Despite numerous in-depth studies in acute myeloid leukemia (AML) and development of novel therapeutic strategies, the issues of AML relapses are not resolved, including those observed after hematopoietic stem cell transplantation (HSCT). These relapses are closely related to preservation and activation of leukemia-initiating stem cells (LSC) which are still insufficiently studied. New opportunities for studying these cells in clinical setting appeared after discovery (Tanner et al 2001) of BAALC (Brain and Acute Leukemia, Cytoplasmic), a special gene inducible in the stem cells. BAALC activation may be successfully evaluated by means of standardized real-time quantitative polymerase chain reaction (RT-qPCR). The aim of the present study was to assess the levels of BAALC -expressing leukemia stem cell (LSC) fractions in groups of patients with juvenile myelomonocytic leukemia (JMML) and FLT3-mutations, and to evaluate efficacy of the therapy having been based on their risk stratification.

Materials and methods. The first study group included 25 patients (13 females, 12 men aged 18 to 84 years old) with FLT3-ITD (n=24) and FLT3-TKD mutations (n=1) including seven EVI1-positive cases (24%). Moreover, similar clinical and laboratory parameters were studied in 21 patients with combined FLT3/NPM1 mutations. The second group consisted of 13 pediatric patients (10 boys and 3 girls aged between 0.3 and 6 years) being well characterized for their mutation profiles as assessed by NGS technique. Measurement of BAALC, WT1, EVI1, and ABL1 gene expression levels was performed by means of standardized RT-qPCR.

Results and discussion: Increased BAALC expression in bone marrow samples (over the cut-off levels of 31% were detected in 20/25 (80%) FLT3-mutated patients, ranging from 2377 to 34%. In parallel studies, an increased WT1 gene expression (over 250/10⁴ ABL1 gene copies) was revealed in 22/24 studied patients (range, 8980 to 1246 copies/10⁴ ABL1 gene). On the contrary, the levels of BAALC gene expression in all studied patients with combined FLT3-ITD and NPM1 mutations (n=21) were found to be under the cut-off levels thus, probably, being related to enrichment of NPM1 mutations in CD34− AML cases. Similar studies in the group of 13 pediatric patients with JMML revealed higher levels of BAALC-expression in LSC fractions thus suggesting a novel tool for evaluation of therapeutic efficacy as well as available marker for development of new risk stratification principles in this orphan disorder.

Conclusion: Serial measurements of gene BAALC expression in bone marrow from patients with AML allow quantitative evaluation of therapeutic efficiency based on the relative levels of LSCs.

Keywords: acute myeloid leukemia, flt3 mutations, npm1 mutations, juvenile myelomonocytic leukemia, baalc gene expression, leukemia stem cells, rt-qpcr, clinical applications, biological issues

Our previous studies concerned potential reasons of modern therapy failure in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), evidencing a crucial role of leukemia stem cells (LSCs) for increased incidence of disease relapses both pre- and post-HSCT.^1-5 As known, the idea of links between hematopoiesis and function of highly specialized hematopoietic stem cells (HSCs) belongs to the Great Russian scientist Alexander Maximov, Professor at the St. Petersburg Military Medical Academy. This fundamental concept opened new ways and reached great relevance for better understanding of hematopoiesis system in whole. Step-by-step, simple Maximov's morphological methods are moved to colony-forming assays in lethally irradiated mice⁶ as well as to proliferation assays in semi-solid agar cultures.^7–9 Since these cultural methods were time-consuming and expensive, these techniques could not be implemented in common clinical practice. Meanwhile, new horizons have been opened for development of quantitative assays of leukemic hematopoietic stem cells (LHSCs) in patients with AML and MDS after discovery of BAALC gene (Brain And Acute Leukemia, Cytoplasmic) by Tanner et al.¹⁰ which was expressed mainly by CD34-positive subpopulation of leukemia hematopoietic stem cells (LHSCs).^11–13 In view of these data, we attempted to evaluate the LHCSs population by means of real-time quantitative polymerase chain reaction (RT-qPCR).^{1–5,14, 15} Our recent research concerned the patients with CBF-positive AML variants¹⁵ and those with 5q- myelodysplastic syndrome.¹⁴ The present article is devoted to analysis of still non-published data obtained in patients with FLT3-mutated AML and JMML.

