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  • 11
    In: Blood Advances, American Society of Hematology, Vol. 4, No. 5 ( 2020-03-10), p. 885-892
    Abstract: Therapy-related myeloid neoplasms (tMN) develop after exposure to cytotoxic and radiation therapy, and due to their adverse prognosis, it is of paramount interest to identify patients at high risk. The presence of clonal hematopoiesis has been shown to increase the risk of developing tMN. The value of analyzing hematopoietic stem cells harvested at leukapheresis before autologous stem cell transplantation (ASCT) with next-generation sequencing and immunophenotyping represents potentially informative parameters that have yet to be discovered. We performed a nested case-control study to elucidate the association between clonal hematopoiesis, mobilization potential, and aberrant immunophenotype in leukapheresis products with the development of tMN after ASCT. A total of 36 patients with nonmyeloid disease who were diagnosed with tMN after treatment with ASCT were included as case subjects. Case subjects were identified from a cohort of 1130 patients treated with ASCT and matched with 36 control subjects who did not develop tMN after ASCT. Case subjects were significantly poorer mobilizers of CD34+ cells at leukapheresis (P = .016), indicating that these patients possess inferior bone marrow function. Both clonal hematopoiesis (odds ratio, 5.9; 95% confidence interval, 1.8-19.1; P = .003) and aberrant expression of CD7 (odds ratio, 6.6; 95% confidence interval, 1.6-26.2; P = .004) at the time of ASCT were associated with an increased risk of developing tMN after ASCT. In conclusion, clonal hematopoiesis, present at low variant allele frequencies, and aberrant CD7 expression on stem cells in leukapheresis products from patients with nonmyeloid hematologic cancer hold potential for the early identification of patients at high risk of developing tMN after ASCT.
    Type of Medium: Online Resource
    ISSN: 2473-9529 , 2473-9537
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2020
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  • 12
    In: BMC Cancer, Springer Science and Business Media LLC, Vol. 22, No. 1 ( 2022-12)
    Abstract: Multiple myeloma remains an incurable disease with multiple relapses due to residual myeloma cells in the bone marrow of patients after therapy. Presence of small number of cancer cells in the body after cancer treatment, called minimal residual disease, has been shown to be prognostic for progression-free and overall survival. However, for multiple myeloma, it is unclear whether patients attaining minimal residual disease negativity may be candidates for treatment discontinuation. We investigated, if longitudinal flow cytometry-based monitoring of minimal residual disease (flow-MRD) may predict disease progression earlier and with higher sensitivity compared to biochemical assessments. Methods Patients from the Nordic countries with newly diagnosed multiple myeloma enrolled in the European-Myeloma-Network-02/Hovon-95 (EMN02/HO95) trial and undergoing bone marrow aspiration confirmation of complete response, were eligible for this Nordic Myeloma Study Group (NMSG) substudy. Longitdudinal flow-MRD assessment of bone marrow samples was performed to identify and enumerate residual malignant plasma cells until observed clinical progression. Results Minimal residual disease dynamics were compared to biochemically assessed changes in serum free light chain and M-component. Among 20 patients, reaching complete response or stringent complete response during the observation period, and with ≥3 sequential flow-MRD assessments analysed over time, increasing levels of minimal residual disease in the bone marrow were observed in six cases, preceding biochemically assessed disease and clinical progression by 5.5 months and 12.6 months (mean values), respectively. Mean malignant plasma cells doubling time for the six patients was 1.8 months (95% CI, 1.4–2.3 months). Minimal malignant plasma cells detection limit was 4 × 10–5. Conclusions Flow-MRD is a sensitive method for longitudinal monitoring of minimal residual disease dynamics in multiple myeloma patients in complete response. Increasing minimal residual disease levels precedes biochemically assessed changes and is an early indicator of subsequent clinical progression. Trial registration NCT01208766
    Type of Medium: Online Resource
    ISSN: 1471-2407
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
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  • 13
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 3235-3235
