RESEARCH ARTICLE Open Access
Evaluation of FTIR Spectroscopy as a diagnostic
tool for lung cancer using sputum
Paul D Lewis1*, Keir E Lewis1,2, Robin Ghosal2, Sion Bayliss1, Amanda J Lloyd3, John Wills1, Ruth Godfrey1,
Philip Kloer2, Luis AJ Mur3
Background: Survival time for lung cancer is poor with over 90% of patients dying within five years of diagnosis
primarily due to detection at late stage. The main objective of this study was to evaluate Fourier transform infrared
spectroscopy (FTIR) as a high throughput and cost effective method for identifying biochemical changes in sputum
as biomarkers for detection of lung cancer.
Methods: Sputum was collected from 25 lung cancer patients in the Medlung observational study and 25 healthy
controls. FTIR spectra were generated from sputum cell pellets using infrared wavenumbers within the 1800 to 950
Results: A panel of 92 infrared wavenumbers had absorbances significantly different between cancer and normal
sputum spectra and were associated with putative changes in protein, nucleic acid and glycogen levels in tumours.
Five prominent significant wavenumbers at 964 cm-1, 1024 cm-1, 1411 cm-1, 1577 cm-1and 1656 cm-1separated
cancer spectra from normal spectra into two distinct groups using multivariate analysis (group 1: 100% cancer
cases; group 2: 92% normal cases). Principal components analysis revealed that these wavenumbers were also able
to distinguish lung cancer patients who had previously been diagnosed with breast cancer. No patterns of spectra
groupings were associated with inflammation or other diseases of the airways.
Conclusions: Our results suggest that FTIR applied to sputum might have high sensitivity and specificity in
diagnosing lung cancer with potential as a non-invasive, cost-effective and high-throughput method for screening.
Trial Registration: ClinicalTrials.gov: NCT00899262
Lung cancer is the most common cancer in the world
where 1.3 million deaths are recorded each year . It is
the second most common cancer in the UK and most
common cause of cancer mortality with 34,500 deaths
per annum . Survival time is also poor with over 90%
of patients dying within five years of diagnosis. Besides
co-morbid conditions, poor survival rates primarily
reflect the fact that over two thirds of patients are diag-
nosed at a stage that is currently not amenable to
potentially curative treatment.
A number of reasons exist as to why so many lung
cancers are diagnosed at late stage. The aetiology of
lung cancer is well established with approximately 90%
of tumours occurring in smokers . Smoking is not
just problematic in terms of the causation of lung can-
cer as common symptoms of lung cancer such as
coughing, dyspnoea or haemoptysis are frequently
caused by smoking itself so are dismissed by long term
smokers as simply being a consequence of smoking.
Current diagnostic methods include chest X-ray, com-
puterized tomography (CT) and bronchoscopy but
despite these methods improving the ability to detect
lung cancer they remain less effective for early stage
detection . In reality, the detection of lung cancer at
an early stage would require a national screening pro-
gramme. Targets for screening would be those at high
risk including people over the age of 60 with a history
of smoking, those with a previous history of cancer, and
patients with chronic obstructive pulmonary disease
(COPD). However, a screening programme would
* Correspondence: firstname.lastname@example.org
1School of Medicine, Swansea University, Swansea, SA2 8PP, UK
Full list of author information is available at the end of the article
Lewis et al. BMC Cancer 2010, 10:640
© 2010 Lewis et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
require a technology that is sensitive to early stage lung
cancer and cost effective with the ultimate aim to
Recent randomized controlled trials have focused on
the evaluation of low dose computerised tomography
(LDCT) . It is estimated that, although having accep-
table cost effectiveness, LDCT would still be an expen-
sive screening method . Furthermore, there is debate
as to the sensitivity of LDCT for use on asymptomatic
high risk cases  and recent findings from the DANTE
trial suggest that mortality reduction using LDCT as a
screening tool might be smaller than anticipated .
Molecular markers for early detection of lung cancer
hold much promise and possess a number of advantages
over existing methods. Indeed, markers such as DNA
methylation status of certain genes can be detected in
biofluids such as blood and sputum from people with
lung cancer  as well as broncho-alveolar lavage (BAL)
fluid . As opposed to LDCT, technologies that can
detect molecular biomarkers involve no radiation expo-
sure, are relatively inexpensive and high throughput so
satisfying the need to have a screening process that is
cost effective and rapid.
