Acute myeloid leukemia cells and MSC-derived exosomes inhibiting transformation in myelodysplastic syndrome.

AIMS: To investigate the mechanism of exosomes' role in the transformation of MDS to AML. METHODS: Exosomes in culture supernatants of MDS and AML cell lines, were extracted by ultrafiltration and identified in three ways: morphology, size, and exosome protein surface markers. Exosomes from AML cell lines were then co-cultured with MDS cell lines and their impacts on MDS cell microenvironment, proliferation, differentiation, cell cycle, and apoptosis were analyzed by CCK-8 assay and flow cytometry. Furthermore, exosomes from MSC were extracted for further authentication. RESULTS: The transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and flow cytometry methods all verify that ultrafiltration is a reliable method to extract exosomes in the culture medium. Exosomes from AML cell lines inhibit the proliferation of MDS cell lines, block cell cycle progression, and promote apoptosis and cell differentiation. It also leads to increased secretion of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in MDS cell lines. In addition, MSC-derived exosomes were found to inhibit the proliferation of MDS cell lines, arrest cell cycle progression, promote apoptosis, and inhibit differentiation. CONCLUSION: Ultrafiltration is a proper methodology in extracting exosomes. The exosomes of AML origin and MSC origin may play a role in MDS leukemia transformation via targeting TNF-α/ROS-Caspase3 pathway.

[Impact of CSF3R Mutation on Treatment Response and Survival of Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression of CSF3R mutation in acute myeloid leukemia (AML) and analyze its clinical characteristics and prognosis. METHODS: A retrospective study was conducted in 212 patients with AML who were newly diagnosed in the Second Hospital of Shanxi Medical University from January 1th 2018 to June 30th 2021, including 22 patients with CSF3R mutations as mutation group and 190 patients with CSF3R wild type ï¼»66 cases of them were screened by propensity score matching (PSM), as control groupï¼½. The early efficacy and survival between the two groups were compared. RESULTS: The median age of patients in the mutation group was 50(17-73) years old, and the ratio of male to female was 1.2:1 The main types were AML with maturation (11 cases) and acute myelomonocytic leukemia (9 cases). Prognostic stratification was carried out according to the risk stratification system of the European leukemia network in 2017, with 16 cases (72.73%) in the middle and high-risk group. At the initial diagnosis, the median count of white blood cell (WBC) was 44.75(1.30-368.71)×109/L, among which 15 cases (68.18%) were >10×109/L, and the median count of platelet (PLT) was 24(4-55)×109/L. CSF3R T618I (68.18%) was a common mutation site, which had concomitant gene mutations, in which CEBPA mutation was the most common (10 cases, 45.45%), but only existed in CSF3R T618I mutation. The CR/CRi rate was 68.18% and 71.21% in the mutant group and the control group (P >0.05), the median over all survival time was 15 months and 9 months (P >0.05), and the median disease-free survival time was 8 months and 4 months (P >0.05), respectively. CONCLUSION: Most AML patients with CSF3R mutation are middle-aged patients, the main types are AML with maturation and acute myelomonocytic leukemia, and most of them have middle and high-risk prognosis. CSF3R mutation may not be an independent prognostic marker for newly diagnosed AML patients.

A germline JAK2 exon12 mutation and a late somatic CALR mutation in a patient with essential thrombocythemia.

