with t(8;21) Translocation and Rare Myelodysplastic Features

By Richard Chiu, D.O.,1,2 Rudolf Estess, D.O.,2 Hadi Yaziji, M.D.,1,3 and Farhad Askarian, M.D.1,3

1Department of Pathology, Larkin Community Hospital, Miami, FL 33012 2Department of Family Medicine, Larkin Community Hospital, Miami, FL 33012 3Vitro Molecular Laboratories, Miami, FL 33174

Address correspondence to: Richard Chiu, D.O. Larkin Community Hospital Palm Springs Campus Department of Pathology 1475 W. 49th Place, Hialeah, FL 33012 Phone: (626) 493-3540 Email: [email protected]

Abstract Acute myeloid leukemia (AML) is a diverse group of hematologic malignancies characterized by uncontrolled proliferation of early myeloid progenitors, a process which impairs bone marrow hematopoiesis. The disease is driven by multiple cytogenetic abnormalities and mutations which affect its clinical course and prognosis, and the disease may arise from a pre-existing background of (MDS). In this report, the authors describe a 74-year-old Hispanic man presenting with fatigue and weakness, and labs showing leukocytosis (WBC 29.8), macrocytic anemia, and thrombocytopenia. His blood smear revealed with Auer rods, and his bone marrow cellularity on both core biopsy and flow cytometry, consisted of 22-23% myeloblasts positive for CD34 and CD117. His cytogenetic profile was positive for t(8;21)(q22;q22) translocation and Y chromosome loss. Despite lacking MDS-related cytogenetic findings, myelodysplastic features of pseudo-Pelger-Huet neutrophils, erythroblasts with nuclear budding and megaloblastoid form, ringed sideroblasts, and macrocytic red cells were observed in his bone marrow and blood smear. His molecular profile was negative for mutations in FLT3 and IDH, precluding him for targeted therapy against those mutations. The patient was diagnosed with AML and started on CPX-351 (Vyxeos) involving a combination of cytarabine and daunorubicin for induction chemotherapy. This is a noteworthy AML case given the co-occurrence of the conventional t(8;21) translocation portending favorable prognosis, with myelodysplastic changes portending a poorer prognosis.

Keywords: acute myeloid (myelogenous) leukemia, t(8;21) translocation, myelodysplastic syndrome, myeloblasts, cytarabine, daunorubicin, CPX-351 (Vyxeos)

Introduction Acute myeloid leukemia is a heterogeneous group of malignancies characterized by clonal proliferation of early myeloid progenitors with impaired ability to differentiate.1 The leukemic myeloid progenitors accumulate in the bone marrow and impede production of normal blood cells, causing anemia and thrombocytopenia, and recurrent infection from dysfunctional .2 The disease may arise as a primary, de novo cancer, or as a cancer secondary to prior chemotherapy or underlying pre-leukemic disease, such as myelodysplastic syndrome (MDS).3 AML has multiple subtypes defined by their cytogenetic and molecular profiles, which dictate their prognosis and treatment.3 The World Health Organization (WHO) chiefly classifies AML into four groups: AML with recurrent genetic abnormalities, AML with myelodysplasia- related changes, AML secondary to therapy, and AML not otherwise specified (based on the older FAB system).4 Diagnosis of AML is made by demonstrating 20% myeloblasts in bone marrow or peripheral blood, or by demonstrating Auer rods or one of the three AML-defining translocations: t(8;21), t(15;17), and inv(16).2 AML arising from a pre-leukemic background of myelodysplastic syndrome harbors a different set of cytogenetic abnormalities associated with poorer outcomes than primary, de novo AML.1,3 The prognosis of AML is influenced by their cytogenetic and mutational profiles.1,5 Good prognosis is conferred by translocations t(8;21), t(15;17), and inv(16),1,6 while poor prognosis is conferred by translocations inv(3), t(6;9), and t(9;22), deletions/monosomies of chromosomes 5 and 7, and complex karyotypes.3,5 Prognosis is also affected by multiple mutations, such as those involving fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase (IDH), which currently serve as new targets of therapy. Table 1 lists the most commonly found cytogenetic changes and mutations in AML, with their associated functions and prognoses. The standard treatment for AML consists of a regimen of cytarabine and daunorubicin,1 which more recently have been given as a liposomal formulation (CPX-351/Vyxeos) to patients with AML secondary to prior chemotherapy or hematologic malignancy like MDS.3,7 Targeted therapy against CD33 (gemtuzumab/ozogamicin), mutated FLT3 (e.g., midostaurin, sorafenib), or mutated IDH (e.g., ivosidenib, enasidenib) can also be added to the regimen if the patient tests positive for these mutations.3,5,8 If complete remission is achieved with induction chemotherapy, hematopoietic stem cell transplantation can be administered to qualifying patients with the intent to cure.7

