Chronic Lymphocytic Leukemia - CLL

Chronic lymphocytic leukemia (CLL) is characterized by small lymphocytes in the bone marrow, blood, and lymphoid tissues. CLL is the most common form of leukemia in adults in the U.S. and represents 40% of all adult leukemias in Western countries.

  • Key Points
  • Diagnosis
  • Algorithms
  • Monitoring
  • Background
  • Lab Tests
  • References
  • Related Topics

Chronic Lymphocytic Leukemia Prognostic Markers

The diagnosis of CLL generally requires detection of >5,000 CLL-type cells per microliter of peripheral blood. CLL cells typically express CD19, weak CD20, and CD23 (B-cell antigens), along with CD5 (a T-cell antigen); these are usually assessed by flow cytometry immunophenotyping.

Cytogenetic, molecular, and flow cytometric testing play an important role in prognostication for CLL patients.

Molecular Markers in CLL
Marker Biology Prognosis

Cytogenetics – cytogenomic SNP microarray is preferred test for detection of prognostically important genomic abnormalities (loss/gain of DNA and/or loss of heterozygosity) in hematologic malignancies (CLL, acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], myelodysplastic syndromes [MDS]/AML with normal karyotype)

Microarray test may not detect low-level clones (<15-20%); therefore, ideal testing time is when significant disease is present

ARUP Tests

Cytogenomic SNP Microarray-Oncology 2006325

Chromosome FISH, CLL Panel 2002295

(Includes ATM (11q22.3), Chromosome 12 centromere (Trisomy 12), (D13S319) 13q14.3, p53 (17p13.1)

del(17p) typically involves the TP53 gene and del(11q) contains the ATM gene; both genes are tumor suppressors

Loss of p53 function or its activator, the ATM protein kinase, is associated with treatment resistance and clinically aggressive disease

del(17p) and/or del(11q) correlate with nonmutated IGHV gene

Karyotypic evolution may occur over course of disease

Least favorable outcome associated with del(17p), followed by del(11q), then trisomy 12q

More favorable outcome associated with del(13q) and normal diploid karyotype

Immunoglobulin Heavy Chain Variable Region Gene (IGHV) Mutation Status – determine risk group in newly diagnosed CLL


IGHV Mutation Analysis by Sequencing 0040227

Immunoglobulin heavy variable genes encode the antigen-binding domain of B-cell antigen receptor (surface immunoglobulin)

Somatic hypermutation of IGHV diversifies antigen-binding repertoire in normal B cells

IGHV mutation status of CLL tends to remain constant over course of disease

del(17p) and/or del(11q) correlate with nonmutated IGHV genes

CLL cases with mutated IGHV genes typically have more indolent clinical course, while those with unmutated IGHV genes often behave in an aggressive fashion

CLL cases that employ VH3-21 segment typically have an unfavorable outcome regardless of mutation status

CD38 expression – for initial diagnosis and assessment of levels of CD38 expression

Follow-up studies (CD5, CD19, CD20, CD23, kappa, lambda, FMC7, CD38)


Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003

Transmembrane glycoprotein modulates intracellular signaling

May reflect proliferative status of CLL cells

Cases that express CD38 often have nonmutated IGHV genes

CD38 expression levels may vary over course of disease

Expression of CD38 by CLL cells is associated with an unfavorable outcome
Dohner H, et al., 2001; Furman R, 2010; Van Bockstaele F, et al., 2009

Indications for Testing

  • Adult with persistent, unexplained lymphocytosis of ≥3 months' duration found on CBC; lymphadenopathy

Laboratory Testing

  • Initial testing – CBC with differential, platelets, lactate dehydrogenase (LD) concentration
    • Diagnosis of CLL requires ≥5,000 lymphocytes/µL
  • Serum testing – monoclonal antibody panels (minimal)
    • B cell – kappa/lambda, CD5, CD10, CD19, CD23, FMC7
    • T cell – CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD30
  • Diagnosis based on lymphocytosis, morphology, and immunophenotyping (CD5+, CD19+, CD20 [weak], CD23+, CD10-)
  • CLL FISH panel may aid in diagnosis
    • CLL must be differentiated from mantle cell lymphoma – consider ordering testing for t(11;14) IGH/CCND


