Case of the Quarter – Aug 2013

Daimon Simmons, MD, PhD, Paola Dal Cin, PhD, and Frank Kuo, MD, PhD

Department of Pathology Brigham and Women's Hospital

75 Francis Street Boston, MA 02115

Diagnosis: Acute Myeloid Leukemia, NOS,

with MYC Amplification and Promyelocytic Differentiation

Introduction:

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks

of genomic amplification in cancer, more common in solid tumors than in hematologic

malignancies. Double minute chromosomes are seen primarily in acute myeloid leukemia, with

amplification of the MYC oncogene or, less frequently, the MLL transcription factor (Rayeroux

and Campbell 2009). Both normal and abnormal karyotypes were reported together with

dmin, and rarely with hsr. It is associated with a poor prognosis, rapid disease progression and

short survival , however only in case reports and small series (Thomas et al, 2004, Receveur et al

2004).

Here we present a case of AML with morphologic and immunophenotypic features resembling

acute promyelocytic leukemia (APL) that has amplification of MYC present on dmin but no

translocation, t(15;17)(q22;q12) or fusion of PML-RARA.

Clinical History:

The patient is a 67 year old man who presented with a several month history of shortness of

breath, increased bruising, and acute swelling of his legs and toes. He reported frequent bruises

on his arms and legs, but denied gingival bleeding. Ultrasound evaluation of the lower

extremities for deep vein thrombosis was negative. His CBC with manual differential counts

revealed mild anemia, thrombocytopenia and circulating immature myeloid forms with large

number of promyelocytes (Hgb 12.2 g/dl, Plt 80 k/µl, WBC 11 k/µl with 24% promyelocytes,

26% myelocytes, 3% metamyelocytes, 24% neutrophils, 18% lymphocytes, 3% monocytes) and

coagulation studies were within normal limits (PT 14.0, INR 1.1, PTT 29.1, Fibrinogen 300

µg/L).

After an initial bone marrow examination, the patient was admitted with the presumptive

diagnosis of APL and initiated treatment with all-trans retinoid acid (ATRA) on admission.

After the results of the full acute leukemia workup became available, his chemotherapeutic

regimen was changed from ATRA to 7+3 induction chemotherapy with cytarabine and

daunorubicin. A subsequent bone marrow biopsy revealed persistent disease and re-induction

was initiated. His clinical course has since been complicated by neutropenic fevers and small

bowel obstruction.

Acute Leukemia Workup:

Pathology of bone marrow aspirate and biopsy

Examination of the bone marrow aspirate smear revealed immature myeloid forms, many with

abundant eosinophilic granules (Figure 1A). The bone marrow differential showed 83% blasts

and abnormal promyelocytes, 10% maturing myeloids, 4% erythroids and 3% lymphoid cells.

Auer rods are not seen and unlike classic APL, the abnormal promyelocytes do not have

bilobed nuclear contour. They also lack prominent Golgi and have prominent nuclei and

aggregates of eosinophilic granules. Histochemistry stain showed heavy myeloperoxidase

staining and negative for non-specific esterase (Figure 1B and data not shown). Bone marrow

biopsy revealed hypercellular marrow with background fibrosis and aggregates of immature

myeloid cells with round nuclei and prominent nucleoli, and abundant cytoplasm - often with

numerous granules (Figure 2).

Flow Cytometric analysis

6-color analysis with CD45-gating revealed that the majority of the immature cells with the

following phenotype: CD45(dim), CD34-, CD117-, HLA-DR-, CD33+, CD15(bright), CD64(dim),

myeloperoxidase+, CD13-, and CD11B-.

Cytogenetics and FISH studies

On karyotype analysis, none of the 20 metaphases analyzed had a t(15;17) or other known AML-associated rearrangements. 3 of the 20 had multiple double-minute chromosomes (Figure

3) and other 13 abnormal metaphases contained a paracentric inversion of the short arm of chromosome 1, as the sole aberration (data not shown). FISH evaluation for MYC amplification was performed on nuclei with the MYC Dual Color, Break Apart Probe at 8q24 and MYC amplification was observed in 7/100 nuclei (Figure 4). FISH evaluation for PML-RARA rearrangement with the PML/RARA Dual Color, Dual Fusion Probe for PML at 15q24 and RARA at 17q21.1 showed no rearrangement in 100 nuclei. Molecular Diagnostics

RT-PCR for BCR-ABL or PML-RAR fusion transcripts were both negative. DNA Sequence

analysis FLT3 ITD, FLT3 D835, NPM1 exon 12 insertions and CEBPA mutations are all negative.

