First experience with BAALC-expressing leukemia stem cell fraction in juvenile myelomonocytic leukemia patients treated by hematopoietic stem cell transplantation

Introduction

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative pediatric malignancy [1, 2]. It is initiated by activation of the RAS signal transduction pathway caused by germline or somatic mutations of RAS-genes (NRAS, KRAS), or any genes regulating RAS-pathway (presumably PTPN11 and, less frequently, NF1 or CBL). These mutations provide higher sensitivity of myeloid progenitors to granulocyte/monocyte colony-stimulating factor (GM-CSF) [3]. Approximately 90–95% of patients with JMML are characterized with canonical mutations in PTPN11 regulator gene of RAS signaling pathway (35%), or, actually, NRAS and KRAS genes (20–25%). Less frequent mutations are found in two other genes- regulators of RAS signaling pathway, e.g., NF1 (10-15%), or CBL (10-15%) [4, 5]. A transient myeloproliferative disorder with a good prognosis is observed in cases of germline NRAS, KRAS, PTPN11, or CBL gene mutations. In contrast, a more aggressive clinical course is typical of JMML with somatic mutations of genes controlling RAS signaling pathway [5]. One should add that unexpectedly high incidence of prognostically poor EVI1-positive variants is seen among the patients with this disorder [6, 7]. Hence, one may suggest a modern risk-stratification classification of JMML which could include this molecular marker [5].

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the only curative therapy for most of these cases, however, followed by relapse in about 35% of treated patients, repeated HSCT may be a necessary option [1, 8-12]. Working Group of Japan for Hematopoietic Cell Transplantation enrolled 129 HSCT-treated children with JMML, wherein 5-year overall survival (OS) rate reached 73%, whereas the cumulative incidences of relapse and transplantation-related mortality to be only 26% and 9%, respectively. The authors attributed their successful treatment to usage of preparation for HSCT and myeloablative conditioning regimen consisting of busulfan, fludarabine and melphalan, as well as development of chronic graft-versus-host disease (GvHD) [10].

Hence, a conclusion was drawn towards successful cure of significant proportion of children with JMML by means of HSCT conditioned with busulfan/fludarabine/melphalan. Another important suggestion presumes pre-transplant chemotherapy under careful molecular control [5, 13, 14]. To achieve these purposes, NGS analysis of the chosen gene methylation, or decrease in RAS gene mutation levels were suggested. However, this kind of monitoring may be problematic in clinical setting. Moreover, haploidentical HSCT was shown to be effective in JMML treatment [15]. Finally, the second allogeneic HSCT proved to be available for significant proportion of the patients relapsing after first allograft [16]. Meanwhile, chemotherapy in JMML patients is used quite often as a bridge to HSCT [16, 17].

Despite in-depth studies of clonal leukemia-initiating cells, their nature was not elucidated yet [18]. Theoretically, this role may be fulfilled by a subpopulation of BAALC-expressing stem cells (SCs) which have been previously revealed by us in several clinical variants of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) [19-21]. In turn, this approach may be useful for the following tasks: a) elucidation of JMML pathogenesis; b) development of JMML risk-stratification system; c) quantitative assay of treatment efficacy, including HSCT, on the level of BAALC-expressing stem cell fraction.

Figure 1. Graphic presentation of RAS signaling pathway with regulatory PTPN11, N/KRAS, NF1 and CBL genes affected by mutations, which provide higher sensitivity of hematopoietic precursors to granulocyte-macrophage colony stimulating factor (GM-CSF) and contribute to formation of main myeloproliferative component in JMML patients. [Extracted from Gupta et al. [4]

Patients and methods

The study enrolled thirteen patients (10 boys and 3 girls, aged 0.3-6 years, a mean of 2.8 years) with proven JMML disorder, with mutation profile confirmed by means of Sanger sequencing analysis. Mutation in PTPN11 gene-regulator of RAS signaling pathway was detected in 8 patients; NRAS, in four cases, whereas CBL and NF1 gene mutations were characteristic for 2 one patients (each on one case). Serial counts of WBCs, numbers of blasts and monocytes in bone marrow and peripheral blood were performed in parallel samples. Where possible, the levels of gene BAALC, WT1 and EVI1 expressions were measured by means of RT-qPCR using standard protocols. The cut-off values for lower vs higher gene expression levels were 31% and 10% for BAALC and EVI1 genes, respectively. The cut-off value for WT1 gene was 250 gene copies per 10⁴ copies of ABL1 reference gene [21].