Mutations of FLT3 (FMS-like tyrosine kinase 3) gene are among the most frequent gene mutations in AML, being closely associated with a negative outcome.^16–18 Approximately one-third of AML patients carry the FLT3 gene mutations.^19,20 The latter gene is mapped at the 13q12 locus encoding type III receptor tyrosine kinase. FLT3 gene contains 24 exons and may undergo alternative splicing thus giving rise to several isoforms. The primary isoform, FLT3-ITD, is more frequent in AML being formed by insertion of different-length tandem duplications within the gene’s coding region, with juxtamembrane domain.

In view of these findings, several attempts were done to develop new targeted agents for FLT3 receptor.^21–31 However, the actual clinical responses to Midostaurin, Gilteritinib, or Quizartinib proved to be limited in time, due to primary or acquired resistance to these drugs. Tyrosine kinase domain closely linked to target places was considered the common mechanism of acquired resistance.²¹ Karl Levis, the known expert in the field of stem cells has been published article unusually entitled “FLT3 dancing on the stem cells” proposing a theoretical explanation of therapy failure by many FLT3-targeted drugs in patients with FLT3–mutated AML.^32–35

Worth of note, the levels of BAALC gene expression in patients with this disorder are found to be increased.^11,36 These studies did not show any significant difference between patients with low and high BAALC expression with respect to their pre-treatment age, gender, WBC counts and percent of bone marrow blasts Moreover, the authors revealed high BAALC expression associated with higher percentage of blast cells in peripheral blood (P=0.004) and with more immature subtypes M0/M1 of AML (P=0.001), whereas, in monocytic FAB M5b leukemia subtype, it correlated with low BAALC expression (p=0.001). Importantly, patients with BAALC overexpression had a higher cumulative indices of relapses (CIR) than the patients with low BAALC expression (3 year CIR: 50% v 32%; P=0.018). Finally, high BAALC expression was predictive for shorter OS (3 year OS, 36% v 54%, P=0.001), being an independent risk factor for poor prognosis. On the contrary, the mode of post-remission treatment did not influence OS rates (P=0.059). Taken together, the findings presented in this large study confirmed an independent adverse prognostic significance of BAALC overexpression in AML patients with normal cytogenetics. Nevertheless, upon multivariate statistical analysis, an increased BAALC expression remained the only independent prognostic factor in this cohort.

Another group under study includes pediatric patients with Juvenile Myelomonocytic leukemia (JMML) which presents a rare and aggressive myelodysplastic /myeloproliferative malignancy^37,38 closely tied with activation of the RAS signal transduction pathway, due to 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 may be main cause of higher sensitivity of myeloid progenitors to granulocyte/monocyte colony-stimulating factor (GM-CSF).³⁹ About 90 to 95% of patients with JMML are characterized with such canonical mutations as PTPN11 regulator gene of RAS signaling pathway (35% of cases), or NRAS and KRAS genes (20–25% each). Less frequent mutations are found in two other genes: regulators of RAS signaling pathway, e.g., NF1 (10–15%), or CBL (10–15%).^40,41 A transient myeloproliferative disorder with good clinical 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.⁴¹

One should add that unexpectedly high incidence of prognostically poor EVI1 positive variants was seen among the patients with this disorder.^40,42,43 Hence, one may develop a modern risk-stratification classification of JMML based on these genetic markers.⁴¹ Allogeneic hematopoietic stem cell transplantation (alloHSCT) is considered the only curative therapy for most of these cases, however, with relapses observed in about 35% of treated patients.^37,44–53

Despite in-depth studies of clonal leukemia-initiating cells, their biological nature is not elucidated so far.¹ Theoretically, this role may be given to a subpopulation of BAALC-expressing stem cells (BAALC-e SCs) which have been previously tested by us in several clinical variants of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).^2–4,15 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, at the level of BAALC-e LSC fraction.

