B-ALL With t(5;14)(q31;q32); IGH-IL3 Rearrangement and Eosinophilia: A Comprehensive Analysis of a Peculiar IGH-Rearranged B-ALL

Background: B-cell acute lymphoblastic leukemia associated with t(5;14)(q31;q32); IGH-IL3 is an exceptional cause of eosinophilia. The IGH enhancer on 14q32 is juxtaposed to the IL3 gene on 5q31, leading to interleukin-3 overproduction and release of mature eosinophils in the blood. Clinical, biological and outcome data are extremely scarce in the literature. Except for eosinophilia, no relevant common feature has been highlighted in these patients. However, it has been classified as a distinct entity in the World Health Organization classification. Cases Presentation: Eight patients with t(5;14)(q31;q32) treated by French or Austrian protocols were retrospectively enrolled. Array comparative genomic hybridization, multiplex ligation-dependent probe amplification or genomic PCR search for IKZF1 deletion were performed in 7. Sixteen patients found through an exhaustive search in the literature were also analyzed. For those 24 patients, median age at diagnosis is 14.3 years with a male predominance (male to female ratio = 5). Eosinophilia-related symptoms are common (neurologic in 26%, thromboembolic in 26% or pulmonary in 50%). Median white blood cells count is high (72 × 109/L) and linked to eosinophilia (median: 32 × 109/L). Peripheral blasts are present at a low level or absent (median: 0 × 109/L; range: 0–37 × 109/L). Bone marrow morphology is marked by a low blast infiltration (median: 42%). We found an IKZF1 deletion in 5 out of 7 analyzable patients Outcome data are available for 14 patients (median follow-up: 28 months): 8 died and 6 are alive in complete remission. Some of these features are concordant with those seen in patients with other IGH-rearranged B-cell acute lymphoblastic leukemias: young age at onset, male sex, low blast count, high incidence of IKZF1 deletion and intermediate prognosis. Conclusion: Based on shared epidemiological and biological features, B-cell acute lymphoblastic leukemia with t(5;14)(q31;q32) is a peculiar subset of IGH-rearranged B-cell acute lymphoblastic leukemia with an intermediate prognosis and particular clinical features related to eosinophilia.

Background: B-cell acute lymphoblastic leukemia associated with t(5;14)(q31;q32); IGH-IL3 is an exceptional cause of eosinophilia. The IGH enhancer on 14q32 is juxtaposed to the IL3 gene on 5q31, leading to interleukin-3 overproduction and release of mature eosinophils in the blood. Clinical, biological and outcome data are extremely scarce in the literature. Except for eosinophilia, no relevant common feature has been highlighted in these patients. However, it has been classified as a distinct entity in the World Health Organization classification.
Cases Presentation: Eight patients with t(5;14)(q31;q32) treated by French or Austrian protocols were retrospectively enrolled. Array comparative genomic hybridization, multiplex ligation-dependent probe amplification or genomic PCR search for IKZF1 deletion were performed in 7. Sixteen patients found through an exhaustive search in the literature were also analyzed.
For those 24 patients, median age at diagnosis is 14.3 years with a male predominance (male to female ratio = 5). Eosinophilia-related symptoms are common (neurologic in 26%, thromboembolic in 26% or pulmonary in 50%). Median white blood cells count is high (72 × 10 9 /L) and linked to eosinophilia (median: 32 × 10 9 /L). Peripheral blasts are present at a low level or absent (median: 0 × 10 9 /L; range: 0-37 × 10 9 /L). Bone marrow morphology is marked by a low blast infiltration (median: 42%). We found an IKZF1 deletion in 5 out of 7 analyzable patients Outcome data are available for 14 patients (median follow-up: 28 months): 8 died and 6 are alive in complete remission. Some of these features are concordant with those seen in patients with other IGH-rearranged B-cell acute lymphoblastic leukemias: young age at onset, male sex, low blast count, high incidence of IKZF1 deletion and intermediate prognosis.
Conclusion: Based on shared epidemiological and biological features, B-cell acute lymphoblastic leukemia with t(5;14)(q31;q32) is a peculiar subset of IGH-rearranged B-cell acute lymphoblastic leukemia with an intermediate prognosis and particular clinical features related to eosinophilia.
Keywords: acute lymphoblastic leukemia, cytogenetics and molecular genetics, clinical and molecular epidemiology, eosinophilia, IKZF1 rearrangement BACKGROUND B-cell acute lymphoblastic leukemia (B-ALL) with translocation t(5;14)(q31;q32); IGH-IL3 is an exceptional cause of massive eosinophilia recognized by the World Health Organization (WHO) classification of hematological malignancies as a distinct entity (1,2). The translocation t(5;14)(q31;q32) juxtaposes the IGH enhancer located on 14q32 to the IL3 gene on 5q31. The subsequent production of interleukin-3 (IL3) induces the maturation and release of eosinophils in the blood stream (3)(4)(5). The relationship between IL3 and eosinophilia is also supported by the constant eosinophilia in all patients treated with recombinant IL3 for Blackfan-Diamond anemia in one study (6). The IL3-receptor has been hypothesized to be present on blast cells and to induce JAK-STAT-dependent cell survival and proliferation in an autocrine manner (4,7). The other part of the translocation involves the IGH gene, which is frequently rearranged via chromosomal translocations in mature B-cell neoplasms such as Burkitt leukemia/lymphoma, mantle cell or follicular lymphomas with MYC, CCND1, or BCL2 as partner genes, respectively, whose expression is upregulated (8). IGH translocations are also reported in B-ALL and usually involve other partners such as members of the CEBP gene family, ID4, CRLF2 or EPOR (9-13). Due to the rarity of t(5;14)(q31;q32) associated B-ALL, little is known regarding clinical and molecular features. To further characterize this very rare entity, we have analyzed in detail 8 new patients and combined these data to those of 16 patients from the literature over 35 years.

