Geeta Shetty

Raman spectroscopy: elucidation of biochemical changes in carcinogenesis of oesophagus

Several techniques are under development to diagnose oesophageal adenocarcinoma at an earlier stage. We have demonstrated the potential of Raman spectroscopy, an optical diagnostic technique, for the identification and classification of malignant changes. However, there is no clear recognition of the biochemical changes that distinguish between the different stages of disease. Our aim is to understand these changes through Raman mapping studies. Raman spectral mapping was used to analyse 20-μm sections of tissue from 29 snap-frozen oesophageal biopsies. Contiguous haematoxylin and eosin sections were reviewed by a consultant pathologist. Principal component analysis was used to identify the major differences between the spectra across each map. Pseudocolour score maps were generated and the peaks of corresponding loads identified enabling visualisation of the biochemical changes associated with malignancy. Changes were noted in the distribution of DNA, glycogen, lipids and proteins. The mean spectra obtained from selected regions demonstrate increased levels of glycogen in the squamous area compared with increased DNA levels in the abnormal region. Raman spectroscopy is a highly sensitive and specific technique for demonstration of biochemical changes in the carcinogenesis of Barretts oesophagus. There is potential for in vivo application for real-time endoscopic optical diagnosis.

Raman spectroscopic biochemical mapping of tissues

Advances in technologies have brought us closer to routine spectroscopic diagnosis of early malignant disease. However, there is still a poor understanding of the carcinogenesis process. For example it is not known whether many cancers follow a logical sequence from dysplasia, to carcinoma in situ, to invasion. Biochemical tissue changes, triggered by genetic mutations, precede morphological and structural changes. These can be probed using Raman or FTIR microspectroscopy and the spectra analysed for biochemical constituents. Local microscopic distribution of various constituents can then be visualised. Raman mapping has been performed on a number of tissues including oesophagus, breast, bladder and prostate. The biochemical constituents have been calculated at each point using basis spectra and least squares analysis. The residual of the least squares fit indicates any unfit spectral components. The biochemical distribution will be compared with the defined histopathological boundaries. The distribution of nucleic acids, glycogen, actin, collagen I, III, IV, lipids and others appear to follow expected patterns.

CHAPTER 8:The Prospects for Real‐Time Raman Spectroscopy for Oesophageal Neoplasia

Raman spectroscopy can provide exquisite sensitivity for molecular analysis of degenerating pre malignant changes in the oesophagus. We are interested in the prospect of rapid endoscopic diagnosis during endoscopy using Raman. The early changes of dysplasia are invisible to white‐light inspection. If detected, the dysplastic area can then be immediately removed and the surrounding area treated. The problem has been that histopathological classification, which is very challenging in these very early abnormalities. We have built prognostic models to classify these areas of abnormality and related the spectral classification to the patient prognosis and development of invasive cancer. The hypothesis is that the Raman signature allows biochemical detection at a biochemical and molecular level prior to morphological changes within the tissue. It is becoming clear that the dependence on the histological appearance of cells to establish a diagnosis of these early changes is subject to great variation and can be highly subjective. In addition, pathological analysis of tissue is very time consuming, expensive, and requires tissue biopsy. Kerkhof et al. demonstrated a poor level of interobserver agreement between expert histopathologists (K=0.58) in the grading of low‐grade (LGD) and high‐grade (HGD) oesophageal dysplasia. This distinction has vital consequences for patient management as LGD can be monitored by serial surveillance endoscopy, whereas HGD necessitates early endoscopic therapy or even surgery, and carries a significant risk of malignant progression. As well as being difficult to classify histologically, dysplasia can be extremely difficult or even impossible to recognise at endoscopy. Raman spectroscopy (RS) could remove the subjectivity from the histopathological assessment by measuring precise biochemical information about the target tissue. A Raman fibre‐optic probe could also enable real‐time diagnosis facilitating immediate treatment of suspicious areas of tissue during endoscopy, and could be used as a surgical adjunct.