Fourier transform infrared spectroscopy (FTIR) is a
non-invasive technology that can detect a change of
functional group in molecules from tissue or cells. Such
changes can be visualized using a spectrum of wave-
numbers usually taken from the mid infrared range
(4000 to 400 cm-1). FTIR has shown promise as a sensi-
tive diagnostic tool to distinguish neoplastic from nor-
mal cells in cancers such as colon , prostate ,
breast , cervical , gastric , oral  and oeso-
phageal . In these and other studies, biochemical
changes are often observed between tumour and normal
cells within a wavenumber range known as the “finger-
print region” (encompassing 1800 to 950 cm-1).
There have been surprisingly few attempts to apply
FTIR for diagnostic purposes in lung cancer tissue. This
likely reflects the fact that access to lung tissue is diffi-
cult, highly invasive and most patients are diagnosed at
a late stage. The few results that have emerged though
are encouraging where FTIR wavenumbers in the finger-
print region were interpreted to suggest that elevated
glycogen levels significantly discriminate both squamous
cell carcinoma and adenocarcinoma tumours from nor-
mal tissue [18,19]. A related infrared technique, Raman
spectroscopy, has also demonstrated success in distin-
guishing lung tumour from normal bronchial tissue 
and has more recently been combined with a broncho-
scope for potentially diagnosing lung cancer ex vivo 
FTIR has successfully been applied for diagnostic pur-
poses on sputum from COPD patients  and for bac-
teria identification in cystic fibrosis . However,
despite success in detecting cancerous change in cells
from pleural fluid , to our knowledge FTIR has
never successfully been applied to sputum for lung can-
cer diagnosis. The aim of our study was to identify
infrared wavenumbers that significantly discriminate
cancer sputum from healthy control sputum and apply
multivariate analysis to determine whether samples form
sub-groups according to variation in wavenumber signal.
This work lays the foundation for further studies to
evaluate whether FTIR might be used as a cost effective,
high throughput and non-invasive method for screening
biochemical changes in sputum from lung cancer cases.
Study Subjects and Sputum Collection
This study had approval from the loco-regional ethical
committee (05/WMW01/75). Spontaneous sputum was
collected from 25 patients (mean age 66.5 ± 9.2 years; 15
males, 10 females), for the majority,(23/25) it was taken
just prior to bronchoscopy for suspected lung cancer.
Informed consent to provide a sputum sample was
obtained from each patient at a previous clinical appoint-
ment. Lung cancer was subsequently confirmed through
final clinical diagnosis) as part of the Medlung observa-
tional study (UKCRN ID 4682). Final histology was
recorded where known (19 non-small cell lung cancer
(NSCLC): 7 squamous cell carcinoma, 5 adenocarcinoma,
7 unknown histological subtype, 3 small cell carcinoma; 1
large cell carcinoma; 2 radiological diagnosis). The two
patients who had a radiological diagnosis based on clini-
cal presentation and radiological findings were too
unwell for further investigation to determine histological
subtype. Tumour location was also recorded for each of
the 23 patients who underwent bronchoscopy: 11 normal
(i.e. no tumour observed at bronchoscopy); 2 external
compression of bronchus/trachea from the tumour/
nodes; 4 abnormal mucosa; 6 tumour seen endobronchi-
ally. Based on CT scans all patients had a centrally
located tumour. In total, 12 cancer cases (48%) showed
no evidence of a tumour at bronchoscopy although ran-
dom bronchial washings were still taken on all. Smoking
status and pack years (pky) were recorded for each
patient: 11 current smokers (median pky = 40); 11 ex
smokers (median pky = 40); 3 never smokers. Sputum
was also collected from 25 healthy donors (mean age
62.5 ± 11.1 years; 15 males, 10 females) consisting of staff
members at Swansea University with no previous history
of cancer or lung disease other than COPD or asthma.
Smoking status and pack years recorded for controls
were: 12 current smokers (median pky = 30); 5 ex smo-
kers (median pky = 27); 8 never smokers.
