CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
CAP TODAY and the Association for Molecular Pathology have teamed up to bring molecular case reports to CAP TODAY readers. AMP members write the reports using clinical cases from their own practices that show molecular testing’s important role in diagnosis, prognosis, and treatment. The following report comes from the University of New Mexico. If you would like to submit a case report, please send an email to the AMP at amp@amp.org. For more information about the AMP and all previously published case reports, visit www.amp.org.
Brittany Coffman, MD
Brian Menkhaus, MD
Devon Chabot-Richards, MD
April 2019—Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) in which promyelocytes predominate. APL accounts for about 10 percent of AML cases, and although APL can be diagnosed at any age, it is most common among young adults with a slight male predominance.1 APL is defined by the balanced reciprocal translocation (15;17)(q22;q21) between PML and RARA, although variant translocations involving RARA and other partner genes can occur. The fusion results in uncontrolled cell proliferation and inhibition of cell differentiation.
The PML gene, or promyelocytic leukemia gene, is located on chromosome 15.2 Its gene product is a tumor suppressor protein that blocks cell proliferation and regulates apoptosis via FAS ligand and tumor necrosis factor-alpha. The RARA gene, or retinoic acid receptor-alpha, is located on chromosome 17.3 The RARA gene codes for a nuclear receptor that regulates gene transcription, including genes involved in differentiation, apoptosis, and granulopoiesis. The translocation between PML and RARA occurs between the long arms of both chromosomes 15 and 17, respectively. The breakpoint of RARA occurs at intron 2, but the breakpoint of PML can occur at three different locations, resulting in different transcript sizes with similar functions (Fig. 1).4
The most common breakpoint subtype of PML, bcr-1 (breakpoint cluster region) or long form, is at intron 6, which occurs in 45 to 55 percent of APL cases.4 The second most common is the short form, or bcr-3, which occurs in 35 to 45 percent of cases and occurs at intron 3 of PML. Finally, the least common subtype is the variable form, or bcr-2, which occurs in five to 10 percent of cases and occurs at exon 6 of PML. The bcr-2 form is termed variable form because the breakpoints can occur at different sites within exon 6. The resulting PML-RARA fusion gene product prevents normal transcription, which ultimately leads to lack of differentiation of myeloid cells and provides cells with a survival advantage.
Fig. 1. The breakpoint of RARA occurs at intron 2, but the breakpoint of PML can occur at different locations, resulting in three different fusion transcripts termed bcr-1, bcr-2, and bcr-3. Bcr-1 (long form) PML breakpoint occurs at intron 6, resulting in a fusion between PML exon 6 and RARA exon 3. The bcr-3 (short form) PML breakpoint occurs at intron 3, resulting in a fusion between PML exon 3 and RARA exon 3. The PML breakpoint of bcr-2 (variable form) occurs within variable regions of exon 6, resulting in a fusion between PML exon 6 and RARA exon 3. (Image courtesy of Dr. Evelyn Lockhart)
Patients may present with nonspecific symptoms such as fever, fatigue, decreased appetite, and/or weight loss; however, a subset may also present with bleeding due to disseminated intravascular coagulation (DIC), which is associated with APL.5 Rapid diagnosis is imperative in cases of APL due to the increased risk of death secondary to DIC in this patient population. Most patients demonstrate pancytopenia on complete blood count, though some patients present with leukocytosis. Leukocytosis is more common in the microgranular variant of APL.
Two morphologic forms of APL are recognized including hypergranular (or typical) and microgranular variants. The blasts of typical APL have irregular large nuclei that can be bilobed and may have a “sliding plate” morphology. The cytoplasm is densely packed with large granules and occasional Auer rods. Microgranular variant blasts mostly have a bilobed morphology, and cytoplasmic granules are not apparent as they are submicroscopic. Cytochemical stains such as myeloperoxidase and Sudan black are strongly positive in both variants, often to the point that the cytoplasmic granules obscure the nucleus.
Fig. 2. Peripheral blood smear demonstrating blasts with “sliding plate” morphology (right) and bilobed nuclei (left) with coarse cytoplasmic granules.
