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I. INTRODUCTION growths are usually benign, but some may develop into can-
cer over time. The ability to classify these polyps in time
Colon cancer is a major public health problem due to its
could provide warning for their development into cancer.
widespread occurrence and death toll worldwide. According
Even within diagnosed cancer cases, the ability to classify
to the estimation of the National Cancer Institute, approxi-
mately 108 070 colon and 40 740 rectal cases were reported between early and severe cases is highly important and could
in 2008, of which 49 960 caused mortality. Of the estimated influence the treatment strategy.
5.2 106 mortalities from cancer per year throughout the The use of FTIR microscopy in the field of cancer diag-
world, 655 000 cases are caused by colorectal malignancies. nosis has shown encouraging trends over the past 20 years.2
It is the second leading cause of cancer-related death in the The wavelength of infrared radiation which is absorbed de-
Western world.1 Despite the improvement in diagnostic tech- pends on the nature of the covalent bond i.e., atoms in-
niques, more than 90% of colon cancer cases have either volved and the type of bond and the strength of any inter-
advanced or metastasized by the time they are diagnosed. molecular interactions van der Waals interactions and H
Hence, there is an urgent need to develop novel digital diag- bonding.3 Various biomolecular components of the cell give
nostic methods to detect the malignancy in the earliest stage a characteristic IR spectrum, from which structural and func-
possible. Many colorectal cancers are thought to arise from tional aspects4 of the cell can be inferred. The differences in
adenomatous polyps in the colon. These mushroomlike the absorbance spectra in the mid-IR region between normal
1047 Med. Phys. 37 3, March 2010 0094-2405/2010/373/1047/9/$30.00 2010 Am. Assoc. Phys. Med. 1047
1048 Zwielly et al.: Statistical techniques applied to FTIR colonic spectra 1048
TABLE I. Average and STD of selected biomarkers are represented in the top section. The bottom section
contains t-test values for all six biomarkers and tissue combination pairs. Each square contains six boxes
corresponding to the six biomarkers in the upper part. The nonsignificant values are marked as NS.
1740 cm-1
1.220.32 0.640.17 0.470.14 0.430.13 0.500.17
(x100)
wavenumbers. The bands at 1083 and 1056 cm1 correspond but in reverse order, hence the A1083 / A1056 ratio was consid-
to absorbance of the vs PO2 of phosphodiesters of nucleic ered significant Table I. In IR spectra, the bands at 1025
acids28 and the O u H stretching coupled with C u O bend- and 1045 cm1 correspond to the vibrational modes of
ing of C u OH groups of carbohydrates, respectively.29 uCH2OH groups and the C u O stretching coupled with
These two biomarkers show the same absorbance intensity C u O bending of the C u OH groups of carbohydrates in-
FIG. 5. DCF of normal, polyps mild, moderate, and severe and cancer
tissues. Each class is represented by an array of average values of four
biomarkers.
FIG. 4. Important biomarkers are marked in the FTIR spectra at the each group based on Eq. 3. It can be noted that the mild
1000 1800 cm1 fingerprint region. While good classification for normal and moderate polyps have similar scores, which means that
tissues is apparent, only small changes are noticed in this region among the
other groups. The shaded region represents the asymmetric phosphate
only small detectable spectral changes occur between mild
biomarker. and moderate polyps as would be expected. Generally, the
score values of the tissues starting with the normal group
gradually approach the spectral values of the malignant
cludes glucose, glycogen, etc. Higher intensity is noticed for group as shown in Fig. 5. It is also noticed from Fig. 5 that
the normal and mild polyp compared to moderate, severe the diversity among polyps was larger than for the malignant
polyps, and the cancer groups Table I. and the controls. This is mainly notable between the mild
Previous works have shown that the band at 1121 cm1 and moderate polyps where they appear to overlap.
arises from RNA absorbance, whereas the 1015 cm1 shoul-
der is due to DNA.30,31 It was found that the best discrimi- III.C. LDA classification
nating values were obtained by deriving the intensity ratio of
PCA is a mathematical algorithm that reduces the large
these two vibrational modes i.e., A1121 / A1015 as can be seen
dimension of the measured spectrum that is being dealt with,
in Table I.
i.e., instead of using many variables, the variability in the
data is described by only few PCs. The reduction is achieved
III.B. Grading the samples using DCF by finding the correlation between the variables. Figure 6
Although the normal, benign polyps, and malignant tis- shows the scores of PC1 versus PC2 for all the measure-
sues constitute three separate main groups, an interesting ments. It can be seen that all normal data points are com-
analysis would be to examine a possible digital grading of pletely separated from carcinoma, while some overlap ap-
the tissues based on a chosen set of biomarkers. Based on pears between the polyps and the carcinoma and between the
these biomarkers, an acuteness ladder could be formed and polyps and normal tissue. PC1 contributes almost solely to
the groups can be classified. Each case was characterized the separation between normal and carcinoma groups, while
using an array of biomarkers, which were arranged as fol- PC2 contributes mainly to the separation between the polyps
lows: and the other two groups. This partial separation obtained by
PCA is not satisfactory and further procedures should be
A2848/A2958 carried out in order to distinguish between all five groups.