Sputum collection and processing
After collection, sputum samples were kept frozen at
-80°C. Prior to processing, sputa were defrosted at room
Lewis et al. BMC Cancer 2010, 10:640
Page 2 of 10
temperature for 12-24 hours. Sputum cells were isolated
by breaking down the mucus with a solution consisting
of 2.5 g Dithiothreitol (DTT) in 31 mL Cytolyt (Fluka
Biochemika Sigma-Aldrich Chemie GmbH Switzerland)
which digested mucus in the specimen. Samples then
underwent centrifugation at 3000 rpm for 10 min and
supernatant poured off leaving a cell pellet. An aliquot
of cells was taken to create a second pellet that was sub-
sequently formalin fixed and wax embedded prior to
sectioning and staining with haemotoxylin and eosin
(H&E). Residual pellets were freeze-dried over night,
diluted in 200 μL of sterile distilled water, agitated for
5 min and split into 20 μL aliquots, which were frozen
immediately in liquid nitrogen and stored at -80°C. To
confirm samples were of bronchial origin, H&E stained
sections were assessed by a consultant histopathologist
for presence of bronchial epithelial cells.
A FTIR mid-infrared spectrometer instrument (Buker
VERTEX 80/80 v, Ettlingen, Germany) was used to
obtain spectra which comprised a KBr Beamsplitter, a
diffuse reflectance absorbance scanning accessory
equipped with a mercury-cadmium telluride (MCT)
detector and a sampling compartment fitted with a hori-
zontal attenuated total reflectance (HATR) sampling
accessory. In this study, we used the MCT detector for
reflectance IR measurements. To reduce noise, the
MCT detector has to be operated at liquid nitrogen
temperatures. Ninety-six well silicon plates (LNC Tech-
nology Ltd., Ystrad Mynach, Hengoed, UK) were
cleaned in warm 0.5% SDS, rinsed with dH2O, soaked
overnight in 5 M nitric acid, rinsed again with dH2O
and air dried. Samples were applied randomly, in tripli-
cate, across a plate, permitting possible variations within
or between plates to be taken into account during analy-
sis. Loaded sample plates were oven dried at 50°C for 30
min (Sanyo Gallenkamp plc., Loughborough, UK) to
remove extraneous moisture prior to FTIR analysis. Pre-
pared plates were allowed to cool and then inserted
onto the motorized stage of the diffuse reflectance
absorbance scanning accessory connected to the FTIR
spectrometer. FTIR spectra were obtained in reflectance
mode. Interference peaks in the mid-infrared region of
spectra were collected to minimise noise from CO2and
H2O vapour. The sampling compartments and micro-
scope stage were purged with dry CO2free air produced
from a Peak Scientific compressor (Peak Scientific Ltd.
Paisley, UK). Data points were collected at a resolution
of 2 cm-1from the 4000 to 600 cm-1wavenumber
range. Each spectrum represented the average of 256
scans for improved signal to noise ratio. Sample absor-
bance spectra were calculated from the ratio of IS/IR,
where IS was the intensity of the IR beam after it has
been absorbed by the sample and IR was the intensity of
the IR beam from the reference. The absorbance spec-
trum was calculated as: -log10(IS/IR).
Data Processing and Analysis
All data processing, analysis and visualization were per-
formed using the R statistical computing environment
 using in-built algorithms and code developed by
our group. We were interested in assessing change in
the fingerprint spectral region so absorbance values
between wavenumbers 950 and 1800 cm-1(442 data
points) were pre-processed prior to further analysis.
Raw spectra were were pre-processed using a simple
two point linear subtraction baseline correction method.
Two points, 900 and 1850 cm-1were selected outside
the wavenumber region of interest that showed no var-
iation across all samples. Spectra were then vector nor-
malised. Second derivative spectra were then calculated
after smoothing using the Savitzky-Golay algorithm with
nine points. We applied the Shapiro-Wilk test to assess
whether second derivative absorbance values for each
number followed a normal distribution. As no wave-
number was found to be normally distributed the
Mann-Whitney U test was used to determine wavenum-
bers that were significantly different (P = 0.05) between
cancer and control spectra. Furthermore, due to multi-
ple testing, a wavenumber p-value was only retained as
significant if it remained so after applying Holm’s
sequential Bonferroni correction.
