Chronic Lymphocytic Leukemia - CLL

Key Points

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.

For molecular markers in CLL, see table below.

Molecular Markers in CLL

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 and ZAP-70 expression

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

ZAP-70 expression – determine risk group in newly diagnosed CLL


ZAP-70 Analysis by Flow Cytometry 0092392

Intracellular protein tyrosine kinase mediates antigen receptor signaling in normal T-cells and appears to enhance B-cell antigen receptor signaling in CLL cells

Expression of ZAP-70 is associated with an unfavorable outcome 

ZAP-70 expression is strongly associated with nonmutated IGHV mutation status in CLL but may provide additional prognostic information in discordant cases

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
  • Serum testing – monoclonal antibody panels (minimal)
    • B cell – kappa/lambda, CD5/CD19, CD23, CD10, FMC7
    • T cell – CD3/CD4, CD3/CD8, CD5, CD7, 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
  • Prognostic factors for CLL





    Stage (Rai and Binet systems)

    • Rai 0, I
    • Binet A
    • Rai II, III, IV
    • Binet B or C

    Lymphocyte morphology



    Lymphocyte doubling time



    Serum markers

    • LD



    • Beta-2 microglobulin



    • Thymidine kinase



    • Soluble CD23



    Cytogenetics (FISH, SNP microarray)

    Genetic abnormalities

    • Normal diploid
    • del(13)(q14) only
    • del(17)(p13.1)
    • del(11)(q22.3)
    • Trisomy 12+
    Molecular Markers (FISH, PCR)
    • CD38 expression



    • ZAP-70 expression



    • IGVH gene mutation



    Note: p53 and KRAS mutations are associated with therapy resistance to alkylating agents, fludarabine, and tyrosine kinase inhibitors (eg, rituximab)

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

Clinical Background

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.


  • Incidence – ~15,000 new cases annually in U.S. (American Cancer Society, 2015)
  • Age – median 79 years
    • 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

Clinical Presentation

  • Symptoms
    • 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
    • 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
  • 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

Indications for Laboratory Testing

  • 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
Test Name and Number Recommended Use Limitations Follow Up
Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
Method: Flow Cytometry

Aid in evaluation of hematopoietic neoplasms (ie, leukemia, lymphoma)

Specimens include peripheral blood, bone marrow or tissue

Monitor therapy in patients with established diagnosis of hematopoietic neoplasms

Markers selected based on provided clinical history and/or previous test results

Antigens included

T cell: CD1, CD2, CD3, CD4, CD5, CD7, CD8, TCR α-β, TCR γ-δ,, cytoplasmic CD3

B cell: CD10, CD19, CD20, CD22, CD23, CD103, kappa, lambda, FMC7, cytoplasmic kappa, cytoplasmic lambda

Myelo/Mono: CD11b, CD13, CD14 (Mo2), CD14 (MY4), CD15, CD33, CD64, CD117, myeloperoxidase

Misc: CD11c, CD16, CD25, CD30, CD34, CD38, CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin, TdT, bcl-2, ALK-1, CD123, CD138, CD200, CD26, CD45

Clinical sensitivity – limit of detection 0.01-1.0% depending on phenotype and disease

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

Preferred test at time of diagnosis to detect prognostically important genomic abnormalities (loss/gain of DNA and/or loss of heterozygosity) in hematologic malignancies

Useful in patients with <20 normal metaphases or with no mitosis to consider on conventional cytogenetics

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)

Detect chromosome abnormalities in bone marrow aspirate

Reflex pattern – if chromosome analysis is "normal" or "no growth," then genomic microarray testing will be added

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

Alternate test for detection of prognostically important genomic gains and losses in CLL

Use for CLL prognostication

Includes ATM (11q22.3); chromosome 12 centromere (trisomy 12); D13S319 (13q14.3); p53 (17p13.1)

Not as sensitive as cytogenomic SNP microarray assay

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

Determine risk group for newly diagnosed CLL

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

ZAP-70 Analysis by Flow Cytometry 0092392
Method: Flow Cytometry

Determine risk group for newly diagnosed CLL

Assay results should not be used for diagnosis but may help in the clinical management of an established diagnoses of CLL

Results should always be correlated with morphologic and clinical information

p53 with Interpretation by Immunohistochemistry 0049250
Method: Immunohistochemistry

Determine risk group for newly diagnosed CLL

Stained and resulted by ARUP 

Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Initial test to determine degree of lymphocytosis

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic

Use for CLL prognostication

Chromosome Analysis, Bone Marrow 2002292
Method: Giemsa Band

Detect chromosome abnormalities in bone marrow

Complex abnormalities and unbalanced translocations are associated with a poorer prognosis

Chromosome Analysis, Leukemic Blood 2002290
Method: Giemsa Band

Detect chromosome abnormalities in peripheral blood

Complex abnormalities and unbalanced translocations are associated with a poorer prognosis

Chromosome Analysis, Leukemic Blood with Reflex to Genomic Microarray 2007131
Method: Giemsa Band/Genomic Microarray (Oligo-SNP array)

Detect chromosome abnormalities in leukemic blood

Reflex pattern – if chromosome analysis is "normal" or "no growth," then genomic microarray testing will be added

CD19 by Immunohistochemistry 2005114
Method: Immunohistochemistry

Aid in histologic diagnosis of B-cell leukemia/lymphoma

Stained and returned to client pathologist for interpretation; consultation available if needed