The first group in our study included 25 patients (13 females, 12 males aged 18 to 84 years old) with FLT3-ITD (n=24) and FLT3-TKD mutations (n=1). Of them, seven patients (24%) were EVI1-positive. Abnormal karyotypes were revealed in 13/25 (52%), with complex karyotypes in 5/13 cases (38.5%). Overall survival (OS) after primary diagnosis ranged from 6 to 2148 days (a mean of 575 days). This survival index in the patients with EVI1- positive AML ranged from 30 to 503 days (mean, 241 days) compared to OS terms in patients with complex karyotypes (6, 18, 371, 882+, and 2184 days). It should be mentioned, that, for treatment of two long-term survivors, we have performed haploidentical and allogeneic nonrelated HSCT, respectively. Further on, to improve characterization of this disorder, we have also included 20 patients with combined FLT3 and NPM1 mutations into this group.

Routine laboratory counts of blood and bone marrow cells were performed on regular basis. Cytogenetic studies were carried out using standard criteria to detect chromosome aberrations. Simultaneous serial measurements of BAALC, WT1 and EVI1 (selectively) gene expressions levels were performed by means of quantitative real time polymerase chain reaction (RT-qPCR), as described elsewhere.⁵ In brief, genomic RNA was isolated from fresh bone marrow samples by guanidine-phenol-chloroform extraction (“Ribozol-DF” kit reagent, InterLabService, Russia), according to the manufacturer's instructions. Aliquots of extracted RNA (11 µL) were used for reverse transcription with cDNA Synthesis Kit (LifeTechnologies, USA). The multiplex PCR of BAALC, WT1, EVI1 and ABL genes was performed for each cDNA sample. Reaction conditions were as follows; 10 µcL of PCR reaction mixture (“Syntol”, Russia), containing dNTP mix of 2.5 mM each, 10xPCR buffer, 5 Units of Taq-DNA polymerase and 2.5 µcL of 25 mM MgCl_2, 7 pmol of each gene-specific primers, 5 pmol of Taqman probes for all tested BAALC, WT1, EVI1 and ABL genes. The cut-off values of 31% and 10% were established for BAALC and EVI expression levels. The threshold level for WT1 gene was 250 copies/10⁴ copies of BCR/ABL1 gene.

The initial qPCR data (Table 1), show quite different BAALC expression levels ranging from 2377% to 2%, with mean value of 77%. The highest BAALC expression level was detected in a 54-year old woman with EVI1-positive AML, harboring normal karyotype. Of interest, WT1 expression level in this patient was also very high (8980 copies/10⁴ ABL1 gene copies). Meanwhile, increased levels of WT1 gene expression were also found in some patients with moderate and lower levels of BAALC gene expression (respectively, #15 and #20). In general, the levels of WT1 gene expression in all patients from this group exceeded 1000/10⁴ ABL1 copies. It should be also noted, that 7 of 25 tested patients (28%) showed increased levels of EVI1 gene expression, being not associated with BAALC or WT1 expression, or cytogenetic features. Further on, we have found increased numbers of bone marrow blast cells as well as WBC counts in the most studied patients. The highest WBC count (522x10⁹/L) was registered in young female (#5) with EVI1-positive variant of AML and abnormal karyotype.

