Acute promyelocytic leukemia with t(15;17): a case study

Introduction

Acute promyelocytic leukemia with t(15;17) is a cancerous disease of the white blood cells in the bone marrow and peripheral blood. It is a subtype of the acute myelogenous leukemia (AML), and is commonly seen in adults. An adolescent male was admitted by referral to a large hospital with symptoms of thrombocytopenia and questionable circulating immature cells. Upon referral, the patient was found to have over 80% circulating atypical promyelocytic cells. Bone marrow biopsy and flow cytometry results indicated features of acute promyelocytic leukemia, confirmed by a positive result for the PML/RARA fusion gene with quantitative PCR. Fluorescent in situ hybridization revealed no findings of the typical fusion gene. However, the patient was treated with a routine chemotherapy of high dose cytosine arabinoside, idarubicin, arsenic trioxide, and all-trans retinoic acid with subsequent bone marrow observations

First recognized in the 1950s, acute promyelocytic leukemia with t(15;17) continues to be one of the most studied and researched leukemias.^1,2 Referred to as APL or APML, it is classified under the French-American-British system of nomenclature as AML-M3 and the World Health Organization as a subtype of AML with characteristic genetic abnormalities. This includes APL with translocation of [t(15;17)] (ICD-0 9866/3); RARA; PML. Most instances of the disease occur in middle-aged adults, representing 10% to 15% of AML cases.^3,4 Approximately 1,000 new cases are diagnosed each year, with equal incidence among males and females.⁴ APL with t(15;17) presents with several clinical symptoms, including fatigue, anemia, shortness of breath, thrombocytopenia, and severe bleeding syndrome. The mortality rate is high if not treated early.^3-5 Complications with secondary fibrinolysis and disseminated intravascular coagulation, or DIC syndrome, often occur due to the release of cellular content from promyelocytic cells in the blood.^4,5 This risk is more often associated with APL with t(15;17) than other acute myeloid leukemias.⁵ Due to the risk of rapid hemolysis, early aggressive treatment and diagnosis is important in ensuring the patient's survival.⁶

Cases of APL with t(15;17) are confirmed through laboratory testing and bone marrow observations combined with molecular techniques. Cellular antigenic profiles and chromosomal and genetic mapping are important in determining cells that have the abnormal fusion transcript. The abnormal transcript is one of the first examples of oncogene targeted therapy, and is present in as many as 90% of APL with t(15;17) cases.³ Currently patients are treated with a combination therapy of retinoic acid with high dose chemotherapeutic drugs or arsenic trioxide. However, new treatment methods are in development.^1,2

Pathogenesis

Acute promyelocytic leukemia arises out of a defect in normal granulocytic cell maturation and apoptosis. Interestingly, APL with t(15;17) diagnosis is usually accompanied by recognition of a common genetic mutation that characterizes the disease. A gene translocation occurs in an abnormal cell between chromosome 15 and 17.⁴ pt between the retinoic acid receptor, or RARA (RARα) and the promyelocytic gene, or PML gene, which is active in cell death and tumor suppression.^2,7 This unique break point allows for three isoforms of the gene, the most typical of which is t(15;17) (q22; q21).^1,2,8 The retinoic acid gene normally dissociates repressor complexes that, once dissolved, allow transcription of genes leading to cell maturation. The PML/RARA or variant version of a RARA fusion protein not only blocks binding of retinoic acid to its receptor but also activates genes that aid in cell self-renewal.^1,2

Survival rates for patients diagnosed with APL with t(15; 17) are good. This type of leukemia is considered preferable or favorable compared to the other variations of AML.⁹ However, patients are at higher risk of death by bleeding. Approximately 10% of patients will die from hemorrhage and 40% of untreated cases will show cerebral or lung bleeding. This underscores the need to begin therapy early.^2,5,6 Statistics show a survival and remission rate of approximately 70% to 90% can be achieved in patients who receive therapies for APL with t(15;17).^5,10

Clinical presentation

Upon initial diagnosis, laboratory results typically reveal a lowered hemoglobin and hematocrit with anemia. Differential results indicate a significant population (at least 20%) of large promyelocytes of the hypergranular or microgranular bilobed form.^2,9 The WBC count may indicate leukocytosis or pancytopenia.^3,5 The presence of Auer rods is also a significant clue. Chemistry results can also show no abnormalities, depending on the patient's clinical status, though liver function, electrolyte, and renal function tests should be performed.^2,6 Thrombocytopenia, bruising, and peticheia of the mouth and other parts of the body are also common.^2,4,5

