Utilising non-consensus pathology measurements to improve the diagnosis of oesophageal cancer using a Raman spectroscopic probe.

The application of semi-supervised methodology to improve the classification performance of a Raman spectroscopic probe for the diagnosis of oesophageal cancer is described. It is well known that gold standard histopathology diagnosis can be highly subjective, particularly for diseases which have several stages, such as cancer. A 'consensus' pathology decision can be obtained to ensure a robust gold standard by obtaining a diagnosis from several experts and samples are then only included in standard classification models if they have been assigned the same pathology by all experts. This can result in a significant number of samples that are excluded from the analysis as no consensus was reached. In this work semi-supervised methodology was used to extend Principal Component Analysis followed by Linear Discriminant Analysis (PCA-LDA) to incorporate samples without consensus pathology when discriminating between benign and oesophageal cancer specimens measured using a Raman endoscopic probe ex vivo. We demonstrate that a fully semi-supervised approach improved sensitivity and specificity from 73% and 78% (PCA-LDA) to 78% and 84% (semi-supervised) for discriminating between intestinal metaplasia and dysplasia and from 44% and 66% (PCA-LDA) to 63% and 72% (semi-supervised) when discriminating between intestinal metaplasia and low grade dysplasia.

Rapid endoscopic identification and destruction of degenerating Barrett's mucosal neoplasia.

There are distinct challenges implicit to the development of minimally invasive endoscopic surgery for the eradication of early neoplasia in Barrett's oesophagus. Endoscopic resection and ablation of high-grade dysplasia and mucosal cancer offer alternative therapeutic options to those unsuitable or unwilling to contemplate radical surgical excision. It may also become the treatment of choice in the future. Technological developments enable the instantaneous and non-invasive diagnosis of microscopic tissue abnormalities in vivo. This is made possible by improving the level of information that can be obtained from the tissue. As well as the two-dimensional surface morphology image, which the traditional endoscope can view, we have used new techniques to enable structure at depth, using Optical Coherence Tomography, to be imaged in high resolution. Other advances, using Raman spectroscopy, enable the early endoscopic detection of biochemical and molecular changes in tissue that precede any changes in morphology, thus enabling earlier diagnosis of tissue abnormalities. This King James IV lecture details our recent work, to develop advanced imaging for the diagnosis of malignancy and pre-malignancy. After detection endoscopic photodynamic therapy and endoscopic mucosal resection can provide eradication of mucosal neoplasia. Following photodynamic therapy there was complete eradication of all high-grade dysplasia and intramucosal carcinoma in 40 of 42 patients with a maximum endoscopic follow-up period of 72 months. Following endoscopic resection of 95 patients, the mean survival for intramucosal adenocarcinoma and high-grade dysplasia was 40.6 and 60.8 months respectively.

Evaluation of Raman probe for oesophageal cancer diagnostics.

Early detection of (pre-)cancerous changes improves prognosis, therefore in the UK patients at high risk of developing gastrointestinal cancers are enrolled on endoscopic surveillance programmes or the Bowel Cancer Screening Programme. The current gold standard technique for the detection of pre-cancerous changes in the gastrointestinal tract is histopathological analysis of biopsy tissue collected at endoscopy. This relies upon subjective assessment of morphological changes within the excised tissue samples and poor targeting of pre-malignant lesions. Raman spectroscopy offers a number of potential advantages for in vivo assessment of tissue at endoscopy. The performance of a custom built Raman probe as a biopsy targeting tool has been evaluated using excised biopsy material. Multivariate classification models have been used to demonstrate the likely ability of a miniature, confocal, fibre optic Raman probe to be used as an optical biopsy tool at endoscopy to provide spectral information in clinically practicable timescales. This technique could facilitate improved targeting of excisional biopsy with associated clinical benefits.

Raman spectroscopy--a new method for the intra-operative assessment of axillary lymph nodes.

Sentinel Lymph Node Biopsy has become the standard surgical procedure for the sampling of axillary lymph nodes in breast cancer. Intra operative node assessment is currently not offered to the majority of patients but would allow definitive axillary surgery to take place immediately. This would confer benefits both to the patient and to the healthcare system. Our experimental study aims to demonstrate that a Raman spectroscopy probe device could overcome many of the disadvantages of current intra-operative analysis techniques. 38 axillary lymph nodes, 25 negative and 13 positive from 20 patients undergoing breast surgery for invasive breast cancer were assessed using a commercially available Raman spectroscopy probe. Spectra were assessed using principal component fed linear discriminant analysis trained by the histopathology results. Leave one node out cross validation achieved a sensitivity of up to 92% and a specificity of up to 100% in differentiating between normal and metastatic lymph nodes.

