BACKGROUND:In acute myeloid leukemia (AML), the most sensitive measurable residual disease (MRD) methods are single-gene approaches, but these are applicable only in ~60% of AML cases. METHODS:We applied multi-omics single-cell analysis on diagnostic and first remission samples to identify leukemia-specific molecular markers for subsequent MRD monitoring in six AML patients lacking AML-defining variants. RESULTS:Five selection criteria were defined to identify suitable MRD markers. Markers of primordial leukemic clones were identified by combining data from single-cell sequencing and immunophenotyping. Specific markers suitable for use in MRD follow-up were identified in 6/6 patients, in some cases in myelodysplasia-related genes and clonal hematopoiesis-related genes usually not recommended for use in MRD determinations. Patient-specific ddPCR (limits of detection: 0.06-0.0011%) or EC-NGS assays correlated with therapeutic responses: 0/4 markers displayed molecular relapses in three non-relapsing patients, contrary to 4/4 markers of three relapsing patients. Of these, 3/4 and 1/4 markers detected molecular relapses earlier than or simultaneous with conventional methods, respectively (-115 to -338 days). CONCLUSIONS:Our results demonstrate that single-cell subclonal mapping at diagnosis and during first remission enables selection of reliable MRD targets for personalized disease surveillance in patients lacking conventional MRD markers.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34 + CD38 − bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

Clonal hematopoiesis (CH) denotes somatic mutations in genes related to myeloid neoplasms present at any variant allele frequency (VAF). Clonal hematopoiesis is associated with increasing age and with a factor 6 increase in the risk of developing therapy-related myeloid neoplasms (tMNs) following autologous stem cell transplantation (ASCT). However, the impact of specific mutations on progression from CH to tMN has yet to be unraveled, and it remains unclear whether mutations directly impact or even drive the development of tMN. We performed deep sequencing in longitudinal samples from a cohort of 12 patients with either multiple myeloma or lymphoma who developed tMN following ASCT. Nine patients had one or more mutations that could be tracked longitudinally. Seven patients had clonal expansion from time of ASCT to diagnosis of tMN. Of these, six patients had CH at VAF < 2% at baseline. The median VAF of non- DNMT3A clones increased from 1% (IQR 0.7%–10.0%) at time of ASCT to 37% (IQR 17%–47%) at tMN diagnosis ( P = 0.002), while DNMT3A clones showed quiescent trajectories ( P = 0.625). Our data provide evidence to support the hypothesis that the development of tMN following ASCT is likely instigated by CH present at VAFs as low as 0.5%, detectable years before tMN onset.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34(+)CD38(-) bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level > 0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45(low)CD34(+)CD38(-)CD123(-)CD7(-)) and not in the putative leukemic stem cells (CD34(+)CD38(-)CD123(+)CD7(+)) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS-MRD assays. (c) 2022 Published by Elsevier Inc. on behalf of ISEH - Society for Hematology and Stem Cells.