Case Presentation The patient is a 74-year-old Hispanic man referred to Larkin Palm Springs Hospital by his primary physician after his lab work showed leukocytosis (WBC 29.8), macrocytic anemia (Hgb 6.7, Hct 20, MCV 109), and thrombocytopenia (Plt 44). He reported experiencing fatigue and weakness but denied fever, chills, night sweats, weight loss, bruising or bleeding, skin rashes, or body pain. Physical exam revealed some pallor, but no cachexia or lymphadenopathy. He denied smoking, prior chemotherapy, and family history of cancers or blood disorders. The patient had concurrent type 2 non-ST segment elevation myocardial infarction (NSTEMI) due to cardiac ischemia from his anemia, with elevated troponins (2.20) but no ST changes on EKG or concurrent chest pain. Chest x-ray and abdominal CT scan were unremarkable. Table 2 presents the patient’s laboratory results from his hospital stay. The patient’s CBC showed elevated monocytes (45.5%, 13.58 x 103/uL), immature granulocytes (13.8%, 4.13 x 103/uL), and nucleated RBCs (0.06 x 103/uL), with low neutrophil percent (17%). Given these findings, a diagnosis of chronic myelomonocytic leukemia was initially favored, but a manual differential count of the peripheral blood smear instead revealed a significant presence of blasts (>50% of white count) and normal monocyte percentage (5%). Several blasts on blood smear showed Auer rods (Figure 1), characteristic of acute myeloid leukemia. Red cells and platelets were morphologically normal. The monocytosis initially shown by CBC, was caused by the automated analyzer misinterpreting the blasts as monocytes (Table 2). Bone marrow core biopsy showed hypercellular marrow (95%) for the patient’s age, and its 500-cell differential count showed excess myeloblasts positive for CD34 and CD117 comprising 22% of bone marrow cellularity (Figure 2A-C). The differential cell count also revealed left-shifted myeloid spectrum with 14% promyelocytes, 19% myelocytes, and 15% mature granulocytes; in contrast, monocytes consisted of only 1% of the marrow cell population. Flow cytometry performed on the bone marrow aspirate showed a similar cell distribution as the manual differential count, with myeloblasts (Figure 3A) positive for CD34, CD117, and HLA- DR comprising 23% of bone marrow cellularity. A few neutrophils in the bone marrow showed pseudo-Pelger-Huet anomaly (Figure 3B), but no hypersegmentation or was observed. The bone marrow core yielded a high myeloid-erythroid ratio of 5:1, with erythroid precursors accounting for 15% of its cellularity. The erythroid precursors exhibited normal maturation, but rare cells with nuclear budding, megaloblastoid forms, karyorrhexis, and bi- lobed nuclei (Figure 3C-F) were also seen, as were sideroblasts and iron-laden erythroid progenitors (Figure 2D) on Prussian blue stain. Red cells showed with elevated RDW-SD (70.2), but no was seen on blood smear, and reticulocyte count and percentage were within normal range. Reticulin stain of bone marrow did not reveal increased marrow fibrosis. Megakaryocytes were present in normal numbers and morphology. Cytogenetic analysis revealed the presence of the t(8;21)(q22;q22) translocation, a commonly seen chromosomal rearrangement in AML that carries relatively favorable prognosis, and loss of Y chromosome (45X,-Y) in all 20 karyotyped cells. To determine if the myeloblasts developed from a prior background of myelodysplastic syndrome (MDS), fluorescence in situ hybridization was conducted on some of the most commonly seen cytogenetic abnormalities in MDS – inv(3), del(5q), del(7q), monosomy 5 and 7, trisomy 8, del(17p), del(20q), and 11q23 – which returned negative. In addition, no mutations were detected in isocitrate dehydrogenase (IDH1 and IDH2 isoforms) and FMS-like tyrosine kinase 3 (FLT3-ITD, FLT3-TKD) genes which carry prognostic significance in AML. To assess for monoclonal gammopathy, serum protein electrophoresis with immunofixation was performed on the patient’s serum, which revealed normal quantities of albumin, globulin, kappa and lambda light chains, normal albumin/globulin and kappa/lambda ratios, and absence of M-spike. To rule out iron, folate, or vitamin B12 deficiency, or fecal occult bleeding as causes of the patient’s anemia, additional lab tests were performed which revealed normal levels of iron, transferrin, transferrin saturation, vitamin B12, and folate. Fecal occult blood test was negative. Due to risk of tumor lysis syndrome, disseminated intravascular coagulation, and hyperviscosity syndrome (assessed from his elevated levels of LDH, D-dimer, ferritin, fibrinogen, ESR, CRP), the patient was maintained on allopurinol and hydroxyurea. His daily uric acid levels were within normal range. Daily coagulation profile was also within normal range. The patient was diagnosed with acute myeloid leukemia, given his findings of leukocytosis (WBC 29.8), percent of >20% on bone marrow biopsy and flow cytometry, and presence of Auer rods and the t(8;21)(q22;q22) translocation. Although his cytogenetic profile did not show MDS-associated chromosomal abnormalities, the presence of dysplastic neutrophils with pseudo-Pelger-Huet morphology, erythroid progenitors with nuclear budding and megaloblastoid forms, sideroblasts with increased iron storage, and of red cells raised concern for AML with myelodysplasia-related changes, a subtype of AML with relatively poor prognosis. The patient was therefore started on CPX-351 (Vyxeos) composed of cytarabine with daunorubicin in a 5:1 ratio in liposomal formulation, approved for AML with MDS-related changes, and also for AML with favorable or intermediate risk cytogenetics as an alternative to the standard “7+3” regimen with cytarabine and daunorubicin. Because his leukemic blasts were not positive for CD33 or for mutations in FLT3 and IDH, the patient was not a candidate for the anti-CD33 antibody-drug conjugate gemtuzumab/ozogamicin, FLT3 inhibitors midostaurin and sorafenib, or IDH inhibitors ivosidenib and enasidenib. Given that the patient has good functional status (ECOG score 0), he is a candidate for curative bone marrow transplantation if induction chemotherapy achieves complete remission.