Conventional Cytogenetics
Karyotypes were analyzed on bone marrow samples at diagnosis and relapse according to the International System for Human Cytogenetic Nomenclature (ISCN, 2016). Whenever possible, at least 20 metaphases were analyzed.

Fluorescence in situ Hybridization
Fluorescence in situ hybridization (FISH) was performed on cytogenetic preparations using an IGH dual-color breakapart probe (Vysis, Abbott Molecular Inc.) according to the manufacturer's instructions.

Informed Consent
The study was approved by the local ethics committee (Comité Local d'Ethique pour la recherche clinique des HUPSSD Avicenne-Jean Verdier-René Muret (CLEA), December 13th, 2018, CLEA-2018-58). According to French legislation, due to the retrospective observational design of the study, informed consent from patient was waived.

CASES PRESENTATION
Firstly, we report here on our 8 patients selected for the presence of the specific t(5;14)(q31;q32). Clinical characteristics are detailed in Table 1, main biological features in Table 2 and outcome in Table S1. Patients #3 and #7 were previously reported (14,15), but are yet further described here because of availability of new relevant biological data and because of occurrence of events during longer follow-up. We chose to highlight hereunder the main clinical features of these patients according to age at onset: pediatric/adolescent onset (8-18 years old) and young adult onset (21-39 years old). We then analyzed them together with the 16 patients from the literature in a third section.

Pediatric Onset
Six patients were children or adolescent (8, 10, 11, 14, 14, and 18 year-old) including one with Down syndrome (patient #7). Two patients presented severe neurologic involvement (encephalopathy with pyramidal syndrome and acute confusion). Magnetic resonance imaging (MRI) showed scattered involvement of white matter and myelitis in one case, multiple cortical and subcortical ischemic lesions in another. Symptoms disappeared after the first courses of chemotherapy. No thrombo-embolic event was recorded. Peripheral blast cells count was increased in 2 (36.6 and 18.7 × 10 9 /L) but was low in others (range: 0-2 × 10 9 /L) and even absent in 10. Peripheral eosinophilia was increased (range: 17.5-96 × 10 9 /L). All patients reached complete remission after induction therapy. All patients are in first (n = 3), second (n = 1), or third (n = 1) continuous complete remission (CCR) for more than 3 years, except for the patient with Down syndrome who died from disease progression after a second relapse.