AML patients with FLT3 mutation

Our prospective study included 25 adult AML patients (13 females, 12 men aged 18 to 84 years) with FLT3-positive AML, harboring ITD (n=24) or TKD (n=1) mutations (Table 1). The levels of BAALC and WT1 gene expression, as well as WBCs counts and number of bone marrow blasts have been measured in parallel. Seven of 25 (24%) of these patients were EVI1-positive. Further on, abnormal karyotypes were detected in 13/25 cases (52%), with complex-type aberrations in 5 patients (38.5%), i.e., cases #2, 8, 16, 18 and 19. Overall survival (OS) rate after the diagnosis ranged from 6 to 2148 days compared with 30 to 503 days in the group with EVI1-positive AML variant. Finally, OS terms in patients with complex karyotypes were 6, 18, 371, 882+ and 2148 days. It should be noted, that the treatment of the two patients with higher OS included haploidentical and allogeneic HSCT from unrelated donor, respectively.

The BAALC gene expression in bone marrow samples exceeding the cut-off values (31%) was revealed in 20/25 FLT3 mutated patients (80%) ranging from 2377 to 34%. Parallel testing of WT1 gene expression showed increase in 22/24 studied patients over appropriate cut-off levels (250/10⁴ABL1 gene copies), at a range of 8980 to 1246/10⁴ ABL1 gene copies (Table 2).

In our opinion, failure of applied therapies in this case of AML with standard FLT3-ITD mutation might be partly explained with: a) too late detection of this molecular marker; and b) isolated therapy with Gilteritinib despite presence of prognostically poor values of BAALC (119%), WT1 (4396 copies), FLT3-ITD relation of pathologically changed to wild alleles relation as well as high bone marrow blast counts (20%). Therefore, an urgent haploidentical HSCT did not provide favorable effect, being followed by severe complications, e.g., graft failure, and septic shock.

Another patient with poor outcome was diagnosed with EVI1-positive AML and FLT3-ITD mutation (Table 3). Clinical and laboratory data demonstrated a dismal clinical course, despite a modern combination of intensive chemotherapy with FLT3-ITD inhibitors followed by allogeneic unrelated HSCT.

The last clinical case concerns a middle-aged patient with secondary AML developing post-MDS, with a prognostically favorable non-standard FLT3-TKD mutation. In contrast to the above demonstrated cases with severe clinical course of FLT3-ITD variants, this case indicates great efficacy of performed therapy which was based on molecular monitoring data with serially measured BAALC and WT1 expression levels (Table 4).

Despite high initial increase of BAALC-e LSCs fraction (229%), as well as high marrow blast counts (60%), morphological remission was achieved, due to combined chemotherapy with FLT3 inhibitors. The clinical success was reinforced by allogeneic HSCT and several courses of Gilteritinib. As a result, the patient is in good health. Currently he demonstrates all features of complete remission, whereas his OS reached 682 days.

Patients with combined FLT3- and NPM1 mutated AML

In contrast to above data for AML patients with isolated FLT3-mutation, the BAALC expression levels in all 21 studied patients with combined FLT3-ITD/NPM1 mutations were under cutoff values (Table 5) whereas those of WT1 gene expression were higher cutoff (250 copies/10⁴ copies of gene ABL1, which may be explained hypothetically with earlier evidenced decrease of CD34 positive Stem Cells wherein genes BAALC and EVI1 should be localized.

Patients with juvenile myelomonocytic leukemia

This clinical group included 13 pediatric patients (10 boys and 3 girls at the age of 0.3 to 6 years, a mean of 2.8 years) with proven JMML, and specific mutation profiles confirmed by means of NGS analysis (detailed clinical and laboratory data will be published later). Mutation in PTPN11 gene-regulator of RAS signaling pathway was detected in 8 patients; NRAS mutation, in four cases, whereas CBL and NFI gene mutations were found in single patients (Table 6A). 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.⁵

The longest OS terms, ranging from 1178 to 2462 days (a mean of 2028 days) were detected in the infants under 1 year, all of whom are alive at present. Further on, the BAALC expression levels in bone marrow ranged from 73 to 9%. The highest levels (73% and 64%) were registered in two patients (#1 and #2). Meanwhile, high levels of BAALC expression (63% to 50%) were determined also in patients #3, #5, #11 and #12. In contrast, 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 where 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) developed JMML transformation to acute myeloid leukemia (AML). It should be noted that similar transition to AML was diagnosed also in two other patients (#5 and 13), one of whom was treated successfully with haploidentical related HSCT.

