New Approaches in Biomedical Spectroscopy - ACS Publications

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Applications of Fourier-Transform Infrared Imaging in Cancer Research Don McNaughton and Bayden R. Wood Centre for Biospectroscopy and School of Chemistry, Monash University, Clayton, Victoria 3800, Australia

False color images of cervical tissue sections, malignant glioma rat brain sections and melanoma sections have been constructed from FTIR hyperspectral data. The images are directly compared with stained sections and shown to be useful in distinguishing cervical pathology, malignant glioma and class I human lymphocyte antigen ( H L A ) expression in melanoma. Infrared spectra extracted from the images are shown to be useful for determining the macromolecular changes that accompany disease. Techniques for building three dimensional images to determine the extent of tissue change are described.

Introduction Over the last 10-15 years Fourier Transform Infra-Red (FTIR) spectroscopy and more particularly infrared micro-spectroscopy, has been investigated and shown to have a role in the diagnosis and understanding of a wide range of cancers, including colon, breast, liver, skin, lung and brain. A recent review details much of the progress to date and indicates the current breadth of the field (/), although a full review of the field is not yet available. Most of these studies have concentrated on single point spectroscopy, either in the laboratory with large apertures, or using synchrotron IR sources to approach diffraction limited resolution (2). For tissue samples point to point mapping has been used in an attempt to emulate and extend visible pathology. Although this methodology showed some promise, the time involved in obtaining sufficient data was

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© 2007 American Chemical Society

In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


15 prohibitive (5). However the advent of focal plane array (FPA) based imaging spectrometers (4) and other instruments based on linear arrays has provided instrumentation capable of rapidly obtaining IR hyper-spectral maps of thin tissue sections at close to diffraction limited resolution. These systems now provide the basis for the further development of infrared spectroscopy as a tool in the diagnosis of cancer and in following the effects of cancer progression and treatment. We outline here some recent work on the use of FPA imaging in cervical cancer diagnosis, brain cancer and in the investigation of H L A expression in melanoma. We also briefly describe the development of 3D imaging and its application to pathology. In this work all spectral images have been collected with a Varian Stingray imaging system in fast scan mode, using a M C T 64 χ 64 array and a 15x objective with a theoretical pixel resolution of 5.5 μπι. Unless otherwise stated spectra were collected at 6cm" resolution with 16 co-added scans. A number of strategies were used to achieve images of larger areas: A 2x magnifying objective was placed in the instrument to give a theoretical pixel resolution of 11 μπι; larger images were built by constructing tiled images from a number of individual tiles; for computer intensive data processing pixel aggregation was necessary to generate files that could be processed in real time. Tissue samples were formalin-fixed in paraffin blocks, sectioned at 4-5 μπι, deposited on "Kevley MirrlR low e microscope slides" and then washed in xylene to remove paraffin. Spectra were recorded in absorption/reflection. As we have shown in a previous publication (5) the spatial resolution using F P A technology with this methodology approaches 10 μπι rather than the diffraction limit. Spectral preprocessing and processing was carried out using mainly Cytospec (6), purpose built routines using Matlab for 2D image stitching and Scirun (7) for 3D imaging. 1

Once hyperspectral data blocks have been collected the data processing, using imaging programs such as Cytospec, can be divided into a number of phases. Firstly, poor quality spectra, resulting from too thin or thick sample areas, noisy pixels or gaseous water contamination, need to be removed. Secondly, a pre-processing routine consisting of baseline correction, or spectral derivativization and normalization is required to eliminate thickness effects and baseline variation. When using absorption-reflection slides at diffraction limited resolution, dispersion and scattering artifacts also become important and these must also be eliminated or minimized (8,9) in the preprocessing phase. The final phase is image construction. FTIR image data can be processed in a univariate mode where chemical maps (also called functional group maps) based on peak intensity, peak area or peak ratios can be routinely generated with the software supplied or using Cytospec. While these methods can provide information on the distribution and relative concentration of a particular functional group and hence a specific major biomolecule, they are not very useful in terms of classifying



