Immunophenotyping, Cytogenetic, and Mutational Analysis


Immunophenotyping refers to tests used to identify unique cell types based on the types of antigens or markers on the cell’s surface.1 Such tests are employed at the initial diagnostic workup and are essential to confirm a diagnosis of FL.2

Immunophenotype, in addition to histology and cytogenetic studies, helps establish the diagnosis of FL.2 The immunophenotype characteristic of FL is shown in the table below.

Subtype Typical immunophenotype2
Follicular lymphoma (FL) CD10+, BCL2+, CD23+/-, CD43-, CD5-, CD20+, BCL6+

Two common immunophenotyping tests are immunohistochemistry (IHC) and flow cytometry (FC).2

Immunohistochemistry (IHC)3

  • A lab test that uses labeled antibodies to test for certain antigens (markers) in a tumor sample
  • Antibodies are usually linked to an enzyme or a fluorescent dye
  • When the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can be seen under a microscope

Flow Cytometry (FC)3

  • A method of characterizing the antigens in a tumor sample by staining them with a light-sensitive dye, placing them in a fluid, and passing them in a stream before a laser or other type of light
  • The measurements are based on how the light-sensitive dye reacts to the light
  • FC also measures the number of cells in a sample, the percentage of live cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface

Additional IHC Markers in FL

In addition to assessing chromosomal rearrangements and the markers required to establish a diagnosis of FL, IHC can be used to measure the expression of different molecular markers, and may be an important part of the workup for a patient with FL.2

Some examples of IHC markers that are tested in a patient with suspected FL include2:

  • IRF4/MUM1 (for FL grade 3)
  • Cyclin D1 (to exclude MCL)
  • Ki-67 (Ki-67 expression is associated with a high proliferation index. A Ki-67 fraction of >30% may be associated with more aggressive clinical behavior; however, there is no evidence that Ki-67 expression should be used to guide treatment)


Cytogenetic tests, which combine principles of cytology (the study of cells using a microscope) and genetics, may be useful in certain circumstances for the diagnosis of FL. Genetic abnormalities may be detected by karyotyping (examination of chromosomes in a sample of cells) or fluorescent in-situ hybridization (FISH).2,3

  • Karyotyping: a method by which chromosomes are visualized, allowing detection of large rearrangements or abnormalities4,5
  • FISH: a method that detects the presence of specific proteins in a biologic sample using fluorescently-tagged antibodies that bind to antigens of interest6

During the initial diagnostic workup of a patient, FISH and/or karyotyping may help establish the diagnosis of FL.1 In FL, the translocation between chromosomes 14 and 18—t(14;18)—occurs in ~90% of all cases.7

t(14;18) detected by FISH

Reproduced with permission from Atlas of Haematological Cytology

t(14;18) detected by Karyotype

Reproduced with permission from Atlas of Genetics and Cytogenetics in Oncology and Haematology.

Molecular Profiling

Molecular analysis may be useful during diagnosis of FL.2 If these tests are ordered, DNA is isolated from a patient and its entire sequence is determined or a panel of known cancer-related genes are sequenced.8,9 Such profiling will detect changes in specific genes in the tumor cells, which may inform diagnosis, prognosis, and treatment.8,10

  • While mutations have been observed in FL that may be related disease biology, these are still being elucidated. There are currently no known mutations that can be used to define FL11

References: 1. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms Blood. 2008;111(8):3941-3967. 2. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for B-Cell Lymphomas V.4.2019. © National Comprehensive Cancer Network, Inc. 2019. All rights reserved. Accessed July 10, 2019. To view the most recent and complete version of the guideline, go online to NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way. 3. National Cancer Institute. NCI Dictionary of Cancer Terms. Accessed June 12, 2019. 4. Gulley ML et al. Genetic Tests To Evaluate Prognosis and Predict Therapeutic Response in Acute Myeloid Leukemia. J Mol Diagn. 2010;12(1):3-16. 5. O'Connor, C. Karyotyping for chromosomal abnormalities. Nature Education. 2008;1(1):27. 6. Bishop R. Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance. Bioscience Horizons. 2010;3(1):85-95. 7. Leich E, et al. Follicular lymphomas with and without translocation t(14;18) differ in gene expression profiles and genetic alterations. Blood. 2009; 114:826-834. 8. Johansen Taber KA, Dickinson BD, Wilson M. The promise and challenges of next-generation genome sequencing for clinical care. JAMA Intern Med. 2014;174(2):275-280. 9. Rosenquist R et al. Clinical impact of recurrently mutated genes on lymphoma diagnostics: state-of-the-art and beyond. Haematologica. 2016;101(9):1002-1009. 10. Ioannidis JPA. Is molecular profiling ready for use in clinical decision making? Oncologist. 2007;12:301-311. 11. Krysiak K, et al. Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma. Blood. 2017;129(4):473-483.