Case Studies

Acute Myelogenous Leukemia with Cuplike

Nuclei
Erica Robinson, BS, MLS(ASCP)CM 1* and Carleen Van Siclen, MS, MLS(ASCP)CM  2

Downloaded from https://academic.oup.com/labmed/article/46/4/e93/2937907 by guest on 28 February 2021

Lab Med Fall 2015;46:e93-e97

DOI: 10.1309/LMXC433INJAYLSTD

Clinical History
Patient: 75-year-old white man.
Chief Compliant: Shortness of breath, coughing. After several weeks
with an unrelenting infection and cough, he was referred to the
emergency department by his primary care physician.
History of Present Illness: None reported.
Medical History: Osteoarthritis.
Family History: Noncontributory.
Physical Examination: Positive for shortness of breath, dyspnea on
exertion, chest pain, generalized weakness, and malaise, with no other
significant findings.
Principle Laboratory Findings: Table 1.
Additional Diagnostic Testing: Flow cytometry and cytogenetic
testing (tachycardia revealed via electrocardiogram [ECG]; chest
X-ray with left pleural effusion; upper right lobe and right middle lobe
pulmonary emboli revealed via computed tomography [CT] angiogram
of chest).
Keywords: acute myelocytic leukemia, acute myeloid leukemia
without differentiation (M1), cuplike nuclei, flow cytometry,
cytogenetics, FLT3 and NPM1 mutation, PML/RARα

Questions
1. What are the clinically significant laboratory findings for
our patient?
Abbreviations:
2. What do the patient’s peripheral blood smear and bone-
ECG, electrocardiogram; CT, computed tomography; M1, acute myeloid
leukemia without differentiation; WBC, white blood cells; WHO, marrow differential reveal?
World Health Organization; CK-MB, creatine kinase–myocardial band;
CBC, complete blood count; APL, acute promyelocytic leukemia; 3. What is the differential diagnosis?
AML, acute myelocytic leukemia; ALL, acute lymphocytic leukemia;
CD, cluster of differentiation; HLA-DR, human leukocyte antigen–D 4. What do the flow cytometry results reveal?
related; RARα, retinoic acid receptor alpha; FISH, fluorescence in situ
hybridization; PCR, polymerase chain reaction; NPM1, nucleophosmin
5. What other laboratory tests are useful in making a
1; FLT3, FMS-like tyrosine kinase; ITD, internal tandem duplication;
RBC, red blood cells; MCV, mean corpuscular volume; MCH, mean definitive diagnosis?
corpuscular hemoglobin; MCHC, mean cell hemoglobin concentration;
RDW, red blood cell distribution width; MPV, mean platelet volume; PB, 6. What is the patient’s definitive diagnosis, and what
peripheral blood; PT, prothrombin time; INR, International Normalized is the relationship between cuplike nuclei and FLT3 and
Ratio; PTT, partial thromboplastin time; ..., not applicable; Tdt, terminal
deoxynucleotidyl transferase. PML, promyelocytic leukemia; FAB, NPM1 mutations?
French-American-British classification
1
Medical Lab Science, Department of Biology, University of North
Florida Medical Laboratory Science, Jacksonville, FL
Possible Answers
2
Department of Laboratory Medicine and Pathology, Mayo Clinic
College of Medicine, Jacksonville, FL 1. Table 1 demonstrates the patient’s principal laboratory
*To whom correspondence should be addressed. findings. The most clinically significant findings are the
erica.l.robinson22@gmail.com critical laboratory values, including critically high white

www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  e93

Case Studies

central pallor that is clefted, indented, folded, or “fish-

Table 1. Principal Laboratory Findingsa
mouthed,” with loose chromatin and 1 to 3 nucleoli.
Patient
Mehtap et al,2 Kussick et al,3 and Bennett et al4 described
Test Results Reference Interval Interpretation
cuplike nuclei as having a nuclear invagination spanning
Hematology
WBC 354.14 4.50-11.00 x 103/μL Critical
25% or greater of the nuclear diameter and in more than
RBC 3.11 4.30-5.90 x 106/μL L 10% of the blasts. Immunophenotypically, this unusual
Hemoglobin 7.6 13.5-17.5 g/dL L variant is similar to the microgranular variant of acute
Hematocrit 24.6 41.0%-53.0% L promyelocytic leukemia (APL). However, promyelocytes

