Predicting leukemic transformation in myelodysplastic syndrome using a transcriptomic signature.

Background: For prediction on leukemic transformation of MDS patients, emerging model based on transcriptomic datasets, exhibited superior predictive power to traditional prognostic systems. While these models were lack of external validation by independent cohorts, and the cell origin (CD34+ sorted cells) limited their feasibility in clinical practice. Methods: Transformation associated co-expressed gene cluster was derived based on GSE58831 ('WGCNA' package, R software). Accordingly, the least absolute shrinkage and selection operator algorithm was implemented to establish a scoring system (i.e., MDS15 score), using training set (GSE58831 originated from CD34+ cells) and testing set (GSE15061 originated from unsorted cells). Results: A total of 68 gene co-expression modules were derived, and the 'brown' module was recognized to be transformation-specific (R2 = 0.23, p = 0.005, enriched in transcription regulating pathways). After 50,000-times LASSO iteration, MDS15 score was established, including the 15-gene expression signature. The predictive power (AUC and Harrison's C index) of MDS15 model was superior to that of IPSS/WPSS in both training set (AUC/C index 0.749/0.777) and testing set (AUC/C index 0.933/0.86). Conclusion: By gene co-expression analysis, the crucial gene module was discovered, and a novel prognostic system (MDS15) was established, which was validated not only by another independent cohort, but by a different cell origin.

Acute fibrinous and organizing pneumonia complicated with hemophagocytic lymphohistiocytosis caused by chronic active Epstein-Barr virus infection: a case report.

BACKGROUND: Acute fibrinous and organizing pneumonia (AFOP) is a rare lung condition that is associated with acute lung injury. Its etiology may be idiopathic or secondary to a series of conditions, including immune-related diseases, unclassified connective tissue diseases, hematopoietic stem cell transplantation, infections, hematological diseases and drug induced lung toxicity. We report for the first time a case of AFOP complicated with hemophagocytic lymphohistiocytosis (HLH) caused by chronic active Epstein-Barr virus (CAEBV) infection. CASE PRESENTATION: A 64-year-old man was admitted with a complaint of fever and dyspnea for 2 weeks. The patient presented with elevated serum aminotransferase levels, splenomegaly, progressive decrease of red blood cells and platelets, hyperferritinemia, hypofibrinogenemia, and elevated of Soluble interleukin-2 receptor (sCD25). His chest computed tomography (CT) scan revealed multiple patchy consolidation in both lungs and multiple lymphadenopathy in the mediastinum and hilum. The serology for antibodies of VCA-IgG was positive, EBV-DNA in peripheral blood was elevated, and EBV nucleic acid was detected in the alveolar lavage fluid. Histopathology of the lung tissue showed a dominant of intra-alveolar fibrin and organizing pneumonia. Hemophagocytic cells was found in the bone marrow smear and biopsy. EBV-DNA was detected in lung tissue and bone marrow using in situ hybridization with an EBV-encoded RNA (EBER) probe. After 50 days of hospitalization, he was improved in lung and hemogram. CONCLUSION: We report a case of AFOP with HLH caused by CAEBV in an immunocompetent adult, suggesting that AFOP may be a rare but serious complication caused by CAEBV, and glucocorticoid therapy may improve short-term prognosis.

MAPK14 over-expression is a transcriptomic feature of polycythemia vera and correlates with adverse clinical outcomes.