Relapse remains a major therapeutic challenge in children with acute myeloid leukemia (AML). Outcome after relapse may improve if preemptive therapy is initiated at first evidence of leukemia regrowth. Early detection of imminent relapse requires molecular measurable residual disease (MRD) monitoring after therapy completion. Today, this is possible only in about 40% of children with AML that harbor genetic abnormalities applicable for quantification using standardized qPCR assays. To enable disease surveillance for all patients, we developed patient-tailored deep sequencing (DS) MRD analysis, which provides highly sensitive detection of leukemia-specific mutations. We investigated the potential of this method for early relapse detection in peripheral blood (PB), the only easily accessible source for MRD sampling in children. PB samples were collected at monthly intervals during follow-up from 45 children diagnosed with AML and treated according to The Nordic Society of Pediatric Haematology and Oncology (NOPHO)-DBH AML 2012 protocol between January 2013 and May 2016 in Denmark, Norway, Sweden and Finland (508 samples, median 11 samples/patient, range 3-27). Nine patients with relapse (median age 5 years, range 0-8) had available diagnostic and relapse material and were included in this study. The patients displayed core binding factor abnormalities (n=3), KMT2A-rearrangements (n=3), monosomy 7 (n=1) or normal karyotype (n=2) at AML diagnosis. Leukemia-specific single nucleotide variants (SNVs) were identified with exome sequencing (ES) of sorted leukemic cells with lymphocytes or remission PB as constitutive DNA template. A variant allele frequency (VAF) with 95% confidence interval including 50% indicates presence of the mutation in all leukemic cells at diagnosis. With the exception of 2 cases with only subclonal mutations at diagnosis, leukemia-specific SNVs with VAF of 50% at diagnosis and persistence at relapse were selected as MRD targets. MRD target mutations were quantified in PB samples preceding overt relapse using patient-tailored DS assays with sensitivity of VAF 0.02%. In diagnostic samples, ES identified 53 leukemia-specific SNVs (median 4 SNVs/patient, range 2-12) of which 33 were also present at relapse (median 2 SNVs/patient, range 1-9). The number of mutations identified at diagnosis increased with age (Rs 0.83, p=0.006). All patients had at least one leukemia-specific SNV detected at both diagnosis and relapse. Twenty-one MRD target mutations (median 2 SNVs/patient, range 1-3) were quantified in PB (55 samples, median sampling interval 28 days, range 11-80) using DS. In 8/9 patients, at least one SNV was detected in PB before overt relapse occurred. The first PB sample showing MRD positivity (median VAF 0.14%, range 0.03-0.44) preceded hematological relapse at a median interval of 3 months (range 0-7.9). In 6 patients not preemptively treated, the median doubling time based on VAF increments was 7 days, with great variability between individuals and genotypes (range 4-28 days). Three patients had molecular relapse diagnosed by qPCR used in clinical diagnostics and received individualized preemptive treatment. In these 3 patients, DS detected mutations in PB for >100 days preceding overt relapse and the doubling times were 14, 25 and 36 days. In conclusion, DS of leukemia-specific mutations at frequent intervals in PB enables early detection of relapse and ES at diagnosis may identify SNVs applicable for such longitudinal MRD monitoring. This approach facilitates molecular disease surveillance and initiation of preemptive therapy in AML patients without established qPCR targets. Disclosures No relevant conflicts of interest to declare.

Overexpressed genes may be useful for monitoring of measurable residual disease (MRD) in patients with childhood acute myeloid leukemia (AML) without a leukemia-specific target. The normal expression of five leukemia-associated genes (SPAG6, ST18, MSLN, PRAME, XAGE1A) was defined in children without hematologic disease (n = 53) and children with suspected infection (n = 90). Gene expression at AML diagnosis (n=50) and during follow-up (n = 21) was compared with child-specific reference values. At diagnosis, 34/50 children (68%) had high expression of at least one of the five genes, and so did 16/31 children (52%) without a leukemia-specific target. Gene expression was quantified in 110 peripheral blood (PB) samples (median, five samples/patient; range, 1 to 10) during follow-up in 21 patients with high expression at diagnosis. All nine patients with PB sampling performed within 100 days of disease recurrence displayed overexpression of SPAG6, ST18, PRAME, or XAGE1A at a median of 2 months (range, 0.6 to 9.6 months) before hematologic relapse, whereas MSLN did not reach expression above normal prior to hematologic relapse. Only 1 of 130 (0.8%) follow-up analyses performed in 10 patients in continuous complete remission had transient expression above normal. SPAG6, ST18, PRAME, and XAGE1A expression in PB may predict relapse in childhood AML patients and facilitate MRD monitoring in most patients without a leukemia-specific target.

BACKGROUND:Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents.RESULTS:We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45-65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias.CONCLUSIONS:These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.

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.

Hematological malignancies are an aggregate of diverse populations of cells that arise following a complex process of clonal evolution and selection. Recent approaches have facilitated the study of clonal populations and their evolution over time across multiple phenotypic cell populations. In this review, we present current concepts on the role of clonal evolution in leukemic initiation, disease progression, and relapse. We highlight recent advances and unanswered questions about the contribution of the hematopoietic stem cell population to these processes.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Objective Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). The assay amplifies both e13a2 and e14a2 BCR-ABL1 transcript variants. Observing distinct variant-dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR-ABL1. Methods We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital [ZUH], Aarhus University Hospital [AU], and Rigshospitalet [RH]) on cell lines expressing either the e13a2 or e14a2 BCR-ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR). Results Although no significant differences were found in amplification efficiencies of the transcript variants, Bland-Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6-, 6.5-, and 1.8-fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7-fold difference between the e13a2 and e14a2 variants. Conclusion Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.