Analysis of mutations in PTPN11, CBL and NRAS genes

Genomic DNA was extracted from whole blood and bone marrow nucleated cells using a Blood DNA Column Kit (Inogene, Russia). The concentration of genomic DNA was measured using the device NanoDrop One (Thermo Fisher Scientific, USA). The mutational analysis of the genes was performed using Sanger sequencing method with the 3500 Genetic Analyzer (Applied Biosystems, USA). The coding sequences of PTPN11 gene (exons 3 and 13), NRAS (exons 2 and 3), and CBL (exons 8 and 9) were evaluated.

Analysis of BAALC, WT1 and EVI1 expression

Total RNA was extracted from blood and bone marrow by using the TriZ Reagent RNA kit (Inogene, Russia). RNA concentrations were assessed with NanoDrop One (Thermo Fisher Scientific, USA). cDNA synthesis was carried out using total RNA (1-10 micrograms) and performed using a random hexamer and REVERTA-L kit (AmpliSens, Russia).

Where possible, the levels of gene BAALC, WT1 and EVI1 expressions were measured by means of Real-time quantitative RT-PCR (Q-PCR) analysis with CFX 96 (BioRad, USA). Expression was calculated using the following formulas:

NCN_BAALC = CN_BAALC/CN_ABL1 * 100%

NCN_WT1 = CN_WT1/CN_ABL1 * 100%

Relative Expression_EVI1 = 2^{Ct(ABL1)-Ct(EVI1)}*100%

Where NCN, normalized copy number; CN, copy number; Ct, cycle threshold.

The primers and protocols were adopted from earlier studies [doi:10.21320/2500-2139-2019-12-3-303-308; 10.1016/j.jmoldx.2020.05.010; 10.1016/j.cell.2014.02.019]. The cut-off values for lower vs higher gene expression levels were 31% and 10% for BAALC and EVI1 genes, respectively. The cut-off value for WT1 gene was 250 gene copies per 10⁴ copies of ABL1 reference gene [21].

The number of transplanted CD34-positive stem cells ranged from median of 5.5*10 (1.5 to 10.6*106) CD34+cells per kg, whereas the myeloablative conditioning regimen with two or three alkylating agents was preferred. Myeloablative conditioning (MAC) consisted of busulfan-based (>12 mg/kg) or Treosulfan-based regimen (30-42 mg/kg); reduced-intensity conditioning (RIC) was used in 3 patients (9%), i.e., Fludarabine (∑150 mg/m²) + Melphalan (∑140 mg/m²). Maximally expressed changes of basic clinical and laboratory values are presented in Table 1. The median follow-up after HSCT was 44 months (1-96 months).

Table 1. Maximal levels of WBC counts and selected genetic markers in 13 JMML patients showing different mutations of RAS and related genes of RAS-signaling pathway

Notes: * transformation to AML, † died; changed clinical and laboratory parameters are indicated in bold, OS, overall survival.

The longest OS terms, ranging from 1178 to 2462 days (a mean of 2028 days) were detected in the patients less than 1 year, all of whom are alive at present. Further on, the BAALC expression levels in bone marrow ranged from 73 to 9%. It was the highest (73 and 64%) in two patients (#1 and #2). Meanwhile, higher levels of BAALC expression (from 63 to 50%) were determined in patients #3, #5, #11 and #12. The minimal BAALC gene expression it was characteristic for a patient with cumulative PTPN11 and NRAS mutations (#5), and a case with single NRAS mutation (#8}. Of seven patients with PTPN11 mutation, the highest level of BAALC expression was noted in a 4-year-old patient (#1) with trisomy 8 in karyotype, wherein the BAALC gene is mapped. This case was also peculiar due to higher expression of WT1 gene (941 copies) and high blast cell counts in bone marrow (up to 16.8%). Moreover, higher levels of BAALC expression (64 and 63%) were detected in 3 patients with similar mutation (#2-4). One of them (#3) revealed JMML transformation to acute myeloid leukemia (AML). It should be noted that similar transformation to AML was diagnosed also in two other patients (#5 and 13). One of whom was treated successfully with haploidentical related grafting.

Of interest, 5 out of 13 JMML patients (##2, 3, 5, 9 and 13) were EVI1-positive. Four of them died relatively soon after transplantation, whereas patient #5 achieved complete clinical and molecular remission associated with lower level (19%) of BAALC-e SC fraction. In general, 6 out of 13 JMML patients (45%) treated with HSCT died with overall survival ranging from 87 till 1024 days. These cases included 4 abovementioned patients with EVI1-positive leukemia as well as two cases (##3 and 13) with transformation to AML. Meanwhile, some patients with single NRAS mutation (#9 and #10) showed BAALC expression levels of 63% and 41%, respectively, thus being close to cutoff level (35%) in the last patient (#8) with cumulative PTPN11/NRAS mutations, and the longest OS registered in the group (2462 days).