A repeat bone marrow biopsy after induction revealed the persistent presence of immature

cells, with an increased number of cells containing granules compared to the prior evaluation

(data not shown).

Discussion:

Since patients with APL frequently present with potentially life-threatening coagulopathies and

require rapid diagnosis and treatment with all-trans retinoic acid (ATRA) (1, 2). Patients with

leukemic cells with morphologic features similar to APL are often given ATRA while waiting

for confirmation of the diagnosis of APL. A translocation of chromosomes 15 and 17 is

identified in the vast majority of cases that present with morphologic and immunophenotypic

features of APL. However, occasional cases present without this classic translocation could have

an alternative form of the PML-RARA fusion or very rarely, the presence of PML-RARA fusion

can not be demonstrated but patients still responded to ATRA clinically. Thus, the use of

multiple methods including both cytogenetic and molecular techniques is critical for

determining whether a cryptic PML-RARA fusion is present in order to guide appropriate

treatment. In this case, both cytogenetic methods and molecular methods did not identify the

PML-RARA fusion but revealed the amplification of MYC that likely plays a key role in

oncogenesis.

This case represents an unusual AML with MYC amplification. MYC is a transcription activator

with many target genes involved in key processes controlling cellular proliferation and

differentiation. Increased level of MYC protein is observed in multiple cancer types and can be

the result of chromosomal rearrangement, gene amplification or transcriptional activation. In

AML or myelodysplastic syndromes (MDS), structural changes of the MYC locus are

uncommon. Increased level of MYC expression is generally attributed to transcriptional up-

regulation through activation of other oncogenic pathways. Review of the literature shows

several reports of AML with alteration of the MYC locus in the forms of amplification, either on

double-minutes or homogenously staining region (Rayeroux KC, Campbell LJ.2009, Thomas et

al 2004, Christacos et al 2005, Frater et al 2006, Villa et al 2008, Lee et al 2009, Bruyer et al 2010,

Angelova et al 2011, Yamamoto et al 20013). On the other hand, trisomy 8 is common in

myeloid neoplasms and one copy increase of MYC is present in many AML and MDS secondary

to trisomy 8.

In most reported cases with MYC amplification, the bone marrow morphology showed AML

with maturation. In this case, the majority of the leukemic cells were arrested at the

promyelocyte stage, but they lacked the characteristic bilobed nuclei and prominent Golgi

apparatus typically seen in cases of APL. Furthermore, although several cells contained

eosinophilic structures suggestive of Auer rods, no classic Auer rods or Faggot cells were seen.

So far , there are a few sparse case reports of patients with APL-like morphology and MYC

amplification, ( old cases reviewed in Frate et al 2006 , Kamath et al 2008, Bruyer et al 2010,

The absence of PML-RARA rearrangement was demonstrare by karyotype, FISH analysis and

/or RT-PCR in all except in two old reports

There are at least two other known case of a patient with APL-like morphology and the

presence of C-MYC amplification but the absence of a PML-RARA rearrangement (4, 16). It is

unclear what characteristics of this cellular genotype results in the morphologic and

immunophenotypic appearance of promyelocytes.

Figures:

Figure 1A. Wright-Giemsa stain of the bone marrow aspirate at 100x magnification. The

majority of marrow cellularity is composed of blasts and abnormal promyelocytes with nucleoli

and abundant azurophilic granules but rare Auer rods.

Figure 1B. Myeloperoxidase stain of the bone marrow aspirate at 100x magnification. Most of

the leukemic cells are intensely positive for myeloperoxidase.

Figure B. Hematoxylin and Eosin stain of the bone marrow biopsy at 100x magnification.

Leukemic cells have round to oval nuclei, distinct nucleoli and moderate amount of cytoplsam

with eosinophilic granules.

Figure 3: Karyotype of cells with multiple double minutes and no t(15;17) on initial

presentation.

Figure 4: Fluorescent in-situ hybridization with MYC Dual Color, Break Apart Probe at 8q24.

The presence of multiple aggregates of green and red signals dispersed in the interface nuclei,

consistent with amplification present on double-minutes.

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