Clonal Evolution In Myelodysplastic Syndromes Research Articles

P749: PROGNOSTIC SIGNIFICANCE OF SERIAL CIRCULATING TUMOR DNA STATUS POST TREATMENT IN MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA

Background: A major advance in our understanding of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) biology has been the implementation of next generation sequencing (NGS) which influenced diagnostic, prognostic, and therapeutic decisions in myeloid malignancies. Circulating tumor DNA (ctDNA) sequencing, as as a novel and minimally invasive measure, was reported to exhibit excellent correlations with matched bone marrow NGS in MDS and AML. However, the clinical relevance of dynamic ctDNA monitor during active treatment is unknown. Aims: In this study, we assessed the role of ctDNA as a biomarker to monitor therapeutic response and clonal evolution in MDS and AML. Methods: Thirty-one MDS and AML patients who had both bone marrow NGS and ctDNA at baseline were included for concordance analysis. Twenty-seven patients who had at least two serial ctDNA assessment with at least one month interval were included for dynamic ctDNA analysis. Targeted deep sequencing was performed on bone marrow and ctDNA using a customized panel of 165 genes known to be recurrently mutated in MDS and acute myeloid leukemia. Results: Thirty out thirty-one patients were identified with ctDNA mutation at baseline. Diagnostic ctDNA and matched bone marrow DNA exhibited excellent correlations with variant allele frequencies (VAFs) (R2=0.721, p<0.001). For 27 patients who had ctDNA mutation in baseline and treated with HMA or chemotherapy, the average mutation VAFs at the end of treatment was lower in patients achieving a CR/CRi versus those with mCR, mLFS or SD (2.7% vs 24.1%, respectively, p<0.001). As expected, mutation VAFs from all patients with treatment failure did not decrease during treatment. (Figure 1). Similarly, the average decrease in mutation VAFs from pretreatment to end of treatment was greater in patients achieving a CR versus those who did not (87.7% vs 35.5%, respectively, p= 0.001). ctDNA negative post treatment was associated with longer PFS (median PFS not reached (NR) vs. 5.6 (95% CI 4.08-7.13) months; P=0.009)(Fig 2A) and OS (median OS NR vs. 12.0 (95% CI 9.08-15.0) months; P=0.023) (Fig 2B). Seven of ten MDS patients who transformed to AML were identified with lately acquired subclones harboring FLT3 or NF1, mutations involving in signaling pathway. Moreover, new subclones were detected 0.5 to 4 months prior to AML transformation in 4 cases, indicating that genetic progression can predate morphological progression. Image:Summary/Conclusion: ctDNA status post treatment was relevant to clinical response and was of prognostic significance for disease progress and relapse. Dynamic ctDNA changes revealed complex patterns of clonal structure of MDS and AML in response to treatment. ctDNA sequencing could be an attractive, prospective measure for disease monitoring.

Screening for myeloid mutations in patients with myelodysplastic syndromes and AML with myelodysplasia-related changes

IntroductionOne of the most critical complications in myelodysplastic syndromes (MDS) is the progression to acute myeloid leukemia (AML). The dynamics of clonal evolution in MDS and how acquired mutations can be used as biomarkers to track disease progression remains under investigation. Objective and methodHerein, we investigated the frequency of common myeloid clonal mutations (FLT3, NPM1, JAK2, IDH1 and IDH2) in 88 patients with MDS and 35 AML patients with myelodysplasia-related changes, followed at a single reference center in northeastern Brazil. ResultsOverall, 9/88 (10%) of the MDS patients and 9/35 (26%) of the secondary AML patients had at least one mutation. While the JAK2 V617F mutation was the most frequent in the MDS patients, the FLT3, NPM1, IDH1 and IDH2 mutations were more frequently found in the secondary AML group. Furthermore, there was a higher frequency of FLT3, NPM1, IDH1 and IDH2 mutations in MDS patients classified as high-risk subtypes than in those of lower risk. ConclusionDespite the limited sample size, our data suggest that mutations in FLT3, NPM1, IDH1 and IDH2 genes could be potential biomarkers to detect early disease progression in MDS.