BACKGROUND: The transcriptomic signature has not been fully elucidated in PV, as well as mRNA markers for clinical variables (thrombosis, leukemic transformation, survival, etc.). We attempted to reveal and validate crucial co-expression modules and marker mRNAs correlating with polycythemia vera (PV) by weighted gene co-expression network analysis (WGCNA). MATERIAL AND METHODS: The GSE57793/26014/61629 datasets were downloaded from Gene Expression Omnibus (GEO) database and integrated into one fused dataset. By R software and 'WGCNA' package, the PV-specific co-expression module was identified, the pathway enrichment profile of which was obtained by over-representation analysis (ORA). Protein-protein interaction (PPI) network and hub gene analysis identified MAPK14 as our target gene. Then the distribution of MAPK14 expression in different disease/mutation types, were depicted based on external independent datasets. Genome-scale correlation analysis revealed the association of MAPK14 and JAK/STAT family genes. Then gene set enrichment analysis (GSEA) was performed to detect the activated and suppressed pathways associating with MAPK14 expression. Moreover, GSE47018 dataset was utilized to compare clinical variables (thrombosis, leukemic transformation, survival, etc.) between MAPK14-high and MAPK14-low groups. RESULTS: An integrated dataset including 177 samples (83 PV, 35 ET, 17 PMF and 42 normal donors) were inputted into WGCNA. The 'tan' module was identified as the PV-specific module (R2 = 0.56, p = 8e-16), the genes of which were dominantly enriched in pro-inflammatory pathways (Toll-like receptor (TLR)/TNF signaling, etc.). MAPK14 is identified as the top hub gene in PV-related PPI network with the highest betweenness. External datasets validated that the MAPK14 expression was significantly higher in PV than that of essential thrombocytosis (ET)/primary myelofibrosis (PMF) patients and normal donors. JAK2 homozygous mutation carriers have higher level of MAPK14 than that of other mutation types. The expression of JAK/STAT family genes significantly correlated with MAPK14, which also contributed to the activation of oxidated phosphorylation, interferon-alpha (IFNα) response and PI3K-Akt-mTOR signaling, etc. Moreover, MAPK14-high group have more adverse clinical outcomes (splenectomy, thrombosis, disease aggressiveness) and inferior survival than MAPK14-low group. CONCLUSION: MAPK14 over-expression was identified as a transcriptomic feature of PV, which was also related to inferior clinical outcomes. The results provided novel insights for biomarkers and therapeutic targets for PV.

The landscape of gene co-expression modules correlating with prognostic genetic abnormalities in AML.

BACKGROUND: The heterogenous cytogenetic and molecular variations were harbored by AML patients, some of which are related with AML pathogenesis and clinical outcomes. We aimed to uncover the intrinsic expression profiles correlating with prognostic genetic abnormalities by WGCNA. METHODS: We downloaded the clinical and expression dataset from BeatAML, TCGA and GEO database. Using R (version 4.0.2) and 'WGCNA' package, the co-expression modules correlating with the ELN2017 prognostic markers were identified (R2 ≥ 0.4, p < 0.01). ORA detected the enriched pathways for the key co-expression modules. The patients in TCGA cohort were randomly assigned into the training set (50%) and testing set (50%). The LASSO penalized regression analysis was employed to build the prediction model, fitting OS to the expression level of hub genes by 'glmnet' package. Then the testing and 2 independent validation sets (GSE12417 and GSE37642) were used to validate the diagnostic utility and accuracy of the model. RESULTS: A total of 37 gene co-expression modules and 973 hub genes were identified for the BeatAML cohort. We found that 3 modules were significantly correlated with genetic markers (the 'lightyellow' module for NPM1 mutation, the 'saddlebrown' module for RUNX1 mutation, the 'lightgreen' module for TP53 mutation). ORA revealed that the 'lightyellow' module was mainly enriched in DNA-binding transcription factor activity and activation of HOX genes. The 'saddlebrown' module was enriched in immune response process. And the 'lightgreen' module was predominantly enriched in mitosis cell cycle process. The LASSO- regression analysis identified 6 genes (NFKB2, NEK9, HOXA7, APRC5L, FAM30A and LOC105371592) with non-zero coefficients. The risk score generated from the 6-gene model, was associated with ELN2017 risk stratification, relapsed disease, and prior MDS history. The 5-year AUC for the model was 0.822 and 0.824 in the training and testing sets, respectively. Moreover, the diagnostic utility of the model was robust when it was employed in 2 validation sets (5-year AUC 0.743-0.79). CONCLUSIONS: We established the co-expression network signature correlated with the ELN2017 recommended prognostic genetic abnormalities in AML. The 6-gene prediction model for AML survival was developed and validated by multiple datasets.

