AML with inv(16)/t(16;16) and high-risk cytogenetic abnormalities: atypical features and unfavorable outcome

ABSTRACT Objectives Acute myeloid leukemia (AML) with inv(16)/t(16;16) is among the most frequent AML subtypes. It is recognized by the detection of the CBFB-MYH11 fusion which confers a favorable prognosis, irrespective of the presence of secondary cytogenetic abnormalities. However, the effect of additional genetic anomalies on the behavior of inv(16) AML is debatable. Recent case reports describe an unfavorable prognosis for those patients, characterized by early relapse and death. In this study, we present a series of patients with CBFB-MYH11 fusion and high-risk rearrangements to increase knowledge about this potentially distinct subgroup. Methods All cases with inv(16)/ t(16;16) and one or more high risk abnormalities were reviewed at two tertiary healthcare centers between years 2006 and 2020 in terms of demographics, biological and clinical data. Results Among the total 1447 and 1283 AML cases, the frequency was found to be 0,2% and 0.3%. Clinical data could be retrieved for 5 patients. Detected high-risk abnormalities included TP53 and 5q deletion, complex and monosomal karyotype. The median age was 67 years, with a majority of females (M:F = 1:1.5). Two out of 5 patients presented with therapy related AML, with short latency periods. All patients presented with thrombocytopenia and/or leukocytopenia. Bone marrow aspirates revealed atypical morphology and the detection of rare CBFB-MYH11 fusion transcripts. All 5 patients died, with a short mean overall survival of 5.8 months. Discussion and Conclusion Our series suggests that the presence of high risk abnormalities confers distinct biological features and poor prognosis to inv(16) AML.


Introduction
Core binding factor (CBF) acute myeloid leukemia (AML) are among the most frequent subtypes of AML in pediatrics and adults, constituting around 15% of AML cases [1]. CBF AML include cases with t (8;21)(q22;q22.1) and inv(16)(p13q22) or t(16;16)(p13; q22) (later referred to as AML inv (16) in the manuscript). Identification of the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively, is essential for the clinical management of those patients, as treatment with high-dose cytarabine-based chemotherapy results in the established good prognosis of this disease entity [2]. AML inv (16) is characterized by the proliferation of myeloid blasts accompanied by abnormal eosinophils and a cell population with granulocytic or monocytic differentiation. Peripheral white blood cell (WBC) counts tend to be high at presentation. In up to 40% of cases, secondary cytogenetic abnormalities are detected by karyotype analysis. The most common abnormalities include trisomy 22 (known as a cytogenetic clue for the presence of a CBFB-MYH11 fusion), trisomy 8, trisomy 21 and deletion of the long arm of chromosome 7 [2]. The role of these additional anomalies on the prognosis of inv(16) AML remains unclear, with conflicting data published in the literature [2,3]. The gain of an additional copy of chromosome 8 was long thought to be associated with a poor prognosis in inv(16) AML, whereas trisomy 22 was associated with a more indolent course and better survival [2]. However, recent studies reveal a better survival with trisomy 8 and no prognostic effect for trisomy 22 [3]. The effect of an associated complex karyotype is also contradictory [3][4][5]. Recent single reports describe a high risk behavior of inv(16) AML carrying high-risk cytogenetic abnormalities, characterized by early relapse and death [6][7][8][9]. This controversy constitutes a management dilemma on the appropriate treatment strategies of patients with AML inv (16) and a secondary cytogenetic abnormality.
In the aim of shedding light on the behavior of AML inv (16) with secondary high-risk aberrations, we performed a retrospective analysis of all AML cases with inv (16) and at least one high-risk anomaly presenting to two French referral centers over a 14 years period. A review of the diagnostic challenges, cytologic and molecular features, clinical characteristics and prognosis was conducted.

Materials and methods
All cases with confirmed AML diagnosis referred to the cytogenetics laboratories of two French tertiary healthcare centers-Centre Hospitalier de Versailles (CHV) and Centre Hospitalier Universitaire de Grenoble Alpes (CHU-GA)-between years 2006 and 2020, were reviewed. Cases harboring inv(16)/t(16;16) in addition to one or more high-risk cytogenetic abnormalities (as defined by the European Leukemia Network [10]) were retrieved from the laboratory information system. Patient information was collected from the electronic health records including demographics (age and gender), biological (complete blood count, bone marrow aspirate, cytogenetic/molecular findings) and clinical data (presentation, treatment, clinical course).
All patients presented with thrombocytopenia (platelet count < 150000 platelets/uL), and 3 out of 5 patients had low white blood cell counts. Atypical morphology on bone marrow aspirates, with monocytic features and absence of eosinophilia or atypical eosinophils, was described in 4 out of 5 patients.
2 out of 5 cases carried classic (type A) CBFB-MYH11 fusion transcripts. Rare transcripts were detected in the remaining cases (one type B, one E and one F). Highrisk abnormalities varied between patients and included 5q deletion, 17p/TP53 rearrangement, complex and monosomal karyotypes. Associated point mutations, as diagnosed by molecular testing methods, were not uniform among our cohort and included mutations in FLT3-TKD, WT1 and KRAS.
Three out of five patients presented with de novo AML. Two received conventional induction therapy with daunorubicin and cytarabine (3 + 7 schedule), in combination with gilteritinib in 1 patient, driven by the presence of a FLT3-TKD mutation. Only one patient achieved adequate response, but subsequently relapsed. Based on her age and clinical status, the remaining patient received 5-azacytidine, but did not achieve complete remission. The two other patients initially presented with myelodysplasia, one secondary to multiple chemotherapy regimens and radiotherapy for solid tumors (laryngeal, palate, oropharyngeal and pulmonary carcinomas) and the other associated with an excess of blasts. Both received 5-azacytidine before transformation into therapy related CBF AML with complex karyotypes. Palliative care was initiated for both patients.
All 5 patients died, including 4 after disease progression or relapse. The mean overall survival was strikingly low (5.8 months), with a 6-month survival rate of 40% (2 out of 5 patients). A comprehensive summary of patients' information can be found in Table 1.