Downloaded from https://academic.oup.com/labmed/article/46/4/e93/2937907 by guest on 28 February 2021

MCV 85 80-100 fL N
MCH 26.2 25.0-35.0 pg N
do not resemble the blasts because morphologically, they
MCHC 30.8 30.0-37.0 g/dL N have cuplike nuclei.
RDW 19.8 11.5%-15.5% H
Platelets 15 150-450 x 103/μL Critical The bone marrow aspirate and biopsy (Image 2) revealed
MPV 9.5 7.0-11.0 fL N similar cellular distribution and blast morphology (Table
PB Differential
2). We did not perform cytochemical stains.
Neutrophils 1 40%-75% L
Lymphocytes 3 15%-45% L
Bands 2 0-2% N
3. The differential diagnosis includes acute myelocytic
Blasts 94 0% Critical leukemia (AML) and acute lymphocytic leukemia (ALL).
Coagulation Based on the morphology of the blasts, it is difficult
PT 15.5 11.8-15.0 sec H to determine cell lineage. Therefore, we performed
INR 1.2
immunophenotyping by flow cytometry on the peripheral
PTT 30.4 22.9-37.8 sec N
Chemistry blood specimen. The results are displayed in Table 3.
CK-MB 18.4 0-6.3 ng/mL H
Troponin I 4.73 0-.05 ng/mL Critical 4. ALL can be ruled out from the patient’s diagnosis
WBC, white blood cells; L, Low; RBC, red blood cells; MCV, mean corpuscular because of the lack of cell markers for cluster of
volume; N, Normal; MCH, mean corpuscular hemoglobin; MCHC, mean cell differentiation (CD)2, CD3, CD5, CD7, CD8, CD10, CD19,
hemoglobin concentration; RDW, red blood cell distribution width; H, High;
MPV, mean platelet volume; PB, peripheral blood; PT, prothrombin time; INR, CD20, and CD22. The aberrant population of cells
International Normalized Ratio; PTT, partial thromboplastin time; CK-MB, creatinine
kinase–myocardial band.
expresses CD33, whereas a subpopulation expresses
a
Our patient is a 75-year-old white man. CD13. Due to an absence of ALL markers and the
presence of CD33 and CD13, the abnormal cells can be
assigned to the myeloid lineage.
blood cell (WBC) count, critically low platelet count, 94%
blasts in the peripheral blood smear, and critically elevated Table 41,5-8 compares the patient’s immunophenotype
troponin I level. Leukocytosis and thrombocytopenia to the World Health Organization (WHO) classification
accompanied by a finding of 20% or greater blasts is of acute myeloid leukemia (AML) subtypes. The
indicative of acute leukemia, according to the World patient’s immunophenotype is most often observed
Health Organization (WHO) classification.1 Elevated in acute promyelocytic leukemia (APL), in which the
troponin I and creatine kinase–myocardial band (CK-MB) immunophenotypic expression of the abnormal myeloid
levels indicate cardiac muscle damage. The portable chest population is positive for CD33 and negative for human
x-ray and computed tomography (CT) angiogram results leukocyte antigen–D related (HLA-DR) and CD34.1
showed right upper lobe and right middle lobe pulmonary
emboli. However, the electrocardiogram reading was However, APL is typically associated with promyelocytes
normal, revealing no cardiac damage. in the peripheral blood smear. The promyelocytes typically
have heavy granulation and bundles of Auer rods or can
2. The complete blood count (CBC) findings are described be hypogranular with bilobed, angel-winged nuclei.9 APL
in Table 1. The erythrocytes were normocytic and is a subtype of AML characterized by a predominance
normochromic, with moderate anisocytosis. The peripheral of promyelocytes and a diagnostic gene rearrangement,
blood smear revealed approximately 94% blasts. We retinoic acid receptor alpha (PML/RARα).9
observed no Auer rods in the blasts (see Image 1, parts
A-C). The blasts have scant, pale blue-gray, agranular 5. Also, a reference laboratory performed fluorescence
cytoplasm and cuplike nuclei. The cuplike nuclei have a in situ hybridization (FISH) and cytogenetic studies on

