Simultaneous Analysis of Phenotype and Cytogenetics Using Imaging Flow Cytometry: Time to Teach Old Dogs New Tricks

The importance of cytogenetic abnormalities in clinical diagnosis and response has been well established in particular for hematological malignancies with the diagnostic tools being microscopy‐based karyotyping or fluorescence in situ hybridization (FISH). For chronic lymphocytic leukemia (CLL), the associated genetic abnormalities are heterogeneous with trisomy 12 (+12) among the most common ¹ and 17p deletions (del(17p)) among the most adverse prognostic parameters for response and survival in CLL ². Immunophenotypically, CLL cells are typically characterized by co‐expression of CD5, CD19, CD20, and CD23 with low expression of surface immunoglobulin, CD20 and CD79b compared to normal B cells and malignant clone‐specific kappa or lambda immunoglobulin light chain restriction ³. Following remission induction, flow cytometry plays an important role in the detection of minimal residual disease with sensitivity of detection in the range of 1 CLL cell in 10,000 leukocytes (0.01%) ³. In contrast, conventional slide‐based FISH applied to detect chromosomal abnormalities typically has a sensitivity of 5–7%, several orders of magnitude lower than that of flow cytometric phenotyping.

The development of suspension fluorescence hybridization approaches quantifiable by flow cytometry was first reported in the context of measuring telomere lengths ⁴. A drawback of this approach, however, particularly in the context of spot counting is that the number hybridization sites is being assessed only by fluorescence intensity on the premise that intensity is directly correlated with number of hybridization spots/sites. However, without specific fluorescence spatial information, this approach does not account for potential non‐specific reactions outside the nuclear area of the cell.

The development of imaging flow cytometers combined the high‐throughput capability of flow cytometry with the capability of microscopy to provide high image content information on each individual event acquired. This technology thus had the potential to perform image analysis on rare cell populations in statistically robust cell numbers in principle limited only by the number of events acquired. The application of imaging flow cytometry to detect aneuploidy using a FISH in suspension protocol ⁵ demonstrated that spot‐counting algorithms alone were insufficient to derive accurate results. The source of the inaccuracy was the occurrence of super‐imposed hybridization spots related to the 2D projection of the 3D cells in suspension, which could lead to underscoring of the spot counts. Additional artifacts were overscoring of spot counts due to disaggregation of hybridization spots. It was determined and validated that these artificial spot count results could be corrected by incorporating a spot intensity measurement with a true single hybridization spot having 50% of the intensity of a spot that resulted from two superimposed spots and that the total intensity of a true trisomy had 1.5‐fold the intensity of a true disomy ⁵. It was further demonstrated that sensitivity of detection of trisomy 8 in AML was as low as 0.5% ⁵. The first imaging flow cytometry application of FISH in suspension in CLL was published in 2017 in which the method was validated against conventional slide‐based FISH and laser scanning cytometry with excellent correlations between these methods ⁶.

The next major advancement in this area was the combination of FISH with immunophenotyping. This had been successfully achieved in the field of microscopy/imaging with the introduction of the so‐called Fluorescence Immunophenotyping, and interphase Cytogenetics as a Tool for the Investigation Of Neoplasms technique (FICTION) ⁷ which combined phenotyping with genotyping on paraffin sections and cytological specimens. In the field of flow cytometry, this combination was tested with limited success in the context of assessment of telomere lengths ⁸. The first report combining FISH with immunophenotyping using imaging cytometry was published by Fuller et al. ⁹ in which the method was validated on cell lines and healthy donor peripheral blood. The first application of the thus established method on clinical material was published by Hui et al ¹⁰ in the context of CLL. In this issue of Cytometry A (Page 521), the same group expands on their previously published work by including the analysis of del(17p) and introducing a novel analysis approach for the detection of loss or gain of hybridization spots by comparing the hybridization spot ratios of phenotypically normal and malignant cells. From a methodological perspective, the del(17p) analysis is significant because it demonstrates that with the current method, imaging flow cytometry is sufficiently sensitive to quantify the hybridization of a commercially available locus‐specific probe. Previously, it was reported that the hybridization signals from commercially available locus‐specific probes in ATM/TP53 CLL kits were too dim for detection by imaging flow cytometry, but that this could be improved upon by increasing the target hybridization size through the use of customized bacterial artificial chromosomes (BACs) contig FISH probes ¹¹.

The applied spot count ratio metric in the current Hui et al. study between phenotypically normal and malignant cells demonstrates that for the samples analyzed, ratios >1 correlate with gain of hybridization sites (due to +12) and ratios <1 correlate with loss of hybridization sites (due to del(17p)). There is now an opportunity to establish clinically relevant correlates using this metric with regards to detection of minimal residual disease, remission duration, and response in this disease. In addition to the expected detection of chromosomal abnormalities of +12 and del(17p), the authors demonstrate a high sensitivity of detection at levels below those attainable by conventional microscopy‐based FISH as well as, in agreement with Do et al. ¹¹, the discovery of intraclonal heterogeneity due to the ability to detect previously unrecognized or unappreciated cell populations. The clinical relevance of these newly detected populations will need to be established, but the availability of the new tool is enabling these types of studies.

The next challenge in this field will be the application to FISH approaches that rely on the exact co‐localization of hybridization sites, for example, for the detection of translocations or inversions. The aforementioned problem of distinguishing superimposed but spatially distinct hybridization spots from truly spatially co‐localized ones is a formidable problem that may only be solved through 3D imaging approaches such through the application of light‐sheet fluorescence imaging ¹² or the design of different hybridization probes such as the use of dual color break apart probes. Depending on the specific application, the latter will have to be critically validated as was recently demonstrated for the EML4‐ALK fusion ¹³.

It is important to re‐emphasize that for the immuno‐flowFISH method, the sensitivity of detection is primarily affected by the number of events acquired. In the present study, the acquisition was limited to 10,000–20,000 cells. Higher numbers of acquisition are feasible but are limited by the computational power of the analysis computers. It should be anticipated that with the continuing technological research developments in which we are able to evaluate increasingly more parameters on increasingly more cells, the developments in computational hardware and analysis software to support these advancements will stay in step or at least will be able to catch up.

The technological and methodological advancements present several challenges not only with regards to data management and analysis but also coming to grips with the realization that existing long relied upon analyses can be improved upon. The latter is the case with the development of the immuno‐flowFISH method. The combination of two significant diagnostic tools in hematological malignancies and the improved sensitivity of detection have the potential to lead to discoveries of new prognosticators of disease and response. The challenge will be to have these new tools be accepted in a well‐established field of diagnostics and to “teach old dogs new tricks.” The paper by Hui et al in this issue of Cytometry A together with the published body of work on FISH analysis by imaging flow cytometry should warrant further development of this application not only in the field of CLL but other hematological malignancies that can be evaluated by loss or gain of probe‐specific hybridization sites as well.