Young Adult Onset
Two patients were young adults (31 and 39 years of age). Both presented with severe thrombo-embolic events (lower limb venous thrombosis with pulmonary embolism and multiple venous thrombosis with transient strokes, respectively). No encephalopathy was documented. Multiple but asymptomatic subcortical lesions on MRI were present in 1 patient. Peripheral blast cells count was 0 and 0.2 × 10 9 /L, respectively. Eosinophilia was much lower (7.9 and 6 × 10 9 /L). Prognosis was poor: none reached complete remission after induction therapy, one is in second complete remission (CR2) 1 month after hematopoietic stem cell transplantation and one is deceased after relapse.
Eosinophilia-related symptoms were common at onset. Ten out of 20 (50%) patients had lung involvement (dyspnea, cough, chest pain, pleural effusions with eosinophilia, interstitial alterations on chest X-ray). Four patients presented multiple venous thromboses (pulmonary embolism in 3 patients and cerebral venous thrombosis in 1), 1 patient had arterial thrombosis of the lower limb [i.e., 5 out of 19 (26%)]. Nine out of 19 (47%) patients had cardiac involvement, e.g., newly acquired ECG abnormalities, global cardiac failure (n = 1), Loeffler myocarditis on MRI (n = 3), restrictive cardiomyopathy (n = 1), or ventricular hypertrophy (n = 1), leading to death due to ventricular fibrillation in one patient. Five out of 19 (26%) patients had neurologic symptoms (decreased consciousness, cerebral palsy, generalized seizure, and transient ischemic attack). MRI data were available for 3 of our patients (described in text above) and in Kobayashi et al. (34). No patient had blasts in the cerebrospinal fluid at initial presentation. Five out of 19 (26%) patients had an erythematous urticaria-like skin involvement.
An IKZF1 deletion was searched only in our newly described patients and detected in a large proportion [5/7 (71%), Table S4]. The isochromosome 7q in Knuutila et al. (26) also leads to the loss of IKZF1.
(3) Eosinophilia-related symptoms are common and severe. It is the expected consequence of the underlying translocation: IL-3 overproduction induces maturation and release of eosinophils in the blood from the bone marrow. (4) Lymphoblastic infiltration of the bone marrow is partial and explains that peripheral blasts are almost undetectable in a vast proportion of patients. This partial infiltration may also account for normal to slightly decreased hemoglobin and platelet count. In cases with <20% of infiltrating blast cells, the specific translocation permitted a diagnosis of t(5;14)(q31;q32) associated B-ALL (2). The frequent CNS-related symptoms with no ALL blast infiltration suggests a direct eosinophil toxicity on neuronal, glial and endothelial cells caused either by eosinophilic proteins release or eosinophil cells infiltration (36,37). Depending on the damaged cell type, symptoms may include signs of encephalopathy or focal symptoms due to local thrombosis and multiple infarcts (38). (5) IKZF1 deletions are frequently detected. (6) Response to treatment is poor: induction failure and high levels of MRD are frequently observed, and may account for the intermediate prognosis.
These particularities suggest new diagnostic strategies, notably because (a) peripheral blasts are often absent (even by flow cytometry analysis) and (b) the translocation t(5;14)(q31;q32) may be missed owing to the low rate of abnormal metaphases on standard karyotype and metaphase FISH. It is worth noting that the 2 patients with a prolonged interval between blood eosinophilia and the appearance of blast cells in the bone marrow could fulfill criteria of idiopathic hypereosinophilic syndrome (39). Persistent and/or massive eosinophilia should thus be investigated by repeated bone marrow aspirations, including cytogenetic investigations and IGH-FISH on cytogenetic pellet on a sufficient number of metaphases and interphase nuclei. FISH analysis on bone marrow smears (patients #2 and #6) or on leukemic cytometer-sorted cells (34) (patient #8) can be used. As suggested in (35,40), a bone marrow biopsy may reveal focal clusters of blasts missed by bone marrow aspiration. Next generation sequencing may be an alternative to detect IGH-IL3 rearrangement (35) . Finally, careful monitoring of eosinophils should be performed during and after treatment as increased eosinophils may suggest a relapse. Of note, t(5;14)(q31;q32); IGH-IL3 B-ALL shares remarkable features with other IGH-rearranged B-ALL (5-11% of B-ALL) (9, 10, 12, 41) ( Table 4). (1) IGH-rearranged B-ALL also affects especially adolescents and young adults [median age of onset: 25 years (12) or 16 years (9)]. (2) A male predominance is present even if male to female ratio is lower (1.44) (12). (3) In both entities, a majority of patients with low blast count is observed (9,10,41). (4) A high incidence of IKZF1 deletion (40%) is frequent in patients with IGH-rearranged B-ALL (9). (5) Only 6 out 14 evaluable patients with t(5;14)(q31;q32); IGH-IL3 B-ALL are alive which mirrors the 27-30% overall survival observed in 3 studies (9,12,41).
In conclusion, t(5;14)(q31;q32); IGH-IL3 B-ALL constitutes a very rare subset of IGH-rearranged B-ALL. The IL3 partner gene is responsible for the specific symptomatology and increased awareness of this B-ALL may lead to a correct diagnosis of B-ALL when facing a severe symptomatic eosinophilia. International collaboration should provide new insights into its biology and bring new treatment strategies.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Comité Local d'Ethique pour la recherche clinique des HUPSSD Avicenne-Jean Verdier-René Muret (CLEA), December 13th, 2018, CLEA-2018-58. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
BF, EB, AA, ML-P, and AB: conception of the study. All authors: collection and assembly of data, final approval of manuscript, and accountable for all aspects of the work. BF, EB, ML-P, and AB: data analysis and interpretation and manuscript writing.