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 which was associated with lower level (19%) of BAALC-e SC fraction. In general, 6 out of 13 JMML patients (45%) treated with HSCT died with OS terms ranging from 87 to 1024 days. These cases included 4 mentioned 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. The longest OS registered in the studied group was 2462 days.

Since HSCT is considered the main therapeutic approach in JMML, we analyzed this treatment option more carefully. Table 3 shows that single alloHSCT was performed in four patients, whereas its combination with Haplo-HSCT was carried out in three cases. Moreover, a 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, but some patients demonstrated it repeatedly (#2). In general, 6 out of 13 studied patients (45%) died, 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 group of comparison enrolled six surviving patients aged 0.3-6 years. Their OS terms ranged from 978 to 2363 days (mean – 1697 days).

To illustrate our original findings in JMML patients, a part of them are presented here additionally, wherein main laboratory parameters were closely associated with treatment performed. The first case concerns a 1.5-year old boy (Table 6B, #2) harboring PTPN11 mutation. His data 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 resulted in clinical effect. It was also accompanied by fast normalization of BAALC and WT1 expression being enforced later by the two-step treatment with HSCT and haplo-transplant. Unfortunately both transplants were not quite effective because of fast transplant rejection. As a result, the last donor chimerism value decreased to 20-29% and the OS terms after first HSCT reached 1024 days.

The second case concerns a 2-year old male patient (#5, Table 7) with similar EVI1 positive JMML also harboring PTPN11 mutation who had laboratory signs of transformation to AML treated according to ADE protocol followed by haplo-related HSCT (14.09.20). In this case, molecular monitoring was for a long time performed by measurement of WT1 gene expression which was increased at diagnosis to 502 WT1/10⁴ ABL copies (Table 6). Prior to transplantation, this laboratory index 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 also that HSCT was performed with myeloablative conditioning regimen, 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. Earlier 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.

The BAALC expression levels in bone marrow ranged from 73 to 9%, being the highest (73 and 64%) in two patients. In 4 other patients, higher levels of BAALC expression (from 63 to 50%) were also determined. Minimal BAALC gene expression was found in a patient with cumulative PTPN11 and NRAS mutations, and in a case with single NRAS mutation. Of seven patients with PTPN11 mutation, the highest level of BAALC expression was noted in a 4-year old child with trisomy 8 in karyotype (BAALC is mapped on chromosome 8). This case was also peculiar due to higher expression of WT1 gene (941 copies/10⁴ copies of ABL1) 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 other patients with similar mutation. One of them revealed JMML transformation to acute myeloid leukemia (AML), although similar transformation to AML was also diagnosed in two other patients, one of whom was successfully treated with haploidentical related grafting.

Transplant failure was registered in five cases, but some patients demonstrated it repeatedly. In general, 6 out of 13 studied patients (45%) died 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 group of comparison enrolled six surviving patients aged 0.3-6 years. Their OS terms ranged from 978 to 2363 days (mean, 1697 days).

