Patricia Minehart Miron, Ph.D., FACMG is Quest Diagnostics' Scientific Director, Advanced Diagnostics-Genetics, Genomics and R&D and Cytogenetics and Genomics Associate Professor Pathology/Pediatrics at UMass Medical School
B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world,1
and has a highly variable clinical course, ranging from indolent to highly aggressive. Stratification is important to guide treatment and currently relies on the Rai and Binet clinical staging systems, as well as genetic factors.2,3,4
Cytogenetic analysis is well-established as playing a key role in both diagnosis and prognosis of disease;5,6
however, historically, conventional cytogenetics (metaphase chromosome analysis) has been hampered by failure of mature B cells to divide readily in culture.
Fluorescence in situ Hybridization (FISH)
To overcome this limitation, fluorescence in situ hybridization (FISH) assays are used to detect targeted abnormalities in interphase cells, removing the requirement of cell culture to obtain an adequate number of metaphase cells for chromosome analysis. Since the publication of the Dohner hierarchical classification in 2000, interphase FISH has been the gold standard for cytogenetic evaluation in CLL. With the use of four FISH assays (for detection of trisomy 12 and deletions of 13q14.3, ATM and TP53), FISH has an approximately 80% abnormality detection rate. This FISH panel also provides useful prognostic information; deletion of 13q14.3 as the sole abnormality is associated with a favorable prognosis, while deletions of ATM (11q22) and TP53 (17p13) are associated with unfavorable prognosis. Trisomy 12 carries an intermediate prognosis; patients with none of these aberrations have a prognosis less favorable than 13q deletion, but more favorable than trisomy 12. Given the high detection rate and the prognostic utility of these 4 FISH markers, this panel was quickly adopted in the clinical setting and many laboratories came to rely solely on FISH for cytogenetic assessment of CLL.
Quest Diagnostics CLL FISH PanelAberration Risk Prevalence (%)
Deletion 13q14 Favorable (if sole abnormality) 50Trisomy 12 Intermediate 20Deletion ATM (11q23) Unfavorable 15-25Deletion TP53 (17p13.3) Unfavorable 5-10Deletion 6q21 Possibly unfavorable/Not fully established 5 FISH, however, is targeted and as such, has significant limitations. FISH cannot provide the comprehensive genomic analysis provided by cytogenetic analysis of banded chromosomes. In addition to missing specific abnormalities not included on the panel, FISH cannot reliably detect complex karyotypes (three or more abnormalities) or multiple clones (tumoral herogeneity), both of which are associated with aggressive disease in CLL.7,8
Lastly, targeted FISH does not allow discovery of new prognostic abnormalities.
Metaphase Chromosome Analysis with New Protocols
In the past 5 to 10 years, clinical cytogenetics laboratories have started to use new protocols to culture CLL cells. The development of these protocols largely stemmed from previous research showing that synthetic CpG oligonucleotides induce proliferation, cytokine production, and high-affinity interleukin 2 (IL-2) receptor expression in CLL cells through interaction with toll-like receptor 9 on B cells.9,10
In the clinical laboratory setting, use of CpG oligonucleotides as a mitogen has proven extremely effective at stimulating CLL cells to divide readily in culture. Analyzable metaphase cells are typically obtained in over 90% of specimens with these newer protocols,11,12,13
and detection of metaphase chromosome abnormalities has increased significantly.14,15,16
The detection rate of abnormalities that are not targeted by the FISH panel ranges from 25%-35%, and new abnormalities are being identified that were not previously appreciated as being important in the CLL landscape.16,17,18
Before the adoption of these new mitogens, translocations were not thought to play a role in CLL. However, translocations are now reported in 30%-40% of cases. Originally thought to independently confer poor prognosis, more recent studies suggest the poor prognosis associated with translocations may be limited to unbalanced translocations or to translocations seen as part of a complex karyotype.19,20
Although uncommon (5%), IGH (14q32) translocations are also seen in CLL; the most common partners are BCL2 (18q21) and BCL3 (19q13). As a group, IGH translocations are associated with poor prognosis. In particular, IGH-BCL3 translocations are associated with aggressive disease and poor prognosis. The one exception is IGH-BCL2, which likely has a more favorable prognosis than other IGH partner genes.21,22
MYC (8q24.1) translocations are also reported in approximately 1% of cases, and can involve IG or non-IG partner genes. These translocations are associated with poor prognosis.23
Complex karyotypes, defined as having 3 or more chromosome abnormalities, are present in approximately 15% of patients and are now well established as conferring poor prognosis in CLL.17,20,24
They are associated with both shortened treatment-free and shortened overall survival after chemotherapy. A recent publication reports the presence of a complex karyotype as being even more significant than deletion of TP53,25
and independent of IGHV mutation status, Binet stage, and serum b-2 microglobulin.Although FISH can hint at the presence of a complex karyotype, metaphase chromosome analysis is required for detection of most cases. Similarly, metaphase analysis aids in the detection of multiple clones (intratumoral heterogeneity).
