A different kettle of FISH: the revolution of leukaemia testing

Scientists at the University of Western Australia have achieved the “impossible” (1) by combining morphology, immunophenotyping, and fluorescence in situ hybridisation (FISH), to create a single, novel method that promises to transform laboratory investigations for leukaemia. 

Figure 1 – Simplified schematic diagram of immuno-flowFISH

The ground-breaking invention, called “immuno-flowFISH”, uses an imaging flow cytometer to rapidly analyse more than 10,000 cells to detect chromosomal abnormalities in a chosen cell population [figure 1]. The method has been dubbed the “holy grail of leukaemia testing”(1), as it combines three established tests for leukaemia into a single automated, powerful platform with high sensitivity – which could potentially provide results for diagnosis, prognosis, and minimal residual disease monitoring in less than two days.

The potential for its clinical application was shown during method development by Hui et al. (2), through the evaluation of chromosome 12 in patients with chronic lymphocytic leukaemia (CLL). CLL is characterised by massive clonal proliferation of mature B-cells. Trisomy 12 (an additional copy of chromosome 12) is one of the most common chromosomal aberrations found in the disease [figure 2].

The World Health Organisation (WHO) has determined that a combination of clinical features, morphology, immunophenotyping, and genetics should be used to diagnose CLL. Cell immunophenotypes and chromosomal aberrations are identified respectively using flow cytometry (4) and FISH (5). 

In CLL, immunophenotyping often forms the basis of the diagnosis by identifying the abnormal presence of a B-cell population expressing the characteristic CLL markers (CD19+CD20+CD5+), while chromosomal aberrations are used to stratify patients into prognostic risk categories, according to the chromosomal defects present. Genetic mutations are present in over 80% of CLL cases, with certain mutations being associated with poor overall survival and treatment response.

Figure 2 – Chronic Lymphocytic Leukaemia (CLL). Risk stratification table adapted from Puiggros et al. (2014) (3)

Although trisomy 12 is one of the most common mutations in CLL, it is associated with an intermediate risk prognosis and does not change management or treatment beyond the standard of care – it was used as a “proof-of-principle” to illustrate the clinical application of the method during its development. 

More importantly – and perhaps more strikingly – following method development, Hui et al. then went on to investigate whether immuno-flowFISH could be used to accurately detect del(17p) (deletion of the short arm of chromosome 17) in CLL (6). Though the del(17p) mutation is much rarer than trisomy 12, its association with very poor prognosis, short survival, and relapse, means that its detection is critical for clinicians to provide appropriate risk-adapted treatment and management beyond the standard of care for patients. 

The investigation was a success. Both studies by Hui et al. using immuno-flowFISH showed 100% concordance for all chromosome 12 and 17p mutations, which were confirmed using standard FISH. Furthermore, the increased specificity and sensitivity from analysing tens of thousands of cells meant that even a low amount of del(17p) mutations could be detected, beyond the current capabilities of slide-based FISH. This means that this method could also be used for minimal residual disease monitoring, as well as to detect and monitor the clonal evolution of CLL, in which multiple neoplastic “sub-clones” arise due to different genetic mutations, or the acquisition of additional mutations over disease progression. This is not currently possible in CLL due to the limitations of FISH [figure 3] (7).

Figure 3 – Limitations of FISH; adapted from Hui (2018) (7)

In the UK, if a patient is suspected of having CLL, a peripheral blood sample is sent to a central laboratory that specialises in investigating haematological malignancies (sometimes called Haematological Malignancy Diagnostic Service or Haemato-Oncology Diagnostic Service). Immunophenotyping and FISH tests are carried out independently of each other – and results can take approximately a week to be reported back to the clinician, with FISH results often taking longer due to the intensive manual labour and specialist skills required for the method. 

Therefore, it comes as no surprise that the development of the immuno-flowFISH method is hugely exciting news in the world of leukaemia testing. There are many advantages over the gold-standard FISH method, including automation, increased sensitivity, and a quicker turnaround time [figure 4] (7). 

Figure 4 – Advantages and disadvantages of immuno-flowFISH; adapted from Hui (2018) (7)

The application of immuno-flowFISH is not just limited to CLL, however. Chromosomal aberrations and abnormal cell phenotypes are found in many other malignancies and disorders. Immuno-flowFISH could be extended to assess other abnormal chromosome numbers (e.g. trisomy 21 in Down Syndrome) and abnormal chromosome structures (e.g. BCR-ABL in chronic myeloid leukaemia and PML-RARA in acute promyelocytic leukaemia). Numerous antibody designs and probe combinations are possible, meaning immuno-flowFISH could even be used to evaluate abnormal chromosome numbers in solid tumours – in which tumour tissue-biopsied cells are analysed in suspension, which is already being done with standard flow cytometry.

There is still a long way to go before immuno-flowFISH can be introduced into routine clinical laboratories. Further studies are in progress for other leukaemias, including acute lymphoblastic leukaemia in children, and multiple myeloma. But there is no denying that the potential positive impact on patients, laboratories, clinicians, and the NHS could be incredible – ultimately improving outcomes and overall survival for our patients.

References

1. Hiatt B. Eureka for UWA scientists in cancer ‘holy grail’ news.com.au2018 [Available from: https://www.news.com.au/national/western-australia/eureka-for-uwa-scientists-in-cancer-holy-grail/news-story/668f83cda79451cef0d4b23a4ebf8d98.

2. Hui H, Fuller KA, Chuah H, Liang J, Sidiqi H, Radeski D, et al. Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia. Methods. 2018;134-135:32-40.

3. Puiggros A, Blanco G, Espinet B. Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go. Biomed Res Int. 2014;2014:435983.

4. mitedustar. Flow Cytometry Animation YouTube2015 [Available from: https://www.youtube.com/watch?v=EQXPJ7eeesQ.

5. Bioassay C. Fluorescent in situ hybridization (FISH) Assay YouTube2018 [Available from: https://www.youtube.com/watch?v=b81DcJC1jAs.

6. Hui HYL, Clarke KM, Fuller KA, Stanley J, Chuah HH, Ng TF, et al. "Immuno-flowFISH" for the Assessment of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia. Cytometry A. 2019;95(5):521-33.

7. Hui H. Development of immuno-flowFISH by imaging flow cytometry for the analysis of haematological maligancies: The University of Western Australia; 2018.