Gary D. Mackenzie

Elastic scattering spectroscopy accurately detects high grade dysplasia and cancer in Barrett's oesophagus

Background and aims: Endoscopic surveillance of Barrett’s oesophagus currently relies on multiple random biopsies. This approach is time consuming, has a poor diagnostic yield, and significant interobserver variability. Elastic scattering spectroscopy is a real time in vivo optical technique which detects changes in the physical properties of cells. The aim of this study was to assess the potential for elastic scattering to detect high grade dysplasia or cancer within Barrett’s oesophagus. Methods: Elastic scattering spectroscopy measurements collected in vivo were matched with histological specimens taken from identical sites within Barrett’s oesophagus. All biopsies were reviewed by three gastrointestinal pathologists and defined as either “low risk” (non-dysplastic or low grade dysplasia) or “high risk” (high grade dysplasia or cancer). Two different statistical approaches (leave one out and block validation) were used to validate the model. Results: A total of 181 matched biopsy sites from 81 patients, where histopathological consensus was reached, were analysed. There was good pathologist agreement in differentiating high grade dysplasia and cancer from other pathology (kappa = 0.72). Elastic scattering spectroscopy detected high risk sites with 92% sensitivity and 60% specificity and differentiated high risk sites from inflammation with a sensitivity and specificity of 79%. If used to target biopsies during endoscopy, the number of low risk biopsies taken would decrease by 60% with minimal loss of accuracy. A negative spectroscopy result would exclude high grade dysplasia or cancer with an accuracy of >99.5%. Conclusions: These preliminary results show that elastic scattering spectroscopy has the potential to target conventional biopsies in Barrett’s surveillance saving significant endoscopist and pathologist time with consequent financial savings. This technique now requires validation in prospective studies.

Image cytometry accurately detects DNA ploidy abnormalities and predicts late relapse to high-grade dysplasia and adenocarcinoma in Barrett's oesophagus following photodynamic therapy

Background and aims:DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barretts oesophagus, independent of histology grade. Image cytometric DNA analysis (ICDA) is an optical technique allowing visualisation of abnormal nuclei that may be undertaken on archival tissue. Our aim was to determine the accuracy of ICDA vs FC, and evaluate DNA ploidy as a prognostic biomarker after histologically successful treatment with photodynamic therapy (PDT).Methods:Nuclei were extracted from 40 μm sections of paraffin-embedded biopsies and processed for ICDA at UCL and FC at UW using standardised protocols. Subsequently, DNA ploidy was evaluated by ICDA on a cohort of 30 patients clear of dysplasia 1 year after aminolaevulinic acid PDT for high-grade dysplasia (HGD). The results were correlated with long-term outcome.Results:In the comparative study, 93% (41 out of 44) of cases were classified identically. Errors occurred in the near-diploid region by ICDA and the tetraploid region by FC. In the cohort study, there were 13 cases of late relapse (7 cancer, 6 HGD) and 17 patients who remained free of dysplasia after a mean follow-up of 44 months. Aneuploidy post-PDT was highly predictive for recurrent HGD or cancer with a hazard ratio of 8.2 (1.8–37.8) (log-rank P=0.001).Conclusions:ICDA is accurate for the detection of DNA ploidy abnormalities when compared with FC. After histologically successful PDT, patients with residual aneuploidy are significantly more likely to develop HGD or cancer than those who become diploid. DNA ploidy by ICDA is a valuable prognostic biomarker after ablative therapy.

