Flash Talk & Poster Presentation 32nd Lorne Cancer 2020

“Immuno-flowFISH”: Next-generation cytogenetic assessment provides unique insight into Chronic Lymphocytic Leukemia (#108)

Henry Hui 1 , Kathy Fuller 1 , Kathryn Clarke 1 2 , Jason Stanley 1 , Hun Chuah 1 3 , Chan Cheah 1 4 5 , Dejan Radeski 4 6 7 , Wendy Erber 1 6
  1. Translational Cancer Pathology Laboratory, School of Biomedical Sciences, The University of Western Australia, Crawley, WA, Australia
  2. Haemato-Oncology Diagnostic Service, Department of Haematology, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
  3. Department of Haematology , Royal Perth Hospital, Perth, WA, Australia
  4. Department of Haematology, Hollywood Private Hospital , Nedlands, WA, Australia
  5. Department of Haematology, Sir Charles Gairdner Hospital, Nedlands, WA, Australia
  6. PathWest Laboratory Medicine, Nedlands, WA, Australia
  7. Department of Haematology, Joondalup Health Campus, Joondalup, Perth, WA, Australia

Aim: “Immuno-flowFISH” is a world-first, award-winning automated imaging flow cytometry invention that integrates fluorescence in situ hybridisation (FISH) on immunophenotyped cells in suspension. We aimed to demonstrate the clinical utility of immuno-flowFISH for cytogenetic assessment in chronic lymphocytic leukaemia (CLL), specifically during disease monitoring post-therapy.

Methods: Peripheral blood mononuclear cells were isolated from the blood of CLL patients during routine diagnostic monitoring. Immunophenotyping was performed with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Double-stranded DNA was denatured and hybridised with chromosome 12 or 17 enumeration (CEP 12, CEP17) and 17p12 locus-specific FISH probes. Cells were analysed on the Amnis ImageStreamX Mark II to assess the number and percent FISH-positive CD5/CD19-positive CLL cells and the ratio of FISH spot counts for CLL cells to healthy internal control CD3/CD5-positive T cells (FISH “mean spot ratio”). 

Results: The protocol was initially established on healthy controls and cases of CLL (n = 60) with trisomy 12 (+12) or del(17p) and found to have 100% concordance with initial standard diagnostic FISH. A total of 10,000 – 100,000 cells (mean = 20,000) was analysed per case. This study identified two clinically significant cases of CLL at the single-cell level; one with minimal residual disease (0.13% +12 CLL cells) and an atypical, clonally evolved case harbouring CD5 negative cells with aberrant morphology, tetrasomy 17 and del(17p).

Conclusions: Immuno-flowFISH accurately detected both numerical (+12) and structural (del(17p)) chromosomal abnormalities in phenotypically identified CD5/CD19-positive CLL cells. Both imagery and quantitative data, including spot count ratio, were used in the analysis. The sensitivity is more than 100-fold greater than standard FISH due to the large number of cells analysed, and that they are identified by their immunophenotype. This invention was found to detect at least 1 abnormal cell in 1,000 normal cells, thereby demonstrating a clinical application for low-level disease monitoring (e.g. disease relapse) and early detection of sub-clonal heterogeneity/evolution to guide therapeutic decision-making.