Background: It has been discovered that Janus kinase 2 (JAK2) exon12 mutations lead to the polycythemia vera (PV) phenotype, while somatic mutations of calreticulin (CALR) are associated with essential thrombocythemia (ET) or primary myelofibrosis. In this article, we report a case of ET with coexistence of JAK2 exon12 and CALR mutations. The objective of this study was to elucidate the pathogenicity mechanism of a JAK2 exon12 mutation (JAK2N533S) and the role of the coexistence of mutations on the hematological phenotype. Methods: We designed a colony analysis of tumor cells obtained from this patient, and attempted to identify mutant genes using DNA from hair follicles. Mutation impairment prediction and conservative analysis were conducted to predict the mutation impairment and structure of JAK2N533S. In addition, we conducted a functional analysis of JAK2N533S by constructing Ba/F3 cell models. Results: Three distinct tumor subclones, namely JAK2N533Shet+/CALRtype1het +, JAK2N533Shet+/CALR wt, and JAK2N533Shet+/CALRtype1hom +, were identified from the 17 selected erythroid and 21 selected granulocyte colonies. The analysis of hair follicles yielded positive results for JAK2N533S. According to the bioinformatics analysis, JAK2N533S may exert only a minor effect on protein function. Functional studies showed that JAK2N533S did not have a significant effect on the proliferation of Ba/F3 cells in the absence of interleukin-3 (IL-3), similar to wild-type JAK2. Notably, there were no increased phosphorylation levels of JAK2-downstream signaling proteins, including signal transducer and activator of transcription 3 (STAT3) and STAT5, in Ba/F3 cells harboring the JAK2N533S. Conclusion: Our study revealed that the JAK2N533Shet+/CALRtype1het+ subclone was linked to a significant expansion advantage in this patient, indicating that it may contribute to the development of the ET phenotype. We further demonstrated that JAK2N533S, as a noncanonical JAK2 exon12 mutation, is a germline mutation that may not exert an effect on cell proliferation and protein function. These results and the present body of available data imply that certain noncanonical JAK2 mutations are not gain-of-function mutations leading to the development of myeloproliferative neoplasms.

BCR/ABL1ΔE7-8-9 isoform contributes to tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) patients, the involvement of the BCR/ABL1 isoform in tyrosine kinase inhibitors (TKIs) resistance has attracted lots of attention. In this work, a novel isoform that encoded truncated protein due to the deletion of ABL1 exon7, 8, and 9 was reported and named BCR/ABL1ΔE7-8-9 here. This isoform was detected only in 10.2% of CML patients with inadequate responses to TKIs. BCR/ABL1Δexon7-8-9 isoform promoted S phase cell proliferation and reduced the expression of fusion gene and ABL1 phosphorylation level more slowly than that of control cells after TKIs treatment. The novel isoform has the qualities of a functional tyrosine kinase, localized in the cytoplasm, and could not be imported into the nucleus by TKIs. These results indicated that BCR/ABL1Δexon7-8-9 showed poorer sensitivity to imatinib and nilotinib than wild-type BCR/ABL1. According to molecular docking studies, nilotinib and imatinib present different binding sites and have a lower binding capacity with BCR/ABL1ΔE7-8-9 protein than the wild type. Our findings suggested that the novel isoform BCR/ABL1ΔE7-8-9 may contribute to TKIs resistance in CML due to its weakened TKIs binding ability. It enriched the mechanism of spliceosome involved in TKIs resistance. Monitoring the expression of BCR/ABL1ΔE7-8-9 helps guide the treatment of CML patients in the clinic.

The clinical characteristics and prognosis of Chinese acute myeloid leukemia patients with CSF3R mutations.