HELQ and EGR3 expression correlate with IGHV mutation status and prognosis in chronic lymphocytic leukemia.

BACKGROUND: IGHV mutation status is a crucial prognostic biomarker for CLL. In the present study, we investigated the transcriptomic signatures associating with IGHV mutation status and CLL prognosis. METHODS: The co-expression modules and hub genes correlating with IGHV status, were identified using the GSE28654, by 'WGCNA' package and R software (version 4.0.2). The over-representation analysis was performed to reveal enriched cell pathways for genes of correlating modules. Then 9 external cohorts were used to validate the correlation of hub genes expression with IGHV status or clinical features (treatment response, transformation to Richter syndrome, etc.). Moreover, to elucidate the significance of hub genes on disease course and prognosis of CLL patients, the Kaplan-Meier analysis for the OS and TTFT of were performed between subgroups dichotomized by the median expression value of individual hub genes. RESULTS: 2 co-expression modules and 9 hub genes ((FCRL1/FCRL2/HELQ/EGR3LPL/LDOC1/ZNF667/SOWAHC/SEPTIN10) correlating with IGHV status were identified by WGCNA, and validated by external datasets. The modules were found to be enriched in NF-kappaB, HIF-1 and other important pathways, involving cell proliferation and apoptosis. The expression of hub genes was revealed to be significantly different, not only between CLL and normal B cell, but also between various types of lymphoid neoplasms. HELQ expression was found to be related with response of immunochemotherapy treatment significantly (p = 0.0413), while HELQ and ZNF667 were expressed differently between stable CLL and Richter syndrome patients (p < 0.0001 and p = 0.0278, respectively). By survival analysis of subgroups, EGR3 expression was indicated to be significantly associated with TTFT by 2 independent cohorts (GSE39671, p = 0.0311; GSE22762, p = 0.0135). While the expression of HELQ and EGR3 was found to be associated with OS (p = 0.0291 and 0.0114 respectively).The Kras, Hedgehog and IL6-JAK-STAT3 pathways were found to be associating with the expression of hub genes, resulting from GSEA. CONCLUSIONS: The expression of HELQ and EGR3 were correlated with IGHV mutation status in CLL patients. Additionally, the expression of HELQ/EGR3 were prognostic markers for CLL associating with targetable cell signaling pathways.

LINC00649 underexpression is an adverse prognostic marker in acute myeloid leukemia.

BACKGROUND: Long noncoding RNAs (lncRNA) play a role in leukemogenesis, maintenance, development, and therapeutic resistance of AML. While few studies have focused on the prognostic significance of LINC00649 in AML, which we aim to investigate in this present study. METHODS: We compared the expression level of LINC00649 between AML patients and healthy controls. The Kaplan-Meier curves of AML patients expressing high versus low level of LINC00649 was performed. The LINC00649 correlated genes/miRNAs/lncRNAs and methylation CpG sites were screened by Pearson correlation analysis with R (version 3.6.0), using TCGA-LAML database. The LINC00649 associated ceRNA network was established using lncBase 2.0 and miRWalk 2.0 online tools, combining results from correlation analysis. Finally, a prediction model was constructed using LASSO-Cox regression. RESULTS: LINC00649 was underexpressed in bone marrow of AML group than that in healthy control group. The patients of LINC00649-low group have significantly inferior PFS and OS. A total of 154 mRNAs, 31 miRNAs, 28 lncRNAs and 1590 methylated CpG sites were identified to be significantly correlated with LINC00649. Furthermore, the network of ceRNA was established with 6 miRNAs and 122 mRNAs. The Lasso-Cox model fitted OS/PFS to novel prediction models, which integrated clinical factors, ELN risk stratification, mRNA/miRNA expression and methylation profiles. The analysis of time-dependent ROC for our model showed a superior AUC (AUC = 0.916 at 1 year, AUC = 0.916 at 3 years, and AUC = 0.891 at 5 years). CONCLUSIONS: Low expression of LINC00649 is a potential unfavorable prognostic marker for AML patients, which requires the further validation. The analysis by LASSO-COX regression identified a novel comprehensive model with a superior diagnostic utility, which integrated clinical and genetic variables.