Two different multivariate analysis approaches were
used to determine and visualize the pattern of similari-
ties within and between cancer and normal spectra.
Hierarchical cluster analysis (HCA) dendrograms were
produced using the ‘hcluster’ function in the R ‘amap’
package with a correlation distance matrix and
unweighted pair-group tree building method. Principal
components analysis (PCA), via the R ‘prcomp’ package,
allowed further visualization and interpretation of spec-
Evaluation of sputum cell pellet FTIR spectra
FTIR was used to generate absorbance spectra in the
frequency region 950 to 1800 cm-1to establish potential
metabolic differences in cells extracted from sputum
between 25 lung cancer and 25 normal control samples.
Representative spectra for cancer and control samples
are shown in Figure 1 A-B. By repeating the procedure
multiple times (and on different dates) we found that
spectra generated for each sample were highly reprodu-
cible. Normality tests revealed that none of the indivi-
dual wavenumber absorbance levels followed a normal
distribution. Thus, for each wavenumber, median(rather
than mean) absorbance levels were used for analysis.
Lewis et al. BMC Cancer 2010, 10:640
Page 3 of 10
Figure 1C shows median spectra for cancer and nor-
mal sputa highlighting key regions of wavenumber
absorbance where differences exist between the two
groups. The prominent absorbance bands observed in
both cancer and normal spectra are characteristic of
vibration modes assumed to represent functional
groups in cellular molecules including proteins, carbo-
hydrates and nucleic acids [26,27,24]. In all spectra,
major peaks were observed for absorptions in the
amide I and amide II regions at 1656 cm-1and 1577
cm-1respectively. Another broad region of peaks
assumed to be associated with proteins was seen
between 1400 cm-1and 1450 cm-1. A fourth prominent
set of peaks was observed in the putative glycogen and
nucleic acid associated regions between 1000 cm-1and
1100 cm-1. In these latter two regions there were clear
differences in peak heights between the cancer and
normal median spectra although potential band overlap
meant it was difficult to quantify the number of peaks
or ascertain exact peak location.
Second order derivative spectra were generated from
the raw cancer and normal median data (Figure 1D).
This approach allowed us to resolve broad, overlapping
bands into individual bands thus increasing the accuracy
of analysis. By applying the Mann-Whitney U test to
each second derivative wavenumber signal, and adjusting
each p-value because of multiple testing, we were able
to determine a set of 92 significant wavenumbers that
differentiated cancer sputum from normal. Significant
wavenumbers were then ranked according to p-value
and plotted against the second derivative median spectra
for each group (Figure 2). This approach allowed us to
quickly filter out significant wavenumbers which were
actual peak centres. By analysis of the spectral alignment
we determined 6 highly significant wavenumbers that
could be associated with prominent and interpretable
second derivative peaks in both groups (labelled as A-F
in Figure 2). Table 1 summarizes each of these wave-
numbers along with the proposed vibrational modes and
primary molecular source.
Figure 1 Raw example FTIR spectra for cancer and normal sputum. Raw example FTIR spectra between wavenumbers 950 cm-1and 1800
cm-1for (A) cancer sputum and (B) normal sputum. (C) Median raw spectra for cancer and normal sputa. (D) Second derivative spectra for
cancer and normal sputa.
Lewis et al. BMC Cancer 2010, 10:640
Page 4 of 10
The small but significant band at 964 cm-1in normal
sputa shifts to the 966 cm-1position in cancer sputa.
This band is thought to result from vibrational modes of
nucleic acids  and symmetrical stretching of phos-
phate monoesters of phosphorylated proteins . Two
of the most significant changes between cancer and nor-
mal sputa were seen at peaks 1024 cm-1and 1049 cm-1in
the glycogen rich region. Absorbance levels were
increased at these positions in all cancer cases relative to
controls. Both these wavenumbers are attributed to C-O
stretching and C-O bending typical of glycogen .