A1740 Thus, LDA was applied to discern between the five groups.
. 5 LDA is a statistical multivariate supervised method. It
A1083/A1056
searches for the variables containing the largest and the
A1021/A1015
smallest interclass variances, and constructs a linear combi-
To further examine the gradual spectral changes encountered nation of the variables to discriminate between classes. The
in the above tissue samples, we utilized the discriminant rule is to construct a training set of samples, which is further
classification function. This statistical tool enables to im- tested using the test set. The large number of valid variables
prove discrimination among normal, polyp stages, and ma- in the infrared spectra is an obstacle for this approach, which
lignancy by representing an adequate quantitative follow up needs more observations than variables. Using PCA unsu-
of transformations versus group type. DCF generates a clas- pervised method prior to the LDA analysis was helpful in
sification score for each group or premalignant stage using a reaching this goal of variable reduction. The results from the
linear combination of a previously derived array of biomar- LDA iteration are summarized in Table II, which shows the
kers with weight coefficients.23 Figure 5 shows the scores of percentage of success of each data set within all the possible
FIG. 6. PCA model employed on the database reducing the 512 valid measured variables in the spectrum to 13 PCs, which describe 98.4% of the data
variance. Full separation was achieved between the normal group plus symbols and the malignant group squares, while partial overlaps exist between the
three polyp groups circles. The solid black circles represent the corresponding groups centroids.
groups included in the study. We performed the LDA analy- in order to examine the classification potential of this optical
sis with two strategies: First, where all five groups were in- methodology in tandem with advanced statistical techniques.
cluded Table IIa and the second with only three groups, The gradual changes shown by the DCF score Fig. 5
namely, normal, polyp, and cancer, where the polyp group present a digital illustration how benign polyps evolve to-
consists of all polyp subgroups mild, moderate, and severe ward carcinoma. This trend was further studied using spe-
Table IIb. The results when all the five groups are in- cific biomarkers which clearly verify this gradual transition.
cluded show relatively lower success rates indicating that The main biomarker that dictates the DCF fitting and fully
many cases were classified within the neighboring groups. shows the gradual behavior is the CH2 / CH3 ratio
This picture dramatically improved when only three groups A2848 / A2958. This is due to its extraordinary high t-value
were assumed, where most of the group members were clas- Table I and the highly ordered gradual absorbance intensity
sified correctly. In both cases, none of the normal was as- of the normal, polyps, and carcinoma groups Fig. 3. These
signed as cancer and vice versa. The largest misidentification results suggest that the lipid/protein ratio gradually increases
occurred between normal and mild polyp 24%, which is with the severity of the disease. Since proteins contain, on
frequently a difficult problem even for an expert pathologist, the average, an equal amount of methyl and methylene
since mild polyps are intrinsically very close in their cell groups, a protein change alone should modify the CH3
morphology to normal tissues. This misidentification im- stretching as well as the CH2 stretching to the same extent.
proves dramatically when normal is misidentified as moder- The precise origin of the increase in the methyl/methylene
ate or severe polyps with only 2% and 1%, respectively. ratio remains to be determined; it may arise from an increase
When all three polyp stages are treated as a single class, the in lipid content, but can also be associated with the modifi-
percentage of normal misidentified as polyp is reduced to cation of the membrane composition during cancer.
14% Table IIb. In our previous studies, changes in the lipid region were
It is encouraging that in both strategies the false negative described differently.32 We believe that the main reason for
and the false positive rates both remain at 0%, as can be seen this is the normalization procedure in each case study. Our
from Tables IIa and IIb. previous study in this subject used min/max normalization of
the entire measured spectra with respect to the amide I in-
tensive band. This approach cannot detect subtle changes in
IV. DISCUSSION the lipid region. In order to reduce the contribution of close
Previous studies have provided evidence that infrared bands and to focus on the relevant regions, we now use a
spectroscopy is a useful and powerful tool to classify tissues different approach by bisecting the entire spectrum to three
and cells. The aim of this work was to test the potential of regions and vector normalized each segment separately as
FTIR spectroscopy on colon cancer patients where five dif- explained in the experimental section Fig. 2. Another ben-
ferent tissue groups stages can be identified. We used a efit over our previous normalization technique is the removal
statistical approach to analyze the large database of IR spec- of the uninformative region of 1800 2800 cm1, which is
tra that was measured. The complete database was analyzed highly dependent on the CO2 surrounding of the measured
TABLE II. True/false identification percentage of each tissue type based on ment may arise from the fact that phosphate level is the sum
the averaged LDA iterations a assuming five groupsNormal, mild, mod- of a larger number of biomolecules containing phosphate
erate, severe polyps, and cancer b assuming only three groups where all
polyp subgroups are treated as a single polyp group.
groups.