INTRODUCTION: The colony-stimulating factor 3 receptor (CSF3R) controls the proliferation of myeloid progenitors and differentiation into neutrophils. However, the clinical features and prognostic significance of CSF3R mutations in primary acute myeloid leukemia (AML) patients are still unclear. METHODS: 158 newly diagnosed AML patients were retrospectively evaluated in our study. Amplicon-based next-generation sequencing (NGS) and multiplex-nested reverse-transcription polymerase chain reaction (RT-PCR) were used to investigate the 34 genes and 43 fusion genes associated with leukemia. In addition, clinical features, mutation incidence, and survival outcomes were compared between patients with CSF3R mutation and patients with wild-type CSF3R. RESULTS: In our study, CSF3R mutations were found in 7.6% (12/158) cases. The membrane-proximal amino acid substitution T618I (58.3%) was the most frequent mutation. CSF3R mutations were associated with higher WBC counts (P = .035). CEBPA mutation, TET2 mutation, and RUNX1-RUNX1T1 translocation were the most common co-mutations of CSF3R. The CSF3R gene was mutually exclusive with signal transduction genes (P = .029), while positively associated with TET2 mutations (P = .014). CSF3R mutations had no effect on CR1 (P = .935), R (P = .625) and OS (P = .1172). Patients with CSF3R mutations had a worse DFS (P = .0352) than those with wild-type CSF3R. Multivariate survival analysis showed that CSF3R mutation was an independent risk factor for DFS of primary AML patients (HR=2.048, 95%CI: 1.006-4.170, P = .048). CONCLUSION: AML patients with CSF3R mutations had unique clinical features and gene co-mutation spectrum. CSF3R mutation was an independent risk factor for DFS and could be a potential prognostic marker and therapeutic target for Chinese primary AML patients.

[Quantitative Analysis on Immunophenotype of CD34+ Myeloid Precursor Cells in Myelodysplastic Syndrome and Its Correlation with Clinical Features].

OBJECTIVE: To investigate the quantitative expression of immunophenotype of CD34+ myeloid precursor cells in myelodysplastic syndrome (MDS) patients and its correlation with clinical characteristics, and understand the effect of quantitative expression of CD7 and CD117 on the prognosis of low-risk MDS patients. METHODS: Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34+ myeloid precursor cells in 79 MDS patients. The correlation between the expression level of each immune marker and clinical characteristics was compared. The effects of quantitative expressions of CD7 and CD117 on the overall survival rate of low-risk patients were explored. RESULTS: Bone marrow blast cell proportion (P<0.01), RBC level (P<0.01), and Hb level (P<0.05) of high-risk MDS patients were higher, while EPO level (P<0.05) was lower than those of low-risk patients. The proportion of CD34+ blast cells (P<0.01), the proportion of CD117 (P<0.05) and the MFI of CD7 (P<0.05) were higher in high-risk patients than those in low-risk patients, but the MFI of CD123 was lower (P<0.05). In high-risk MDS patients, CD15/CD34 (MFI) and CD19/CD34 (MFI) positively correlated with the proportion of total T cells (r=0.458; r=0.505), while CD19/CD34 (%) and CD19/CD34 (MFI) negatively correlated with WBC levels (r=-0.469; r=-0.503). In low-risk MDS patients, CD34+ (%) positively correlated with bone marrow erythrocyte proportion, PLT level and neutrophil level (r=0.426; r=0.486; r=0.495), but negatively correlated with LDH level (r=-0.421); WT1 expression level was positively correlated with CD10/CD34 (%), CD10/CD34 (MFI) and CD117/CD34 (MFI) (r=0.745; r=0.800; r=0.434), while negatively correlated with CD11b/CD34 (%)(r=-0.457); CD19/CD34 (%) and CD71/CD34 (MFI) negatively correlated with NK cell proportion (r=-0.786; r=-0.514); CD10/CD34 (%) positively correlated with Th/Ts, while CD7/CD34 (MFI) negatively correlated with the proportion of Th cells (r=0.738; r=-0.513); HLADR/CD34 (%) and HLADR/CD34 (MFI) negatively correlated with PLT level (r=-0.461; r=-0.445), while HLADR/CD34 (MFI) positively correlated with bone marrow NAP fraction (r=0.552). The quantitative expression of CD7 and CD117 had no significant effect on the overall survival rate of low-risk MDS patients. CONCLUSION: The immunophenotype of CD34+ myeloid precursor cell in different risk groups in MDS patients is related to clinical characteristics. Bone marrow cell morphology, clinical and laboratory features and immunophenotype will be of great significance to the diagnosis, clinical classification and prognosis evaluation of MDS patients.

Monitoring Treatment-Free Remission by Droplet Digital PCR in CML Patients with Deep Molecular Response to Tyrosine Kinase Inhibitor: An Analysis Based on Real-World Data.

