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68 C. CONTI ET AL.
The samples were grouped as follows: Warthin tumor SIMCA correlations (by using classes and parame-
(A), oral epithelium with dysplasia (B), lymphoma (C), ters for HCA procedures) enabled us to discharge out-
polymorphous low-grade adenocarcinoma (D), adenoid liers spectra. The remaining spectra were resubmitted
cystic carcinoma (E), and its corresponding healthy tis- to HCA and the final grouping was used to extract rep-
sue (F). resentative spectra of each subcluster by means of
Tissues were cut into sections of 5 lm thickness by Standard Normal Variate Correction. These spectra
using a cryo-microtome. Three adjacent sections were were compared with those extracted point–point from
used for both histopathological and FTIR analysis. The selected regions of the map (by following the sugges-
first section was stained with hematoxylin-eosin tions of the pathologist); with a satisfactory compari-
(H&E) for histological examination to identify various son, the average spectra were used for the spectral
regions, whereas the second and the third were depos- analysis.
ited on steel and silicon supports for infrared analysis On bands of interest, frequency and absorbance val-
(Conti et al., 2007b). ues were obtained and analyzed statistically.
Data Processing for IR Microspectroscopy
Spectral data were achieved with the following spec- RESULTS AND DISCUSSION
trometers: Perkin Elmer Spectrum One and Perkin IR microspectroscopic imaging generates molecular
Elmer Spotlight 400, equipped with a Perkin-Elmer tissue maps providing a spectral signature of the inten-
Autoimage microscope (with a photoconductive sity and a spatial location of the chemical components
HgCdTe, MCT, array detector, operating at liquid nitro- of diseased tissues. In the following, the spectral analy-
gen temperature which covers the entire IR spectral sis of a representative section (well featuring the spec-
range from 4,000 to 700 cm21). The spectral resolution tral behavior of the remaining sections) will be
was 4 cm21. The spatial resolution, depending on the described in detail for each group of specimens.
heterogeneity, ranged from 50 3 50 to 6.25 3 6.25 lm2. The microphotograph of a salivary gland tissue inter-
The spectra were the result of 256 scans. Background ested by Warthin tumor (A), H&E stained, is shown in
scans were acquired and ratioed against the sample Figure 1.
spectrum. On the thin sections, deposited on steel or Following histological assessments, two different
silicon supports, reflectance and transmission spectra zones were examined where the H&E staining revealed
were obtained, respectively. Specific areas of interest a diversified histological composition: a yellow-signed
were selected by means of the microscope television zone with cystic component, lymphoid tissue and focal
camera. necrosis and a red signed zone with mainly solid tumor.
Baseline (polynomial line fit) was performed in all By the use of HCA (not reported) and PCA analysis,
cases, whereas Second Derivative (9 points), Fourier the 873 and 2,219 spectra, collected from cystic (yellow
Self Deconvolution and Curve Fitting (Gaussian char- square area with neoplastic lesions, CK and CK0 ) and
acter) procedures were used to determine the absorb- solid (red rectangular area, SK) regions, were nicely
ance ratio between bands of interest. All spectra were grouped in subclusters (Figs. 2a,b).
scaled for equal intensity in the Amide II absorption. In both regions, necrosis (Ne), lymphoid tissue (Ly),
Attribution of the bands was done according to litera- and vessels (V) were identified too. Representative
ture data (Jackson and Mantsch, 2002; Steudel, 1995). spectra are reported in Figures 3a,b.
Spectral images were produced by Perkin Elmer In the region 3,000–2,800 cm21, differences are evi-
Auto IMAGE 5.0.1 and by Spectrum IMAGE 1.6.1. dent in the spectral profiles of the six components
For data handling, the following software packages (bands normalized to the intensity of the phosphate
were used: Spectrum v.6.3.1 (Perkin-Elmer), Grams AI stretching mode at 1,080 cm21) (Fig. 3a): (i) the bands
(Galactic Corp.), and Pirouette 4.0 (Infometrix Corp.) shapes of SK and CK are more or less the same and
for multivariate analysis. Pirouette has already been resemble the one exhibited by cellular components; in
used in spectral data treatment of spectra from oral both spectra, the lower masymCH3/msymCH2 intensity ratio
cavity tissues (Conti et al., 2007b; Tobin et al., 2004). with respect to the CK0 one, can be ascribed to a hypo-
Hyper View Images Perkin-Elmer software was used to methylation process during carcinogenesis (Table 1)
generate pseudo color maps. and confirmed also by the same intensity ratio between
On a number of sections, supervised analysis was SK and the healthy tissue F (Fig. 4); (ii) a discrete pres-
undertaken following histopathological suggestions, ence of cellular component in CK0 and, mainly in Ne,
whereas on the remaining specimens blind tests (unsu- can be argued by band broadening; (iii) the lymphoid
pervised analysis) were performed. For each whole sec- tissue (Ly) shows the mCH2sym mode red shifted to 2,858
tion, a number of spectra from 10,000 to 40,000 could cm21; (iv) the vessels show proper spectral profiles
be extracted. The spectra from selected areas were sub- where the mCH3sym is hidden by the broad mCH2sym mode
mitted to hierarchical clustering analysis in the region (Conti et al., 2005, 2008; Wong et al., 1991; Yamada
1,800–900 cm21 where the main absorptions of biomo- et al., 2002).
