MLL-SEPT6 Positive Acute Myeloid Leukemia Patients Often Co-occur With NRAS Mutations?

Background The MLL-SEPT6 fusion gene is a relatively rare genetic event in leukemia. Its clinical characteristics, prognosis, especially the prole of co-occurring gene mutations remain unclear. Methods We retrospectively analyzed four rare leukemia cases carrying MLL-SEPT6 in our hospital from laboratory examination, diagnosis, treatment and prognosis, and provided a comprehensive and detailed description on clinical prole of MLL-SEPT6-positive AML patients in the literature. Results All the four patients were diagnosed with acute myeloid leukemia (AML) and harbored X chromosome and 11 chromosome rearrangements. Three of four cases occurred NRAS mutation while the rest one with congenital AML did not. Of the four cases, one developed drug-resistant, one suffered relapse after bone marrow transplantation (BMT) and one died. Combined with other cases reported in literatures, we found that of all patients diagnosed with AML, 90.9% were children ( ≤ 9 years old) and 54.5% were infants ( ≤ 1 year old). The survival time between infant group ( ≤ 1 year old) and pediatric group (>1 and <18 years old), patients that received BMT and that received chemotherapy alone did not show signicant differences (P>0.05). Conclusions MLL-SEPT6 was more commonly observed in pediatric AML patients, some of which may co-occur with NRAS mutations. The prognosis was inconclusive and may not be related to age or BMT. More information needs to be accumulated and summarized from additional cases to conrm the underlying connection between NRAS mutations and MLL-SEPT6 in order to better understand the prole in MLL-SEPT6-positive AML.

All MLL-SEPT6 positive cases (case 1-4) were male with ages ranging from 0 to 57 years and a median of 5.2 years, their detailed information was summarized in Table 1. All cases had manifestations of fever and pale complexion. The three pediatric cases (cases 2, 3, and 4) were accompanied by hepatosplenomegaly, and two cases (cases 3 and 4) had scattered petechiae and ecchymoses. The case 1 is an elderly male patient with perianal abscess and diabetes in addition to the above symptoms. The case 2 was accompanied by pain in both lower limbs, tenderness of the sternum and hypertrophy of tonsils. In the case 3, multiple lymph nodes were palpable on bilateral neck, he was positive of sternal tenderness, he also had hyperuricemia and acute bronchial pneumonia. The case 4 is a newborn delivered by cesarean section due to "decreased fetal heart rate". The birth weight was 3100g. The infant had no spontaneous breath at birth and was generally cyanotic, he restored spontaneous respiration under assisted ventilation, he developed persistent pulmonary arterial hypertension and neonatal pneumonia. His parents were healthy and with no history of genetic disease. Laboratory results showed that three cases (cases 1, 3, and 4) had high white blood cells (WBC) count, anemia, and low platelet count. One case (case 2) had low level of hemoglobin and WBC count but normal platelet count, and two cases (case 2 and 3) had increased level of serum lactate dehydrogenase. The Ddimer level of all four patients were increased.
All cases exhibited morphological characteristics of AML ( Figure 1). The French American British (FAB) morphological classi cation of each case was M5 (case 1 and 4), M2 (case 2) and M4 (case 3). Three cases (cases 1, 3, and 4) showed hypercellular bone marrow and the other case (case 2) had severe hypocellular bone marrow. The case 1 and case 4 were evaluated as M5 and revealed of 92.0% and 20.4% blasts in marrow aspirate, 69.0% and 17.0% in peripheral blood respectively (Table 1, Figure 1A and 1D). The case 2 was evaluated as M2, the marrow aspirate revealed 27.5% myeloblasts and the peripheral blood exhibited 2.0% blasts (Table 1, Figure 1B). The case 3 was evaluated as M4, the marrow aspirate showed 56.0% blasts and the peripheral blood exhibited 52.0% blasts (Table 1, Figure 1C).

Flow cytometric analysis
The ow cytometric analysis revealed the presence of myeloid blasts in bone marrow samples from all four patients (Table 1, Figure 2). The percentage of blasts was highest of 92% in the case 1 and lowest of 4.2% in the case 3. All cases were positive for CD33, CD15, CD64 indicating myeloid lineage, CD13 was positive only in the case 1 and case 2. All the cases were positive for CD38 and HLA-DR. CD117 was positive only in the case 3, CD34 was negative in all the four cases.