    Abstract: Introduction Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases marked by dysplasia in the bone marrow (BM) causing cytopenias in the peripheral blood and with an inherent tendency to progress to acute myeloid leukemia (AML). Myelodysplastic syndrome has generally been regarded a stem cell disease, which very recent research has emphasized to hold true also for low-risk MDS (Woll, P. S. et al Cancer Cell. 2014). In AML, we have identified the human Myeloid Inhibitory C-type lectin-like receptor (hMICL) (also named CLL-1 or CLEC12A) as a stable and reliable marker at diagnosis and as a tool for assessing minimal residual disease (Roug, A. S. et al Br. J. Haematol. 2014). Furthermore, hMICL has been proposed as a marker of the leukemic stem cell (LSC) since it is present on CD34+CD38- cells in the majority of CD34-positive AMLs, while it is absent on the CD34+CD38- cell compartment in normal controls (van Rhenen, A. et al Leukemia. 2007). Previous studies have shown aberrant marker-expression on CD34+CD38- cells in MDS, e.g. CD7 or CD2, but only in minor fractions of patients (Xie, W. et al. Cytometry A. 2010). Using multicolor flowcytometry, we have examined the aberrant expression of hMICL on the CD34+CD38- stem cell compartment from 19 untreated MDS patients. We correlated the aberrant hMICL positive cells as a percentage of the total CD34+CD38- compartment (CD34+CD38-hMICL+/CD34+CD38-) to follow-up data. Materials and methods Bone marrow (BM) samples were obtained from 19 MDS patients diagnosed and followed at the Department of Hematology, Aarhus University Hospital from January 2010 to January 2011. Diagnosis was determined by morphology according to the 2008 WHO classification. Risk stratification of patients was done using the International Prognostic Scoring System (IPSS). All samples were collected before treatment with a cytoreductive agent or 5-azacytidine. Normal bone marrow (NBM) from 11 healthy volunteers served as controls. Following lysis of red blood cells, BM was stained with the following monoclonal antibodies: anti-CD34 APC, anti-CD38 FITC, anti-CD45 PerCP-Cy5.5 and anti-hMICL PE. Data acquisition was performed on a FACSCanto II and subsequently analyzed using FACSDiva Software. Using the back-gating strategy, the CD34+CD38-hMICL+ cell subset was shown to cluster together in an FSC-SCC plot. A total number of 5 clustered events were considered significant for further analyses. Results and perspectives The median age at sampling was 69 years (range 44-83 years). According to IPSS, 1 patient was classified as low-risk, 11 patients as intermediate-1 and 3 patients as intermediate-2. Four patients could not be classified. Consistent with previous reports, hMICL was not found on CD34+CD38- cells in any of the 11 NBM examined (0.0%) whereas aberrant hMICL expression was present on the CD34+CD38- stem cells in 16/19 (84%) of MDS cases. In MDS, the CD34+CD38-hMICL+ cells amounted a median of 5.68% (range 0,0-56.94%) of the CD34+CD38- subset, which was significantly different from NMB (p 〈 0.0001) (Figure 1A). The median follow-up period for the cohort was 3.7 years. Patients with fractions of hMICL positive CD34+CD38- cells above the median (5.68%) showed a significantly shorter progression free survival (PFS) compared to patients with hMICL+ fractions below the median (p=0.01) (Figure 1B). The same trend was evident with regards to overall survival (OS), although borderline significant (p=0.06) (Figure 1C). The aberrant expression of hMICL on CD34+CD38- stem cells from MDS patients belonging to both low- and intermediate risk groups identifies hMICL as a potential LSC-marker in this heterogeneous group of diseases. A considerable advantage of hMICL is its complete absence on normal hematopoietic stem cells and the fact that it was present in the majority of MDS cases studied. Despite the small patient-number in our cohort, the impact of a high hMICL+ stem cell fraction on PFS and OS is evident. Thus, hMICL marking of the CD34+CD38- stem cell compartment might serve as a prognostic indicator in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 14
    Online Resource
    Online Resource
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 5643-5643
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 5643-5643
    Abstract: Markers indicative of prognosis play a consequential role in the clinical management of patients suffering from chronic B-cell lymphocytic leukemia (B-CLL). Soluble CD163 (sCD163) has been shown to be a useful biomarker in a wide variety of disease entities (Moller, 2012), however its presence in B-CLL has not been addressed. Using an enzyme-linked immunosorbent assay the concentration of sCD163 was measured in peripheral blood of 30 B-CLL patients at diagnosis. The results were related to the course of disease for up to two years post diagnosis. The median level of sCD163 in the plasma was not significantly higher in the B-CLL patients (2.085 mg/L, range 0.77- 9.01 mg/L) than in an age-matched control group (1.800 mg/L, range 0.97-2.45 mg/L) (n=10) (p=0.157). As CD163 is a monocyte/macrophage specific membrane protein, the relationship between the percentage of monocytes and the level of sCD163 was relevant. The level of sCD163 did not correlate with the percentage of monocytes in peripheral blood of the patients, but as previously described, there was an inverse correlation between measured sCD163 and the CD163 surface expression as measured by mean fluorescence intensity on the monocytes (r=0.088, p= 0.658 and r= -0.476, p=0.01, respectively) (Davis et al. 2005). Elevated levels of sCD163 