16 anatomical and histopathological features within the tissue matrix and multivariate image reconstruction is required. Typical methodology includes unsupervised hierarchical cluster analysis (UHCA), K-means clustering, principal components analysis, linear discriminant analysis, artificial neural networks and fuzzy C-means clustering. These methods are aimed at classifying spectra based on similarity and thus are used to discern anatomical and histopathological features based on underlying differences in the macromolecular chemistry of the different cell and tissue types that constitute the sample. We and others have found U H C A to be the most useful technique for direct pathological comparison, although this severely restricts the size of images due to the computing overheads involved in calculating the distance matrices. Using a desk top PC with a Windows™ operating system the largest images that can be processed with U H C A are 128 χ 128, hence the need for the pixel aggregation mentioned above.

FTIR imaging for Cervical Cancer Pathology In the early 1990s Wong et al. (10,11) investigated the infrared spectra of exfoliated cervical cells and reported spectral differences between samples from patients diagnosed normal by cytology and those from patients diagnosed with dysplasia or cancer. Subsequent work by the Diem group and independently by McNaughton and co-workers indicated that the spectral changes observed may not be related to the number and molecular composition of dysplastic cells per se but other factors such as inflamation (12-13), the number of dividing versus non-dividing cells (14) as well as the cells overall divisional activity (75). The presence of biological components such as mucin, erythrocytes and leukocytes was also shown to obscure diagnostic regions of the spectra (12,13). Multivariate and artificial neural networks have been applied to the analysis of the spectra of exfoliated cells in an attempt to circumvent these confounding variables.(/6-20) Some of these techniques provide information on the important variables that may distinguish normal from diseased samples, but they are limited in that they do not provide information on the cervical cell types and their stage of differentiation and maturation within the cervix. Another factor that was shown to affect the analysis was the stage of the menstrual cycle at cell collection (21). In summary these studies demonstrated that a detailed understanding of the spectral features of the cell types and spectral variations resulting from differentiation, maturation, and cell cycle stages is a pre-requisite for the interpretation of the spectral differences between normal and dysplastic cytological diagnosed samples.


17 In this study we applied FTIR FPA imaging spectroscopy and unsupervised hierarchical clustering ( U H C A , D-values, Ward's algorithm) to investigate the tissue architecture of the cervical epithelium. U H C A was performed on the whole spectral region (1800 - 950 cm* ), the amide I region (1700-1570 cm* ) and the v^ym P 0 " region (1300-1200 cm" ) in order to ascertain which region or regions are appropriate for analysis. In the final step all spectra in a cluster are assigned the same color and the final image derived from a false color map. Such a map, using the whole spectral region, is presented in grayscale in figure 1 together with the mean 2 derivative spectra for each cluster and an adjacent haematoxylin and eosin (H&E) stained section of cervical epithelium. The mean spectrum of a cluster represents all spectra in a cluster and can be used for the interpretation of the chemical or biochemical differences between clusters. The number of clusters to be used is chosen by the user and we investigated from 2 8 clusters to determine the optimum number for each tissue sample. At five clusters most of the cell types apparent in the stained cervical epithelium tissue section, i.e. superficial, intermediate, parabasal, and connective tissue correlate well with the color coded clusters, although the small stromal inclusion of intermediate cells is not apparent in the parabasal region. At 8 clusters the narrow basal cell region is well defined, the small stromal inclusion is apparent and a region between intermediate and parabasal is apparent as a separate cluster. Results using just the amide I region are similar to those using the whole spectrum but the basal layer and the small inclusion are apparent at lower cluster numbers. There is also further differentiation of the connective tissue apparent at high cluster numbers. Given that only 5 or 6 major cell types are expected in such a normal sample this is what one would expect. Thus by minimizing the spectral region for analysis and using fewer clusters no useful information has been lost. The inclusion of further clusters in the analysis resulted in differentiation based mainly on baseline variation and artifacts introduced by the mosaic nature of the images. Investigation of the nucleic acid spectral region in the same fashion was not as useful. Differentiation of all but the three major cell types was lost even at cluster numbers > 3. This is due to overlying absorptions from glycogen, the amount of which varies with non-specific disease and confounds analysis. The major spectral differences observed in the mean spectra are: changes in the bands due to protein where both spectral shifts, intensity changes and broadening are apparent in amide I; nucleic acids, where the v P0 * intensity shows great variation; glycogen, where the bands vary markedly, possibly due more to baseline effects than to real spectral changes. These changes are as expected and the mean spectra from the analysis using only the protein region are very similar, indicating that using a reduced spectral region and consequently less computer time, gives almost identical discrimination. B y selecting the spectral window for data processing we show that the combination of F P A 1

