Background on AML

To begin, let's learn something about AML.

Click the "next" button to proceed through each section.

Case Introduction

• The following case example will be used to illustrate the application of flow cytometry in the diagnosis of AML.
• As you work through the case, you will be asked questions about key points.
• Give it your best guess, and learn from the response. These case-based questions will not be graded.
• Your knowledge will be tested/graded at the end of the e-learning activity.

Clinical History

• A 29 year-old pregnant female (G3P1A1) presented at ~6 weeks gestation with night sweats and nausea for one week.
• She had also experienced an episode of gum bleeding while brushing her teeth approximately one week prior.
• She was found to have leukocytosis, anemia and thrombocytopenia on routine CBC:
  
  
• Peripheral blood smear review showed blasts.

Flow Cytometry

• A peripheral blood specimen was received in the flow cytometry lab.
• Four color flow cytometry was performed using a BD FACSCalibur™ flow cytometer.
• Ungated, cluster analysis was performed with BD Paint-a-Gate™ software.
• FCS files in FCS2.0 format are also available for download and analysis in your own lab.
• Antibody tubes with the following antibody combinations are included for review.
  
  
  *Intracellular tube

Which antibody combination would you use to identify blasts?

Blast Identification Summary

• Acute leukemias are often first recognized on a CD45 versus SSC plot.
• CD45 versus SSC allows one to generate a rough 4 part differential.
  • Lymphocytes, monocytes, myeloid forms, and “blast” area (dim CD45 and low SSC).
    
• Most blasts have decreased CD45 expression.
• However, cells present in the “blast gate” are not all blasts.
  • Basophils, plasmacytoid dendritic cells (PDC), hypogranular myeloid cells, early monocytic cells.
    
• Therefore, specific markers of blasts are required for definitive identification of blasts (usually CD34 and CD117).
• Additionally, markers are required to differentiate blasts from cells that may be contaminating the blast gate.
• Also of note, not all blast equivalents fall into a standard CD45 versus SSC blast gate.
  • Immature monocytic cells
  • monoblasts, promonocytes
  • Abnormal promyelocytes
    

Blast Cell Gate Video

More information about the blast gate can be found in this video. You can access the full Blast Cell Gate video by logging into your ICCS account and selecting "Member Services".

Enumeration of Blasts

Phenotype of Blasts

What is the phenotype of the blasts?

Summary of Phenotype

This case shows a 15% population of variably-sized blasts with the following immunophenotype:

CD34 (+)
CD2 (few +)
CD13 (+)
CD15 (partial +)
CD25 (few +)
CD36 (few +) CD38 (variably +)
CD45 (moderately +)
CD64 (few +)
CD117 (+)
CD123 (variably +)
HLA-DR (variably +)
MPO (dim +)
TdT (-)
Other myeloid and lymphoid antigens pre-dominantly (-)

Commonly Used Antigens for Flow Cytometric Evaluation of AML

Progenitor cell CD34, CD117 CD38, HLA-DR Myeloid CD13, CD33, CD15, MPO, CD11b, CD16 Monocytic CD33, CD14, CD64, CD36

Erythroid CD235a (glycophorin A), CD71, CD36 Megakaryocytic CD41, CD61, CD36 Lymphoid CD19, CD79a, CD10, cytoplasmic CD3

Recommendations of Bethesda International Consensus Conference.

• Initial assessment of a new acute leukemia requires evaluation of myelomonocytic and lymphoid cells
  • Myelomonocytic cells: CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, and HLA-DR
  • B cells: CD5, CD10, CD19, CD20, CD45, kappa, and lambda
  • T cells: CD2, CD3, CD4, CD5, CD7, CD8, CD45 and CD56
• Additional markers should be used on an as needed basis.

How is Blast Aberrancy Determined?

• Normal myeloblasts show a reproducible maturation pattern, with myeloid antigen expression at distinct levels depending on the stage of maturation.
• Aberrant myeloblasts (such as those found in AML) deviate from the normal immunophenotype.
• This case shows 15% myeloblasts with immunophenotypic aberrancies including an unusual expression pattern for CD15/CD33/CD34 as well as slightly dim expression of CD38 and HLA-DR.
• In addition to the aberrancy identified on the blasts, there is an expanded population of monocytes with increased variability of expression of CD11b, CD13, CD14, CD36, CD64 and HLA-DR, and with a minute proportion showing expression of CD34 and CD117.
• Please see following discussion for further information on normal and abnormal blast immunophenotypes.

Normal Myeloid Blast Maturational Patterns

• Once myeloid blasts have been specifically identified, one needs markers to distinguish normal from abnormal myeloid blasts.
  • Abnormal blast immunophenotypes have been designated leukemia associated immunophenotypes (LAP or LAIP) in the literature.
• Identifying a LAP can be helpful in distinguishing marrsow regeneration from a myeloid stem cell disorder and is particularly important for MRD testing at follow-up post therapy.
• Detecting abnormalities on myeloid blasts requires recognition of normal patterns of antigen expression during myeloid maturation.
• Similar to the changes in morphology noted with maturation, hematopoietic cells demonstrate predictable, conserved changes in antigen expression with normal maturation.
• Such changes are seen at all stages of development with transition from early progenitor to early myeloid blasts to maturing myeloid, monocytic or erythroid elements.
  