Molecular analysis
We performed molecular biology tests including screening of fusion genes and next-generation sequencing (NGS) analysis on all cases ( Table 1)

Clinical Course
The treatment and follow-up information of all cases (case 1-4) was shown in Table 1. Three cases (case 1-3) received chemotherapy, the case 2 subsequently received a bone marrow transplantation (BMT), the case 4 was a newborn and did not receive any chemotherapy. The clinical follow-up period ranges from 0.5 to 38 months with a median of 21.5 months. The case 1 initially received IA (idarubicin + cytosine arabinoside) regimens, which didn't make him reach CR.
Then he began a new chemotherapy regime with decitabine combined with half dose CAG (cytarabine + aclacinomycin + granulocyte colony stimulating factor) and achieved CR. He also received an intrathecal injection (cytarabine + dexamethasone + methotrexate) for central nervous system in ltration prevention. After the chemotherapy, the bone marrow aspiration of the patient revealed a normal karyotype and negative molecular results of MLL-SEPT6 rearrangement and NRAS mutation. The case 2 received MAE (mitoxantrone + cytarabine + etoposide) regimens and reached CR one month later. In the following six months, the patient received BMT. Nineteen months after the transplantation he was re-admitted for headache. A ow cytometry analysis of the cerebrospinal uid revealed 81.9% leukemia cells which indicated central nervous system leukemia, following ve courses of intrathecal injection, the child's headache relieved and no leukemia cells were detected in his cerebrospinal uid. The case 3 received HA (homoharringtonine + cytarabine) regimens and intrathecal injection (cytarabine + dexamethasone + methotrexate), he reached CR four weeks after the diagnosis. This patient relapsed 27 months later, after receiving HAI (homoharringtonine + cytarabine + idarubicin) regimen he reached and remained CR status. The case 4 had dyspnea at birth, he was on assisted ventilation and given blood infusion to improve anemia and thrombocytopenia. The newborn's condition did not improve during the treatment. His parents refused the follow-up treatment, and the child died a week later.

Literature review
A total of 22 MLL-SEPT6 positive cases were included in this literature review, including four cases in our report and eighteen cases from literatures [6-16]. Table 2 and Table 3 listed the detailed laboratory results and clinical information of all cases. were children (≤ 9 years old), including twelve (54.5%) infants (≤1 year old). The majority of the patients manifested leukocytosis (range 1-608 × 10 9 /L), anemia (range 41-109 g/L) and low platelet counts (range 9-254 × 10 9 /L). According to the high WBC index [18], nine (60.0%) of the fteen cases with WBC count information were de ned as high WBC levels. Twelve cases were not provided with description of clinical features. Of the remaining ten cases, ve children (50.0%) were observed of splenomegaly and hepatomegaly and three patients (30.0%) had lymphadenopathy. Central nervous system involvement was observed in three children (30.0%) and skin involvement was observed in two (20.0%).
All patients were diagnosed with AML (twenty children and two adults) according to the former FAB classi cation: ve patients (three children and two adults, 22.7%) of M5, ve children (22.7%) of M4, eight children (36.4%) of M2, one child (4.5%) of M1, and three children (13.6%) unknown.
All the cases had available cytogenetic information, chromosomal translocations (eleven cases) were the most common chromosomal rearrangements, followed by chromosomal insertions (nine cases). Among them, Xq24 (nine cases) and 11q23 (fourteen cases) were the most frequently involved chromosomal bands. Seven cases (31.8%) demonstrated complex abnormalities.
Of all 22 cases, 18 cases had clinical follow-up with median period of 27.7 months (0.5-101.5 months). Table 3 showed the clinicopathologic features of evaluable patients. Eight patients (42.1%) received chemotherapy alone. Nine patients (50%) received BMT. Six of eighteen patients died during the period of follow-up. Kaplan-Meier survival analysis was performed on eighteen cases with complete follow-up information ( Figure 4A). As of the nal follow-up, median survival time has not been reached. In order to understand the impact of age and BMT on survival time, the patients were divided into infant group (≤1 year old, n=10), pediatric group (>1 and <18 years old, n=7), and adult group (≥18 years old, n=1). At the time of the last observation, there was no statistically signi cant differences in survival time between infant group and pediatric group (hazard ratio for infant-pediatric = 0.26, 95% con dence interval = 0.07 to 1.67, P = 0.1822, Figure 4B). The adult group was not included in the statistical analysis because there was only one case with complete follow-up information in this group. Meanwhile, the patients were also divided into receiving chemotherapy alone (n=6, one without survival information was excluded) and receiving BMT (n=9) treatment groups according to the treatment protocol. Survival time between the patients received chemotherapy alone and BMT did not show signi cant differences neither (hazard ratio for chemotherapy alone-BMT = 1.04, 95% con dence interval = 0.18 to 6.19, P = 0.9647, Figure 4C).