  • Bone marrow sampling – unnecessary if characteristic phenotype, consistent cytology is present, and CBC is normal (based on IWCLL, 2008, recommendations)
    • If performed, typically hyper- or normo-cellularity, nodular or diffuse pattern of lymphocytic infiltration in >10% of nucleated cells
    • Consider immunophenotyping, if marrow obtained
    • Immunohistochemistry – p53 (KRAS) positivity may correlate with TP53 gene mutation


  • SNP microarray testing – preferred test for detection of genomic abnormalities; may not detect low-level clones
  • Fluorescence in situ hybridization (FISH) testing also used

Differential Diagnosis

  • Flow cytometry and fluorescence in situ hybridization (FISH) used for follow-up to detect minimal residual disease (MRD)
    • Cytogenomic microarray not recommended for MRD
  • Infectious disease screening prior to and during therapy
    • Hepatitis B – identify infection prior to initiating CD20 monoclonal antibody therapy
      • Monitor all patients with positive test result who are receiving therapy
    • Cytomegalovirus (CMV) antibodies – high risk of CMV reactivation with tyrosine kinase inhibitor (TKI) therapy
      • Monitor every 2-3 weeks in patients using alemtuzumab
    • Herpes simplex virus (HSV) and Pneumocystis jirovecii risk of reactivation of these infections in patients with HIV who undergo therapy
      • HSV and P. jirovecii testing as indicated


  • Incidence – 4/100,000 (Hallek, 2015)
  • Age – median 67-72 years (Hallek, 2015)
    • 80% diagnosed ≥60 years
    • Rare in patients <50 years (~10% of cases)
  • Sex – M>F, 2.8:1
  • Ethnicity – lower risk in Chinese, Japanese, and Filipino ethnicities

Risk Factors

  • Family member with CLL
    • First-degree relative has threefold risk of developing CLL or other lymphoid neoplasm


  • Specific subtypes
    • Small lymphocytic leukemia (SLL) – different manifestation of CLL if nodes are principally involved
      • SLL has similar biology and  same treatment approach
    • Monoclonal B-cell lymphocytosis (MBL) – asymptomatic patient with <5,000 circulating CLL phenotype cells per microliter and no node involvement
      • Precedes development of CLL
      • MBL estimated to be present in 5% of adults ≥50 years; however, general screening for MBL is not recommended
      • MBL with lymphocytosis >4,000 CLL phenotype cells per microliter progresses into CLL at a rate of 1% per year

Clinical Presentation

  • CLL principally involves bone marrow and blood
    • Majority of patients present as asymptomatic with lymphocytosis found on CBC
    • Disease may remain indolent for several years until treatment is required
  • Symptoms
    • Constitutional symptoms – night sweats, weight loss, fatigue
    • Adenopathy – most patients have some degree of adenopathy at presentation
      • Hepatosplenomegaly
      • Lymph nodes – most commonly cervical, supraclavicular, axillary
    • Extranodal disease – uncommon
    • Complications
Tests generally appear in the order most useful for common clinical situations. Click on number for test-specific information in the ARUP Laboratory Test Directory.

Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
Method: Flow Cytometry

Cytogenomic SNP Microarray - Oncology 2006325
Method: Genomic Microarray (Oligo-SNP Array)


Low-level mosaicism (<15-20%) may not be detected; test may not be appropriate for individuals with expected lower levels of malignant cells

Does not detect balanced rearrangements; FISH should be used to evaluate specific balanced rearrangements in the following

  • ALL – balanced translocation 9;22 (ABL1-BCR), translocation 12;21 (ETV6-RUNX1), and balanced rearrangement of MLL, IGH, MYC, and TCF3
  • AML – balanced translocation 15;17 (PML-RARA),  translocation 8;21 (RUNX1T1-RUNX1), and balanced rearrangement of the MLL or the CBFB gene