Discussion In this report, the authors described an AML patient harboring the t(8;21) translocation with Y chromosome deletion. His labs showed findings of leukocytosis, anemia, and thrombocytopenia characteristic of AML. The diagnosis of AML can be made from the t(8;21) translocation alone, but the patient’s bone marrow also showed myeloblasts comprising 20% of its cellularity and containing Auer rods, which are two more diagnostic features of AML. Despite testing negative for MDS-related cytogenetics, the patient’s blood and bone marrow showed dysplastic cells characteristic of MDS, including neutrophils with pseudo-Pelger-Huet anomaly, macrocytic red blood cells, sideroblasts, and erythroblasts with nuclear budding, megaloblastic change, karyorrhexis, and bi-lobed nuclei. His molecular profile was negative for mutations in FLT3 and IDH, and, therefore, he was not a candidate for targeted therapy against those mutations. Other normal labs ruled out deficiencies of vitamin B12, folate, and iron, and gastrointestinal bleeding as causes of his red cell macrocytosis and anemia. Similarly, normal immunoglobulin quantities/ratios and lack of paraproteins ruled out monoclonal gammopathy as the cause of his hematologic findings. This is a noteworthy case because of the dual presence of t(8;21) translocation and MDS-related dysplastic features, which yield opposing prognostic effects. The t(8;21) translocation harbored by this patient, is the most common cytogenetic subtype within the WHO category “AML with recurrent genetic abnormalities.” It is classically associated with AML with maturation (M2) of the French-American-British (FAB) system, and confers good prognosis with high remission rates and survival.5,6 It creates the fusion protein RUNX1-RUNX1T1, which causes impaired hematopoiesis via the suppressive effects of its RUNX1T1 repressor on its RUNX1 transcription factor unit.9 The patient’s Y chromosome loss is likely an age-related phenomenon with benign significance, given that Y chromosome deletion in bone marrow cells increases with age, and the leukemic AML clone may have developed from a cell that has already lost its Y chromosome.10,11 AML arising from pre-existing MDS has worse prognosis and response to chemotherapy. In this case, the patient tested negative for some of the most common cytogenetic anomalies in MDS, including inv(3), del(5q), del(7q), monosomy 5 and 8, trisomy 8, del(17p), del(20q), and 11q23 rearrangement. The criteria for diagnosing “AML with myelodysplasia-related changes,” another category in the WHO system, involves 20% blasts in bone marrow or blood, plus at least one of three criteria: a previous diagnosis of MDS, an MDS-related cytogenetic anomaly, or dysplasia in > 50% of cells in at least two myeloid lineages without having an NPM1 or bi-allelic CEBPA mutation.12 The presence of dysplastic myeloid and erythroid cells in this case, although not reaching 50% of observed cells in either lineage, was concerning for MDS and unusual in the presence of a t(8;21) translocation. However, earlier reports of AML have documented cases of t(8;21) with coexistent MDS dysplastic features without MDS-related cytogenetic findings.13-17 These reports have leaned towards concluding that the prognosis and outcomes of such patients are not significantly affected by dysplasia in the absence of MDS-related cytogenetic changes.13-17 A case report described an AML patient with t(8;21) and coexistent MDS dysplasia but without MDS cytogenetics, who achieved complete remission with standard chemotherapy.13 Another study suggested that similar patients should be treated like de novo AML unless MDS-related cytogenetics like del(5q)/-5 and del(7q)/-7 are detected.14 A separate study reported that such patients showed lower numbers of pseudo-Pelger-Huet neutrophils, micro-megakaryocytes, and multi-nuclear erythroblasts, compared to those with AML that evolved from prior MDS.15 Taken together, these reports suggest that morphologic dysplasia alone in AML cases with t(8;21), does not impact prognosis or clinical outcomes, but only has an impact when there are MDS-related cytogenetic changes. The patient in this case also tested negative for mutations in FLT3 and IDH, which are two prognostically important genes in AML that can also serve as targets of therapy.8,18 FLT3 is a tyrosine kinase receptor that can be inhibited with midostaurin, sunitinib, and sorafenib,19 while IDH is a Krebs cycle enzyme that can be inhibited with ivosidenib and enasidenib (which targets IDH1 and IDH2 isoforms respectively).3,8 Patients with these mutations may benefit from having such inhibitors added to induction chemotherapy with cytarabine and daunorubicin to improve clinical outcomes.18 For patients with AML with myelodysplasia-related changes, cytarabine and daunorubicin delivered via the liposomal formulation CPX-351 (Vyxeos) yield better remission and survival than the conventional “7+3” regimen in which cytarabine is given continuously over seven days and daunorubicin is given as three daily boluses.1 Because CPX-351/Vyxeos can also be used for AML with the t(8;21) translocation, the patient in this case received this formulation as his induction chemotherapy without inhibitors for FLT3 or IDH, given that he tested negative for their mutations.