Since HSCT is considered the main therapeutic approach in JMML, we analyzed this treatment option more carefully. As shown in Table 1, single allo-HSCT was performed in four patients, whereas its combination with Haplo-HSCT was carried out in three cases. Further on, single Haplo-HSCT was performed in 6 patients, being repeated in two cases (##3 and 9). It should be noted again, that a third of these malignancies were EVI1-positive, whereas transformation to secondary AML was diagnosed in two cases (##3 and 13). The conditioning regimens were not identical in this group, and the number of transplanted HSCs ranged widely.

Transplant failure was registered in five cases, being registered again in patient #2. In general, 6 out of 13 studied patients (45%) deceased, and their OS ranged from 87 to 1024 days (mean, 470 days). Among them, 4 out of 5 patients exhibited EVI1-positive malignancy including two cases with subsequent transformation to secondary AML. The comparison group enrolled six surviving JMML patients aged 0.3-6 years. Their OS terms ranged from 978 to 2363 days (mean – 1697 days).

To illustrate typical findings in JMML patients, we present some cases in details, showing close association between the main laboratory markers and therapies performed. The first case concerns a 1.5-year old boy (Table 1, #2) harboring PTPN11 mutation. His data (Table 2) show that the initial level of BAALC gene expression was relatively high (64%) as well as EVI1 gene expression (17%). The initial induction treatment included cytarabine and 6-MP followed by 6 cycles of hypomethylating agents. The resulting clinical effect was also accompanied by fast normalization of BAALC and WT1 expression. The therapy was enforced later by the two-step treatment with HSCT and haplo-transplant. Both transplants were not quite effective because of fast transplant rejection. Subsequently, the last donor chimerism value decreased to 20-29%. In sum, the OS terms after first HSCT reached 1024 days.

Table 2. Serial laboratory parameters in the 1.5-year-old patient with EVI1-positive variant of JMML (#2) and PTPN11 mutation

The second case concerns a 2-year old male patient (#5, Table 3) with a similar EVI1-positive JMML harboring PTPN11 mutation who had laboratory signs of transformation to AML treated according to ADE protocol followed by haploidentical related HSCT.

In this case, a long-term molecular monitoring was performed by WT1 gene which showed high expression at diagnosis (502 per 10⁴ copies of ABL1 gene) as seen from Table 3. Prior to transplantation, this laboratory parameter reached its maximal level (3795/10⁴ ABL1 copies) which may be a sign of clinical relapse. In particular, the blast cells counts were also maximal (21%] in bone marrow at this time. Despite clinical evidence of AML, the subsequent haploidentical HSCT was successful, since all molecular markers have been returned to normal for a long time after transplantation. It should be noted that HSCT was performed with myeloablative conditioning regimen (12 mg/kg busulflex + fludarabine, melphalan), at optimal total number of transplanted CD34+ cells (6.7×10⁶/kg of body mass). To prevent GvHD, a multi-component immunosuppressive therapy was applied with everolimus and tacrolimus since day 5, being combined with cyclophosphamide on the days +3 and +4 posttransplant. Engraftment was achieved on day +21. Restaging of disease (day +21) revealed mixed donor chimerism (<97%), complete hematologic, cytogenetic and molecular responses. Early post-transplant period was complicated on day +3 by febrile neutropenia and Grade 1 oral mucositis. Examination carried out 1 year after haplo-HSCT revealed a sustained complete remission. Current survival term reached 1103 days.

Table 3. Serial changes of basic clinical and laboratory findings in the patient #11 with EVI1-positive JMML and PTPN11 mutation

The next patient was a 2.5-year old boy with JMML (#6) harboring a similar PTPN11 mutation. At diagnosis, his initial WBC count was 45×10⁹/L, with 1.2% of blast cells in bone marrow (Table 4). Pathological changes of molecular markers were presented by suboptimal of WT1 gene expression (296 copies), whereas sequential BAALC gene testing did not show any increase. As expected, HSCT carried out in this clinical setting, was successful. The patient is alive, and his current survival time reached 1090 days posttransplant.

Table 4. Serial changes of basic laboratory parameters in the 2.5-year old male patient with JMML (#6) and PTPN11 mutation

The next case presents the youngest patient in our group (#10, Table 5) with NRAS mutation associated with higher WBC count, splenomegaly and hepatomegaly, increased number of blast cells and higher levels of BAALC-expressing LSCs soon after birth. However, the infant has reached stable complete clinical and molecular remission, probably, harboring the NRAS mutation. The latter suggestion may be proven by detection of similar NRAS mutation in other tissue types. The initial BAALC-expression in bone marrow of this patient was 41%, with WBC count 24.3×10⁹/L and number of blast cells ranging from 5 to 6%. The patient was successfully treated by HSCT, currently being alive with OS reaching already 1179 days.