Abstract Background: Even though children with acute myeloid leukemia (AML) receive a very intensive chemotherapy and most achieve a complete remission (CR) ~30% of patients suffer from relapse. Post-treatment monitoring of measurable residual disease (MRD) can allow detection of a re-emerging leukemic clone several months before clinical relapse, and studies are in progress that aim at treating children with a molecular relapse. Specific genetic aberrations can be used for disease monitoring after therapy completion, but even though oncogenic fusion transcripts are more common in childhood than adult AML, NPM1 mutation is much rarer and consequently MRD measurements based on these aberrations are clinically applicable in only ~40% of childhood AML patients. Thus, a considerable fraction of patients do not have a suitable leukemia-specific molecular MRD target, but genes with an abnormally high expression in the leukemic cells might be candidate MRD targets in those patients. WT1 overexpression in childhood AML is well described, and if distinctly overexpressed at diagnosis, serves as a suitable MRD target in a large proportion of patients. Gene expression profiling has identified several other genes with an abnormally high expression in the leukemic blasts compared to normal hematopoietic cells. We investigated the applicability of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) as targets for early detection of relapse in peripheral blood (PB) in a Danish cohort of childhood AML patients, defined child-specific reference values of gene expression based on a large material of PB and BM samples from hematologically healthy children, and investigated gene expression levels under the presence of infection. Methods: We investigated the expression of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) in hematologically healthy children (n=53) and during suspected infection in febrile but otherwise healthy children (n=90). Gene expression in de novo AML at diagnosis (n=50) and during follow-up (n=20) was compared with child-specific reference values. We defined the 95th percentile of expression levels in hematologically healthy children as the upper limit of normal expression. RT-qPCR analyses were performed in compliance with EAC protocols and due to concordant qPCR efficiencies the ΔΔCq method for relative quantification could be applied. Results: At AML diagnosis, 64% had high expression of at least 1 of the 4 genes defined as >20-fold overexpression compared to hematologically healthy children. Nine out of 10 patients (90%) without established molecular MRD targets or high WT1 expression had high expression of at least 1 of the 4 genes. All 7 children with t(9;11) had GAGED2>1000-fold overexpressed. Gene expression was quantified in 99 PB samples (163 RT-qPCR analyses) during follow-up in 20 patients with distinct overexpression at diagnosis. All 10 patients with PB sampling performed within 100 days of disease recurrence displayed expression above normal by a median of 1.6 months (range 0.5-6 months) before hematological relapse. Patients with CBF-AML had a significantly longer interval between molecular relapse and hematological relapse than patients with non-CBF-AML (2.5 months (range 0.8-6 months) vs. 0.9 months (range 0.5-2 months), p=0.047). One patient with PB sampling performed only once at 119 days prior to hematological relapse did not show any molecular evidence of disease recurrence before hematological relapse. Only 1 of 96 (1%) post-therapy follow-up analyses performed in 9 patients in continuous CR for >5 years after diagnosis had expression above normal. In this case, a 9-year-old girl in continuous CR had an increase in ST18 expression, however the increase was transient and returned to normal level in the following samples. We found no clinically relevant influence of fever on gene expression levels, except for GAGED2, where 21% of febrile children had expression above normal. Conclusions: Sequential post-therapy monitoring of overexpressed genes in PB can predict relapse in childhood AML patients and facilitates molecular MRD monitoring in 90% of patients without a leukemia-specific target or WT1 overexpression. Frequent PB sampling (every 4-6 weeks) is necessary to detect an upcoming relapse, however in the post-treatment follow-up setting PB serves as an attractive and easily accessible source of preference compared to BM aspiration. Disclosures No relevant conflicts of interest to declare.

Introduction: Diagnosing BCR-ABL negative myeloproliferative neoplasms (MPN) may be challenging due to overlapping features and lack of robust discriminatory parameters, especially between essential thrombocythemia (ET) and prefibrotic myelofibrosis (MF). Circulating immature hematopoietic cells are variably present in polycythemia vera (PV), ET, and MF. The C-type lectin hMICL is aberrantly expressed on hematopoietic stem cells in the majority of acute myeloid leukemia patients. However, the hMICL expression in MPN, having varying propensity of leukemic transformation, is unsettled. We hypothesized that enumeration of immature cells by flow cytometry (FCM) could be a discriminatory tool in MPN diagnostics. Methods: By FCM, we quantified circulating stem cells with aberrant hMICL expression in 39 MPN patients, 10 age-matched controls, and in leukapheresis products from 10 patients with lymphoproliferative neoplasms. The utility of the FCM assay for discriminating MPN entities was evaluated by applying ROC curve analysis. Results: While hMICL was absent in control samples, MF patients had significantly more hMICL+ stem cells (median 15.2%) than PV and ET (0.0%, P=.001 and 0.0%, P=.002, respectively). By ROC curve analysis, the presence of hMICL+ stem cells (>0 cells) in peripheral blood reliably discriminates MF from ET and PV with a sensitivity of 80% and a specificity of 97%. Conclusion: Enumeration of circulating hMICL+ stem cells by FCM can discriminate between MPN phenotypes and holds potential for monitoring disease evolution.