Conclusion In this report, the authors described a case of AML with coexistent t(8;21) and MDS dysplastic features, a finding that has been reported in literature. The combined literature leans towards treating such cases like de novo AML if only occasional dysplastic features (<50% of cells in each myeloid lineage) but no MDS-associated cytogenetics are found, and like AML with MDS-related changes if MDS-associated cytogenetics are found. The patient in this case was treated with CPX-351, a new form of chemotherapy intended mainly for AML with MDS- related changes; however, the treatment can also be used for de novo AML with prognostically favorable cytogenetics, such as the t(8;21) translocation.

Acknowledgements: Special thanks to Carlos Dominguez, M.D., and his fellow Michelle El- Hajjaoui, D.O. in hematology/oncology for their care and management of this patient.

Disclosures: Informed consent was obtained from the patient and all participants in this study.

Conflict of Interest: The authors declare no conflicts of interest regarding this case.

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Table 1. (A) The most common cytogenetic abnormalities found in AML with their associated prognoses, gene products, and AML FAB subtypes. *Denotes 11q23 rearrangements other than t(9;11). Aside from del(Y) and t(11q23), the other rearrangements/translocations are entities in the WHO category, “AML with recurrent genetic abnormalities.” (B) The most common MDS- related cytogenetic anomalies found in AML arising from pre-existing MDS, associated mostly with poor prognosis. Complex karyotype denotes 3 cytogenetic abnormalities. (C) The most common mutations found in AML with their associated prognoses and gene functions. IDH1/2 yields an oncogenic metabolite, 2-hydroxygluarate, which blocks myeloid differentiation and causes epigenetic dysregulation.

Table 2. Laboratory results of our patient presented in this report. The initial CBC generated by the automated hematology analyzer reported a monocyte percentage and absolute count of 45.5% and 13.58 x 103/uL respectively. A manual differential cell count of a peripheral blood smear was performed to verify the CBC results, and instead yielded a blast cell percentage of 90%, and monocyte and neutrophil percentage of 5% each. Coags, AST, ALT, and uric acid are reported in terms of the highest value from the daily values obtained, which still fall within normal range.

Figure 1. Myeloblast with Auer rod in our patient’s peripheral blood smear (Auer rod is circled in red).

Figure 2. Patient’s bone marrow core biopsy showing: (A) bone marrow hypercellularity at 95% on H&E stain (patient is 74 years of age); and positive CD34 staining of myeloblasts at low (B) and high (C) magnification, constituting about 22% of bone marrow cellularity. (D) Iron- laden erythroid progenitors on Prussian blue stain in bone marrow aspirate, reflecting defective hemoglobin synthesis.

Figure 3. Patient’s bone marrow aspirate findings, showing (A) myeloblast, characteristic of AML, with increased nuclear-to-cytoplasmic ratio, open chromatin, and prominent nucleoli; (B) dysplastic neutrophil with pseudo-Pelger-Huet anomaly (bi-lobed nucleus); and also dysplastic erythroid progenitors with (C) karyrrhexis (fragmenting nucleus), (D) megaloblastic change with enlarged nucleus and cell size from failure of nuclear maturation, (E) nuclear budding, and (F) bi-lobed nucleus. Findings in figures B-F are characteristic of myelodysplasia.