Despite removing mucus during the process of
extracting cells from sputum we assumed that mucins
might still be present in each sample pellet used for
FTIR. The presence of mucins has been shown to cause
peaks at 1040 cm-1, 1076 cm-1and 1120 cm-1in FTIR
spectra due to C-O stretching . It is possible that
patients with lung cancer might simply produce more
mucus leading to peak changes at these positions. Sig-
nificant peaks, although less prominent, were observed
between 1062 cm-1and 1066 cm-1(Figure 2B) but no
peak was present at or around 1076 cm-1or 1120 cm-1
in any spectrum. Similarly, no peak was apparent at
1040 cm-1however this position lies within a broad
peak (Figures 1C, D) with a maxima at 1049 cm-1. Thus,
without further analysis, it is difficult to rule out a
mucin related peak being present at 1040 cm-1.
A peak at 1411 cm-1, associated with COO- stretch-
ing and C-H bending, was significantly increased in
cancer spectra relative to normal. Furthermore, there
was a shift in peak position from 1417 cm-1seen in
normal spectra. Peaks at 1577 cm-1in the amide II
region and around1656 cm-1in the amide I region
were both elevated in cancer spectra relative to
Table 1 Frequency (cm-1) assignment, proposed vibrational mode and molecular source of 6 prominent significant
Peak Peak position from second derivative (cm-1) Proposed vibrational modeProposed primary source
PO4=stretch; C-C stretch
C-O stretch, C-O bend
C-O stretch, C-O bend
COO- stretch, C-H bend
Amide II, NH bend, C-N stretch, C = N imidazole ring stretching Protein and nucleic acid
Amide I, C = O stretch
Protein and nucleic acid
Assignments taken from references: [22,24-26].
Figure 2 Median second derivative spectra and significant wavenumbers. (A) Median second derivative spectra for cancer and normal
sputum. Six major significant peaks are identified (A-F) as described in Table 1. (B) Positions of 92 peaks statistically significant between cancer
and normal spectra. Each peak is ranked according to p-value where the lowest p-value attained the highest rank of 1.
Lewis et al. BMC Cancer 2010, 10:640
Page 5 of 10
control. These bands characteristically reflect bending
of N-H bonds and stretching of C-N bonds as well as
stretching of C = O bonds . In cancer spectra
there was also a peak shift from 1656 cm-1to 1654
cm-1. Interestingly, none of these peaks were signifi-
cantly different between adenocarcinoma and squa-
mous cell carcinoma spectra
Multivariate analysis of sputum FTIR spectra
The second derivative absorbance values at 964 cm-1,
1024 cm-1, 1411 cm-1, 1577 cm-1and 1656 cm-1were
further subjected to multivariate analysis (MVA). The
1049 cm-1peak was not considered due to the potential
association with mucin presence. We used two MVA
methods to determine the underlying structure of how
cancer and normal sputa samples grouped according to
their spectra of significant wavenumbers. Application of
MVA performed in this way would allow us to visualize
the causal effects of significant wavenumbers on the pat-
terns of differences existing both within and between
cancer and normal sputum groups. The first method,
hierarchical cluster analysis (HCA) would allow visuali-
zation of the overall grouping structure and conse-
quently sub-groups of spectra. The second method,
principal components analysis (PCA), would provide
further information on how spectra group indicating
which wavenumbers cause such groupings.
The HCA dendrogram for all 25 cancer and 25 normal
sputum is shown in Figure 3. Two large sub-clusters are
clearly visible in the dendrogram suggesting that these
wavenumbers discriminate the two groups with high
accuracy. Working from left to right, the first major sub-
cluster contains all but two of the normal spectra. Seven-
teen (68.0%) normal spectra grouped tightly in a smaller
sub-cluster. Interestingly, 5 out of 6 cases grouping away
from this sub-cluster recorded a cough prior to providing
a sputum sample. The second major cluster contained all
cancer spectra as well as one normal spectrum and
another that is an obvious outlier within this group. This
cancer cluster had a distinct sub-cluster containing 15
(60.0%) cases. Interestingly, the four cases that had pre-
viously been diagnosed with either colon or breast cancer
grouped outside this sub-cluster. Furthermore, the two
cases with previous breast cancer grouped together as a
pair. No sub-clustering was evident for final histology
and no cluster patterns emerged for smoking status in
either the cancer or normal groups. The ‘tightness’ of
spectra within the main sub-clusters described indicates
a high degree of similarity across wavenumber absor-
bance patterns within these groups.