Background The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. Results By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. Conclusions In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.

Congenital hypoplastic bone marrow failure is a rare condition in neonates. The genetics and mechanisms behind are largely obscure. Here we characterize a neonate presenting with congenital thrombocytopenia and anemia. During the first 2-4 weeks after birth the neonate developed severe neutropenia while the lymphoid lineages were unaffected. The neonate was without dysmorphic signs. A de novo mono-allelic constitutional microdeletion of 175.1 kb at 3q26.2 affecting exon 2 of MECOM, involving MDS1 but not EVI1, was identified as the only copy number alteration by oligo-based array-CGH analysis. Expression analysis showed profoundly reduced expression of multiple MECOM transcripts in the bone marrow cells. Whole exome sequencing detected no pathogenic mutations in genes known to be associated with inherited bone marrow failure syndromes. The patient was successfully treated with hematopoietic stem cell transplantation at 5 months of age. Interstitial deletions encompassing the 3q26.2 region are very rare. A literature search revealed two previous cases with microdeletions involving this region, and the cases were associated with congenital thrombocytopenia and anemia, but unaffected lymphopoiesis. Together these data indicate that MECOM may be important for normal myeloid hematopoiesis in humans but dispensable for lymphoid differentiation. We suggest that partial deletion in MECOM may be a primary event associated with congenital pancytopenia.

Targeted therapy directed against rare disease-propagating leukaemic stem cells (LSCs) is a promising prospect for improving the outcome of acute myeloid leukaemia (AML) patients. Thus, distinguishing LSCs from normal haematopoietic stem and progenitor cells (HSPCs) is essential. The CLEC12A receptor has been proposed as a specific marker of LSCs, and consequently as an appealing treatment target. To explore the role of CLEC12A in further detail, we investigated whether a sorting strategy based on the activity of aldehyde dehydrogenase and CLEC12A expression could separate residual normal HSPCs from LSCs in bone marrow from 5 AML patients. We demonstrate that this distinction was possible in 2/5 cases, however with evidence of pre-leukaemic mutations in the CLEC12A- stem cells in one case. In contrast, cytogenetic and/or molecular aberrations were detected in both the CLEC12A+/- cell subsets in 3/5 AML cases studied. Furthermore, targeted next generation sequencing (NGS) of the sorted cell subsets revealed a pronounced clonal heterogeneity in the CLEC12A- cells suggestive of the leukaemia often originating in this immature cell subset. In conclusion, we provide proof-of-concept that precision diagnostics employing targeted cytogenetic/NGS-based analyses on highly purified cell subsets could be a powerful tool for selecting patients eligible for LSC-directed therapy.

Introduction: Therapy-related myeloid neoplasms (tMN) are high-risk conditions evolved after exposure to a number of agents, including cytotoxic therapy, and include myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). As such cytoreduction as part of autologous stem cell transplantation (ASCT) increases the risk of developing tMN. Importantly, both the use of ASCT and the incidence of tMN are rising. The recent characterization of clonal hematopoiesis of indeterminate potential (CHIP) has, in preliminary reports, been shown to increase the risk of developing de novo hematological disease as well as tMN. We hypothesized that patients with non-myeloid primary disease who develop tMN after ASCT, had detectable myeloid mutations at time of transplantation, and that these may represent a risk factor in the development of tMN. This study characterizes tMN patients previously subjected to ASCT and investigates whether CHIP mutations are present in hematopoietic stem cells at time of ASCT.

Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL), are generally considered mutually exclusive, but several reports have suggested that they coexist in a small subgroup of patients. In this study, we retrospectively searched for CALR mutations in 136 suspected MPN patients with low allelic burden (< 5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutations were identified, of whom 10 were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR-mutated MPN cases. Type 1 is generally considered the most common mutation, but, interestingly, our double-mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of coexisting JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F. (C) 2018 ISEH Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.