Elastic scattering spectroscopy for detection of cancer risk in Barrett's esophagus: experimental and clinical validation of error removal by orthogonal subtraction for increasing accuracy

Elastic scattering spectroscopy (ESS) may be used to detect high-grade dysplasia (HGD) or cancer in Barretts esophagus (BE). When spectra are measured in vivo by a hand-held optical probe, variability among replicated spectra from the same site can hinder the development of a diagnostic model for cancer risk. An experiment was carried out on excised tissue to investigate how two potential sources of this variability, pressure and angle, influence spectral variability, and the results were compared with the variations observed in spectra collected in vivo from patients with Barretts esophagus. A statistical method called error removal by orthogonal subtraction (EROS) was applied to model and remove this measurement variability, which accounted for 96.6% of the variation in the spectra, from the in vivo data. Its removal allowed the construction of a diagnostic model with specificity improved from 67% to 82% (with sensitivity fixed at 90%). The improvement was maintained in predictions on an independent in vivo data set. EROS works well as an effective pretreatment for Barretts in vivo data by identifying measurement variability and ameliorating its effect. The procedure reduces the complexity and increases the accuracy and interpretability of the model for classification and detection of cancer risk in Barretts esophagus.

Radiofrequency ablation is effective for the treatment of high-grade dysplasia in Barrett's esophagus after failed photodynamic therapy.

Endoscopic radiofrequency ablation (RFA) is an effective treatment for high-grade dysplasia in Barretts esophagus in ablation-naïve patients, but no studies have evaluated its use in patients in whom ablative therapy has previously failed. We describe 14 patients with residual high-grade dysplasia following aminolevulinic acid or Photofrin (porfimer sodium) photodynamic therapy (PDT). An overall complete reversal of dysplasia was achieved in 86 % with a combination of RFA and rescue endoscopic mucosal resection. The median total follow-up is 19 months. The rate of strictures was 7 % (1/14) and there was a low rate of buried glands (0.5 % follow-up biopsies). These data suggest RFA is both safe and effective for eradication of high-grade dysplasia in patients in whom PDT has failed.

In-vivo detection of pre-cancerous changes in Barrett's esophagus using elastic scattering spectroscopy (ESS)

We present the results of a clinical study using ESS to detect dysplasia in the esophagus. We focus on the use of novel statistical techniques and the clinical benefits this technique provides.

A Randomised Controlled Trial of ALA V Photofrin PDT for High Grade Dysplasia in Barrett's Esophagus

Introduction Sodium porfimer photodynamic therapy (sp-PDT) is a licensed minimally invasive treatment for Barrett9s Oesophagus (BO) with high grade dysplasia (HGD). Complete reversal (CR)-HGD is 50% at 5 years. sp-PDT is associated with a significant risk of strictures and prolonged photosensitivity up to 3 months. 5 aminolaevulinic acid (ALA) is an alternative treatment with 36 h photosensitivity. Non-randomised data suggests 85% CR-HGD and low risk of side effects. The aim of this study was to compare the side effect profile between the drugs and outcomes of therapy. Methods Single centre randomised controlled trial. Presence of HGD was confirmed on two occasions by two specialist GI pathologists. All patients had Endoscopic Ultrasound and endoscopic resection of visible lesions prior to PDT. Stratification was by length of BO ( 6 cm) and extent of dysplasia. Standard protocols for ALA and sp-PDT were followed. Endoscopic follow-up with 2 cm four-quadrant biopsy was at 6 weeks, 4 months, then annually. All adverse event data were collected. Results Sixty-four patients were randomised, 34 ALA and 30 sp-PDT. Median follow-up is currently 23 months. Strictures and skin photosensitivity were significantly more common with sp-PDT than ALA-PDT (33% vs 9% and 43% vs 6% respectively) p 2 = 4.34, p=0.037. For BO length >6 cm there was no significant difference. Rate of buried glands was significantly higher post PDT (48%) than pre PDT (20%), no significant difference between groups. Conclusion ALA-PDT has a better risk profile than sp-PDT. In patients with BO length ≤6 cm, preliminary results show ALA-PDT is associated with significantly higher CR-HGD. In longer segments of BO neither PDT drug was sufficiently efficacious to warrant use.