PCA returned three components that collectively
explained 96.1% of the variation within the 5 wavenum-
bers (where each component comprised at least 5% var-
iation). The scree plot in Figure 4A shows that these
three components (PC1, PC2 and PC3) explained 71.9%,
19.2% and 5.0% of the variation each. A scatterplot of
the correlation of spectra on PC1 and PC2 (Figure 4B)
reveals how these components explain variation existing
between cancer and normal cases. Cancer spectra had a
higher negative correlation on PC1 compared to normal
spectra. An assessment of wavenumber loadings on PC1
revealed that this was primarily due to high negative
and positive loadings of 1024 cm-1and 1411 cm-1
respectively on PC1. With the exception of an outlier,
all normal spectra had a negative correlation on PC2
and this component had a high positive loading of 1656
cm-1and high negative loading of 1577 cm-1. The two
cases previously diagnosed with breast cancer are sepa-
rated from all other spectra due to a high negative cor-
relation on PC3. Again, this can be explained by a high
negative loading of 1656 cm-1on PC3.
This study is the first (to our knowledge) to generate
FTIR spectra from sputum and derive chemical finger-
prints for the purpose of diagnosing lung cancer.
With the knowledge that FTIR yielded excellent repro-
ducibility for sample spectra, our primary objective in
Figure 3 HCA of prominent significant wavenumbers.
Dendrogram showing general and sub-clusters of lung cancer (C)
and normal (N) sputum spectra produced by HCA using significant
panel of wavenumbers. Supplementary information for samples are
provided at the bottom of the plot: y = smoker, n = never-smoker,
x = ex smoker, Previous diagnoses of other cancers are also
labelled: * = breast, ‡ = larynx and bladder, † = colorectal. Normal
cases who had stated that they had a cough prior to providing
sputum are labelled with ■.
Lewis et al. BMC Cancer 2010, 10:640
Page 6 of 10
this study was to determine which wavenumbers were
significantly different between sputum of cancer and
normal controls. Prominent significant wavenumbers
could then be used to explore the structure of patterns
of similarities within and between both cohorts using
MVA techniques. The data analysis strategy we
employed was robust and took into consideration the
data distribution at each wavenumber. We have found
that many FTIR studies apply parametric tests to wave-
number data with no evidence of data distribution yet
we found that data at all wavenumbers in this study did
not follow a normal distribution. Interestingly, White-
man et al.  also made the same observation in their
study on FTIR spectra in sputum of COPD patients.
The six peaks described in Table 1 all arose due to an
increase in absorbance at that spectral position in cancer
relative to normal controls often with a noticeable posi-
tion shift. A rise in absorbance at a wavenumber in one
sample relative to another can be due to different reasons
including an increase in the frequency of a bond vibra-
tion mode . It should also be noted that the non-uni-
form distribution and degree of compaction of molecules
within cells can also have a non-linear affect on absor-
bance level as considered for chromatin within dividing
and non-dividing cells . Stronger intermolecular
interactions at bonds, such as C-O in carbohydrates or
COO- in proteins, result in higher absorbance levelsIt is
possible that an increase in absorbance levels at 1656 cm-
1and 1577 cm-1is due to an increase in protein levels
between cancer and normal cells in sputum .
Band shifts and significant differences in absorbance
intensity at 1024 cm-1and 1049 cm-1may signify an
increase in cancer sputum of levels in glycogen. A pre-
vious study by  using FTIR suggested that a glyco-
gen band at 1045 cm-1was increased in lung tumours
(squamous cell carcinoma and adenocarcinoma) relative
to normal tissue. A subsequent study using FTIR micro-
scopy confirmed increased levels of glycogen in lung
tumour cells . FTIR showed glycogen levels were
also increased in lung tumour cells of pleural fluid due
to an increase in absorbance at 1030 cm-1in the glyco-
gen rich region . An increase or decrease in glyco-
gen levels varies according to cancer type, for example
levels are higher in colon tumour tissue relative to nor-
mal but a reduction is seen in tumours of the cervix
and liver . Indeed, the significant increase in absor-
bance levels at 1024 cm-1and 1049 cm-1in lung cancer
sputa observed in our study contrast the decreased
levels for these wavenumbers in tumours of the cervix
compared to normal tissue . Importantly, if differ-
ences in absorbance at 1024 cm-1and 1049 cm-1wave-
numbers between lung cancer and normal sputa cells
represent glycogen levels then our results confirm pre-
vious studies and suggest that increased glycogen levels,
via detection by FTIR, could prove to be a powerful
diagnostic factor for lung cancer.