Overexpression of HOXA10 is associated with unfavorable prognosis of acute myeloid leukemia.

BACKGROUND: HOXA family genes were crucial transcription factors involving cell proliferation and apoptosis. While few studies have focused on HOXA10 in AML. We aimed to investigate the prognostic significance of HOXA10. METHODS: We downloaded datasets from GEO and BeatAML database, to compare HOXA expression level between AML patients and controls. Kaplan-Meier curves were used to estimate the impact of HOXA10 expression on AML survival. The differentially expressed genes, miRNAs, lncRNAs and methylated regions between HOXA10-high and -low groups were obtained using R (version 3.6.0). Accordingly, the gene set enrichment analysis (GSEA) was accomplished using MSigDB database. Moreover, the regulatory TFs/microRNAs/lncRNAs of HOXA10 were identified. A LASSO-Cox model fitted OS to clinical and HOXA10-associated genetic variables by glmnet package. RESULTS: HOXA10 was overexpressed in AML patients than that in controls. The HOXA10-high group is significantly associated with shorter OS and DFS. A total of 1219 DEGs, 131 DEmiRs, 282 DElncRs were identified to be associated with HOXA10. GSEA revealed that 12 suppressed and 3 activated pathways in HOXA10-high group. Furthermore, the integrated regulatory network targeting HOXA10 was established. The LASSO-Cox model fitted OS to AML-survival risk scores, which included age, race, molecular risk, expression of IKZF2/LINC00649/LINC00839/FENDRR and has-miR-424-5p. The time dependent ROC indicated a satisfying AUC (1-year AUC 0.839, 3-year AUC 0.871 and 5-year AUC 0.813). CONCLUSIONS: Our study identified HOXA10 overexpression as an adverse prognostic factor for AML. The LASSO-COX regression analysis revealed novel prediction model of OS with superior diagnostic utility.

[Detection and Diagnostic Values of JAK2, CALR, MPL Gene Mutations in 208 Cases of BCR/ABL1 Negative Chronic Myeloproliferative Diseases].

OBJECTIVE: To detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value. METHODS: Two hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing. RESULTS: among 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia. CONCLUSION: Combined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

Non-malignant T-cells lacking multiple pan-T markers can be found in lymph nodes.

In order to observe and ascertain the properties of a sub-group of T cells in the lymph node (LN) from seven patients who did not suffer from T cell lymphoproliferative disorders (T-LPDs), the expression levels of several pan-T markers were evaluated by multiparameter flow cytometry (FC) and the clonality of these T-cells was evaluated by both FC analysis and PCR assessment. It turned out that multiple pan-T-cell markers such as CD2, CD5 and CD7 were found to be lost in these T cells. The majority of them were positive for TCRαß, only a minority of them being positive for TCRγδ. A subset of these T-cells were positive for CD4 or CD8 or dual-negative for CD4 and CD8. Oligoclonality was detected in one case by FC, while clonal TCR rearrangement was detected in three cases. Absence of multiple pan-T-cell markers could be found in benign T cells in LNs.

Successful eltrombopag treatment of severe refractory thrombocytopenia in chronic myelomonocytic leukemia: Two cases reports: A CARE-compliant article.

