Over the last few years, IR absorption spectroscopy has grown up as a potential analytical method in tissue and cell studies for cancer diagnosis. The application could be twofold: as a tool to assess the presence or absence of malignant cells in biopsies; and as an aid to help pathologists to classify those cells that are suspicious but not diagnostic for cancer. The last application has the problem that in order to assess these cells pathologists would have already dealt with stained samples. Up to recently, IR microspectroscopy of tissue and cytology samples had been carried out using non stained samples. However, pilot work carried out at the IR microspectroscopy beamline at Diamond proved that it is possible to obtain IR spectra of good quality from already stained tissue samples. Staining caused mainly changes in the lipid region. This has clear important implications for future clinical applications.
|Figure 1. Bronchoscopy image of a lung tumour appearing as a white mass. The tumour has been circled for clarity .
The diagnosis of cancer is based, amongst other things, on obtaining a biopsy sample. However, it is crucial to obtain a good quality tissue or cytology sample that would allow clinicians to confirm or rule out the presence of cancer. This is not such a problem when patients present with big tumours that are easily amenable to biopsies. However, in the case of small tumours and centrally located in the body, the matter becomes more complicated. In the case of lung cancer, patients usually have a bronchoscopy. This technique entails introducing a tube through the windpipe down to the lung in order to obtain a tissue sample. Sometimes it is easy to see a lung tumour and obtain a tissue sample (Figure 1), in other cases this becomes more difficult. On the other hand, the lung biopsy might contain only a small number of cells that might be abnormal but the amount of material is not enough to reach the diagnosis of cancer (Figure 2). This means that, in these cases, patients have to undergo further biopsies, facing further risks and side effects, delayed treatments, and the costs have to be covered by the hospitals and the National Health Services (NHS). Therefore, it would be ideal to have a technique that could help clinicians in obtaining the diagnosis of cancer and, more importantly, to be able to (sub)classify the type of cancer from a very small number of cells. Such a technique could be Fourier Transform IR (FTIR) spectroscopy.
|Figure 2. Histopathological image of abnormal cells suspicious of lung cancer but not diagnostic. The abnormal cells have been circled for clarity .
Differently from conventional IR methods, the possibility of using synchrotron-based microFTIR spectroscopy allows us to measure spectra of single cells at both cellular and subcellular level  with an excellent signal to noise and in a short time. At this point, the clinical applications in the pathological diagnosis of cancer could be twofold. First, micro-FTIR spectroscopy could be used as a technique to screen tissue and/or cytology samples for the presence or absence of cancer cells. The second application in pathology is the further characterization a posteriori of those cells deemed abnormal by pathologists/cytologists and confirming whether or not these cells are malignant or even the subtype of cancer present. This application has however one drawback. These samples already studied by a pathologist/cytologist will have previously been stained with either haematoxylin and eosin (H&E) or Papanicolau (Pap) stains, each one of these are widely used in pathology departments. Pathologists require samples to be stained in order to better identify cells and tissue structure under the gold standard technique of visible microscopy (Figure 3). To our knowledge, the work carried at the beginning of December 2009 on B22, has charaterised B22 for the first time, the effects induced by staining in tissues . Figure 4 shows that the main differences revealed were a decrease in the amount of lipids due to the staining process [2,3]. These studies are the first step in setting the background for the standardization of IR microspectroscopy in this medical study, and hold promise toward a clinical application of IR fingerprinting of stained single cells .
|Figure 3. Microscopy image of lung cancer cells CALU-1 stained with Papanicolau. The red square is to shown the area probed by the IR beam on the sample.
The characterization of spectral biomarkers for the diagnosis of cancer will require a strong collaboration and multidisciplinary approach between spectroscopists and clinicians, amongst others. It is very important to carry out studies of a large number of samples from a large number of patients in order to increase the probabilities of finding these spectral markers. Multicentre collaborations will be required in order to include hundreds if not thousands of patients. Another crucial point is the standardization of both sample preparation and instrumentation. In other words, the same sample should give the same results in different centres using the same protocols and their IR instrumentation. Only when studies including large numbers of cases and standardization have been achieved will the medical community be more willing to take IR microspectroscopy directly into the clinical practice. The proof of concept is done, now we need to further characterise the IR spectral biomarkers with a deeper biological insight of the cellular processes, which include cross validation of the spectroscopical results with other biochemical methods applied on the samples.
|Figure 4. Representative FTIR spectra of a lung cancer cell in an already H&E stained lung tissue sample obtained at Diamond IR beamline B22. Spectrum is offset for clarity.
The Cancer Centre, UHNS Charitable Fund
 Dumas P., Sockalingum G. D., and Sulé-Suso J., Trends in Biotechnology, 25: 40-44; 2007.
 Pijanka J, et al. Spectroscopy, 24: 73-78; 2010.
 Pijanka J, et al. Lab Invest, 90: 797-807; 2010.
 Sulé-Suso J. and Cinque G. Microsc Anal, 140: 25-28; 2010.
 Moss, D. (Ed.). Biomedical Applications of Synchrotron Infrared Microscopy. RSC Publishers, Cambridge; 2010.
Principal Publications and Authors
Sulé-Suso J. and Cinque G. Infrared Microspectroscopy in Cancer Diagnosis. Do We Need Synchrotron Light?, Microsc Anal, 140: 25-28; 2010.
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