e94  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com


the patient’s bone marrow to differentiate the diagnosis
between APL and another AML subgroup. FISH analysis
demonstrated a negative presence for the fusion of
promyelocytic leukemia and retinoic acid receptor alpha
genes (PML/RARα). This fusion is present in approximately
97% of patients with APL. Due to a lack of this fusion
and a lack of promyelocytes displayed in the patient’s
peripheral blood and bone-marrow biopsy, APL was thus
excluded from the patient’s diagnosis.10
Further genetic aberrations, NPM1 and FLT3, were
investigated. Cytogenetic mutation analysis results
via polymerase chain reaction (PCR) were positive for
www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  e95
Case Studies
Downloaded from https://academic.oup.com/labmed/article/46/4/e93/2937907 by guest on 28 February 2021
Image 1
Acute leukemia as seen in a peripheral blood smear from our
patient, a 75-year-old white man. The smear demonstrates pre-
dominance of blasts, with Auer rods not seen (A; original magni-
fication, ×100); blasts with central indentation (B; original magni-
fication ×1000); and multiple blasts with cuplike nuclei (C; original
magnification, ×1000).
nucleophosmin 1 (NPM1) insertion mutation at position
959 of exon 12 and negative for FMS-like tyrosine
kinase (FLT3) mutation. The presence of NPM1 and
FLT3 mutations usually co-occur and accompany the
variant form of AML that has cuplike nuclei. NPM1 by
itself has a positive predictive value of approximately
86%.11 Approximately one-third of adults with AML
carry the NPM1 mutation, which is now included in the
fourth edition of the World Health Organization (WHO)
classification.5 The immunophenotypes associated with
this mutation are controversial, but Chen et al11,12 have
discovered in patients with M1 polymorphism who have
the NPM1 mutation an absence of human leukocyte
Case Studies

Table 3. Immunophenotype of Peripheral Blood

via Flow Cytometry
Predominant Approximate %
Variable Reactivity Cells Positive Interpretation
CD3 Mature T cells 0 Negative
CD4 Helper T cells 1 Negative
CD5 T cells 0 Negative
CD7 T cells 1 Negative

Downloaded from https://academic.oup.com/labmed/article/46/4/e93/2937907 by guest on 28 February 2021

CD8 Suppressor T cells 0 Negative
CD10 Common ALL antigen 0 Negative
CD13 Panmyeloid cells 5 Negative (5%+)
CD14 Monocytes 2-3 Negative
CD15 Monocytes 4 Negative (4%+)
Image 2 CD16 Natural killer cells 1 Negative
CD19 Pre-B and B cells 0 Negative
Acute leukemia, as seen in a bone-marrow biopsy smear from our
CD20 B cells 0 Negative
patient, a 75-year-old white man. The smear expresses predomi- CD22 B cells 2 Negative
nantly blasts, which are morphologically similar to those observed CD23 B cells 1 Negative
in the patient’s peripheral blood (original magnification, ×100). CD33 Panmyeloid cells 75 Positive
CD34 Stem cells/blasts 0 Negative
CD36 Monocytes 1 Negative
CD38 General activation/ 46-63 Positive
Table 2. Bone-Marrow Differential in Our plasma cells
Patient, a 75-Year-Old White Man CD45 Total leukocytes 69 Positive
(subpopulation)
Differential % Cells
CD56 Natural killer cells 3 Negative
Blasts 79 HLA-DR … 2 Negative
Granulocytic precursors 17 Tdt … 0 Negative
Lymphocytes 3
CD, cluster of differentiation; ..., not applicable; ALL, acute lymphoblastic leukemia;
Erythrocytes 1 HLA-DR, human leukocyte antigen–D-related; Tdt, terminal deoxynucleotidyl
Segments and bands 0 transferase.

