiate between
collagen types I, II, and III in the 390–410 nm emission
range. Although classification of different collagen types is
difficult, most studies have reported higher collagen fibrils
and thus longer collagen lifetimes in diseased states in
comparison to healthy states, verifying our results of
significantly higher tumor lifetime (4.3 ns) compared to
normal collagen at (3.5 ns) at the wavelength of 400 nm
corresponding to collagen emission, as shown in Table 7.
NADH has a mean fluorescence lifetime ranging from
0.2 to 0.4 ns in its free state. However, when NADH is
protein bound, it will exhibit longer lifetimes typically in
the 2.5–3.4 ns range. As per Table 7, our measured
response at 460 nm, attributed mainly to NADH, was 6.3
and 5.1 ns for normal and tumor breast tissue respectively,
higher than the reported protein bound NADH lifetime. A
study by Skala et al. reported significantly decreased
(P<0.05) protein-bound NADH in tumor tissue compared
TABLE 5. Absorption Coefficient at 545 and 575 nm for Fresh and Pre-Frozen Normal and Tumor Breast Tissue
(n¼10)
Normal Tumor
Mean
(cm
1
)
Standard
error P-value
Power
analysis
Mean
(cm
1
)
Standard
error P-value
Power
analysis
Fresh absorption coefficient
545 nm
5.735 1.281 P¼0.434
a
0.078 5.208 0.624 P¼0.128
a
0.216
Pre-frozen absorption
coefficient 545 nm
6.598 1.188 7.141 1.528
Fresh absorption coefficient
575 nm
6.033 1.389 P¼0.591
a
0.065 5.229 0.623 P¼0.139
b
0.252
Pre-frozen absorption
coefficient 575 nm
6.702 1.265 7.507 1.651
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
TABLE 6. Reduced Scatter Coefficient at 545 nm and 575 nm for Fresh and Pre-Frozen Normal and Tumor Breast
Tissue (n¼10)
Normal Tumor
Mean
(cm
1
)
Standard
error P-value
Power
analysis
Mean
(cm
1
)
Standard
error P-value
Power
analysis
Fresh reduced scatter
coefficient 545 nm
15.814 1.745 P¼0.210
a
0.118 17.310 1.415 P¼0.05
b
0.583
Pre-frozen reduced scatter
coefficient 545 nm
17.756 1.869 21.952 1.607
Fresh reduced scatter
coefficient 575 nm
15.180 1.675 P¼0.210
a
0.119 16.616 1.358 P¼0.05
b
0.584
Pre-frozen reduced scatter
coefficient 575 nm
17.044 1.794 21.072 1.542
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Significance tested at P<0.05.
6 SHALABY ET AL.
to normal tissue and attributed this change to a shift in
metabolic conditions from oxidative phosphorylation to
glycolysis in tumor prognosis [29].
Table 7 shows lower FAD lifetime in tumor of 5.2 ns
compared to the normal 7.1 ns breast samples, which
falls within the range of published data as free state
FAD has been reported to have a decay time of 5 ns, with
a faster lifetime of about 1 ns in the bound form [30].
Any changes in the NADH/FAD and their relative
amounts of free and protein-bound states depend on the
glycolysis and oxidative phosphorylation ratio. Since
tumor progression resultsinashiftfromoxidative
phosphorylation to glycolysis, comparing short and long
lifetime fluorescence decays of NADH and FAD can be
used to discriminate between different metabolic
conditions.
Fluorescence intensity. The increase of collagen
deposition in malignant breast tissue has been observed
in many previous studies [6,27,31–34]. Many have
confirmed that increased density in breast correlates to
higher incidences of breast cancers. This increase in breast
density can be attributed to increased fibril collagen
deposition. All previous literature matches our results of
significantly increased collagen content (integral area of
15.778 a.u.) at 400 nm in tumor compared to the normal
(integral area of 9.746 a.u.) breast samples as per Figure 5
and Table 8.
Another major fluorescence component displaying sig-
nificant differences between tumor and normal breast
tissue at 460 nm is the emission of NADH. Like collagen,
NADH is also found to be significantly higher in tumor
compared to the normal breast samples [4,8,12,29,30].