The significant band shift at 964 cm-1has been asso-
ciated with symmetrical stretching in bonds of phos-
phorylated proteins but has also been associated with
cancer related structural change in nucleic acids .
The band increase (and shift) at 1411 cm-1in cancer
sputum spectra might be an indicator of a change in
nucleic acid level  alternatively, this change could
also suggest COO- stretching and C-H bending due to
proteins. Our results suggest that further work should
explore the contribution different molecules that differ-
entiate lung cancer from normal sputum spectra so
accurate assignments of the causation of absorbance
changes can be made.
An important consideration when generating FTIR
spectra from biospecimens such as sputum is that cells
will often be derived from mixed tissue types which can
lead to spurious results . In this study, we ensured
that each sputum sample was assessed by a pathologist
for sufficient presence of bronchial epithelial cells.
Indeed, validation of our work could involve analysis of
cell pellets by FTIR microspectroscopy to generate spec-
tra for pre-identified normal bronchial and tumour cells.
Using 5 significant wavenumbers we were able to
visualize the underlying structure of how all sputum
Figure 4 PCA of prominent significant wavenumbers. Plots
produced after application of PCA to the panel of significant
wavenumbers. (A) Scree plot showing the number of components
to retain explaining at least 5% of the variation. The first 3
components explain 95% variance. Scatterplots of the loadings of
each cancer (C) and normal (N) sputum spectrum on: (B)
components 1 (PC1) and 2 (PC2); (C) components 1 and 3 (PC3); (D)
components 2 and 3.
Lewis et al. BMC Cancer 2010, 10:640
Page 7 of 10
samples grouped according to patterns of differences in
absorbance levels. HCA and PCA in combination
allowed visualization and interpretation of spectral sub-
groups. The observation from the HCA dendrogram
(Figure 3) that no sub-groupings emerged due to histo-
logical type suggests that the 5 significant wavenumbers
are not type specific. Perhaps a much larger set of sam-
ples of NSCLC subtypes and especially small cell carci-
noma cases may reveal FTIR spectral differences
according to tumour sub-type.
PCA gave further insight into the causal relationship
between groupings of spectra and individual significant
wavenumbers with 3 components explaining 96.1% of the
variation. The first two components (PC1 and PC2) show
that cancer spectra clearly separate from normal spectra
according to the loadings on the protein, glycogen and
DNA associated wavenumbers 1577 cm-1, 1024 cm-1and
1411 cm-1. These wavenumbers are thus important
potential diagnostic markers for lung cancer. PC3 was
highly associated with the two spectra for patients who
had previously been diagnosed with invasive ductal carci-
noma of the breast. This result is interesting as both
cases had a final histology of NSCLC yet, PCA reveals
that both spectra have a high similarity to each other but
are separated from other lung cancer spectra. Although
data is extremely limited one might hypothesize that
FTIR has the potential to further discriminate metastatic
tumours where the primary arose in the breast.
Throughout the analysis we were mindful of con-
founding variables that might lead to misinterpretation
of differences between cancer and normal sputum spec-
tra. It is suggested that inflammation plays a key role in
the pathogenesis of lung cancer . From the patient
medical histories recorded we noted conditions that
could contribute to inflammation in the bronchial tubes.
For example, a number of cancer cases had also been
diagnosed with COPD or asthma according to standard
criteria. Furthermore, the control group also included
cases with COPD and asthma. However, an inspection
of the grouping patterns of HCA and PCA did not
reveal any similarities either within group or between
groups due to the presence of these conditions. It is
interesting to note however that spectra of nearly all the
normal cases who had presented with a cough (prior to
sputum acquisition) were more dissimilar to the large
sub-cluster of normal spectra in the HCA dendrogram.
We were not however able to find any association of
wavenumbers with these few cases using PCA.
The spectra of cancer and COPD from sputum can be
further compared in detail. Whiteman et al.  com-
pared the FTIR spectral profiles from sputum of 15
COPD patients and 15 healthy volunteers. That study
yielded reproducible spectra from sputum with no sig-
nificant difference between patterns in smokers and
non-smokers, factors that are mirrored in our study.