Diagnostic challenges
High-risk rearrangements and CBF fusions were traditionally thought to be mutually exclusive in AML [11], and the combination was not later considered as a separate entity in disease classification schemes [2,10]. Our 14 years review in 2 referral centers reveals a low prevalence of around 0.3%, indicating the rarity of this co-occurrence. However, this AML inv(16) subgroup could be affected by underdiagnosis. inv(16) is a subtle, often cryptic, cytogenetic anomaly which can be missed by karyotyping alone [2]. Visualization of the inversion becomes more difficult in the presence of numerous additional cytogenetic abnormalities (complex karyotype), especially in low quality metaphases [6]. In 2 of our cases, the inversion was missed by conventional karyotyping and required FISH analysis for detection (cases 3 and 5), due to the involvement of chromosome 16 in rearrangements with other chromosomes. As such, reflex FISH or RT-PCR testing for the detection of a CBFB-MYH11 fusion in the setting of a complex karyotype and a chromosome 16 rearrangement (or loss of one chromosome 16) might be beneficial to increase the diagnostic rate and improve knowledge on the clinical behavior of this AML subtype.

Molecular characteristics
inv (16) results in the fusion of CBFB at 16q22 to MYH11 at 16p13.1, leading to the formation of a classic type A transcript in 85% of cases [12]. Types D and E transcripts constitute 5-10%, while the rare fusions B, C, F-K were only described in individual case reports [12]. Interestingly, rare transcripts (B, E and F) were detected in the majority of our cases (60%), supporting previous observations associating the presence of rare CBFB-MYH11 transcripts with 'atypical' additional genetic rearrangements (i.e rearrangements not involving the numerical gain of chromosomes 22, 8 or 21). Rare fusion transcripts were found to be associated with lower white blood cell counts and atypical cytomorphology but were not identified as independent prognostic parameters [13].

Hematologic characteristics and cytomorphology
As opposed to patients with inv(16) as a sole abnormality [2], our patients presented with leukopenia and thrombocytopenia, in addition to a cytomorphology not typical of the FAB AML-M4 (absence of increased numbers or morphologically aberrant eosinophils). Low WBC counts have been previously reported in association with rare CBFB-MYH11 transcripts, in addition to atypical morphology [13]. The presence of thrombocytopenia in the peripheral blood of our patients, however, has not yet been described nor associated with specific molecular characteristics of AML inv (16).

Treatment related AML inv(16)
Treatment with RT, chemotherapy and topoisomerase inhibitors was shown to be a precursor for the development of t-AML with balanced translocations, including inv (16) [14,15]. The presence of additional cytogenetic abnormalities and complex karyotypes are also known to be more frequent in therapy related myeloid neoplasms, [15] with TP53 mutation constituting the most common alteration [16]. The genetic profiling of t-AML cases with inv(16) should thus prompt the investigation for a 17p/TP53 aberration, taking into account the potential effect on prognosis.
In our t-AML cases, the latency period was found to be shorter (median: 10 months) than the period described for inv (16) t-AML without high risk abnormalities (median: 22 months) [15], indicating a potential effect of the adverse secondary abnormalities.

Effect on prognosis
AML with inv(16) is associated with a good prognosis, characterized by high complete remission rates and long-term overall survival [2]. Favorable outcomes in AML inv (16) are affected by the presence of FLT3-TKD mutations and previous therapy, [2] but not rare fusion transcripts [13]. The clinical behavior of our patients seems to parallel the poor outcome of the additional high-risk cytogenetic abnormalities [10] rather than the favorable prognosis of inv (16). As previously stated, the effect of secondary abnormalities on the prognosis of CBFB-MYH11 positive AML is still debatable. While some studies observed a poor outcome of inv(16) AML with additional monosomy 7/del7q [9], tp53 mutation [17] or complex karyotype [5], others do not report a worse outcome [3,[18][19][20]. These studies, however, considered each secondary abnormality as a single factor in univariate analysis. To date, no published study has compared inv(16) AML with high risk abnormalities to AML with inv(16) as a sole abnormality or other secondary cytogenetic abnormalities. In addition to our case series, 2 published case reports describe a high-risk behavior of this CBF AML subgroup [6,7]. In their refinement of the ELN2017 classification, Herold et al describe a case of AML inv (16) and mutated tp53 treated with HSCT at first intention [17].
In conclusion, data about inv (16) and high risk cytogenetic abnormalities is still scarce. Our case series, in addition to few case reports, suggests a potential clinical behavior as a distinct entity, mimicking the prognosis of high-risk anomalies rather than the CBFB-MYH11 fusion. Larger cohort studies are required to assess the prognostic impact of adverse cytogenetic abnormalities on inv(16) AML.

Availability of data and material
All data is available at the medical records departments of the Centre Hospitalier de Versailles and Centre Hospitalier Universitaire de Grenoble Alpes.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Ethics approval
This study was approved by the ethics committee at the Centre Hospitalier de Versailles, along with informed consents and waivers for deceased patients.