have been linked to bacterial infection however in neither the B-CLL cohort nor the healthy control group, a correlation was found between the levels of sCD163 and CRP concentrations (r=0.24 and p=0.23 in B-CLL, and r=0.10 and p=0.81 in healthy controls) (Knudsen et al., 2007). To test the prognostic impact of sCD163 the B-CLL patients were divided into two subsets using the highest level of sCD163 measured in the age-matched healthy control group as a cut-off. Hence, 11 of 30 B-CLL patients were assigned to an sCD163high group. In total, 7/30 (23%) patients experienced disease progression defined as need for cytoreductive treatment within the two years follow-up period. In more detail, 5/11 sCD163high patients (45%) and 2/19 patients (11%) in the sCD163low group received therapy (p=0.068). When analyzing the relevance of sCD163 in terms of predicting disease progression, there was a significant difference between patients in the two groups, indicating that patients with high concentrations of sCD163 in the plasma progressed more rapidly (p=0.029) (Figure 1A). In this cohort, CD38 expression was also of prognostic value for progressive disease (p=0.006) (Figure 1B), while b2M was of borderline significance (p=0.051) (Figure 1C). The role of mutational status as predictor of short time to treatment was not significant (p=0.21) (Figure 1D). When performing the log-rank univariate analyses on the dataset, there was an obvious increased hazard of receiving treatment for patients in the sCD163high group compared to the sCD163low patients (Table I). As expected, CD38 expression, b2M, and IgVH mutational status were albeit to varying degrees covariates in the time to treatment analyses (Table I). The data strongly indicate that sCD163 is a prognostic marker in B-CLL. The study is limited by the cohort size, however, indubitably, the results lay the ground for a larger study. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 15
    Online Resource
    Online Resource
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 2626-2626
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 2626-2626
    Abstract: Flow cytometry constitutes a cornerstone in the diagnosis and follow up of acute myeloid leukemia (AML) and it is based on the identification of leukemia-associated immunophenotypes (LAIPs). We have recently demonstrated that the C-type lectin hMICL in combination with CD123 constitute a highly stable and reliable LAIP marker combination at diagnosis and relapse. In addition, we have shown that an hMICL/CD123-based assay is an effective tool for the monitoration of minimal residual disease (MRD) in AML. To what extent hMICL/CD123 marking identifies early leukemic cells is, however, not established. We hypothesized that this could be addressed by studying molecular aberrations in leukemic cell subsets according to their expression of hMICL and CD123. Employing cell sorting and mutational analyses, we here establish the leukemic origin of hMICL and CD123 expressing cell populations. Analyzing diagnostic AML samples with homozygous FLT3-ITD aberrations allowed for verification of pure malignant clones. Five patients with FLT3-ITD allelic burden of 〉 50% (range 77-93%, median 85%) as measured by DNA fragment analysis by capillary electrophoresis on mononuclear cells (MNC) were identified in our local database of 600 cases. We found that 5/5 patients displayed a normal karyotype and carried NPM1 mutations (NPM1 allelic burden 42-48%, median 46%). In contrast, mutations in FLT3-D835, IDH1-R132, c-KIT-D816V or indel mutations in CEBPA and WT-1 exon 7 were absent. From samples of cryopreserved mononuclear cells (bone marrow (n=4) and peripheral blood (n=1)), CD45low/SSClow blast cell subsets with the following immunophenotypes were sorted by FACS: CD34+/hMICL+/CD123+, CD34+/hMICL+/CD123-, CD34+/hMICL-/CD123+, and CD34+/hMICL-/CD123-. In one case of CD34 negative AML the sorted subsets were CD34-/hMICL+/CD123+, CD34-/hMICL+/CD123-, CD34-/hMICL-/CD123+, and CD34-/hMICL-/CD123-. Sorted cell subsets were analyzed for FLT3-ITD and NPM1 mutations using fragment analysis by capillary electrophoresis. The results of the fragment analyses are tabulated in the table below. In all cases the hMICL and CD123 expressing subsets of interest closely approximated 100% FLT3-ITD allelic burden. In contrast, hMICL-/CD123- cells approximated only a 50% FLT3-ITD allelic burden. Of note, an extended search in our AML database, revealed only 9 of 600 patients to have an FLT3-ITD allelic burden 〉 50% (range 52-94%, median 81%) hence indicating a state of either homo- or hemizygosity. Interestingly, with the exception of one case carrying a chromosome 13 duplication, each of these 9 patients also harbored a mutation in the NPM1 gene as the only other known aberration. In conclusion using AML patients with high FLT3-ITD allelic burdens we have been able to show that blasts expressing hMICL and/or CD123 at diagnosis are indeed malignant thus further substantiating the use of these antigens in AML MRD detection. Additionally, a direct relationship between NPM1 and FLT3-ITD homo-/hemizygosity may be suggested in the evolution of the malignant clone.Phenotype of sorted cell subsetNumber of patientsFLT3-ITD allelic burden (%) Min-max (median)NPM1 allelic burden (%) Min-max (median)MNC577-93 (85)42-48 (46)CD45low/SSClow/CD34+/hMICL+/CD123+495-100 (98)48-50 (49)CD45low/SSClow/CD34+/hMICL+/CD123-1*9248CD45low/SSClow/CD34+/hMICL-/CD123+497-100 (99)47-51 (48.5)CD45low/SSClow/CD34+/hMICL-/CD123-436-68 (47)16-38 (25)CD45low/SSClow/CD34-/hMICL+/CD123+110046CD45low/SSClow/CD34-/hMICL+/CD123-19448CD45low/SSClow/CD34-/hMICL-/CD123+110047CD45low/SSClow/CD34-/hMICL-/CD123-17735*Subset only present in one of four patients Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 16