The aim of our present work was to encourage the researchers for active clinical implementation of our novel molecular approach based on serial measurements of BAALC-expressing LSC fraction which is useful for efficacy therapy evaluation and recognition of AML relapses on the level of stem cells. Meanwhile, this important indicator of Stem Cells being combined with that of WT1 which is characteristic for common lineage - differentiated hematopoietic precursors (l-dHP) and blasts compartment allows to organize a new molecular approach for power serial assay of therapy efficacy in clinical sets and its correction in a case of necessity. Our recent studies suggest that this concept may be available for further elucidation of two-step changes in pathological AML hematopoiesis which is really evident in several AML types, including orphan JMML. Unfortunately, there is still no direct evidence for BAALC-expressing cell fractions in leukemic hematopoiesis, although one of them is presented here. It concerns the low (under-cutoff) levels of BAALC-expressing LSCs in patients with combined FLT3 and NPM1 mutations, wherein CD34 positive stem cells are shown to be decreased.^32,33 Since our original data demonstrate a higher fraction of BAALC-e LSCs in 80% patients with sole FLT3 mutations, and in 8/13 (70%) of studied patients with JMML thus enabling further development of risk stratification system for these categories of the patients. Moreover, such molecular monitoring is able to evaluate efficacy of treatment, including HSCT. It has been mentioned that one-fourth of FLT3-mutated and one-third of JMML patients to be EVI1 positive thus making their prognosis more risky. Hence, our positive experience with measurement of BAALC-e LSCs fractions in patients with EVI1-positive leukemia patients^2,54 as well as CBF-positive variants of AML¹⁵ may be now extended towards to FLT3 AML and JMML cohorts. Of note, the group of our JMML patients was rather heterogeneous with regard of laboratory and clinical findings as well as BAALC expression levels by LSCs. Despite it, higher levels of BAALC-e LSCs were present in most of these cases (##1-4, 8-10, 12 and 13). Since this finding is more characteristic for JMML group with prevailing myelodysplastic pattern, this laboratory index may be available for the risk-stratification programs. In particular, only two of our patients (##6 and 11) might be assigned to more favorable myeloproliferative category on the basis of this genetic marker. Meanwhile, the risk classification of patients ## 5 and 7 seems to be impossible on this basis. I.e., one of them had the increased number of blasts in bone marrow (21%) which is characteristic to AML. Since the fraction of BAALC-expressing LSCs in this case was not elevated, thus being a probable reason of longitudinal complete clinical and molecular remission achieved, despite EVI1 positive variant of this leukemia. On the other hand, 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. Despite it, the haploidentical related HSCT was very successful.

The available diagnostic tools for these disorders were recently improved due to implementation of NGS technology. Meanwhile, the treatment approaches are still relying on allogeneic HSCT. In contrast to adult AML, the molecular monitoring in JMML therapy, both prior to and after transplantation, is still not perfect. It may be based on such intricate approaches as: (a) detection of hypermethylated gene patterns, or (b) changes in relative levels of RAS pathway gene mutations, which are problematic for many clinical labs. As for , the presented positive results on serial measurements of BAALC, WT1 and EVI1 expression levels their obtaining seems to be more simpler and cheaper way. 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. Further on, 5 out of 13 tested JMML patients belonged to prognostically poor EVI1 positive variants, as previously shown by several researches,^42,43 who presented the first evidence of EVI1 gene activity directly in stem cells.⁴³ Hence, this gene is expected to be an important regulator in biology of stem cells and their functional status in JMML and other blood malignancies. In general, all these findings might be useful for improvement of expected risk stratification system in JMML.⁴¹ 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 mortality in this category of patients is still high. Under these conditions, serial measurement of BAALC-e HSC fraction seems to be available too with regard to treatment of JMML patients with new targeted agents.⁵⁵

Molecular monitoring of treatment results in AML patients based on serial measurements in bone marrow of BAALC-expressing LSCs fractions allows to evaluate efficacy of this therapy at the level of active stem cells and may be even more perspective in clinical setting in combination with another molecular indicator of WT1-expressing blasts and l-d HP, The findings of lower levels of BAALC gene expression in all studied AML patients with combined FLT3 and NPM1 mutations is quite important since the CD34- positive cells are not characteristic for this clinical pathology. Finally, it seems to us that investigations on the stem cells biology are increasingly important and need further collective work, involving both clinical researches, and molecular biologists as well.

The authors would like to acknowledge the assistance of Professor Alexey Chukhlovin in the preparation of this manuscript.

The author declares that there are no conflicts of interest.

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