FISHing in the dark: How the combination of FISH and conventional karyotyping improves the diagnostic yield in CpG‐stimulated chronic lymphocytic leukemia

Abstract

Despite significant advances in molecular genetic approaches, fluorescence in situ hybridization (FISH) remains the gold standard for the diagnostic evaluation of genomic aberrations in patients with chronic lymphocytic leukemia (CLL). Efforts to improve the diagnostic utility of molecular cytogenetic testing have led to the expansion of the traditional 4-probe CLL FISH panel. Not only do these efforts increase the cost of testing, they remain hindered by the inherent limitations of FISH studies -namely the inability to evaluate genomic changes outside of the targeted loci. While array-based profiling and next generation sequencing (NGS) have critically expanded our understanding of the molecular pathogenesis of CLL, these methodologies are not routinely used by diagnostic laboratories to evaluate copy number changes or the mutational profile of this disease. Mitogenic stimulation of CLL specimens with CpG-oligonucleotide (CpG-ODN) has been identified as a reliable and reproducible means of obtaining a karyotype, facilitating a low-resolution genomewide analysis. Across a cohort of 1255 CpG-ODN-stimulated CLL specimens, we describe the clinical utility associated with the combinatorial use of FISH and karyotyping. Our testing algorithm achieves a higher diagnostic yield (~10%) through the detection of complex karyotypes, well-characterized chromosomal aberrations not covered by the traditional CLL FISH panel and through the detection of concurrent secondary malignancies. Moreover, the single cell nature of this approach permits the evaluation of emerging new clinical concepts including clonal dynamics and clonal evolution. This approach can be broadly applied by diagnostic laboratories to improve the utility of traditional and molecular cytogenetic studies of CLL.

Examination of clonal evolution in chronic lymphocytic leukemia

“…The results obtained showed that conventional karyotyping should be included in the observation of clonal evolution in addition to the FISH technique. These observations confirm the suggestions of many researchers that the assessment of risk of the clinical course of the disease and monitoring the treatment of patients with CLL indicate the need to include conventional karyotyping for screening these patients, similarly to those routinely performed in other hematological disorders [21,28].…”

“…The detection of these changes has increased dramatically in recent years by the introduction of stimulating agents, such as DSP30/IL2, in cell cultures. [21]. The phenomenon of clonal evolution was more frequently observed with the use of a more sensitive method, namely the FISH technique [20,22,23].…”

Quality Assurance/Quality Control of Fluorescence in Situ Hybridization Tests in Hematologic Malignancies

“…Fluorescence in situ hybridization (FISH) is simple, reliable and cost-effective and therefore FISH is a major technology that is widely applied for clinical diagnosis, especially for hematologic malignancies, even in the era of next-generation sequencing (NGS) [1][2][3][4]. In the Clinical Cytogenetics Laboratory at MD Anderson Cancer Center, over 15,000 FISH tests are performed each year, including approximately 1,000 BCR-ABL1 and 500 MYC FISH tests, respectively.…”

Cytogenetic landscape in 1012 newly diagnosed chronic lymphocytic leukemia

“…Taking into account the CBA in LR-FISH group, 28% of cases might be reclassified in intermediate risk category (n = 90) and 7% in defavorable risk category (n = 24). The observation of additional chromosome abnormalities in LR-FISH category had previously been noticed 19,20. Indeed, one to two additional chromosomal anomalies were reported to be sufficient to modify time to first treatment and even overall survival independently if the number of anomalies is exclusively superior to 3 21.…”