    Online Resource
    Online Resource
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 4969-4969
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 4969-4969
    Abstract: Introduction Despite the discovery of new genetic alterations the cytogenetically normal acute myeloid leukemia (CN-AML) subset is still insufficiently characterized. As mRNA-sequencing (RNA-seq) is becoming more accessible, sequencing of the transcriptome could reveal not only information regarding deregulated expression patterns in leukemias, but also enable mutational evaluation of expressed alleles. To address this issue we performed RNA-seq of 5 AML patients from diagnostic bone marrow aspirates to assess the validity of the following: 1) remission samples can be used as control, 2) concordance between standard clinical laboratory analysis and RNA-seq data, and 3) implementation of existing microarray data repository to infer expected mutational status of the sequenced samples based on expression patterns. Methods Diagnostic samples were selected among patients with high leukemic blast fraction (mean of 75%) and paired remission samples with very low, or no detectable, molecular minimal residual disease. A panel of recurrent somatic mutations was assessed by means of quantitative PCR (qPCR) and fragment analysis for sequencing comparisons. Sequencing was performed on single HiSeq lane aimed at minimum 66 million reads per sample (AROS Applied Biotechnology, Aarhus, Denmark). Biomedical Genomics Workbench 2 was employed for alignment, mutation calling and analysis of differential expression (Robinson-Smyth exact test, Bonferroni corrected). R and Mathematica were used for comparison of expression patterns from the individual samples in conjunction with CN-AML (251) and control bone marrow (73) data from microarray repositories (Gene Expression Omnibus, GSE15434 and GSE13159). Results 259 genes were differentially expressed as defined by the following thresholds: normalized fold-change 〉 2, expression range values 〉 10 RPKM and p 〈 0.05. Of these, 41 genes were upregulated in AML samples (p 〈 0.01 subset heatmap is shown in fig. 1A). Supervised clustering of the samples on the basis of differential expression patterns efficiently divided the data into 2 groups according to diagnosis and remission status (fig. 1A, dendrogram). A median of 61 mutations per sample was observed (35 to 675). Thirty-four mutations occurred twice or more (fig. 1B, size reflects transcript fraction of mutated allele). A close correlation between routine molecular diagnostics and sequencing data was found. As expected, routine minimal residual disease marker WT1 was in agreement and found to be clearly expressed at diagnosis, but not at time of remission. By comparing patient expression patterns with NPM1, FLT3 or CEPBA mutation specific microarray expression signatures we were largely able to deduce expected mutational status for each patient (fig. 1C, showing the individual matching fraction of mutation specific gene expression signature in terms of upregulated or downregulated) in favor of qPCR analysis shown in table 1. Table 1. Comparison of mutational status from qPCR/RNA-seq FLT3 mut NPM1 mut IDH1 mut WT1 mut CEBPA mut KIT mut #1 +/+ +/+ +/+ -/- -/- -/- #2 +/+ +/+ -/- -/- -/- -/- #3 +/+ +/- -/- -/- -/- -/- #4 -/- +/- +/+ +/- -/- -/- #5 +/+ -/- -/- -/- -/- -/- Conclusions This approach serves as a proof of the concept that RNA-sequencing can be directly implemented in the routine laboratory. Moreover, transcriptome data such as these can extend the molecular survey in a dynamic manner by aiding in therapy-related decision-making for the application of targeted therapy and for delineating the reasons for treatment. While publicly available repositories of RNA-seq data are being generated for referencing, it is possible to include microarray data to support molecular classification of the individual patients, as is shown here. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 17
    In: Leukemia Research Reports, Elsevier BV, Vol. 3, No. 2 ( 2014), p. 94-97
    Type of Medium: Online Resource
    ISSN: 2213-0489
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2014
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  • 18
    In: Journal of Hematology, Elmer Press, Inc., Vol. 9, No. 3 ( 2020), p. 89-92
    Type of Medium: Online Resource
    ISSN: 1927-1212 , 1927-1220
    Language: English
    Publisher: Elmer Press, Inc.