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Figure 1. Cervical epithelium section (a) H&E stained section showing the five major cellular tissue types; (b) UHCA image using 5 clusters and (c) Mean spectra from each cluster in the image.


19 technology and U H C A is a rapid non-subjective analytical tool for the identification of anatomical features in cervical tissue. The same type of analysis can be carried out on a diseased tissue section (22) and Figure 2A and B , depict a H & E stained section from a patient diagnosed with high-grade dysplasia showing a potential metastatic inclusion within the glandular endometrium, whilst 2C and D are U H C A images of the outlined areas. The spectra of this particular section were taken by point to point mapping with a 20 χ 20 μιη aperture size chosen to allow sufficient S/N for further analysis within a reasonable timeframe (5.5hr). From these spectra an image of the ca 1200 χ 1200 / i m section along with another image recorded over a 300 χ 300 μη have been constructed for comparison with the H & E stained section. Even though the images are in gray scale the correlation of the cell types and dysplastic regions within the stained section and the cluster maps is easily apparent. The mean extracted spectra for each of the clusters shown in Figure 2E display a broad range of differences across the spectrum, indicating significant molecular differences between the different cell types. One spectrum shows bands indicative of collagen at 1229, 1239 and 1250 cm" while the other spectra seem to be relatively devoid of collagen but show an increase in the symmetric and asymmetric phosphate modes indicative of nucleic acids. In particular the cells associated with the cancer cells have quite pronounced v ym(P0 ") and v y (P0 ") at 1081 and 1240 cm" wavenumber values, respectively. With the glycogen region precluded from the analysis as described above, correlation with the major anatomical and histopathological features observed in the H & E stained section and the 5 clusters is excellent. For these samples the overlying "confounding variables" such as regions of leukocytes, erythrocytes and the underlying connective tissue appear as distinct clusters in the image. 2


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Although the use of an F P A FTIR imaging system reduces the time taken for obtaining images to a matter of minutes, the U H C A takes considerable processing time, as mentioned above, and becomes the limiting step in analysis. To overcome this we have explored A N N imaging as a means of reducing this time. We have built a database of spectra from each cell type and this is used to train an A N N . This has greatly decreased the processing time and produces results to date that are as good as the U H C A images presented above.

FTIR imaging of glioblastoma multiforme in rat brain Brain tissue and tumors have rarely been studied using FTIR, although Salzer et al (23) have recently described the use of FTIR mapping as a basis for the classification of human astrocytic gliomas. Glioblastoma multiforme (GBM) is a highly malignant human brain tumor and numerous clinical studies and



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In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007. 1 600

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Figure 2. A) H&E stained section of a high grade dysplastic lesion. B) "Blow up " ofpotential micro-metastacy. C) UHCA map generatedfrom spectra recorded over the area shown in A. D) UHCA map generatedfrom spectra recorded over area shown in Β. E) Mean extracted spectra from UHCA map D. (Reproducedfrom reference 22)

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Figure 3. Coronal H&E stained sections of (a) healthy rat brain and (b) with malignant glioma, FC: site of Falx cerebri (interhemisphericalfissure), AC: anterior commissure, CC: corpus callosum; LV: lateral ventricle; SN: septal nucleus, R: right hemisphere; L: left hemisphere. Chemimage (amide I) of (c) healthy and (d) malignant. UHCA (6 cluster) image of (e) healthy and (f) malignant, (adaptedfrom reference 24)


