Chronic Lymphocytic Leukemia: Updates in Diagnosis and Testing

Special Issues, The Changing Chronic Lymphocytic Leukemia Treatment Paradigm, Volume 1, Issue 1

There have been updates in chronic lymphocytic leukemia prevalence and diagnosis, with an emphasis on testing options.

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults in Western countries, with an estimated 20,720 people in the United States expected to be diagnosed in 2019.1,2 The disease is characterized by an accumulation of leukemic cells often appearing as small, mature lymphocytes in the peripheral blood, bone marrow, and lymphoid tissues.1

Over the past decade, diagnosis, testing, and prognostic stratification have substantially improved,3 leading to revisions in guidelines from both the International Workshop on Chronic Lymphocytic Leukemia and the National Comprehensive Cancer Network (NCCN). Among the most significant changes is the increasing essential prognostic role of the immunoglobulin heavy-chain variable (IGHV) mutational status and the clinical relevance of genomic alterations found in CLL.4 This article reviews updates in CLL prevalence and diagnosis, with an emphasis on testing options.


The majority of patients diagnosed with CLL receive diagnosis when absolute lymphocytosis is discovered incidentally during a routine blood count.3 Some patients, however, may exhibit painless lymphadenopathy or present with symptoms caused by splenomegaly or cytopenias.3 The median survival of patients with low-risk disease is 150 months, which is similar to the survival rate of age-matched controls.1 Intermediate-risk disease is associated with a median survival of 71 to 101 months, while high-risk disease is associated with a poorer prognosis and a median survival of 19 months.1

Patients with asymptomatic early-stage disease (low-risk Rai 0 or Binet A) or intermediate-risk disease (Rai I-II or Binet B) can be closely observed without therapy.1,4 For this patient population, evidence of progressive disease or the development of symptomatic disease would be indications for initiating treatment.1,4 Therapy is necessary for patients with advanced-stage or high-risk CLL with progressive cytopenia, while patients with mild, stable cytopenia may continue to be monitored in selected cases.1

Disease-related symptoms include severe fatigue, weight loss, night sweats, and fever without evidence of infection.1,4 Additional signals for treatment include progressive or symptomatic splenomegaly or lymphadenopathy, progressive anemia or thrombocytopenia, autoimmune complications, or thrombocytopenia unresponsive to corticosteroids.1,4 According to the NCCN guidelines, in the absence of other signs or symptoms, absolute lymphocyte count is an indication for treatment only if it is above 200 to 300 x 109/L or if symptoms related to leukostasis develop.1


For a patient with suspected CLL, it is crucial to rule out other lymphoproliferative diseases, such as hairy cell leukemia or leukemic manifestations of various lymphomas, which may appear similar to CLL.4 Assessments of the blood smear, the immunophenotype, and, in some cases, the genetic features of the circulating lymphoid cells are necessary to assess to differentiate CLL from other lymphoproliferative diseases.4 According the NCCN guidelines, the presence of at least 5 x 109/L B lymphocytes in the peripheral blood for 3 or more months is required for a CLL diagnosis, and the clonality of B lymphocytes must be verified by flow cytometry.1,4 Small, mature lymphocytes with a narrow border of cytoplasm and a dense nucleus are distinctive of CLL, as are smudge cells.4

Immunophenotyping and Testing Options

The immunophenotype for CLL is typically CD5+, CD10—, CD19+, CD23+, cyclin D1–, CD43 +/–, CD20 dim, and surface immunoglobulin.1 Flow cytometry of peripheral blood is adequate to establish diagnosis, with biopsy of the bone marrow normally not needed.1 According to the NCCN guidelines, flow cytometric studies should include cell surface markers kappa/lambda, CD19, CD20, CD5, CD23, and CD10.1 It is also recommended, if flow cytometry is used for diagnosis, to include cytospin for cyclin D1 or fluorescence in situ hybridization (FISH) for t(11;14);t(11q;v);1 if the diagnosis is not established by flow cytometry, lymph node biopsy should be performed.1 Immunophenotyping by immunohistochemistry should include CD3, CD5, CD10, CD20, CD23, and cyclin D1.1 A fine needle aspiration or core needle biopsy by itself is typically not suitable to determine initial diagnosis.1 However, a combination of core biopsy and fine needle aspiration biopsy, along with supplemental techniques (ie, flow cytometry and immunohistochemistry), may be a sufficient option to establish a diagnosis if a lymph node is not easily accessible for biopsy.1 See Table 1 for a listing of diagnostic testing options for CLL.1