lecules are located. For the HCA procedure all the spec- It is known that cholesterol, phospholipids and also
tra were converted in absorbance, baseline linear fit- creatine are important cellular metabolites with bands
ted, smoothed (5 points) vector normalized (Wood et al., in the region 3,100–2,800 cm21. In particular, the val-
2004). ues of masymCH2/masymCH3 ratio are related to phospholi-
The suitability of this method was controlled by run- pids peroxidation after oxidative stress and, hence, can
ning the HCA on some sections by using second deriva- be considered cancer targets for carcinogenesis
tive spectra (Savitzky-Golay, 9 points) and comparing (Petibois and Déléris, 2006; Petibois et al., 2007). Also
both clustering results. in our case, the values of the above ratio in tumoral
Fig. 4. Peak fitting in the region 1,800–1,450 cm21 (absorbance in arbitrary units, A.U.) of: (a) SK
(Warthin tumor, tissue A) and (b) control (tissue F).
Fig. 8. Image from cluster of cystic (a) and solid tumor area (b): green (SK), red (CK and CK0 ,
mainly), blue (Ne and Ly), dark green (holes).
Fig. 9. Photo of an H&E-stained section of the C tissue (lymphoma) and relative PCA analysis: green
(connective), red (tumor), and blue (lipids).
72 C. CONTI ET AL.
O groups, from various compositions of proteins, and TABLE 2. dasymCH3/dsymCH3 and AI/AII intensity ratio
from modifications of disulphide links (Fig. 6). in the healthy tissue (F) and tumoral tissues (A–E).
Overall SD 6 0.1–0.3
It is reported that an excessive increase in lactic acid
content can be related to glucose consumption by ma- A B C D E F
lignant cells to supply oxygen decrease during cells car- dasymCH3/dsymCH3 0.8 0.8 0.9 1.5 0.8 1.2
cinogenesis. This behavior can be proved also in our tis- AI/AII 2.0 2.2 2.5 1.9 2.2 1.7
sues, where the presence of the band of lactic acid at
1,127 cm21 is evident only in CK and SK and increases
from the former to the latter (John, 2001; Petibois sheet conformation (a-helix/b-sheet 5 2.9 6 0.2 and 0.8
et al., 2007). 6 0.1 in ialine and eosinophile connective tissue,
A visual inspection on high power 50 3 50 slides of respectively), the profile of CH2 and CH3 bending
limited areas of cyst and solid tumor (Fig. 1), suggested modes, the C OH band found at 1,172 (due to a
that not only biochemical changes but also tissue mor- change of C OH into C OP), a higher intensity of the
phology and packing are responsible for the spectral component bands on the right side of the convoluted
differences on going from Ly to CK0 , CK and, finally, to band centered at 1,080 cm21. In Figure 10, the spec-
the homogeneous and dense SK neoplasia (Figs. 7a–d). trum of the healthy tissue F is also reported. Spectral
In Figures 8a,b, the pseudo color maps are reported, differences of F with the tumoral spectrum are clearly
in which each pixel (6.25 3 6.25 lm2) is characterized evident: the vibrational mode C OH of sugars found,
by a proper spectrum; by using HCA analysis, similar as expected, at 1,164 cm21; a 1,080/1,240 ratio <1 and
spectra are grouped in subclusters (shown in the maps the DNA band at 960 cm21 almost absent (Fig. 10). (Ci
by different colors) assigning a specific zone for various et al., 1999; Conti et al., 2005; Gazi et al., 2004, 2007).