Detection of clonal evolution is critical in CLL, in which many patients may be on a “watch and wait” regime for many years before undergoing treatment. Estimates of the prevalence of clonal evolution range from approximately 10%-40%.26,27
Interphase FISH and metaphase chromosome analysis complement each other in the detection of clonal evolution. FISH analysis remains critical for the detection of small aberrations; it is especially important to detect the high risk deletions of ATM and TP53, which can be too small to be seen by metaphase chromosome analysis. On the other hand, metaphase chromosome analysis casts a wider net and has a higher detection rate.
Impact on Prognosis
Complementing CLL FISH analysis with metaphase chromosome analysis is also critical for stratifying patients with no abnormalities identified by FISH. With the Dohner prognostic scheme, FISH normal patients have a prognosis in between 13q deletion and trisomy 12. However, the FISH normal group is heterogeneous, and can include patients who are cytogenetically normal as well as patients who have abnormalities not included on the FISH panel. In a recent study by Rigolin et al., approximately one-third of FISH-normal patients had abnormalities detected by metaphase chromosome analysis. Also, of note approximately the same percentage of the favorable FISH cohort had additional abnormalities detected by metaphase chromosome analysis. Most importantly, approximately 25% of cases were reclassified into a higher cytogenetic risk by the addition of metaphase chromosome analysis.28
Metaphase chromosome analysis and interphase FISH are complementary techniques, and both are useful in identifying clinically important cytogenetic aberrations in CLL. The identification of specific abnormalities by FISH is a well-established method for identifying abnormalities important to overall prognosis, disease progression and response to treatment. FISH also identifies abnormalities that are too small to be detected by study of metaphase chromosomes: deletion of 13q is often cytogenetically cryptic. However, detection of abnormalities is limited to the probes used and will miss a number of chromosome aberrations. Perhaps most importantly, targeted FISH can miss identifying clonal evolution and complex karyotypes, which confer poor prognosis. Thus, targeted FISH should not be used as the sole test to evaluate cytogenetic abnormalities at time of diagnosis or disease progression, as a favorable FISH profile can be seen in patients with unfavorable cytogenetic profiles.