Although both CLL and mantle cell lymphoma are CD5+ B-cell tumors, the absence of cyclin D1 expression is a key differentiator.1 Assessment of cyclin D1 or FISH for t(11;14), CD200 by flow cytometry, and lymphocyte enhancer-binding factor 1 (LEF1) by immunohistochemistry are useful in distinguishing CLL from mantle cell lymphoma.1,5,6 CD200 has a moderate-to-strong expression in both typical and atypical CLL cells, with a negative expression in cases of mantle cell lymphoma.6 Although LEF1 is a specific diagnostic marker for CLL, it is expressed in only about 30% of positive tumor cells in other lymphoma entities.5

The clinical course of patients with CLL is highly variable.7 Clinical staging of CLL does not transparently interpret the observed variations in response to therapy and survival.7 The mutational status of IGHV, cytogenetic abnormalities detected by FISH, flow cytometry—based prognostic markers, and serum markers may provide additional prognostic information.1,8 However, these prognostic factors were generated using data of patients treated with chemotherapy or chemoimmunotherapy.1 Survival outcomes for patients treated with newer small molecule inhibitor—based therapy have substantially improved, but follow-up data with the use of these treatments are limited.1 Therefore, the NCCN guidelines recommend caution when interpreting the survival data associated with these identified prognostic factors.1

Recommended testing for proper prognosis and/or guidance on therapy selection include FISH, stimulated metaphase karyotype, TP53 sequencing, and molecular genetic analysis (Table 2).1 Although indications for initiating treatment in patients are independent of these results, they are useful for the selection of treatment.1,4 Patient age, performance status or fitness level, assessment of TP53 mutation status, del(17p) status, and IGHV mutation status should be evaluated prior to initiating therapy.1

FISH for Cytogenetic Abnormalities

Approximately 80% of patients with previously untreated CLL present with cytogenetic abnormalities that can be found using FISH,1,9 with the most common irregularities being del(11q), del(13q), trisomy 12, and del(17p).10 Longest median survival (133 months), as well as longest median treatment-free interval (92 months), are observed in patients with del(13q) as the sole abnormality.10 Shorter median survival times are observed in patients with del(17p) or del(11q) in CLL cells, compared with patients with leukemic cells with other abnormalities or a normal karyotype.10 Treatment regimens that contain fludarabine, cyclophosphamide, and rituximab appear to improve the poor prognosis associated with del(11q).11,12

TP53 Sequencing

Del(17p) indicates the loss of the TP53 gene and is often associated with mutations in the remaining allele of TP53.1 Mutations of TP53 have also been observed in the absence of del(17p).13 The presence of del(17p) and/or TP53 mutations in CLL cells is linked with poor prognosis and a relative resistance to standard chemotherapy regimens using alkylating drugs and/or purine analogues.10 The poor prognosis observed with TP53 mutations is independent of the presence of del(17p).13 Patients with del(17p) and/or TP53 mutations in CLL cells experience better outcomes when treated with small molecule inhibitors of Bruton tyrosine kinase, phosphatidylinositol 3-kinase, or BCL-2 protein, compared with standard chemotherapy regimens.4 The treatment algorithms suggested by the NCCN guidelines are based on the presence or absence of del(17p) or TP53 mutation.1

CpG-Stimulated Metaphase Karyotype

The leukemic cells in CLL have a very low in vitro proliferative activity, making conventional metaphase cytogenetics challenging.1 Interphase cytogenetic analysis with FISH represents the standard method to detect specific chromosomal abnormalities.1 Cytosine—phosphate –guanosine (CpG) oligonucleotide stimulation has been used successfully to detect cytogenetic defects, allowing for a more in-depth chromosome analysis compared with FISH.14,15

NCCN guidelines recommend CpG-stimulated metaphase karyotype for the identification of complex karyotype, which is characterized by the presence of at least 3 independent chromosomal abnormalities in more than 1 cell1,16 and is an independent predictor of a substantially shorter overall survival (OS) in patients with CLL.9,16 In addition, complex karyotype may have stronger prognostic implications than del(17p) or TP53 mutations in patients with CLL who are treated with ibrutinib-based regimens.17,18 In a multivariate analysis among patients with relapsed or refractory CLL treated with ibrutinib-based regimens, the presence of complex karyotype was associated with shorter survival, while independent relationships were identified between del(17p) and OS.17