Treatment-free remission (TFR) is emerging as a new therapy goal for chronic myeloid leukemia (CML) patients in the tyrosine kinase inhibitors (TKI) era. Data indicates the unfavorable success rate of TFR. This study aimed to compare and evaluate the clinical value of dd-PCR in predicting relapse in CML patients entering TFR. Using dd-PCR and RT-qPCR technology, dynamic BCR/ABL transcripts were detected in 13 CML patients who discontinued TKI treatment after sustaining undetectable BCR-ABL levels for a median time of 25 months. The results showed that in 13 patients, only 2 cases (22.2%) of 9 patients who executed planned discontinuation achieved TFR within 12 months. In the first 6 months, the detection rate of BCR/ABL transcripts by dd-PCR was higher than that by RT-qPCR and the two methods kept a positive correlation (r=0.9651, P=0.0349). Meanwhile, the time of detectable BCR/ABL by dd-PCR were significantly shorter (P<0.05), which was an average of 2.98 months earlier than RT-qPCR. The total TKI therapy and MR4.5 duration time related with TFR were longer in patients with intermediate or high Sokal risk scores (p<0.05). The dd-PCR could be more sensitive than RT-qPCR for monitoring BCR/ABL transcripts of CML patients with deep molecular response to TKI. The technique can be used as a preferred method to detect the transcripts in the first 6 months after TKI cessation.

Identification of Two Novel Truncated Transcripts of Janus Kinase 2 Gene.

Jak2 is a nonreceptor tyrosine kinase that plays a critical role in signal transduction through an abundance of receptors, such as erythropoietin receptor. In this paper, we report two previously unknown transcripts of Jak2 gene. One transcript deletes the 77nt of 3' end exon 10 of the Jak2 gene, resulting in a frameshift that introduces a stop codon in the downstream exon and produces a truncated protein of 421 amino acids if translated. The other transcript skips the entire exon 10, leading to a premature stop codon in the adjacent exon 11, producing a truncated protein of 414 amino acids if translated. Therefore, the physiological significance of the expression of two novel transcripts in healthy volunteers and patients with myeloproliferative neoplasms, acute leukemia, and chronic myeloid leukemia needs to be investigated further.

Myelodysplastic Syndrome with t(1;14),t(1;17),t(1;19) Transforms to AML-M5: A Case Report and Literature Review.

Chromosomal aberrations play an important role in the incidence of myelodysplastic syndromes (MDS) and development to acute myeloid leukemia (AML). We report a case of a 62-year-old male patient diagnosed with MDS with excess blasts. The karyotype was 45, XY,+1,+1,-7,-10,-22,t(1;14) (q21;q32),t(1;17)(q21;p13),t(1;19)(q21;p13). The patient and his family refused treatment for financial reasons. After 2 months, the patient's MDS transformed into acute myeloid monocytic leukemia (AML-M5). This case of MDS with poor prognosis shows that patients with chromosomal numerical abnormality and balanced translocations should be treated early to prevent transition to AML. Further study of this case will reveal the molecular mechanism of MDS-to-AML transformation and identify new leukemic fusion genes.

Disulfiram/cytarabine eradicates a subset of acute myeloid leukemia stem cells with high aldehyde dehydrogenase expression.

Most patients with acute myeloid leukemia (AML) achieve complete remission (CR) after induction chemotherapy, however, in some patients, the disease subsequently relapses and may lead to death. Leukemia stem cells (LSC) have been identified as the main cause for recurrence. Increased aldehyde dehydrogenase (ALDHhigh) activity in a variety of cancer stem cells prevents effective action of chemotherapeutic drugs. In this study, we found that approximately 50.7% of AML patients had ALDHhigh, and the presence of ALDHhigh stem cells was associated with poor cytogenetic prognosis. Lentiviral vector transduced ALDHhigh leukemia cell lines are insensitive to the conventional chemotherapy drug cytarabine, and inhibition of ALDH activity by disulfiram (DSF) can increase the sensitivity of ALDHhigh leukemia cells to cytarabine. Unlike traditional chemotherapy drugs, DSF is not toxic to healthy umbilical cord blood stem cells. An ALDHhigh leukemia cell xenograft model was established using immunodeficient mice to mimic the disease environment, and DSF and cytarabine were found to eliminate the ALDHhigh leukemia cells in transplanted mice while not affecting the healthy blood cells of mice. These findings suggest that DSF may have therapeutic potential by inhibiting ALDH activity and thereby increasing chemosensitivity.