Case. A 16-year-old male presented with a one-week history of fatigue and easy bruising. Complete blood count revealed a white count of 4.8 × 103/mm3 with left shift of the myeloid lineage with 50 percent blasts, hematocrit of 31 percent, hemoglobin of 10.8 g/dL, and a platelet count of 29 × 103/mm3. Review of the peripheral blood smear showed numerous blasts with increased nuclear-cytoplasmic ratio and bilobed nuclei with sliding plate morphology (Fig. 2). Myeloperoxidase stain performed on the peripheral blood was strongly positive (Fig. 3). The morphologic and clinical findings were concerning for APL and thus the clinical team started ATRA (all-trans-retinoic acid) while awaiting final diagnosis. Flow cytometry revealed the blast population to be dim CD45, bright CD33, subset CD34, dim HLA-DR, dim CD13, CD117, subset CD56, and cytoplasmic MPO positive (Fig. 4). Dual color dual fusion fluorescence in situ hybridization was negative for t(15;17) (Fig. 5).6
Fig. 3. Peripheral blood smear stained with myeloperoxidase (MPO) demonstrating a blast with MPO-positive granules, which obscure the nucleus.
A bone marrow biopsy was then performed and revealed a hypercellular marrow with left shifted maturation in the myeloid lineage with 77 percent blasts (Fig. 6). Blast morphology was similar to the peripheral blood. Flow cytometry performed on the bone marrow was identical to the peripheral blood. Conventional karyotype performed on the bone marrow revealed a normal male chromosome complement with no abnormalities identified. Due to the clinical suspicion of APL along with blast morphology and immunophenotype, despite negative FISH and karyotype, reverse transcription-polymerase chain reaction (RT-PCR) testing was obtained.7 Testing revealed a cryptic t(15;17) with the bcr-3 transcript, and the patient was formally diagnosed with APL (Fig. 7). Despite starting ATRA, the patient developed DIC, which was effectively treated with transfusion. The patient was discharged home one month later and remains in remission more than three years later.
Fig. 4. Flow cytometry performed on the peripheral blood revealed blasts (highlighted red) that are positive for CD33, subset CD34 (top right plot), dim HLA-DR, dim CD13 (bottom left plot), CD117, and cytoplasmic myeloperoxidase (MPO) (bottom right plot).
Discussion. APL is an acute leukemia that is defined by t(15;17) and requires prompt diagnosis because it is associated with DIC. Review of blast morphology and cytochemical stains can aid the pathologist in narrowing the differential diagnosis, but the final diagnosis of APL often requires additional testing. It is common for rapid turnaround FISH to be performed to identify the t(15;17), thus securing the diagnosis. However, in cases such as this one, FISH and karyotype will fail to identify the cryptic translocation. Delay in diagnosis in these cases may be detrimental and even lead to death if ATRA therapy is not begun.
Fig. 5. FISH for t(15;17) revealed no translocation demonstrated by the two separate signals in both cells. In this dual color dual fusion probe, PML is labeled red and RARA is labeled green. In normal cells (and in this case) two red and two green signals will appear reflecting the two normal copies of RML and RARA. In an abnormal cell with the t(15;17), one red, one green, and two fusion signals will appear.
Cryptic translocations of APL account for about two percent to four percent of all APL cases. The majority of cryptic translocations result from submicroscopic insertions of RARA into PML, but complex rearrangements involving numerous chromosomes can also be a cause. In addition, variant translocations between RARA and other genes can occur. Variant translocations may require additional FISH probes as they may not be recognized by usual t(15;17) dual color probes; it is important to recognize, however, that these are not cryptic translocations but variant translocations. Furthermore, all cryptic and variant alterations involve RARA but may not involve PML, highlighting the importance of RARA gene alterations in the development of APL.
Of note, cases of variant translocations involving RARA but not PML may have blasts morphologically resembling APL but with a worse prognosis, specifically t(11;17)(q23;q21), which may also be resistant to ATRA. These cases are best classified as APL with a variant RARA translocation per the 2016 WHO.8 The majority of cryptic cases of APL have similar morphology, immunophenotype, and cytochemical staining patterns, which should raise clinical concern for APL. In these cases, RT-PCR, microarray, and gene sequencing can identify the cryptic translocation and definitively establish the proper diagnosis.
Fig. 6. Bone marrow biopsy demonstrated hypercellular marrow with increased blasts.