components and reproducing the histological biochemi- From tissues B, containing oral displastic epithelium
cal distribution and architecture. together with healthy zones characterized by glands
In the case of marginal B-cell lymphoma (C), the with alveolar shape, two main average spectra were
spectral and subsequent HCA (not reported) and PCA isolated: one well resembling the spectrum of the solid
analysis classified tumor, connective, and lipids compo- tumor, SK (with differences in Amide I and II band
nents (Fig. 9). shapes, only) and the other with a high content of gly-
The spectrum of this neoplasia (see Fig. 12) resulted coproteins (mucine) (Figs. 11a,b).
similar to the one of the solid tumor (A) as regard to For a comparison among the spectra representing
the profile and the absorption of bands in almost the various neoplasia, some spectral windows resulted rel-
entire spectral region, except for the position of AI and evant: (i) the ratios between methyl and methylene
AII (found 9 and 7 cm21 to lower frequencies), for the vibrational modes the position; (ii) the profile and the
peak intensity ratio of CH2 and CH3 at 2,960/2,854 and peak ratio of Amide I and II; (iii) the presence of the
1,452/1,397 cm21 and for a red shift of 6 cm21 of DNA band at 1,171 cm21; (iv) the shape of the band centered
band at 970 cm21 (Fig. 12 and Table 2). at about 1,080 cm21 and its intensity ratio with the one
Also in D tissues (polymorphous low-grade adenocar- at 1,240 cm21; (v) the intensity of the band at 970 cm21
cinoma), HCA and PCA (not reported) showed three (Fig. 12). The comparison of spectral with histological
clusters assigned to the cancer, to the connective and to classification resulted more than satisfactory.
the cancer with a significant presence of mucus. With To further verify the reliability of our classification,
respect to the solid SK, the spectrum of the cancer (see all the spectra of SK were mixed with those representa-
ahead, Fig. 10), showed different 2,960/2,854. AI/AII tive of the other oral cavity tumors, always isolated
and 1,453/1,398 intensity ratios (Table 2). from the corresponding clusters, obtaining a new HCA
In addition, Amide bands appeared broad for the in the spectral region (Fig. 13).
presence of components bands to lower frequencies. From the dendogram, the various classes of neopla-
The presence of mucine is argued by the bands found sia resulted well grouped and in a conceivable
at 1,376, 1,317, 1,120, 1,072, 1,035 cm21. The DNA sequence. The nearness of D with A subclusters is not
band at 970 cm21 is practically absent (Fig. 12) (Lasch strange due, as also stated by pathologists, to the anal-
et al., 2002b, 2004). ogous heterogeneity of the two displasia. The existence
All the sections from patients with an adenoid cystic of a unique subcluster for CK and CK0 is in favor of a
tumor (E) resulted constituted by muscular, connec- very smooth difference in biochemical composition and,
tive, necrotic, lipidic, and tumoral components (multi- probably, of a prevalent incidence of the morphological
variate analysis not reported). The representative assembly to the spectral profile. Owing to the similar-
spectrum of the tumor resulted identical to the one of ity of the representative spectra, the grouping of E
the solid tumor, SK, with some minor dissimilarities with B is also feasible.
mainly for the lower intensity of phosphate bands of
nucleic acids (1,240, 1,080 e 970 cm21) probably due to
the presence of cells with a lower mitotic activity (Fig.
12) (Lasch et al., 2002a; Tobin et al., 2004). CONCLUSIONS
To point out spectral differences between eosino- The results from salivary gland specimens, pre-
philic and ialine connective tissue, produced during sented here, once again demonstrate the capability of
carcinogenesis, high-resolution spectra (6.25 3 6.25 infrared microspectroscopy imaging, in combination
lm2) were taken into consideration (Fig. 10). Contrary with multivariate data analysis, to point out even
to that found in eosinophile connective tissue, analo- subtle biochemical and morphological changes, distin-
gies with the tumoral spectrum were found in ialine guishing various kinds and grades of neoplasia in tis-
connective tissue spectrum: a-helix prevalent on b- sues from a specific region of the human body. To study
Fig. 10. Comparison between representative spectra in the region 1,800–900 cm21 of neoplastic and
connective zones of E tissue (adenoid cystic carcinoma) with the healthy tissue F. [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 11. (a) Microphotograph of a section of B tissue (oral displas- biological systems, a rapid images acquisition at a
tic epithelium) where the displastic (blue panel) from the healthy
(black panel) epithelium can be distinguished and (b) their represen-
high-spatial resolution is requested that can be fully
tative spectra. [Color figure can be viewed in the online issue, which satisfied with the introduction of new conventional
is available at www.interscience.wiley.com.] (thermal) light sources. For example, Spotlight system