Clonal evolution of myelodysplastic syndrome

Clonal evolution of myelodysplastic syndrome

Reprogramming identifies functionally distinct stages of clonal evolution in myelodysplastic syndromes

AbstractMyeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells (HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.

Single-Cell Mutational Profiling Describes the Molecular Heterogeneity of Clonal Evolution in MDS during Therapy and Relapse

Background: Myelodysplastic syndromes (MDS) are a collection of clonal diseases of dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. Increased understanding of the mutational landscape of MDS has led to initial improvements in prognostic models based on clinical and cytogenetic variables. However, bulk sequencing techniques are limited in their ability to delineate clonal complexity and identify rare drug resistant subclones. To better understand clonal heterogeneity and clonal evolution of MDS we applied a high-throughput single cell sequencing technique to both diagnostic and longitudinal MDS samples.Methods: Samples were examined for 5 patients with MDS at diagnosis and, when available, progression. Mutational bulk sequencing was performed by NGS panel sequencing and exon sequencing was available in select cases. Single cell processing was performed using the Tapestri (Mission Bio) platform. Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual MDS samples, genotyping the most clinically relevant loci across upwards of 10,000 individual cells.Results: Single-cell sequencing was able to be performed successfully on all samples tested and recapitulated bulk sequencing data. We observed high concordance between bulk variant allele frequencies (VAFs) and sample level VAFs derived from single cell sequencing data (r2 = 0.98). Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. Single-cell SNVs were able to resolve host and donor cell populations after bone marrow transplant and accurately predict chimerism and disease relapse. Furthermore, we were able to resolve the co-occurance of molecular alterations within subclones and establish zygosity of individual mutations at a single cell level. Rare subclones associated with disease relapse, were able to be identified in initial diagnostic samples that were frequently under the limit of detection of bulk NGS.Conclusions: Our results suggest more molecular complexity in MDS tumor samples than implied from bulk sequencing methods alone and indicates utility of single-cell sequencing for identification of resistant clones and longitudinal therapy monitoring. DisclosuresAleshin:Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Durruthy-Durruthy:Mission Bio, Inc.: Employment, Equity Ownership. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.

Abstract 3004: Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression

Abstract Myelodysplastic syndromes (MDS) are a collection of clonal diseases with dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. MDS is increasingly being recognized as a molecularly heterogeneous disease with variability in clinical phenotype, prognosis, and response to treatment. The clonal evolution in response to therapy remains a challenge in the management of MDS. However, resolution of complex clonal architectures is difficult with current bulk sequencing approaches. High-throughput single-cell genomic profiling enables the resolution of tumor heterogeneity, and may improve clinical diagnosis and treatment monitoring by allowing for characterization and early identification of resistant subclonal populations. To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cells confined to droplets. The barcodes were then used to reassemble the genetic profiles of cells from next-generation sequencing data. We applied this approach to sequential clinical MDS samples, genotyping the most clinically relevant loci across more than 15,000 individual cells. Additionally, to study effects of subclonal mutations on drug sensitivity, ex vivo functional testing was performed on red blood cell-lysed peripheral blood and/or bone marrow aspirate patient samples. Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. Furthermore, the single-cell nature of our approach enabled definitive determination of mutational co-occurrence within the same cell. For examples in one sample, bulk sequencing identified mutations in JAK2 and NRAS both at 3% variant allele frequency (VAF), where single-cell analysis suggested that these mutations were mutually exclusive, each defining a distinct subclone. Single-cell sequencing allowed for serial monitoring of clonal evolution, with analysis of sequential samples from this same patient showing increase of NRAS clone from 2.5% to 24.7% at time of disease progression after hypomethylating agent therapy. Furthermore, single-cell analysis was able to identify a distinct subclone characterized by a KRAS mutation (0.4% at initiation of therapy and 6.7% at relapse), missed by serial bulk sequencing. Taken together, our results suggest a greater degree of heterogeneity in MDS samples than suggested with bulk sequencing methods alone, and demonstrate utility of single-cell sequencing for sequential monitoring and identification of resistant clones prior to therapy initiation. We show here that this approach is a feasible, scalable, and effective way to identify and track heterogeneous populations of cells in MDS. Citation Format: Alexey Aleshin, Peter L. Greenberg, Bruno C. Medeiros, Diane Heiser, Marianne Santaguida, Sacha Prashad, Robert Durruthy- Durruthy, Dennis J. Eastburn. Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3004.