  
  
  
• Early progenitor cells (*) express very high levels of CD34 and low to absent CD38.
• With maturation, these early blasts gradually drop CD34 as they acquire CD38.
• As CD38 reaches maximal “blast” levels, CD34 drops precipitously and markers associated with maturation and differentiation (such as CD15 for myeloid differentiation and maturation), are acquired.
  
  
  
  
• Early progenitor cells (*) express very high levels of CD34, dim CD33 and no CD15.
• With maturation, these early blasts gradually drop CD34 as they acquire CD33 and CD15.
• Blasts are shown in red; granulocytes and monocytes are in green and blue, respectively.
• Small lymphocytes are shown in cyan.
  
  
  
  
• Normal myeloblasts are negative for myeloid maturation markers, CD16 and CD11b.
• The arrow shows the normal granulocytic maturation curve.
• Blasts are shown in red; granulocytes and monocytes are in green and blue, respectively.
• Small lymphocytes are shown in cyan.
  
  

Abnormal Immunophenotype

• Abnormalities seen on myeloid progenitors in AML (and in other myeloid stem cell disorders) generally fall into one of 4 categories. Click on each to view:
  • • Abnormal intensity of antigen expression (increased, decreased or absent)
  • • Asynchronous expression of markers associated with maturity and markers associated with immaturity
  • • Homogeneous expression of an antigen usually expressed at varying levels through blast maturation
  • • Expression of non-lineage antigens
• Identification of abnormal antigen expression is particularly helpful when performing evaluation for minimal residual disease (MRD) post therapy.

Overall Immunophenotypic Findings in Case Example

• 15% population of variably-sized blasts with the following immunophenotype:
• CD34 (+), CD2 (few +), CD13 (+), CD15 (partial +), CD25 (few +), CD36 (few +), CD38 (variably +), CD45 (moderately +), CD64 (few +), CD117 (+), CD123 (variably +), HLA-DR (variably +), MPO (equivocal/dim +), TdT (-), other myeloid and lymphoid antigens pre-dominantly (-).
• 54% monocytes with variable expression of CD11b, CD13, CD14, CD36, and HLA-DR and with a minute proportion showing expression of CD34 and CD117

Final Diagnosis

Diagnosis

Definition

• A Core Binding Factor (CBF) Leukemia:
  • Defined by a recurrent cytogenetic abnormality irrespective of blast count at diagnosis - inv(16)(p13q22) or t(16;16)(p13;q22).
  • This is one of the genetic alterations that allow making a diagnosis of AML even with <20% blasts.
• WHO 2008 Classification Definition: Acute myelomonocytic leukemia with abnormal eosinophils (AML-M4Eo by FAB) in the bone marrow

Morphology

• Heterogeneous neoplastic population of myeloblasts, monoblasts & promonocytes.
• Maturing granulocytes are sparse and do not show dysplasia.
• Hypercellular marrow often with more than 20% blasts
  • May be lower than 20% in some cases
• Auer rods may be seen in blasts.
• Most striking abnormality: Eosinophils and eosinophilic promyelocytes and myelocytes have large immature basophilic granules.

Immunophenotype

• Double population of pathological cells:
  • Myeloid blast cells: CD34, CD117, CD13, CD33, CD15, myeloperoxidase.
  • Monocytic cells: CD4, CD11b, CD11c, CD14, CD64, CD36, lysozyme
• Aberrant coexpression of CD2 in the blast population and monocytic cells occurs in a subset of cases but it is not specific for this type of AML

Prognosis

• Overall good prognosis
• Good response to Cytarabine-based chemotherapy
• KIT mutations are present in approximately 30% of cases
  • Worse overall survival and higher rates of relapse
• Trisomy 22 - Specific and seen in 10-15% cases favorable outcome

Learning Assessment

Now that you’ve completed the e-learning activity, assess your knowledge with the following graded questions.

Summary

• Flow cytometry is a critical part of the diagnosis of AML
• Flow cytometry can facilitate:
  • Blast identification
  • Lineage assignment
  • AML classification
• However, blast enumeration in the marrow should be done by morphology.
• Genetic information also assists with final classification, such as in the example of AML with inv16.

Suggested Reading

• Eds. Steven Swerdlow EC, Nancy Lee Harris, Elaine S. Jaffe, Stefano A. Pileri, Harald Stein, Jurgen Thiele, and James W. Vardiman. World Health Organization Classification of Tumors: WHO classification of tumors of haematopoietic and lymphoid tissues, 4th edition. Lyon, 2008.
• Wood BL. Myeloid malignancies: myelodysplastic syndromes, myeloproliferative disorders, and acute myeloid leukemia. Clin Lab Med. Sep 2007;27(3):551-575, vii.
• Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. Apr 15 2008;111(8):3941-3967.
• Al-Mawali A, Gillis D, Lewis I. The role of multiparameter flow cytometry for detection of minimal residual disease in acute myeloid leukemia. Am J Clin Pathol. Jan 2009;131(1):16-26.
• Wood BL, Arroz M, Barnett D, et al. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia. Cytometry B Clin Cytom. 2007;72 Suppl 1:S14-22.
• Kussick SJ, Wood BL. Using 4-color flow cytometry to identify abnormal myeloid populations. Arch Pathol Lab Med. Sep 2003;127(9):1140-1147.