Table 5. Serial changes of basic laboratory parameters in a child with JMML carrying NRAS mutation (#10)

As another example, we would like to present basic laboratory and therapeutical data of a 2.5-year old boy with JMML and mutated PTPN11 gene combined with additional karyotype alterations (case #3, table 6) who developed non-controllable AML after HSCT. Initial WBC count was 35.8×10⁹/L with 12.8% blasts in bone marrow and 8% in peripheral blood. Initial therapy consisted of 6-mercaptopurine only. The first molecular testing (24.08.20) showed over-expression of all three marker genes exceeding the respective cut-off values. The latter finding was associated with WBC increase (16.6×10⁹/L) and moderate elevation of blast cell ratio in his bone marrow and peripheral blood (7.8% and 2.0%, respectively); WBC – 72.4×10⁹/L and after FLAM 2,01*10⁹/L.

Preparation for HSCT included a course of chemotherapy according to FLAM protocol with Fludarabine and Busulflex. Results of molecular monitoring carried out later revealed only normalized level of WT1 expression (158 copies). The further therapy was not intensified and had a palliative character. The patient died on the day 375 after HSCT.

Table 6. Serial laboratory indexes in the course of therapy in a 2.5-year old patient (#3) with JMML, PTPN11 mutation and progression to AML

Notes: * Date of AML development

Discussion

Despite sufficient advances in elucidation of JMML pathogenesis and positive experience with HSCT in these children, current therapy results in long-term overall survival of only 50-60% of patients [8, 9, 13]. Our original data demonstrate a fraction of LSCs with BAALC over-expression in 8 out of 13 observed JMML patients (70%) thus allowing usage of this parameter both for risk stratification in these patients, and for evaluation of treatment efficacy, including HSCT option.

Our recent positive experience with EVI1-positive leukemia patients [22, 23], as well as CBF-positive variants of adult and pediatric AML [20] may be now extended to JMML cohort. As seen in Table 1, the group of JMML patients shows high heterogeneity, with regard of laboratory and clinical findings as well as BAALC expression levels by LSCs. In this view, higher levels of BAALC-e LSCs were present in most of them (##1-4, 8-10, 12 and 13). This finding appears to be more characteristic for JMML group with prevailing myelodysplastic course and poor prognosis. On the basis of this genetic marker, only two patients (##6 and 11) might be assigned to another myeloproliferative category. Meanwhile, the risk classification of patients ## 5 and 7 seems to be impossible on this basis. One of them had the increased number of blasts in bone marrow (21%) which is characteristic to AML. In this case, the fraction of BAALC-expressing LSCs was not elevated which may a reason of longitudinal complete clinical and molecular remission achieved, despite EVI1-positive variant of JMML. Moreover, the number of blast cells in bone marrow from the second patient (#7) reached 16.6%, whereas the expression level of WT1 gene increased to 3010 copies. However, haploidentical related HSCT became successful again.

Of note, the diagnostic tools for this disorder were recently improved due to implementation of NGS technology. The treatment approaches are still relying on allogeneic HSCT. In contrast to adult AML, the molecular monitoring in JMML therapy, both prior to and post-transplant, is still not perfect being based on some intricate approaches, e.g., (a) detection of hypermethylated gene patterns, or (b) changes in relative levels of RAS pathway gene mutations which could not be done serially in common clinical labs in. Meanwhile, we present here positive results on serial measurements of BAALC, WT1 and EVI1 expession levels which seem to be more simple and cheap. Since the levels of BAALC expression in most JMML patients exceeded appropriate cut-off value (31%), they should be related to the poor prognostic variants of JMML with prevailing myelodysplastic component. Meanwhile, 5 out of 13 tested JMML patients belonged to prognostically poor EVI1-positive variants, as previously shown by several researchers [6, 7], who presented the first evidence of EVI1 gene activity directly in stem cells [7]. Hence, one may suggest that this gene may be an important regulator of stem cell biology and their functional state in JMML and other blood malignancies. In general, these findings may promote an improved system of JMML risk stratification [5]. We believe that, due to dominance of the mentioned aggressive JMML variant with myelodysplastic component in the tested group characterized by higher BAALC-e LSCs fraction, this molecular parameter might be successfully tested in clinical settings both for risk stratification, and for quantitative assay of therapy efficacy, including HSCT. Meanwhile, transplant-related tmortality in this category of patients is still high. Under these conditions, serial measurement of BAALC-e HSC fraction seems to be useful upon treatment of JMML patients with new targeted agents [24].

Conflict of interest

None declared.

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