IGHV Mutational Status

The somatic hypermutation status of the IGHV genes, which distinguishes 2 disease groups with substantially different behavior, is an important prognostic marker in CLL.19 Molecular genetic analysis, by polymerase chain reaction or sequencing, should be performed to detect the mutational status of IGHV.1 Patients with unmutated IGHV have a more dismal clinical course and shorter survival compared with patients with mutated IGHV, irrespective of CLL stage.20,21 In a follow-up study evaluating the outcomes of patients treated with a regimen of fludarabine, cyclophosphamide, and rituximab, progression-free survival (PFS) at 12.8 years was nearly 54% for patients with mutated IGHV, compared with 8.9% for patients with unmutated IGHV.22

Although CD38, CD49d, and ZAP-70 expressions have been suggested as surrogate markers for IGHV-mutation status, NCCN guidelines recommend testing for the mutation as the preferred method.1 Given the challenges associated with standardization and reproducibility of these markers across laboratories, assessment of CD38, CD49d, and ZAP-70 should occur only within the context of clinical trials.1

Additional Tests

The pattern of bone marrow involvement, diffuse versus nondiffuse, was once an identifiable prognostic factor.23 However, markers such as analyzing circulating lymphocytes, IGHV mutational status, and cytogenetic abnormalities detected by FISH have been found to be more reliable prognostic factors.1 Therefore, bone marrow biopsy is no longer required for patients who may have CLL.1 However, this procedure can be a useful method to determine the etiology of cytopenias.1

The level of serum b-2 microglobulin has been identified as an independent prognostic factor for OS in patients with CLL.24 In a multivariable analysis, the achievement of normalized values of b-2 microglobulin after receiving 6 months of ibrutinib-based treatment was linked to superior PFS.25 This association was independent of pretreatment prognostic features.25 The international prognostic index for CLL includes b-2 microglobulin concentration as a parameter, in addition to TP53 mutations, IGHV mutational status, clinical stage, and age.24 The NCCN guidelines suggest the measurement of b-2 microglobulin as part of the CLL workup, as it may provide beneficial prognostic information.1

Additional genomic abnormalities with prognostic implications for patients with CLL, such as mutations in NOTCH1 and SF3B1, have also been identified.4,8 However, the impact of these genomic abnormalities within the context of treatment with newer targeted therapies is unclear.1 The NCCN guidelines recommend against their use as a basis for selecting treatment options.1


Advances in the understanding of CLL have amplified the clinical and prognostic relevance of genetic alterations, which have led to the emergence of novel targeted therapies and monoclonal antibodies. These new therapeutic agents have improved the outcomes of patients with CLL, including those with high-risk disease. Clinicians can individualize the use of prognostic tools as well as therapy options to improve diagnosis and management of patients with CLL. Further research that evaluates prognostic factors within the context of novel targeted therapies will improve the understanding of these factors’ significance in the current treatment environment. 