The shoulder at 1740 cm1 can be assigned to the ester
Identified as Normal Mild Moderate Severe Carcinoma C v O stretching of phospholipids, not present in DNA and
Polyp Polyp Polyp
proteins. The decreased intensities at 1740 are also consistent
Type with the methyl/methylene ratio, except for the cancer group
that shows a higher value than the moderate and severe pol-
Normal 74 24 2 1 0
yps Table I. The 1740 cm1 band is composed of residues
Mild 6 63 14 12 5 (a) of other vibrational modes35 besides phospholipids that may
Moderate 1 22 60 15 2
cause for this inconsistency.
Our LDA results Table II indicate that a high discrimi-
Severe 2 7 7 66 18
nation percentage is achieved when dealing only with the
Carcinoma 0 1 2 17 80 three main groups Table IIb: Normal, polyps, and cancer
tissues. A significant but relatively low percentage is ob-
tained when considering all the five groups: Normal tissue,
Identified as Normal Polyps Carcinoma mild, moderate, advanced polyps, and cancer Table IIa.
False identification can result from several reasons: Built-in
Type
spatial36 and spectral resolution limitation of the FTIR to
(b) distinguish between proximate tissues; cytological identifica-
Normal 86 14 0
tion failureEspecially when dealing within the polyp sub-
Polyps 6 85 9 groups; averaging over different measured sites inside spe-
cific sample can lead to realistic uncertainty of the sample
Carcinoma 0 15 85
type since some regions show borderline behavior; and dam-
aged FTIR measurement due to unstable surrounding and
equipments. All the above reasons except the first confound-
ing factor can be eliminated by consistently acquiring a
tissues. This is especially important when applying math- larger database.
ematical based distinction algorithms such as PCA where it The t-test values Table I revel that besides methyl/
could lead to artifacts in the classification. The two different methylene ratio, the chosen biomarkers have almost no sig-
normalization approaches reveal that different spectral pre- nificant differences between carcinoma severe polyp and
processing techniques may alter the biochemical interpreta- moderate polyp groups. Although the LDA algorithm takes
tion. This bisect technique was widely used,20,33,34 where into account all wavenumbers, this result is also true for the
sections of the spectrum were cut, baseline corrected, and severe polyp case Table IIa. This leads to mixing between
normalized independently from the entire region of the spec- the severe polyp and carcinoma groups. In contrast, the DCF
trum. classification Fig. 5 shows remarkably different scores be-
In complex systems such as tissues, the main absorptions tween carcinoma and severe polyps. DCF weights are influ-
arise from N u H, C v O, C u H, and P v O bonds from enced mainly by the t-test values. Thus, the methyl/
proteins, lipids, and nucleic acids present in the cells. methylene ratio, which has the largest t-value, dictates the
Wavenumbers below 1800 cm1 constitute prominent re- DCF behavior. When comparing the LDA results just for the
gions that contain all the above vibrational modes. This re- normal and malignant samples, excellent separation is
gion shows remarkable differences among normal tissues, achieved in the t-test analysis Table I as well as in the DCF
cancer, and all three polyp groups. This is strongly ostensible scores Fig. 5.
in the 1045 cm1 region, where a decreased absorption is In summary, we conclude that infrared spectroscopy is a
noted for all groups compared to the normal. The normal useful tool to identify different types of colonic tissues. The
cases clearly stand above all pathological tissues polyp and DCF scores formed acuteness ladders, which give further
cancer. This can be explained by the substantial glycogen benefits to the ability of grading the samples in the correct
reduction consumption in the polyps and the cancer tissues. order, namely, normal, polyps, and cancer based on the pre-
Another remarkable different biomarker is the 1072 cm1 viously selected array of biomarkers. We also demonstrated
band that corresponds to carbohydrates. This biomarker de- that PCA combined with LDA is a powerful tool for inves-
creased in the normal group. tigating the global biochemical modifications responsible for
The antisymmetric phosphate levels 1170 1310 cm1 tissue classification. However, we do not claim to replace the
reveal the metabolic turnover, as it consists of energy pro- pathologist. Spectroscopic methods may provide a second
ducers such as ATP and GTP, and other biomolecular com- opinionEspecially in difficult cases where ambiguous as-
ponents which include phospholipids, nucleic acids DNA signments are given by histopathology.
and RNA, and phosphorylated proteins. The difference in We have shown that different normalization techniques
total phosphate level among normal, the three groups of pol- can change the biochemical interpretation, although not the
yps, and cancer was clear in the normal case. This enhance- total changes among spectra.
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This work was supported in part by the Israel Science 17
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