Does not detect base-pair mutations or very small deletions/duplications; imbalances of the mitochondrial genome; low-level clones (not recommended for MRD)

Chromosome Analysis, Bone Marrow with Reflex to Genomic Microarray 2007130
Method: Giemsa Band/Genomic Microarray (Oligo-SNP array)

Chromosome FISH, CLL Panel 2002295
Method: Fluorescence in situ Hybridization


Not as sensitive as cytogenomic SNP microarray assay

IGHV Mutation Analysis by Sequencing 0040227
Method: Polymerase Chain Reaction/Sequencing


Time-sensitive test

Assay is designed for those with a confirmed CLL diagnosis

For diagnoses other than CLL, testing will terminate after amplification and will not include sequencing

p53 with Interpretation by Immunohistochemistry 0049250
Method: Immunohistochemistry

CD200 by Immunohistochemistry 2012844
Method: Immunohistochemistry


Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, Hillmen P, Keating MJ, Montserrat E, Rai KR, Kipps TJ, International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008; 111(12): 5446-56. PubMed

Protocol for the Examination of Specimens From Patients With Hematopoietic Neoplasms Involving the Bone Marrow. Based on AJCC/UICC TNM, 7th ed. Protocol web posting date: Jun 2012. College of American Pathologists (CAP). Northfield, IL [Revised: Jun 2012; Accessed: May 2017]

Protocol for the Examination of Specimens from Patients with Hematopoietic Neoplasms of the Ocular Adnexa. Based on AJCC/UICC TNM, 7th ed. Protocol web posting date: Mar 2010. College of American Pathologists (CAP). Northfield, IL [Revised : Mar 2010; Accessed: Dec 2016]

Protocol for the Examination of Specimens From Patients With Non-Hodgkin Lymphoma/Lymphoid Neoplasms. Based on AJCC/UICC TNM, 7th ed. Protocol web posting date: Oct 2013. College of American Pathologists (CAP). Northfield, IL [Revised: Oct 2013; Accessed: May 2017]

General References

Chiorazzi N. Implications of new prognostic markers in chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program. 2012; 2012: 76-87. PubMed

Cramer P, Hallek M. Prognostic factors in chronic lymphocytic leukemia-what do we need to know? Nat Rev Clin Oncol. 2011; 8(1): 38-47. PubMed

Furman RR. Prognostic markers and stratification of chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program. 2010; 2010: 77-81. PubMed

Hallek M. Chronic lymphocytic leukemia: 2015 Update on diagnosis, risk stratification, and treatment. Am J Hematol. 2015; 90(5): 446-60. PubMed

Inamdar KV, Bueso-Ramos CE. Pathology of chronic lymphocytic leukemia: an update. Ann Diagn Pathol. 2007; 11(5): 363-89. PubMed

Nabhan C, Rosen ST. Chronic lymphocytic leukemia: a clinical review. JAMA. 2014; 312(21): 2265-76. PubMed

Rawstron AC, Bennett FL, O'Connor SJ, Kwok M, Fenton JA, Plummer M, de Tute R, Owen RG, Richards SJ, Jack AS, Hillmen P. Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med. 2008; 359(6): 575-83. PubMed

Schnaiter A, Mertens D, Stilgenbauer S. Genetics of chronic lymphocytic leukemia. Clin Lab Med. 2011; 31(4): 649-58, ix. PubMed

Sun T. Clinical Application, Cases 16, 17, and 18, Ch 6. In Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2011.

Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Harald S, Thiele J, Vardiman J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France: International Agency for Research on Cancer, 2008.