antigen–D related (HLA-DR) and cluster of differentiation
(CD)34, similar to our patient.13-15
6. The definitive diagnosis is acute myeloid leukemia
(AML) without differentiation (M1) with cuplike nuclei.
Similarly, Park et al 9 referred to cuplike nuclei as most
often occurring in AML M1. Although the relationship
between cuplike nuclei and nucleophosmin 1 (NPM1)
and FMS-like tyrosine kinase (FLT3) mutations is highly
debated, some studies have demonstrated a correlation
with these mutations by themselves or in tandem.
Bennett et al4 claim that the NPM1 gene mutation is
the most common genetic alteration in AML. A report
published by the ASCP also reports that as many as
50% to 60% of case individuals with normal karyotypes
carry the NPM1 mutation.5 NPM1 encodes for a
shuttle protein between the nucleolus and cytoplasm,
which controls cell cycle and regulates centromere
duplication to facilitate mitosis.5 Some studies 4,5,11-13
reveal an association of cuplike AML with NPM1 and
FLT3–internal tandem duplication (FLT3-ITD) mutations,
less expression of human leukocyte antigen–D related
(HLA-DR), and an absence of cluster of differentiation
(CD)34 expression.
A high correlation of cuplike nuclei and NPM1 mutation
has been demonstrated by various studies.11-13 NPM1
mutations are further associated with a normal karyotype,
an internal tandem duplication of the FLT3 gene, a higher
white blood cell (WBC) count in the peripheral blood,
a higher fraction of blasts in the bone marrow, lower
expression of CD34 antigen, and female sex.4,9 Many of
these characteristics are similar to our patient’s leukemic
expression.
FLT3-ITD mutations are well documented as being a poor
prognostic indicator rather than a diagnostic factor.5,16
Absence of FLT3-ITD mutations in AML correlates with
an improved outcome.4 NPM1 mutations are favorable
prognostic indicators in patients with normal karyotypes,
whereas those with FLT3 mutations have a poor
prognostic factor in AML.9

e96  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com

 Case Studies

Table 4. Immunophenotypes Associated with the WHO Classification of AML of Myeloid Origin
Compared with the Patient’s Cuplike AML Varianta
Antigen Markers
AML Group AML Subgroup CD13 CD14 CD15 CD33 CD34 CD36 CD38 CD45 HLA-DR
Patient’s cuplike AML variant +/- - +/- + - - + + -
AML with recurrent APL, AML with PML/RARα, and variants + - - + - - - + -
genetic abnormalities (FAB M3)

Downloaded from https://academic.oup.com/labmed/article/46/4/e93/2937907 by guest on 28 February 2021

AML not otherwise AML minimally differentiated (FAB M0) + - - + + - - + +
categorized
AML without maturation (FAB M1) + - - + + - - + +
AML with maturation (FAB M2) + - + + v - - + V
Acute myelomonocytic leukemia (FAB M4) + + + + v + - + -
WHO, World Health Organization; AML, acute myeloid leukemia; CD, cluster of differentiation; HLA-DR, human leukocyte antigen–D-related; APL, acute promyelocytic leukemia;
PML, promyelocytic leukemia; RARα, retinoic acid receptor alpha; FAB, French-American-British classification; V, variable.