Cytogenetic clonal evolution in myelodysplastic syndromes is associated with inferior prognosis.

The karyotype of bone marrow cells at the time of diagnosis is a strong prognostic parameter for overall survival as well as acute myeloid leukemia (AML) progression in patients with myelodysplastic syndromes (MDS). However, to the authors' knowledge, few data exist regarding the prognostic impact of cytogenetic clonal evolution during the course of MDS. The authors evaluated follow-up karyotype analyses in 549 patients from the Dusseldorf MDS Registry. Clonal evolution was detectable in 24% of the entire cohort and in 18% of 294 patients receiving best supportive care. The authors noted a clear adverse effect of clonal evolution on the risk of leukemic transformation (hazard ratio, 2.233; P = .036) and overall survival (hazard ratio, 3.677; P<.001). The authors also analyzed the prognostic influence of subclones detectable at the time of diagnosis. Again, such a finding was associated with a significantly shorter overall survival and a higher 5-year-probability of acute myeloid leukemia progression (30% vs 22%). The results of the current study support the belief that follow-up karyotype analyses should be performed, especially in patients with lower-risk and intermediate-risk MDS, to identify those patients who are at higher risk of disease progression and therefore might benefit from earlier or more intensive treatment. Cancer 2017;123:4608-4616. © 2017 American Cancer Society.

90 - Cytogenetic Clonal Evolution in Myelodysplastic Syndromes (MDS) with Isolated Del(5Q)

Evaluation of megakaryocyte morphology is difficult but can be essential for the diagnosis of myelodysplastic syndromes (MDS) and other myeloid neoplasms. We agreed upon descriptions and provided images of megakaryoblasts and of normal and dysplastic megakaryocytes, which were used as a basis for assessing the concordance of expert morphologists in their recognition. We showed a high rate of concordance for the recognition of micromegakaryocytes and confirmed their strong association with hematologic neoplasia, including MDS. Concordance was also found to be good for the recognition of multinucleated megakaryocytes, which showed a significant association with MDS. However cytoplasmic abnormalities were found not to be useful in MDS recognition. The occurrence of appreciable numbers of nonlobulated and hypolobulated megakaryocytes in individuals without a myeloid neoplasm was confirmed. We demonstrated that subjects without a myeloid neoplasm can have some megakaryocytes that are assessed as ‘dysplastic’ or ‘possibly dysplastic’ and that to avoid over diagnosis of dysplasia, ‘possibly dysplastic’ forms should be excluded from the count of dysplastic cells. Our results demonstrate that the nature as well as the presence of megakaryocyte dysplasia is important in the diagnosis of MDS; although evaluation of 30 megakaryocytes is strongly recommended, it may be possible to recognize diagnostically important dysplasia when fewer megakaryocytes are present but highly diagnostic forms are seen.

Clonal evolution in myelodysplastic syndromes

Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5–11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions.

Dynamics of clonal evolution in myelodysplastic syndromes.