Quest Diagnostics Test Codes:Chromosome Analysis, Hematologic Malignancy-TC14600FISH, B-Cell Chronic Lymphocytic Leukemia Panel-TC16864Chromosomal Microarray, Hematologic Malignancy, ClariSure® Oligo-SNP-TC90961References1. Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med. 1995; 333:1052-1057.2. Rai KR, Sawitsky A, Cronkite EP et al. Clinical staging of chronic lymphocytic leukemia. Blood. 1975; 46:219-34.3. Binet JL, Leporrier M, D’Ighiero G et al. Clinical staging system for chronic lymphocytic leukemia. Cancer. 1977; 40:855-864.4. Zenz T, Eichhorst B, Busch R et al. TP53 mutation and survival in chronic lymphocytic leukemia, J Clin Onco. 2010; 28:4473-9.5. Dohner H, Stilgenbauer S, Benner A et al. Genomic aberrations and survival in chronic lymphocytic leukemia. 2000; N Eng J Med. 343:1910-1916.6. KroSeiler T, Benner A et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. 2002; Blood. 100:1410-1416.7. Ouilette P;Collins R, Shakhan S et al. Acquired genomic copy number aberrations and survival in chronic lymphocytic leukemia. Blood. 2011; 118:3051-61.8. Knight SJ, Yau C, Clifford R et al. Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia. Leukemia. 2012; 26:1564-75.9. Decker T, Schnellearwasser T, et al. Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells. Blood. 2000; 95:999-1006.10. Takeshita F, Leifer CA, I , et al. Cutting edge: Role of toll-like receptor 9 in CpG DNA-induced activation of human cell. J Immunol. 2001; 167:3555-3558.11. Mayr C, Speicher Mr, Kofler DM et al. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood. 2006; 107:742-751.12. Muthusamy N, Breidenbach H, Andritoss L et al. Enhanced detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide in combination with pokeweed mitogen and phorbol myristate acetate. Cancer Genet. 2011; 204(2):77-83.13. Lin X, Chen J, Huang H. Immunostimulation by cytosine-phosphate-guanine oligodeoxynucleotides in combination with IL-2 can improve the success rate of karyotype in chronic lymphocytic leukaemia. Br J Biomed Sci. 2016; Jun 21:1-5 (Epub ahead of print]14. Put N, Konnings P, Rack K et al. Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study. Genes Chromosomes Cancer. 2009; 48:843-853.15. Heerema Na, Byrd, JC, Dal Cin PS et al. Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotype result among laboratories: a CLL Research Consortium (CRC) Study. Cancer Genet Cytogenet. 2010; 203:134-14016. Shi M, Cipollini JM, Crowley-Bish P et al. Improved Detection Rate of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia and Other Mature B-Cell Neoplasms With Use of CpG-Oligonucleotide DSP30 and Interleukin 2 Stimultaion. Am J Clin Pathol. 2013; 139:662-669.17. Haferlach C, Dicker F, Schnittger S et al. Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgVH status and immunophenotypice. Leukemia. 2007; 21(12):2442-2451.18. Rigolin, G, Cibien F, Martinelli S et al. Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with “normal” FISH: correlations with clinicobiologic parameters. Blood. 2012; 119(10): 2310-2313.19. Van den Neste E, Robin V, Francart J el al. Chromosomal translocations independently predict treatment failure, treatment-free survival and overall survival in B-cell chronic lymphocytic leukemia patients treated with cladribine. Leukemia. 2007; 21(8):1725-1722.20. Baliakas P, Iskas M, Gardiner A et al. Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data. Am J Hemat. 2014; 89(3): 249-255.21. Nguyen-Khac F, Chapiro E, Lesty C et al. Specific chromosomal IG translocations have different prognoses in chronic lymphocytic leukemia. Am J Blood Res. 2011; 1(1):13-21.22. Davids M, Vartanov A, WernerL et al. Controversial fluorescence in situ hybridization cytogenetic abnormalities in chronic lymphocytic leukaemia: new insights from a large cohort. Br J Haematol. 2015; 170; 694-703.23. Put N, van Roosbroeck K, Konings P et al. Chronic lymphocytic leukemia and prolymphocytic leukemia with MYC translocations: a subgroup with an aggressive disease course. Annals of Hemat. 2012; 91 (6):863-873.24. Jaglowski S, Ruppert A, Heerema N et al. Complex karyotype predicts inferior outcomes following reduced-intensity conditioning allogeneic transplant for chronic lymphocytic leukaemia. 2012; Br J Haematol 159(1):82-87.25. Herling C, Klaumunzer M, Rocha C et al. Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy. Blood. 2016; 128(3): 395-404.26. Puiggros A, Blanco G, Espinte B. Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go. Biomed Res Int. 2014: 435983.27. Dubuc A, Davids M, Pullugi M et al. FISHing in the dark: How the combination of FISH and conventional karyotyping improves the diagnostic yield in CpG-stimulated chronic lymphocytic leukemia. Am J Hematol. 2016; Jun 24 (Epub)28. Rigolin, Giudice, Formigaro et al. Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia: clinical and biologic correlations. Genes, Chrom & Cancer. 2015; 54: 818-826