249 DNA Ploidy As a Prognostic Biomarker After Ala Photodynamic Therapy for High Grade Dysplasia in Barrett's Oesophagus

Introduction: Photodynamic therapy (PDT) is an approved minimally invasive treatment for high grade dysplasia (HGD) in Barretts oesophagus (BE). Complete reversal of HGD at 1 year post therapy occurs in up to 90% of patients, but late relapse has been documented in 20%. DNA ploidy (aneuploidy/tetraploidy), as analysed by flow cytometry, is a strong predictor of future cancer risk in untreated Barretts oesophagus, independent of histology grade. Image cytometry is a simpler method that allows accurate evaluation of nuclear morphology on parrafin embedded samples. Aims: To determine whether residual aneuploidy after successful treatment with ALA-PDT predicts late relapse to HGD or cancer. Methods: 30 patients (80% male, mean age 66 years (range 45-86)) successfully treated with ALA PDT for HGD in BE between 1998-2006 were included in analysis. All patients had HGD confirmed by two specialist GI pathologists prior to treatment. Four quadrant biopsies were taken every 2cm from the treated segment at 2, 4, 12, 18 and 24 months after PDT, then annually. All were clear of HGD 1 year post PDT. DNA ploidy was analysed by image cytometry of monolayer preparations stained with Feulgen. 405 samples were analysed; 156 prior to PDT; 139 at 4 months post PDT; 110 at 1 year post PDT. Results: 25 patients had aneuploidy prior to treatment. 11/25 remained aneuploid post PDT and 9/11 have subsequently relapsed; 5 to invasive cancer and 4 HGD. The median time to relapse was 30 months (range 14-64). 14/25 patients reverted to diploid on their 4 month follow up and all remained diploid at 1 year. None have relapsed after median 44 month follow up (range 32-60 months) (Fishers exact p<0.001). 5 patients were diploid prior to treatment and 2/5 patients have relapsed, one to cancer (diploid throughout) and one to HGD (aneuploidy at 1 year). There was no significant difference in rates of aneuploidy within this small subset of patients. Conclusions: Following successful Photodynamic therapy for HGD in BE, patients that remain aneuploid are significantly more likely to develop HGD or cancer than those who become diploid. This may allow change in surveillance patterns to be tailored to the patient, with persistent aneuploidy one year post PDT necessitating closer follow up. Moreover, patients that have reverted to diploid after one year may return to 2 yearly follow up and be reassured that risk of cancer after 3 years is very low. These data demonstrate the utility of DNA ploidy as a prognostic biomarker following ablative therapy. This work is supported by the CRUK Experimental Cancer Medicine Centre at UCL and the NIHR Comprehensive Biomedical Research Centre at UCLH.

ALA PDT for high grade dysplasia in Barrett's oesophagus: review of a decade's experience

We have been investigating PDT with 5 aminolaevulinic acid (ALA) for the treatment of high grade dysplasia (HGD) in Barretts oesophagus (BO) for over a decade. This drug has inherent advantages over porfimer sodium (Photofrin), the current approved photosensitiser in the UK and USA, which causes strictures in 18-50% and light sensitivity for up to three months. ALA has a lower rate of oesophageal strictures due to its preferential activity in the mucosa, sparing the underlying muscle, and patients are only light sensitive for 1-2 days. Within a randomised controlled trial, we demonstrated that an ALA dose of 60mg/kg activated by 1000J/cm red laser light is the most effective. Using these values we achieved complete reversal of HGD at 1 year in 89% of 27 patients. A randomised controlled trial of ALA vs porfimer sodium PDT for HGD is currently under way with end points of efficacy and safety. 50 of 66 patients have been recruited. Preliminary data suggest ALA PDT is safer with a trend to higher efficacy. Late relapse can occur in 20% of patients. New prognostic markers, in particular aneuploidy, are helping us to identify and target patients at risk of late relapse. Furthermore optical biopsy techniques such as elastic scattering spectroscopy (ESS) may allow detection of nuclear abnormalities in vivo and enable us to target areas of interest whilst reducing sampling error. PDT faces new challenges for the treatment of HGD in BO, with the recent introduction of balloon based radiofrequency ablation. This technique appears simpler and as effective as PDT, but follow up is currently short and long term safety data is lacking. In our experience ALA PDT is currently the most effective minimally invasive treatment for HGD in BO. This work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Healths NIHR Biomedical Research Centres funding scheme.