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23 animal experiments are under way with the goal of understanding tumor biology and developing potential therapeutic approaches. C6 cell glioma, a commercially available cell line, in the adult rat is a frequently used animal model for the malignant human glial tumor. By combining standard analytical methods such as histology and immunohistochemistry with FTIR microspectroscopy and imaging, we have explored a tumor diagnostic which allows us to obtain information about structure and composition of tumor tissues that is not easily obtained with either method alone (24). Two adult Wistar rats, one serving as a host for the brain tumor and one as an age matched control, were used. The H & E stained sections in figure 3 show all the easily identifiable anatomical structures and these are labeled on the healthy tissue image. In the tissue of the tumor bearing rat the tumor growth is easily seen and the brain morphology is distinctly distorted. Figures 3c and d show chemimages constructed from the integrated intensity of the amide I band (1680-1620cm" ). The good correlation between these chemimages and the stained sections show that the protein concentration alone is useful for delineating anatomical features. By using two clusters in the U H C A , distinction between the tumor and normal tissue was easily apparent, whilst for the healthy tissue the two clusters essentially identified white and grey matter as separate clusters. The white matter spectra showed increased intensity for protein bands and phosphodiester bands (1235 and 1078 cm" ). Figures 3e and f show images constructed from six clusters. For the healthy tissue the anatomical structures are well delineated and the U H C A is able to isolate the spectra of each cell type with much of the difference attributed to the degree of myelination present in the different brain structures. At larger cluster numbers (8 clusters) the U H C A also differentiates layers in the brain cortex which correspond well with the known Brodmann anatomical layers of the neocortex. Much less structural differentiation is apparent in the tumor tissue, although there is distinct clustering of tumor/non tumor tissue. 1

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FTIR imaging for detection of HLA class I expression in Melanoma tumors. Human Lymphocyte Antigen ( H L A ) class I molecules are ubiquitously expressed on the surface of tissues and have a major role in both immunosurveillance and tumor immunity. Tumor cells decrease the expression of H L A class I molecules (termed downregulation) in order to avoid eradication by the immune system (25). This downregulation of H L A class I molecules renders Τ cell- based immunotherapies ineffective because they rely on H L A class I molecules and as a result, downregulation of H L A class I molecules is also found to be associated with poor prognosis (26). Normally H L A class I



24 molecule expression on the surface of melanoma tissues is identified using immunofluorescence and appropriate H L A type I monoclonal antibodies (27). This is a time consuming and costly methodology. We have recently investigated the use of FTIR imaging as an alternative method of determining H L A class I expression on the surface of melanoma samples (28). A grey scale color image of a metastatic melanoma tumor section immunohistochemically stained with H L A class I heavy chain (HC-10) antibody is presented in figure 4a, whilst figure 4b contains a U H C A IR image of an adjacent section. Figure 4b shows there is a separation between H L A non-expressing (light cluster) and H L A expressing (dark cluster) cells which correlates well with the immunochemically stained sections. This excellent correlation was shown in all sections examined in tissue sections originating from a number of different patients.

Figure 4. Digital photograph and 2-D false colour cluster map (grey scale) of consecutive tissue sections obtainedfrom a metastatic melanoma tumour dissectedfrom the lymph node (a) Immunohistochemistry staining of tumorous tissue section using primary antibody specific for free HLA class I heavy chain (HC-10); (b) Cluster image of adjacent tumour tissue section with the tissue edge pixels removed, (adapted from reference 28)

From the cluster average spectra representing H L A expressing and nonexpressing cells there are consistent characteristic spectral differences. The amide I band of the spectra of H L A expressing cells is more intense and spectra from non-expressing cells show a small shift of this band to a lower wavenumber value, indicating differences in protein conformation. The area under the amide I peak is significantly different (by t-test) between expressing/non expressing cells and this could be used in identifying H L A positive and negative areas on tissues. Bands at ca 1400 and 1450cm" , attributed to methyl and methylene deformation modes associated with amino acid side chains also show lower absorbance in the spectra of expressing cells as do the asymmetrical and symmetrical phosphate stretching modes at 1244 and 1080cm" respectively. The latter difference may 1