  1. NCCN Clinical Practice Guidelines in Oncology. Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, version 4.2019. National Comprehensive Cancer Network website. Published March 15, 2019. Accessed May 1, 2019.
  2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7-34. doi: 10.3322/caac.21551.
  3. Sharma S, Rai KR. Chronic lymphocytic leukemia (CLL) treatment: so many choices, such great options. Cancer. 2019;125(9):1432-1440. doi: 10.1002/cncr.31931.
  4. Hallek M, Cheson BD, Catovsky D, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 2018;131(25):2745-2760. doi: 10.1182/blood-2017-09-806398.
  5. Menter T, Dirnhofer S, Tzankov A. LEF1: a highly specific marker for the diagnosis of chronic lymphocytic B cell leukaemia/small lymphocytic B cell lymphoma. J Clin Pathol. 2015;68(6):473-478. doi: 10.1136/jclinpath-2015-202862.
  6. Sandes AF, de Lourdes Chauffaille M, Oliveira CR et al. CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry. Cytometry B Clin Cytom. 2014;86(2):98-105. doi: 10.1002/cyto.b.21128.
  7. Stilgenbauer S, Schnaiter A, Paschka P, et al. Gene mutations and treatment outcome in chronic lymphocytic leukemia: results from the CLL8 trial. Blood. 2014;123(21):3247-3254. doi: 10.1182/blood-2014-01-546150.
  8. Rossi D, Gaidano G. The clinical implications of gene mutations in chronic lymphocytic leukaemia. Br J Cancer. 2016;114(8):849-854. doi: 10.1038/bjc.2016.78.
  9. Blanco G, Puiggros A, Baliakas P, et al. Karyotypic complexity rather than chromosome 8 abnormalities aggravates the outcome of chronic lymphocytic leukemia patients with TP53 aberrations. Oncotarget. 2016;7(49):80916-80924. doi: 10.18632/oncotarget.13106.
  10. Döhner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-1916.
  11. Tsimberidou AM, Tam C, Abruzzo LV, et al. Chemoimmunotherapy may overcome the adverse prognostic significance of 11q deletion in previously untreated patients with chronic lymphocytic leukemia. Cancer. 2009;115(2):373-380. doi: 10.1002/cncr.23993.
  12. Oscier D, Wade R, Davis Z, et al; Chronic Lymphocytic Leukaemia Working Group; UK National Cancer Research Institute. Prognostic factors identified three risk groups in the LRF CLL4 trial, independent of treatment allocation. Haematologica. 2010;95(10):1705-17 doi: 10.3324/haematol.2010.025338.
  13. Rossi D, Cerri M, Deambrogi C, et al. The prognostic value of TP53 mutations in chronic lymphocytic leukemia is independent of del17p13: implications for overall survival and chemorefractoriness. Clin Cancer Res. 2009;15(3):995-1004. doi: 10.1158/1078-0432.CCR-08-1630.
  14. Dicker F, Schnittger S, Haferlach T, et al. Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: a study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression. Blood. 2006;108(9):3152-3160.
  15. Heerema NA, Byrd JC, Dal Cin PS, et al; Chronic Lymphocytic Leukemia Research Consortium. Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) study. Cancer Genet Cytogenet. 2010;203(2):134-140. doi: 10.1016/j.cancergencyto.2010.07.128.
  16. Le Bris Y, Struski S, Guièze R, et al. Major prognostic value of complex karyotype in addition to TP53 and IGHV mutational status in first-line chronic lymphocytic leukemia. Hematol Oncol. 2017;35(4):664-670. doi: 10.1002/hon.2349.
  17. Thompson PA, O’Brien SM, Wierda WG, et al. Complex karyotype is a stronger predictor than del(17p) for an inferior outcome in relapsed or refractory chronic lymphocytic leukemia patients treated with ibrutinib-based regimens. Cancer. 2015;121(20):3612-3621. doi: 10.1002/cncr.29566.
  18. Woyach JA, Ruppert AS, Guinn D, et al. BTKC481S-mediated resistance to ibrutinib in chronic lymphocytic leukemia. J Clin Oncol. 2017;35(13):1437-1443. doi: 10.1200/JCO.2016.70.2282.
  19. Baliakas P, Agathangelidis A, Hadzidimitriou A, et al. Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations. Blood. 2015;125(5):856-859. doi: 10.1182/blood-2014-09-600874.
  20. Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999;94(6):1840-1847.
  21. Hamblin TJ, Davis Z, Gardiner A, et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 1999;94(6):1848-1854.
  22. Thompson PA, Tam CS, O’Brien SM, et al. Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia. Blood. 2016;127(3):303-309. doi: 10.1182/blood-2015-09-667675.
  23. Rozman C, Montserrat E, Rodríguez-Fernández JM, et al. Bone marrow histologic pattern--the best single prognostic parameter in chronic lymphocytic leukemia: a multivariate survival analysis of 329 cases. Blood. 1984;64(3):642-648.
  24. International CLL-IPI Working Group. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. Lancet Oncol. 2016;17(6):779-790. doi: 10.1016/S1470-2045(16)30029-8.
  25. Thompson PA, O’Brien SM, Xiao L, et al. b2-microglobulin normalization within 6 months of ibrutinib-based treatment is associated with superior progression-free survival in patients with chronic lymphocytic leukemia. Cancer. 2016;122(4):565-573. doi: 10.1002/cncr.29794.