Some but not all patients show severe coagulopathy and DIC that affect the patient's clinical outcome of APL with t(15;17).² Patients will often have an abnormal increase in fibrin degradation products, show lowered fibrinogen values, overproduction of plasmin, and increased prothrombin or thromboplastin times.^2,4,5

Patients undergoing ATRA treatment for APL with t(15;17) are at risk for retinoic acid syndrome.^1,5,11 This syndrome is thought to arise from toxicity of ATRA and inflammatory cytokines released from treated cells.¹² Symptoms are fever, shortness of breath, high WBC counts, respiratory distress, pleural and pericardial effusion, weight gain, and renal failure.^11,12 Pediatric patients are particularly at risk for pulmonary edema, which is treated with dexamethasone.¹¹

Secondary infections can also occur in immunocompromised APL with t(15;17) patients. Varicella zoster virus can appear in pediatric patients as well as localized soft tissue infections of herpes simplex virus.^5,11 Patients may also experience gum bleeding and increased amounts of Candida albicans in the oropharyngeal area.⁵

Laboratory role in diagnosis

Genetic and chromosomal testing is used to identify the APL with t(15;17) subtype from other types of AML. Laboratory results are essential in the diagnosis and management of APL cases. Laboratories can monitor a patient's progress through blood and bone marrow testing. The clinical laboratory performs chemistry, coagulation, and hematology testing to monitor disease persistence and adverse complications such as DIC and to identify the circulating cells in the blood. A complete blood count, differential, comprehensive metabolic panel, APTT/PTINR, fibrinogen, D-Dimer, and periodic bone marrow biopsy and aspirate are the most common tests performed to evaluate the patient's condition.^2,6 Bacterial and fungal cultures may be necessary to identify infections. CSF studies may also be recommended for high white blood cell counts to evaluate cellular movement in the body.²

In flow cytometry studies, a single major cluster population with a wide range of side scatter on a scatter dot plot suggests an APL profile.¹³ Cellular antigen (CD33), co-expression (CD13), lack of or weak HLA-DR, and CD34 are also further evidence of the atypical population.¹³ Expression of CD33 gradually decreases as granulocytic cells mature, and is present at all stages.^5,13 Weak expression of other mature cellular markers is also possible. The dot plot cluster for APL may appear as a teardrop or triangle shape, depending on the degree or lack of granularity.¹³ The rarest form of this type may appear similar to a scatter plot for acute myelocytic leukemia (AML) with minimal maturation.¹³

In suspected APL with t(15;17) cases, a bone marrow biopsy and aspirate are quickly obtained for cellular differentials, histology studies, myeloperoxidase activity, and cell antigen marker testing.⁴ Samples are also sent for investigation with FISH probing to search for the typical translocation and also for cytogenetic studies to look for an abnormal chromosomal karyotype.⁹ Quantitative PCR results of the gene transcript ratio also serves as a positive confirmatory procedure. Because the classic t(15;17) translocation creates two fusion genes from one splice, 70% of cases with translocation, harbor a RARA/PML gene, and 100% show the reciprocal PML/RARA gene.² This makes qPCR a useful assay for disease screening.

Treatment

Treatment of acute promyelocytic leukemia is unique and involves a combination regimen. Currently, APL with t(15;17) is different from other variations of AML in that it is the only one with a target specific therapy.⁹ The use of all-trans retinoic acid initiates differentiation of the immature myeloid cells and helps manage coagulopathy.^2,7 First used in clinical trials in China in 1986, this compound is a vitamin A derivative that releases repressor complexes from the RARA receptor fusion gene.^3,11,12 Retinoic acid helps rid the blood of promyelocytes and their destructive granular material that contributes to DIC. ATRA is taken orally in 45 mg/m² doses and must be accompanied with chemotherapy because cell differentiation and remission is not long-lasting.^2,11 Possible chemotherapeutic drugs to support ATRA therapy include anthracyclin, idarubicin, and cytarabine, the latter also referred to as Ara-C.^2,6 Side effects of retinoic acid syndrome are of concern, and patients must also be monitored for risk.¹² Undergoing chemotherapy can also suppress the patient's bone marrow and immune system and contribute to bleeding.⁹ Benefits of administering heparin to APL with t(15;17) patients are uncertain and undergoing debate.⁵

In addition to ATRA and chemotherapy, use of arsenic trioxide has also shown effectiveness in improving patient prognosis. The treatment was associated with APL therapy in China in the 1970s.^1,11 It has become an additional standard of care since 2000, especially in patients with ATRA resistance and relapsing cases.^1,10 Arsenic trioxide is a destructive therapy for the abnormal fusion protein, and aids in marking the oncoprotein for degradation.⁷ It functions similarly in advocating cell differentiation and apoptosis.