The key findings of the COPD study were that major
spectral changes between groups were observed as peak
shifts in the regions of 1559 cm-1, 1077 cm-1and 1458
cm-1. Thus, in sputum, the significant pattern of change
in FTIR spectra of COPD patients is different to that
seen in cancer patients. Whiteman et al. conclude from
their study that the main contributor shaping the het-
erogeneous FTIR spectrum in COPD patient sputa is in
vivo airway inflammation. If this is the case then airway
inflammation is not a major contributor to the lung
cancer sputum spectrum strengthening the argument
that the molecular changes observed are cancer-specific.
It was also important to ensure that absorbance at key
wavenumbers were not changed in cancer sputa simply
due to differing levels of mucus despite the removal
process. Absorbance levels of key mucus related peaks
at 1076 cm-1and 1120 cm-1were either very low or
non-existent. Absorbance levels of another mucus
related peak at 1040 cm-1were more difficult to estab-
lish as this wavenumber was situated in the shoulder of
the glycogen related 1049 cm-1peak. Removal of the
1049 cm-1wavenumber during analysis ensured that dif-
ferences between cancer and normal sputa were not
influenced by presence of mucus.
Although the HCA dendrogram demonstrates a clear
separation between the major cancer and normal clus-
ters two normal spectra did group with cancer spectra.
Thus, an important question arising from this study is:
what are the potential levels of specificity and, more
importantly, sensitivity when using the panel of wave-
numbers to discriminate cancer from normal sputum?
An exact figure should not be estimated from just 50
cases but the grouping patterns observed using MVA
suggests that sensitivity and specificity would be at least
greater than 80% which compares more than favourably
with existing methods of lung cancer detection.
In conclusion, we report the preliminary application of
FTIR to determine biochemical changes in sputum
between lung cancer and normal cases. Our results sug-
gest that FTIR applied to sputum might have a high
sensitivity and specificity in diagnosing the disease using
a small panel of significant wavenumbers. The continu-
ous collection of sputum within the Medlung project
will allow us to generate predictive models for lung can-
cer on much larger datasets. The cases used in this
study were recruited mainly at bronchoscopy so tended
to have more centrally localised tumours. Thus, we are
currently investigating the ability of FTIR to detect per-
ipheral lung tumours using sputum and are encouraged
by the fact that FTIR was able to detect cancer in 48%
of cases where no tumour was visible during
Lewis et al. BMC Cancer 2010, 10:640
Page 8 of 10
bronchoscopy. If biochemical changes in sputum can
also be detected by FTIR in the early stages of lung can-
cer, then the technology might prove to be a non-inva-
sive, cost-effective, high-throughput method for eventual
screening. With this goal in mind, we are also perform-
ing a longitudinal study to determine whether the panel
of infrared wavenumbers can also discriminate patients
deemed at high-risk for lung cancer.
We acknowledge the Welsh Assembly Government and Hywel Dda Health
board for financial support. We would like to thank Dr Rohan Mehta, Dr
Sarah Prior, Oliver Lyttleton, Claire Duggan and Sarah J Jones for their
assistance and advice during collection and processing of samples. Finally,
we are extremely grateful to the three reviewers of this manuscript for their
expert advice and helpful comments.
1School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
2Department of Respiratory Medicine, Prince Phillip Hospital, Llanelli, SA14
8LY, UK.3Institute of Biological, Environmental and Rural Sciences,
Aberystwyth University, Aberystwyth, SY23 2AX, UK.
PDL conceived the study, performed data analysis and participated in its
design and supervision. LUM participated in study design and coordinated
FTIR. KEL, RG and PK coordinated tissue and data collection and provided
clinical input into the study. SB, AJL and JW performed FTIR. ARG provided
intellectual input into the study and helped draft the manuscript. All authors
read and approved the final manuscript.
The authors declare that they have no competing interests.
Received: 26 March 2010 Accepted: 23 November 2010
Published: 23 November 2010
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Cite this article as: Lewis et al.: Evaluation of FTIR Spectroscopy as a
diagnostic tool for lung cancer using sputum. BMC Cancer 2010 10:640.
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