Investigating a New Methodology for the Detection of Low Frequency 17p Deleted Clones in Chronic Lymphocytic Leukaemia (CLL): Impact on Prognostic Outcome and Therapeutic Resistance
Authors: Kuss Bryone J , Do Hoang Cuc , Lower Karen M. , Wannop Jasmine , Macardle Peta C 
Published: October 2019

Abstract
Cytogenetic analysis by standard FISH of malignant CLL lymphocytes has been identified as predictive of disease outcome. However CLL is likely to be a disease of many sub-clones which either evolve post chemo-immunotherapy or are present in very low frequency at the outset. In the contemporary therapeutic environment greater understanding of these drug resistant sub-clones is required to prevent inappropriate treatment options being employed to treatment naive patients. However, this can only be achieved by meaningful and reliable analysis of low frequency clones.
CLL patients with 17p (TP53 gene) deletion, have the poorest outcome with an overall survival of 2-3 years due to chemo-refractoriness and early relapse. Although TP53 gene deletions are infrequent in CLL at initial diagnosis (5%-10%), their frequency increases to 40%-50% in the relapsed refractory patients. Importantly, CLL patients having less than 5% 17p deleted cells by standard FISH are considered normal with good prognosis, while patients with high frequency 17p deleted nuclei have the worst outcomes. A significant number of patients have TP53 deleted clones of the order of 5-40% of total cell number in which we suspect chemotherapy will have a disadvantageous effect on the genetic profile but whom currently receive this treatment.
AIMS/METHODS: We are therefore exploring techniques to reliably quantify low frequency clones carrying 17p and other critical gene deletions using Flow FISH a flow cytometry-based method (Amnis Corp) capturing multiple images by Image Stream X (ISX). Extended Depth of Field (EDF) technology expands the depth of focus over the entire cell to enumerate nuclear FISH probe “spots” accurately. The "spot count/cell” program allows to stratify the number of “spots” which based on the intensity and size parameters counted for 10000 cells. Our current project requires development of unique probe sets with sufficient signal to allow reliable detection of single gene deletions with this technology.
Data obtained with centromeric probes allows separation of single chromosome deletion and separation of 1 spot vs. 2 spots on image analysis. RP11 BAC probes have been selected for the region of 17p loss and individually nick translated. Individual probes have been tested by standard FISH analysis for repetitive DNA and region specificity and then applied in multiplex to intensify signal. Orange channel fluoresce has been selected to eliminate background fluorescence from histones. Preliminary results have demonstrated satisfactory technical outcomes of signal over background for utilization of this approach.
Figure 1 17p probe analysed on IDEAS software, ISX (above). Figure 1. 17p probe analysed on IDEAS software, ISX (above).
Figure 2 17p13.1 region for probe selection (below) Figure 2. 17p13.1 region for probe selection (below)
Figure 3 CLL cells from CLLARC CLL5 with low frequency 17p deleted clones will be analysed for comparison of this approach over standard FISH analysis. Patient samples pre and post therapy are being evaluated before a larger pretreatment cohort is evaluated. Figure 3. CLL cells from CLLARC CLL5 with low frequency 17p deleted clones will be analysed for comparison of this approach over standard FISH analysis. Patient samples pre and post therapy are being evaluated before a larger pretreatment cohort is evaluated.

Disclosures
Kuss: Roche: Research Funding; Sanofi: Research Funding.