NADH is one of the main coenzymes responsible for
metabolic activities and the relative concentration of this
coenzyme changes as the shift from oxidative phosphor-
ylation to aerobic glycolysis is observed in the progression
from normal to malignant state.
The third constituent of the emission spectra peaking
at 510 nm in Figure 5 is att ributed to FAD. Like NADH,
FAD is another coenzyme responsible for cellular
metabolism. The high-energy demands in breast cancer
results in major metabolic reprogramming in the tumor
state, affecting levels of FAD present within the cell. In
normal conditions, NADH, acting as an electron donor,
reduces FAD to FADH
2
. After a series of intermediate
reactions, FADH
2
is oxidized to FAD. However, in tumor
development, the low oxygen concentrations prevent the
conversion of FADH
2
to FAD, thereby resulting in lower
levels of FAD in tumor breast compared to normal breast
samples [35]. This explains the lower FAD content
(integral area of 9.935 a.u.) observed in the tumor breast
tissue samples compared to the normal FAD content
(integral area of 13.659 a.u.) reported in our study
(Table 8).
Although NADH and FAD values provide information
on the metabolic state of the tissue, the ratio of NADH to
FAD is a more accurate measurement as it provides a
control for cell density and accounts for metabolic
variation between a healthy and diseased cellular status.
This red-ox ratio is important to monitor, as it is sensitive
to cellular metabolic changes and oxygen depletion
occurring during the progression of healthy to malignant
state. As observed in this study, the increased NADH and
decreased FAD levels observed in tumor tissue results in
significantly increased NADH/FAD (integral area of
6.854 a.u.) red-ox levels compared to the normal
NADH/FAD content (integral area of 4.88 a.u.) as shown
in Table 8.
Diffuse reflectance. Numerous studies have used
diffuse reflectance as an individual modality to discrimi-
nate between diseased and healthy tissue [7,24,36–38]
and observed higher diffuse reflectance in tumor com-
pared to normal breast tissue. This was consistent with
our findings as shown in Table 9 and Figure 6 where
higher DR was noted for tumor samples (0.409 a.u at
545 nm and 0.395 a.u. at 575 nm) when compared to
normal breast samples (0.312 a.u at 545 nm and 0.306
a.u. at 575 nm).
Absorption coefficient. Although no significant
differences were observed between the average
TABLE 7. Lifetime at 400, 460, 515 nm in Pre-Frozen
Normal and Tumor Breast Samples (n¼40)
Mean (ns) Std. error P-value
Normal lifetime 400 nm 3.567 0.138 P<0.01
,b
Tumor lifetime 400 nm 4.318 0.205
Normal lifetime 460 nm 6.342 0.172 P<0.01
,a
Tumor lifetime 460 nm 5.141 0.097
Normal lifetime 515 nm 7.411 0.238 P<0.01
,a
Tumor lifetime 515 nm 5.261 0.138
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Statistically significant at P<0.05.
Fig. 5. Average fluorescence intensity in normal tumor breast
samples (n¼40). Collagen produces an emission spectrum
peaking at 390–400 nm, whereas reduced nicotinamide adenine
dinucleotide (NADH), and flavin adenine dinucleotide (FAD) emit
at 460 and 510 nm, respectively, when induced with a UV laser
source of 355 nm. Emission peaks of collagen, NADH and FAD
were taken at 400, 460, and at 510 nm, respectively. Errors are SE
of the mean.
TRF AND DRS FOR MARGIN ANALYSIS IN BREAST CANCER 7
absorption coefficient in tumor and normal samples in
our study as shown in section 5.2.4.1 (Fig. 7 and
Table 10), other studies found significantly higher
absorption in normal compared to tumor tissue, espe-
cially at 545 and 575 n m, wavelengths corresponding to
oxygenated hemoglobin, the main absorber present in
breast tissue. Zhu et al. [6] attributed the decrease in
hemoglobin saturation in malignant breast tissue to the
limited oxygen supply in the rapidly proliferating tumor
cells. Zhu also noted significantly higher hemoglobin
TABLE 8. Collagen, NADH, FAD, and NADH/FAD Integral Area in Normal and Tumor Breast Tissue Taken at
400 nm (n¼40)
Mean (a.u.) Std. error P-value
Normal collagen area 9.746 0.661 P<0.01
,b
Tumor collagen area 15.778 0.899
Normal NADH area 60.720 0.718 P¼0.03
,a
Tumor NADH area 62.517 0.849
Normal FAD area 13.659 0.609 P<0.01
,a
Tumor FAD area 9.935 0.429
Normal NADH to FAD area 4.88 0.276 P<0.01
,b
Tumor NADH to FAD area 6.854 0.362
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Statistically significant at P<0.05.