RATIONALE: Thrombocytopenia in chronic myelomonocytic leukemia (CMML) is usually attributed to impaired marrow production resulting from cytotoxic drug use or CMML itself ("CMML-induced thrombocytopenia"). In very rare cases, immune thrombocytopenia (ITP) can be a complication of CMML ("CMML-associated ITP"). However, treatment of severe thrombocytopenia in patients with CMML is still a challenge. PATIENT CONCERNS: Case 1 was a 61-year-old female patient admitted to our hospital because of skin petechiae and purpura for 6 days. She had increased monocyte cell count (1.82 × 10/L), markedly decreased platelet count (2 × 10/L), hypercellularity of the megakaryocyte lineage with many immature megakaryocytes, and ZRSR2(zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) mutation. She failed to the treatment of corticosteroids, intravenous immunoglobulin (IVIg), TPO (thrombopoietin), and cyclosporin A (CsA). Case 2 was a 72-year-old female patient with thrombocytosis and monocytosis for 4 years, and thrombocytopenia for 6 months. After 10 courses of decitabine therapy, she had a persistent severe thrombocytopenia and decreased number of megakaryocytes, TET2 (tet methylcytosine dioxygenase 2) and SRSF2 (serine and arginine rich splicing factor 2) mutations were detected. She was dependent on platelet transfusion. DIAGNOSES: Case 1 was diagnosed as CMML-associated ITP, and case 2 as CMML with decitabine therapy-induced thrombocytopenia. INTERVENTIONS: Both patients were treated with eltrombopag. OUTCOMES: In both patients, the platelet counts returned to the normal within 1 week after eltrombopag therapy. The platelet count in case 1 patient remained stable at 141-200 × 10/L for 20 months with stopping therapy for 3 months. In case 2 patient, eltrombopag was stopped 1 month later. Her platelet count decreased to 41 × 10/L, but was stable at ∼30 × 10/L for 3 months with platelet transfusion independency for 12 months. Both patients had no adverse effects with eltrombopag. LESSONS: CMML-associated ITP is very rare and easily misdiagnosed. To the best of our knowledge, case 1 is the first reported case of the successful treatment of CMML-associated ITP with eltrombopag. Both CMML-associated ITP and decitabine therapy-induced thrombocytopenia in these 2 patients were highly sensitive and safe to eltrombopag therapy.

[Application of Cytogenetic Test for Diagnosis of Bone Marrow Involvement in Patients with Non-Hodgkin's Lymphoma].

OBJECTIVE: To investigate the role of cytogenetic analysis in the detection of bone marrow (BM) involvement in patients with non-Hodgkin's lymphoma (NHL). METHODS: The bone marrow samples of 74 patients with NHL were detection by using morphology, cytogenetic test, flow cytometry and molecular biological assay. The detected results of morphology, cytogenetic test, flow cytometry and molecular biological assay alone and thier combined detection were compared, the detective rate and consistencies of the 4 methods were analyzed. RESULTS: The detection rates of BM involvement by using morphology, cytogenetic, flow cytometry, and molecular biological assays were 21.6%, 17.6%, 23.0% and 33.8% respectively. The detective rate was enhanced to 44.6% by combining the 4 methods. Cytogenetic test showed the result consistent with the other methods. CONCLUSION: Although cytogenetic test shows a lower detective rate than the other methods, but in some patients the cytogenetic test can detect the abnormality of bone marrow which can not be detected by other methods alone, the combination test of 4 detection methods can enhance the detectable rate of BM involvement.

The application of CD73 in minimal residual disease monitoring using flow cytometry in B-cell acute lymphoblastic leukemia.

The expression of CD73 by flow cytometry (FC) in bone marrow (BM) specimens of B-cell acute lymphoblastic leukemia (B-ALL) with or without minimal residual disease (MRD) was studied, and its advantages were evaluated using the MRD assay. This study also detected the expression profile of CD73 in hematogones and mature B cells in BM specimens of 18 healthy donors. Results showed that the mean value of CD73 expression in MRD-positive B cells was 6-fold greater than that in the MRD negative ones. Also, 41.82% MRD-positive B-ALL cases expressed high CD73 and the sensitivity of CD73-based MRD detection reached 10(-4). Since the expression of CD73 increases with the maturation of normal B cells, it is better to mix it with CD34, CD10 and CD20 in one tube to prevent the disturbance of mature B cells. CD73 is recommended as an optional MRD marker for B-ALL patients by using FC.