Vibrational spectroscopy: a clinical tool for cancer diagnostics.

Vibrational spectroscopy techniques have demonstrated potential to provide non-destructive, rapid, clinically relevant diagnostic information. Early detection is the most important factor in the prevention of cancer. Raman and infrared spectroscopy enable the biochemical signatures from biological tissues to be extracted and analysed. In conjunction with advanced chemometrics such measurements can contribute to the diagnostic assessment of biological material. This paper also illustrates the complementary advantage of using Raman and FTIR spectroscopy technologies together. Clinical requirements are increasingly met by technological developments which show promise to become a clinical reality. This review summarises recent advances in vibrational spectroscopy and their impact on the diagnosis of cancer.

Detecting temporal and spatial effects of epithelial cancers with Raman spectroscopy.

Epithelial cancers, including those of the skin and cervix, are the most common type of cancers in humans. Many recent studies have attempted to use Raman spectroscopy to diagnose these cancers. In this paper, Raman spectral markers related to the temporal and spatial effects of cervical and skin cancers are examined through four separate but related studies. Results from a clinical cervix study show that previous disease has a significant effect on the Raman signatures of the cervix, which allow for near 100% classification for discriminating previous disease versus a true normal. A Raman microspectroscopy study showed that Raman can detect changes due to adjacent regions of dysplasia or HPV that cannot be detected histologically, while a clinical skin study showed that Raman spectra may be detecting malignancy associated changes in tissues surrounding nonmelanoma skin cancers. Finally, results of an organotypic raft culture study provided support for both the skin and the in vitro cervix results. These studies add to the growing body of evidence that optical spectroscopy, in this case Raman spectral markers, can be used to detect subtle temporal and spatial effects in tissue near cancerous sites that go otherwise undetected by conventional histology.

Miniature standoff Raman probe for neurosurgical applications.

Removal of intrinsic brain tumors is a delicate process, where a high degree of specificity is required to remove all of the tumor tissue without damaging healthy brain. The accuracy of this process can be greatly enhanced by intraoperative guidance. Optical biopsies using Raman spectroscopy are a minimally invasive and lower-cost alternative to current guidance methods. A miniature Raman probe for performing optical biopsies of human brain tissue is presented. The probe allows sampling inside a conventional stereotactic brain biopsy system: a needle of length 200 mm and inner diameter of 1.8 mm. By employing a miniature stand-off Raman design, the probe removes the need for any additional components to be inserted into the brain. Additionally, the probe achieves a very low internal silica background while maintaining good collection of Raman signal. To illustrate this, the probe is compared with a Raman probe that uses a pair of optical fibers for collection. The miniature stand-off Raman probe is shown to collect a comparable number of Raman scattered photons, but the Raman signal to background ratio is improved by a factor of five at Raman shifts below ∼500 cm(−1). The probe's suitability for use on tissue is demonstrated by discriminating between different types of healthy porcine brain tissue.

Advances in the clinical application of Raman spectroscopy for cancer diagnostics.

Light interacts with tissue in a number of ways including, elastic and inelastic scattering, reflection and absorption, leading to fluorescence and phosphorescence. These interactions can be used to measure abnormal changes in tissue. Initial optical biopsy systems have potential to be used as an adjunct to current investigative techniques to improve the targeting of blind biopsy. Future prospects with molecular-specific techniques may enable objective optical detection providing a real-time, highly sensitive and specific measurement of the histological state of the tissue. Raman spectroscopy has the potential to identify markers associated with malignant change and could be used as diagnostic tool for the early detection of precancerous and cancerous lesions in vivo. The clinical requirements for an objective, non-invasive, real-time probe for the accurate and repeatable measurement of pathological state of the tissue are overwhelming. This paper discusses some of the recent advances in the field.

Exploiting the diagnostic potential of biomolecular fingerprinting with vibrational spectroscopy.