Discussion
The MLL gene is a frequent target of rearrangement in human leukemia, especially in infant and pediatric leukemia [9,19,20]. It is well established that the rearrangement heralds poor prognosis [5,21]. These rearrangements include fusions with many partner genes, but rarely involve the X chromosome. SEPT6 is a member of the septin family of GTPases. Members of this family are involved in cell polarity, cytokinesis and oncogenesis [22,23]. The MLL gene and SEPT6 gene are vulnerable to damage to form translocations associated with infant AML.
In this study, we described four cases of AML with MLL-SEPT6 fusion gene. The FAB subtypes were mainly M2, M4 and M5, which was consistent with literatures. Most cases that have been reported were children. As far as we know, only two adult patients have been reported including one case in our series and 54.5% (12/22) were infant patients (≤1 year old). Among these cases, 60.0% were with high level of WBC, and 30% manifested central nervous system involvement, which were similar to the clinical features of MLL-rearranged AML patients. The ndings of Balgobind BV et al. showed that MLL-rearranged AML patients usually exhibit high tumor burden, including organomegaly, high median WBC and central nervous system involvement [24]. The present study included the largest number of MLL-SEPT6 cases to date. The patients' NGS test results were not provided except ours, and we also tracked the patients' molecular biological examination results. Three of four cases in our series occurred NRAS mutation while the rest one with congenital AML did not.
The NRAS mutations has very important roles in pathogenesis and progression of human leukemia, which have been frequently reported in AML patients [25,26]. NRAS G12V is required in leukemia self-renewal process, independent of its effects on growth and survival [27]. Compared with other subtypes of leukemia, acute leukemia with MLL-translocations (such as MLL-AF4 and MLL-AF9) harbored the fewest number of mutations, in which NRAS mutations commonly co-occur [27,28]. In our series, we identi ed NRAS mutations in MLL-SEPT6 positive AML patients for the rst time, and most of the mutation sites appeared at codon 12 and 145. The former site is a hotspot mutant of NRAS and the latter site has also been reported [25,29]. The VAF of NARS mutation decreased as the patients condition improved. When the patients achieved CR, it also turned negative. The underlying connection between NRAS mutations and MLL-SEPT6 and whether non-congenital MLL-SEPT6-positive AML patients all have NRAS mutations remain to be further studied in a larger cohort in the future.
Of the four cases in our series, one developed drug-resistant at rst, one suffered relapse after BMT and one died, showing unsatisfactory therapeutic effect.
However, whether the outcomes of patients with MLL-SEPT6 were aggravated by the concurrence of NRAS mutations needs a follow-up study. Kaplan-Meier curve demonstrated that pediatric group (>1 and <18 years old) did not show better survival time compared with infant group (≤1 year old). Age may not be an independent prognostic factor for survival. Most of the patients received chemotherapy, nine of them received BMT, but three of them eventually died. The survival time of patients between the chemotherapy alone group and the BMT group did not show a signi cant difference, which suggested that BMT may not improve the survival time of such patients. This was consistent with several studies and meta-analyses that suggesting BMT does not improve survival in patients with MLL rearrangement [34,35]. But the prognosis and its factors of MLL-SEPT6-positive AML patients need further analysis in more cases.
Our study has some limitations. Firstly, this was a retrospective study, coupled with limited number of reported cases and incomplete clinical information, which makes it impossible for us to obtain the median survival time of patients through statistical methods. On the other hand, the small sample sizes of some subgroups may lead to false negative results. Secondly, our NGS detection only covers the twenty most frequently mutated genes in AML, and prognostic effects of some critical genes may be neglected. Thirdly, gene mutation information in the reported cases was not available and cases in our series are detailed but limited by sample size. Therefore, our ndings need to be combined with more cases to further explore the correlation between MLL-SEPT6 and NRAS mutations.

Conclusions
In conclusion, the MLL-SEPT6 fusion gene was more commonly observed in pediatric patients diagnosed with AML. NRAS mutations were observed in these patients, most frequently of the NRAS G12V hotspot mutation. Whether NRAS mutations are related to the occurrence of MLL-SEPT6-positive AML is currently unclear. The prognosis was inconclusive and may not be related to age. BMT may not improve survival in these patients. More cases should be accumulated and summarized to better understand the pro le in MLL-SEPT6-positive AML. Our ndings provide a basis for better understanding the mechanisms of leukemogenesis and the development of potential therapeutic targets for MLL-SEPT6-positive AML.