Fig. 6. Diffuse reflectance (DR) spectra of tumor and normal
breast tissue from fiber 1 (n¼40). Tumor samples showing
significantly higher DR than normal samples. Errors are SE of the
mean.
TABLE 9. Diffuse Reflectance in Normal and Tumor
Breast Samples at 545 and 575 nm (n¼40)
Mean
(a.u.)
Std.
error P-value
Normal diffuse reflectance
545 nm
0.312 0.018 P<0.01
,b
Tumor diffuse reflectance
545 nm
0.409 0.024
Normal diffuse reflectance
575 nm
0.306 0.018 P<0.01
,a
Tumor diffuse reflectance
575 nm
0.395 0.023
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test
.
Statistically significant at P<0.05.
Fig. 7. The average absorption coefficient in normal and tumor
breast samples (n¼40). Errors are SE of the mean.
TABLE 10. Average Absorption Coefficient at 545 and
575 nm of Normal and Tumor Breast Tissue (n¼40)
Mean
(cm
1
)
Std.
error P-value
Normal absorption
coefficient 545 nm
6.282 0.500 P¼0.78
a
Tumor absorption coefficient
545 nm
6.431 0.436
Normal absorption
coefficient 575 nm
6.484 0.531 P¼0.81
b
Tumor absorption coefficient
575 nm
6.654 0.472
a
Paired-Samples t-test.
b
Wilcoxon Signed Ranks test.
Statistically significant at P<0.05.
8 SHALABY ET AL.
saturation in normal and benign tissue compared to
malignant tumor.
Reduced scattering coefficient. The reduced scat-
tering coefficient provides informationonthescattering
centers present in the biological tissue, such as the nuclei.
Since increases in cellular proliferation and cell density is
a hallmark of tumor progression, an increase in the
reduced scattering coefficient is anticipated in tumor
tissue. The reduced scattering coefficient was also shown
to increase as a result of increased nuclear size, DNA
content and hyperchromasia [39]. Significantly higher
reduced scattering coefficient was observed in tumor
(22.162 cm
1
at 545 nm and 21.273 cm
1
at 575 nm)
compared to the normal breast tissue (18.571 cm
1
at
545 nm and 17.826 cm
1
at 575 nm) as per Table 11 and
Figure 8, which was consistent with findings from
previous studies [3,22,40,41]. Zhu et al. [6] noted that
the reduced scatter coefficient was inversely correlated to
the amount of adipose tissue present as well as the
patient body mass index (BMI). The observed increase in
the reduced scatter coefficient observed in this study
could be linked to increased fibro-connective and glandu-
lar tissue content and thus cancer development.
CONCLUSION
The preliminary study indicates the validity of using
banked pre-frozen tissue to study the optical parameters
required in distinguishing between normal and tumor
breast samples. The time-resolved fluorescence and diffuse
reflectance spectroscopy system was also used to discrimi-
nate between normal and tumor breast samples in 40
matches pair cases. The fluorescence intensity was used to
provide information on the endogenous fluorophores
collagen, NADH and FAD. The diffuse reflectance was
used to reveal tissue optical properties; the absorption and
reduced scatter coefficient. Statistical significant variables
(collagen, NADH, FAD, and NADH/FAD integral area, as
well as the diffuse reflectance spectra and the reduced
scattering coefficient) were found.
ACKNOWLEDGMENTS
The authors would like to thank Juravinski Hospital and
Cancer Centre Foundation 2014 Research Development
Grant.
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10 SHALABY ET AL.
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