There is immense clinical need for techniques that can detect the biochemical changes associated with pre-malignancy. The ideal diagnostic test would provide rapid, non-invasive diagnosis at the point of care with high throughput and without prior tissue processing. Over the past decade vibrational spectroscopy techniques have demonstrated their ability to provide non-destructive, rapid, clinically relevant diagnostic information. Biochemical fingerprints of tissues measured using Raman and infrared spectroscopy analysed in conjunction with advanced chemometrics have shown great potential in the diagnostic assessment of biological material. Development of Raman probes is enabling the potential of in vivo clinical measurements to be realised. A novel probe design has been evaluated in clinical studies to identify and classify the subtle pre-malignant biochemical changes related to the carcinogenesis process. Exciting recent developments have enabled the probing of tissue samples at depth with huge potential for breast and prostate cancer diagnostics. Furthermore, the potential of vibrational spectroscopy to provide prognostic information is tantalising. Raman spectral data acquired on oesophageal biopsy samples analysed in conjunction with patient outcome data has shown the power of spectral biomolecular fingerprinting in predicting the outcome of patients with high-grade dysplasia in Barrett's oesophagus. Raman mapping can also be used to analyse thin tissue sections on calcium fluoride slides enabling the distribution of tissue constituents to be realised. The spectral data acquired effectively enables multiplexing of digital tissue stains since a whole array of information is gathered simultaneously. Technological developments are bringing the technologies closer to the clinical reality of spectral pathology and high-throughput non-destructive measurement with high resolution.

Raman spectroscopy: a potential tool for early objective diagnosis of neoplasia in the oesophagus.

There is a profound clinical need for a diagnostic tool that will enable clinicians to identify early neoplastic change in the oesophagus. Raman Spectroscopy (RS) has demonstrated the potential to provide non-invasive, rapid, objective diagnosis of endoscopically invisible precancerous oesophageal dysplasia in vitro. RS analyses biological material to identify highly specific biochemical information that can be used to influence clinical care. Raman spectroscopic mapping could provide automated assessment of tissue biopsies to aid histopathological diagnosis in vitro. Furthermore, the recent development of fibre-optic Raman probes has enabled endoscopic assessment of oesophageal mucosa in vivo. Accurate identification of dysplasia will enable targeted endoscopic resection of early lesions preventing the development of oesophageal cancer. This review summarises the development of Raman systems for use as laboratory based analytical adjuncts and endoscopic diagnostic tools in the distal oesophagus.

Evaluation of linear discriminant analysis for automated Raman histological mapping of esophageal high-grade dysplasia.

Rapid Raman mapping has the potential to be used for automated histopathology diagnosis, providing an adjunct technique to histology diagnosis. The aim of this work is to evaluate the feasibility of automated and objective pathology classification of Raman maps using linear discriminant analysis. Raman maps of esophageal tissue sections are acquired. Principal component (PC)-fed linear discriminant analysis (LDA) is carried out using subsets of the Raman map data (6483 spectra). An overall (validated) training classification model performance of 97.7% (sensitivity 95.0 to 100% and specificity 98.6 to 100%) is obtained. The remainder of the map spectra (131,672 spectra) are projected onto the classification model resulting in Raman images, demonstrating good correlation with contiguous hematoxylin and eosin (HE) sections. Initial results suggest that LDA has the potential to automate pathology diagnosis of esophageal Raman images, but since the classification of test spectra is forced into existing training groups, further work is required to optimize the training model. A small pixel size is advantageous for developing the training datasets using mapping data, despite lengthy mapping times, due to additional morphological information gained, and could facilitate differentiation of further tissue groups, such as the basal cells∕lamina propria, in the future, but larger pixels sizes (and faster mapping) may be more feasible for clinical application.

The potential for histological screening using a combination of rapid Raman mapping and principal component analysis.

Rapid Raman mapping was carried out on 20 microm sections of oesophageal biopsy samples. Contiguous 7 microm sections were stained with haematoxylin and eosin (H&E) with histopathology provided by an expert pathologist. The step size and acquisition times were varied and the resulting spectra, principal component (PC) score maps and loads were compared. Overall mapping times were also compared to traditional Raman point mapping. The principal component loads for each of the maps were seen to be similar despite varying the acquisition time and number of spectra. Gross biochemical information was extracted showing good correlation with the H&E sections even for short overall mapping times (30-90 minutes for a 2 mm biopsy, 0.5 s acquisition time per 25.3 microm Raman pixel). This demonstrates that low signal to noise spectral maps are sufficient for the identification of histologically relevant biochemistry using principal component analysis as long as the spectral dataset is large enough.