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

Abstract

Background and aims: DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barrett's 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 mum 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.

Figure 1

Histograms by FC and ICDA (left to right). (A) Aneuploid case by FC and ICDA. (B) Discordant case, near-diploid aneuploid by FC and diploid by ICDA. Note that both analysis methods show evidence of separate G₂ peaks. (C) Discordant case, diploid by FC and tetraploid by ICDA.

Figure 2

Histograms from a patient who relapsed to cancer at 24 months. (A) Aneuploid histogram before PDT with DI=1.7. (B) Persistent aneuploidy post-PDT with DI=1.7.

Figure 3

Histograms from a patient who was disease free at 42 months. (A) Aneuploid histogram before PDT with DI=1.8. (B) Diploid 4 months post-PDT.

Figure 4

Kaplan–Meier disease-free survival estimates according to DNA ploidy status at (A) 4 months, (B) 1 year and (C) at both time points post-ALA PDT. Blue=diploid post-PDT, red=DNA ploidy abnormalities post-PDT (The colour reproduction of this figure is available on the html full text version of the manuscript).