References from the ARUP Institute for Clinical and Experimental Pathology

Cimino PJ, Bahler DW, Duncavage EJ. Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia T-cells. Exp Mol Pathol. 2013; 94(1): 40-4. PubMed

Gunn SR, Bolla AR, Barron LL, Gorre ME, Mohammed MS, Bahler DW, Mellink CH, van Oers MH, Keating MJ, Ferrajoli A, Coombes KR, Abruzzo LV, Robetorye RS. Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene. Leuk Res. 2009; 33(9): 1276-81. PubMed

Gunn SR, Hibbard MK, Ismail SH, Lowery-Nordberg M, Mellink CH, Bahler DW, Abruzzo LV, Enriquez EL, Gorre ME, Mohammed MS, Robetorye RS. Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia. Leukemia. 2009; 23(5): 1011-7. PubMed

Gunn SR, Mohammed MS, Gorre ME, Cotter PD, Kim J, Bahler DW, Preobrazhensky SN, Higgins RA, Bolla AR, Ismail SH, de Jong D, Eldering E, van Oers MH, Mellink CH, Keating MJ, Schlette EJ, Abruzzo LV, Robetorye RS. Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia. J Mol Diagn. 2008; 10(5): 442-51. PubMed

Ho AK, Hill S, Preobrazhensky SN, Miller ME, Chen Z, Bahler DW. Small B-cell neoplasms with typical mantle cell lymphoma immunophenotypes often include chronic lymphocytic leukemias. Am J Clin Pathol. 2009; 131(1): 27-32. PubMed

Kelley TW, Arber DA, Gibson C, Jones D, Khoury JD, Medeiros BC, O'Malley DP, Patel KP, Pilichowska M, Vasef MA, Wallentine J, Zehnder JL. Template for Reporting Results of Monitoring Tests for Patients With Chronic Myelogenous Leukemia (BCR-ABL1(+)). Arch Pathol Lab Med. 2016; 140(7): 672-4. PubMed

Klinger M, Zheng J, Elenitoba-Johnson KS, Perkins SL, Faham M, Bahler DW. Next-generation IgVH sequencing CLL-like monoclonal B-cell lymphocytosis reveals frequent oligoclonality and ongoing hypermutation. Leukemia. 2016; 30(5): 1055-61. PubMed

Li D, Xu X, Matynia AP. Systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) involving chronic myelogenous leukemia with complex cytogenetics: A case report and literature review.. San Diego Society of Hematopathology. [Published: Mar 2016; Accessed: Apr 2017]

Preobrazhensky SN, Bahler DW. Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2008; 74(2): 118-27. PubMed

Preobrazhensky SN, Szankasi P, Bahler DW. Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies. Cytometry B Clin Cytom. 2012; 82(2): 78-84. PubMed

Salama ME, Swierczek SI, Tashi T, Warby CA, Reading S, Prchal JT. Calreticulin mutated prefibrotic-stage myelofibrosis and PMF represent an independent clone from coexisting CLL. Blood. 2014; 124(10): 1691-2. PubMed

Swierczek S, Nausova J, Jelinek J, Liu E, Roda P, Kucerova J, Jarosova M, Urbankova H, Indrak K, Prchal JT, Divoky V. Concomitant JAK2 V617F-positive polycythemia vera and B-cell chronic lymphocytic leukemia in three patients originating from two separate hematopoietic stem cells. Am J Hematol. 2013; 88(2): 157-8. PubMed

Szankasi P, Bahler DW. Clinical laboratory analysis of immunoglobulin heavy chain variable region genes for chronic lymphocytic leukemia prognosis. J Mol Diagn. 2010; 12(2): 244-9. PubMed

Szankasi P, Schumacher JA, Kelley TW. Detection of BCR-ABL1 mutations that confer tyrosine kinase inhibitor resistance using massively parallel, next generation sequencing. Ann Hematol. 2016; 95(2): 201-10. PubMed

Velusamy T, Palanisamy N, Kalyana-Sundaram S, Sahasrabuddhe AA, Maher CA, Robinson DR, Bahler DW, Cornell TT, Wilson TE, Lim MS, Chinnaiyan AM, Elenitoba-Johnson KS. Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2013; 110(8): 3035-40. PubMed

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Last Update: October 2017