    Publication Date: 2020
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  • 19
    In: British Journal of Haematology, Wiley, Vol. 175, No. 3 ( 2016-11), p. 393-401
    Abstract: Evidence of distinct disease propagating stem cells in myelodysplastic syndrome ( MDS ) has emerged in recent years. However, immunophenotypic characterization of these cancer stem cells remains sparse. In acute myeloid leukaemia ( AML ), we have previously described aberrant expression of the C‐type lectin domain family 12, member A ( CLEC 12A) as a stable and reliable marker of leukaemia blasts and as a tool for assessing minimal residual disease. Furthermore, CLEC 12A has been proposed as a promising marker of leukaemic stem cells in AML . The role of CLEC 12A in MDS , however, remains to be elucidated. In this study, we found CLEC 12A aberrantly expressed on the CD 34 + CD 38 − cell compartment in 71% (22/31) of MDS patients, distributed across all Revised International Prognostic Scoring System risk groups. We showed that the CD 34 + CD 38 − CLEC 12A + cells were indeed malignant and possessed functional stem cell properties in the long‐term colony‐initiating cell assay. As opposed to reported findings in AML , we showed that cancer stem cells from MDS samples derived from both CLEC 12A positive and negative CD 34 + CD 38 − subpopulations. Due to the absence of CLEC 12A on normal haematopoietic stem cells, CLEC 12A stem cell immunophenotyping may contribute to diagnosing and monitoring MDS patients and could furthermore add knowledge about disease propagating cells in MDS .
    Type of Medium: Online Resource
    ISSN: 0007-1048 , 1365-2141
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2016
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  • 20
    In: Human Mutation, Hindawi Limited, Vol. 2023 ( 2023-6-2), p. 1-11
    Abstract: Current estimates suggest that up to 10% of patients with myeloid neoplasms (MN) harbor variants associated with a germline predisposition. A pathogenic variant in the runt-related transcription factor 1 gene (RUNX1) is a frequent cause of germline predisposition to MN. RUNX1 variants detected in tumor tissue at a VAF close to 50% are potentially germline and causative of RUNX1 familial platelet disorder with associated myeloid malignancies. Previous studies have found germline RUNX1 variants in 3% of patients with acute myeloid leukemia; however, the frequency of germline RUNX1 variants in less advanced myeloid neoplasms has not been examined. We screened 590 patients suspected of MN, excluding myeloproliferative neoplasms, for germline variants in RUNX1. We found RUNX1 variants in 83 patients (14%) by targeted sequencing of tumor tissue. In 40 patients (6.8%), the VAF of RUNX1 was above 30%. In 32 of the 40 patients, skin biopsies were available and used for Sanger sequencing to assess the germline status. Two of the tested variants (6.3%) were confirmed as germline, and both variants were curated as variants of unknown significance. To further explore the pathogenicity of these variants, we implemented a novel CRISPR-Select functional genetic assay. The assay demonstrated a profound effect on proliferation in K562 cells for a known pathogenic variant but no effect for the two germline variants detected in the study. We therefore propose that both germline variants are classified as likely benign. In this study, we show that RUNX1 germline variants are rare in Danish patients with MN and use a novel assay for functional classification of germline RUNX1 variants.
    Type of Medium: Online Resource
    ISSN: 1098-1004
    Language: English
    Publisher: Hindawi Limited
    Publication Date: 2023
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