Outpatient care incorporates prophylactic drugs, including antibiotics. Blood product transfusions are given periodically to manage the anemia and lack of platelets, as well as screening for retinoic acid syndrome while patients are taking ATRA prophylaxis.^2,5 Also, 6-mercaptopurine together with methotrexate may improve survival during maintenance therapy while ATRA is being administered.^2,11

Case presentation

A 17-year-old male was referred to a local hospital from an oncology service for evaluation of thrombocytopenia, petechiae, and swelling of a large bruised area in the upper right quadrant. Questionable circulating blasts were seen on the peripheral blood smear at the initial assessment. The patient was in normal health one day prior to admission. Patient history showed no previous fever, bone pain, or malaise, but did indicate easy fatigability after physical activity. Approximately six to eight random donor platelet units were administered before referral.

At initial evaluation, the patient complained of headache in recent weeks but lacked other abnormal symptoms. Hepatosplenomegaly and lymphadenopathy were absent. The skin examinations revealed a small purpuric lesion on the tip of the tongue and diffuse petechiae predominantly on the lower extremities, but also on the torso and upper extremities. Chemistry, coagulation, and hematology tests were ordered, which included a bone marrow aspiration and biopsy. Initial laboratory results are shown in Table 1 . The complete blood count with differential revealed atypical promyelocytic cells with Auer rods and granulation, anemia, and thrombocytopenia with a platelet count of 30 x10³ µL.The WBC count also indicated leukocytosis. The D-dimer and prothrombin time were increased, and the patient's fibrinogen was decreased. Bone marrow and cytogenetic observations are shown in Table 2 . Flow cytometry results showed a spectrum (94% of WBCs) of increased forward and side scatter, including CD33 on 100% of cells, CD13 on 64%, and a dim HLA-DR expression. Fluorescent in situ hybridization studies indicated no presence of the APL fusion gene. This was contrary to qPCR results that were positive for the PML/RARA transcript associated with the disorder. These results, in correlation with all clinical data, confirmed a persistent disease state of acute promyelocytic leukemia. The patient was given a broad-spectrum antibiotic and saline/sodium bicarbonate hydration. Chemotherapy and prophylactic treatment were also scheduled.

Table 1.
Patient laboratory results

Table 2.
Initial immunophenotypic and cytogenetics profile

Case conclusion

The patient was admitted with symptoms of a hypergranular form of acute promyelocytic leukemia. Despite a negative FISH result, qPCR confirmed a transcript for the PML/RARA t(15;17) translocation. Clinicians did not rule out a possible variation on the common translocation. The patient was evaluated repeatedly for abnormal cell clusters in the bone marrow and circulating promyelocytes in the peripheral blood. He was also evaluated for questionable disseminated intravascular coagulation. At one month the vascular lab evaluated a swelling of the upper right limb and found no instance of thrombosis or arterial abnormalities. He was treated with high doses of chemotherapy, cytosine arabinoside (ara-C) and idarubicin. The program was accompanied with all-trans retinoic acid administration for a four-day protocol. At four months, he began arsenic trioxide therapy via intravenous with 8mg/250cc. At five months, he received 1600 mg of cytarabine in 12 hour periods along with dexamethasone and two doses of 16 mg of mitoxantrone daily followed by ATRA. The patient tolerated the treatment well. Results of later bone marrow observations are shown in Table 3.

Table 3.
Changes in the bone marrow profile over time

Over a period of several months, the patient had multiple hospitalizations for fever and chest pain, and also occasionally expressed fluctuating limb pain. He continued to show signs of bruising and petechiae into the sixth month. Prophylactic plateletpheresis and blood transfusions were given over an eight-month period, and the patient's platelet count and periodic pancytopenia were being monitored.

Summary

Acute promyelocytic leukemia (APL with t15;17) is a disease characterized by abnormal proliferation of promyelocytic cells in the peripheral blood and bone marrow, along with coagulopathy and thrombocytopenia. It is a variation of AML that frequently exhibits a typical chromosomal translocation. Laboratory analysis and confirmatory testing for the fusion gene make diagnosis and treatment of APL with t(15;17) possible.

References

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Dr. Floyd Josephat, EdD, MT(ASCP), is an assistant professor of hematology and hemostasis in the Department of Medical Laboratory Science at Armstrong Atlantic State University in Savannah, GA. He has more than 20 years of laboratory medicine experience. Erin Pruitt, BS(MT), a graduate of Clemson University and the Department of Medical Laboratory Science at Armstrong Atlantic State University, is a Medical Technologist (MLS, ASCP) in the core laboratory of Palmetto Health Richland in Columbia, SC.