T-cell blast crisis of chronic myelogenous leukemia presented with coexisting p210 and p190 BCR-ABL transcripts and t(10;11)(q11;p15).

BACKGROUND: Blast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is rare, and the molecular mechanism is still unclear. CASE REPORT: A 28-year-old woman who developed T-ALL with coexpressing both p210 and p190 BCR-ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t(9;22)(q34;q11) and t(10;11)(q25;p15). Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3. CONCLUSION: It is the first T-cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T-lymphocyte transformation depend on potential molecular and pathological mechanism.

The CD9+ CD11b- HLA-DR- immunophenotype can be used to diagnose acute promyelocytic leukemia.

OBJECTIVE: To investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL) and explore the sensitivity and specificity of various antibody combinations for the timely and accurate diagnosis APL. METHODS: A retrospective analysis was performed using morphological, immunological, genetic, and molecular biological data from 92 patients diagnosed with APL and 190 controls diagnosed with non-APL acute myeloid leukemia. RESULTS: For APL diagnosis, the CD9/CD11b/human leukocyte antigen (HLA)-DR antibody combination had 85% sensitivity and 95% specificity, AUC = 0.85. However, the sensitivity and specificity were 39% and 92%, AUC = 0.65, respectively, for the HLA-DR/CD34/CD117 combination, and 80% and 80%, AUC = 0.80, respectively for the CD11b/HLA-DR combination. Significant differences were observed between the different antibody combinations. CONCLUSIONS: The CD9/CD11b/HLA-DR antibody combination displays high sensitivity and specificity and can be used to diagnose APL.

Monitoring of clonal evolution of double C-KIT exon 17 mutations by Droplet Digital PCR in patients with core-binding factor acute myeloid leukemia.

C-KIT gene mutations result in the constitutive activation of tyrosine kinase activity, and greatly affect the pathogenesis and prognosis of core-binding factor acute myeloid leukemia (CBF-AML). C-KIT mutations are often found as single point mutations. However, the rate of double mutations has recently increased in AML patients. In this study, we detected six cases (18.8%) harboring double C-KIT exon17 mutations in 75 patients with CBF-AML. The clone composition and dynamic evolution were analyzed by sequencing and droplet digital PCR (ddPCR). Results revealed that these double mutations can be occurred in either the same or different clones. Different clones of double mutations may result in different sensitivity to the treatment of CBF-AML. The clones with N822 mutation responded better to treatment as compared to those with D816 mutation. Moreover, D816 clone was readily transformed into a predominant clone at relapse. Meanwhile, the predominant clones in the same patient may change during the progression of disease. The emerging mutation can originate from a small quantity of clones at diagnosis or newly acquired during the course of disease. Furthermore, patients with double mutations had better overall survival (OS) and event-free survival (EFS) than those with single mutation, but the differences did not reach statistical significance (P > 0.05). The ddPCR is an effective method for monitoring clonal evolution in patients with CBF-AML.

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression.

OBJECTIVE: This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS: Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS: At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2  ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. CONCLUSION: In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.

Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms.

Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F.

[Identification of a novel aberrant spliceosome of MPL gene (MPLL391-V392ins12)in patients with myeloproliferative neoplasms].

OBJECTIVE: To identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN). METHODS: MPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR). RESULTS: A novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people. CONCLUSION: MPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.