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25 be attributable to higher concentrations of R N A and D N A in the expressing cells. In summary FTIR imaging enables the distinction of H L A class I positive areas from class I negative areas in melanoma tissue in an accurate, rapid and cost effective manner without the use of antibodies. However, more work is required to determine i f the observed changes between class I expressing and non-expressing cells is the direct result of the FTIR technique detecting differences in molecules on the cell surface, or alternatively changes in the underlying molecular architecture responsible for the expression of these molecules.

2D and 3D imaging of tissue There are other areas where FTIR imaging can be extended to provide further useful tools in cancer research. Using a MATLAB® routine developed by our group, four FTIR images of adjacent sections were stitched together side by side to give a single large 2D image frame. Spectra that passed a quality test were converted to second derivative spectra using the Savitsky-Golay algorithm. U H C A (D-values, Ward's algorithm) was performed on second derivative spectra over the 1272-950 cm" spectral window. The resultant cluster map was then divided back into the 4 component 2D cluster maps, each map corresponding to one of the FTIR images. This is particularly useful for the comparison of adjacent sections where the same cell types are present or when sections have been subjected to different treatments and a direct comparison needs to be made. Moreover, the ability to generate and visualize 3D FTIR cluster maps provides a new avenue to assess variation in multiple tissue sections and to determine the extent of penetration of histopathological structures based on the underlying macromolecular structure of the diseased tissue. The thin sections (4 μπι) required for use with the Kevley slides are less than the thickness of a single cervical cell, consequently multiple sections enable the analysis of whole cells and also minimize the effects of orientation artifacts that can arise during tissue sectioning. Figure 5a shows a U H C A image of four adjacent sections of adenocarcinoma containing cervical tissue stitched together, allowing the penetration of histological features to be easily visualized. In general terms the clusters represent red blood cells embedded in the stromal matrix, stroma, lymphocyte exudates and glandular tissue indicative of adenocarcinoma. In tissue sections 3 and 4 there is an increase in the area of connective tissue relative to glandular tissue when compared to sections 1 and 2, indicating penetration of the glandular tissue into the connective layer. Mean spectra can be 1



Figure 5. (a) UHCA five cluster maps of four adjacent cervical sections with adenocarcinoma; (b) Chemimage (amide I intensity) block constructedfromfour adjacent sections of Monkey gut; (c) Sections through the block in (b).



27 extracted from the 3D image and compared to provide biochemical information and hence may be used for diagnostic purposes. Using our Matlab modules the sections can be stacked to provide a three dimensional block as shown in figure 5b for monkey gut tissue where the gut villi can easily be seen projecting into the tissue block. These blocks can then be cut vertically or horizontally to interrogate the tissue architecture as seen in figure 5c. Software products such as Scirunll (7) are also available to allow larger 3D blocks to be constructed and these powerful programs allow the user to manipulate the colors and make the blocks transparent. The SCIRunll software suite provides a graphical user interface for rapid development of "networks" of instruction routines for the stacking and rendering of the input data. For example the 4 cluster maps of figure 5b were "stacked" into a scalar volume field of numbered clusters from which 3D cluster maps were rendered. Using the A N N maps mentioned above to speed up processing the generation of these 3D blocks and visualization of pathological features within a block is possible within a reasonable time scale.

Acknowledgements We thank the National Health and Medical Research Council (NHMRC) of Australia for financial support and our many collaborators who made this work possible in particular Brian Tait, Sock Fern Chew, Keith Bambery, Corey Evans, Max Diem, Elizabeth Schultke and Sarah Rigley MacDonald. Dr Wood is funded by an Australian Synchrotron Research Program Fellowship Grant and a Monash Synchrotron Research Fellowship. M r Finlay Shanks is thanked for instrumental support and M r Clyde Riley (Royal Women's Hospital, Melbourne) for sectioning.

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