7. Nguyen DT, Diamond LW, Braylan RC. Flow Cytometry in

Acknowledgments
We thank Mark Steciuk, MD, PhD, of, Jacksonville
Pathology Consultants for providing photography and
mentoship.
References
1. McKenzie SB, Williams JL. Clinical Laboratory Hematology, 2nd
edition. Upper Saddle River, NJ: Pearson Education. 2010:505-523.
2. Mehtap O, Atesoglu E, Gonullu E, Keski H, Hacihanefioglu A. Are
cup-like blasts specific to AML patients with FLT3 ITD and a normal
karyotype? An ALL case report and review of the literature. Turk J
Hematol. 2011;28:142-145.
3. Kussick SJ, Stirewalt DL, Yi HS, et al. A distinctive nuclear
morphology in acute myeloid leukemia is strongly associated with
loss of HLA-DR expression and FLT3 internal tandem duplication.
Leukemia. 2004;18:1591-1598.
4. Bennett JM, Pryor J, Laughlin TS, Rothberg PG, Burack WR. Is
the association of “cup-like” nuclei with mutation of the NPM1
gene in acute myeloid leukemia clinically useful? Am J Clin Pathol.
2010;134:648-652.
5. Foucar K, Reichard K, Czuchlewski D. Chapter 18: Acute Myeloid
Leukemia. ASCP website. http://www.ascp.org/PDF/Books/
Chapter-18.pdf. Accessed September 21, 2015.
6. Jaffe E, Harris N, Stein H, Vardiman J. World Health Organization
Classification of Tumours: Pathology and Genetics, Tumours of
Haematopoietic and Lymphoid Tissues. Lyon, France. IARC Press.
2001:77-95.
Hematopathology: A Visual Approach to Data Analysis and
Interpretation. Totowa, NJ: Humana Press; 2002:187-191.
8. Ismail MA, Hosny SM. Prognostic significance of progenitor
cell markers in acute myeloid leukemia. Life Science Journal.
2011;8(4):680-686.
9. Park BG, Chi H-S, Jang S, et al. Association of cup-like nuclei in
blasts with FLT3 and NPM1 mutations in acute myeloid leukemia.
Ann Hematol. 2013;92:451-457.
10. Dekking EHA, van der Velden VHJ, Varro R, et al. Flow cytometric
immunobead assay for fast and easy detection of PML-RARA fusion
proteins for the diagnosis of acute promyelocytic leukemia. Leukemia.
2012;26:1976-1985.
11. Chen W, Konoplev S, Medeiros J, et al. Cuplike nuclei (prominent
nuclear invaginations) in acute myeloid leukemia are highly associated
with FLT3 internal tandem duplication and NPM1 mutation. Cancer.
2009;115:5481-5489.
12. Chen W, Rassidakis GZ, Li J, et al. High frequency of NPM1
gene mutations in acute myeloid leukemia with prominent nuclear
invaginations (“cuplike” nuclei). Blood. 2006;108:1783-1784.
13. Kroschinsky FP, Schakel U, Fischer R, et al. Cup-like acute myeloid
leukemia: new disease or artificial phenomenon? Haematologica.
2008;93:283-286.
14. Liu Y-R, Zhu H-H, Ruan G-R, et al. NPM1-mutated acute
myeloid leukemia of monocytic or myeloid origin exhibit distinct
immunopheotypes. Leuk Res. 2013;37:737-741.
15. Syampurnawati M, Tatsumi E, Ardianto B, et al. DR negativity is a
distinctive feature of M1/M2 AML cases with NPM1 mutation. Leuk
Res. 2008;32:1141-1143.
16. Port M, Böttcher M, Thol F, et al. Prognostic significance of FLT3
internal tandem duplication, nucleophosmin 1, and CEBPA gene
mutations for acute myeloid leukemia patients with normal karyotype
and younger than 60 years: a systematic review and meta-analysis.
Ann Hematol. 2014;93(8):1279-1286.

www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  e97