To elucidate differential roles of mutations in myelodysplastic syndromes (MDS), we investigated clonal dynamics using whole-exome and/or targeted sequencing of 699 patients, of whom 122 were analyzed longitudinally. Including the results from previous reports, we assessed a total of 2,250 patients for mutational enrichment patterns. During progression, the number of mutations, their diversity and clone sizes increased, with alterations frequently present in dominant clones with or without their sweeping previous clones. Enriched in secondary acute myeloid leukemia (sAML; in comparison to high-risk MDS), FLT3, PTPN11, WT1, IDH1, NPM1, IDH2 and NRAS mutations (type 1) tended to be newly acquired, and were associated with faster sAML progression and a shorter overall survival time. Significantly enriched in high-risk MDS (in comparison to low-risk MDS), TP53, GATA2, KRAS, RUNX1, STAG2, ASXL1, ZRSR2 and TET2 mutations (type 2) had a weaker impact on sAML progression and overall survival than type-1 mutations. The distinct roles of type-1 and type-2 mutations suggest their potential utility in disease monitoring.

Ips Technology Revealed the Genetic and Functional Diversity Present in a Secondary AML Patient

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and risk of progression to acute myeloid leukemia with poor prognosis. Although massive parallel sequencing studies have revealed a number of genomic alterations associated with myelodysplastic syndromes (MDS), the relationships between genetic architecture and clonal evolution in MDS remain poorly understood, mainly due to a difficulty in the ex vivo culture of primary MDS cells and a lack of good animal models. Induced pluripotent stem cells (iPSCs) from MDS patients are expected to provide a new platform for elucidation of the pathogenesis of MDS. We used reprogramming technology and transplantation into immunodeficient mice to functionally dissect the genetically defined subclones of secondary acute myeloid leukemia (AML) evolving from MDS (MDS/sAML).We attempted to generate iPSCs from bone marrow and peripheral blood mononuclear cells of a MDS/sAML patient with normal karyotype and multiple FLT3-internal tandem duplication (ITD) mutations, utilizing episomal methods. We successfully established more than 30 iPSC lines from sAML clones (sAML-iPSC) and 4 normal iPSC lines from normal clones at the same time. sAML-iPSC lines displayed characteristic morphology and expressed pluripotent stem cell markers at the levels comparable to those in isogenic normal iPSC lines and ES cell lines. Five randomly selected sAML-iPSC lines formed teratomas. Most of the sAML-iPSC lines retained normal karyotype even after 30 passages. About half of the sAML-iPSC lines harbored one out of four types of FLT3-ITDs identical to the primary sAML cells, reflecting the mosaicism of the FLT3-ITDs in the patient's sAML cells. Whole exome sequencing and SNP-CGH analysis revealed that all sAML-iPSC lines shared the chromosome 11p uniparental disomy (UPD) and 14 somatic single nucleotide variants (SNVs) identical to those of the primary sAML cells, indicating that these iPSC lines are derived from clonal sAML cells. Additionally, most of the sAML-iPSC lines had a frameshift mutation of CEBPA. Unexpectedly, 2 of the sAML-iPSC lines without any FLT3-ITD mutations had a FLT3-D835Y mutation, which was only detected by droplet digital PCR (ddPCR) at extremely low levels in the primary bone marrow sample.Hematopoietic differentiation efficiency of sAML-iPSC lines was comparable to that of isogenic normal iPSC lines. However, CD34+CD38-CD45+lineage- hematopoietic stem and progenitor cells (HSPCs) from sAML-iPSC lines displayed two to three times enhanced colony formation ability in myeloid-conditioning semifluid medium. Colonies formed by sAML-iPSC-derived HSPCs were predominantly composed of immature myeloblasts and could be replated for at least five rounds. Both FLT3-ITD- and FLT3-D835Y-positive sAML-iPSC-derived hematopoietic progenitor cells (HPCs) exhibited cytokine-independent growth but only FLT3-ITD-positive sAML cell lines retained in vitro sensitivity to AC220. To functionally evaluate the subclonal diversity in vivo, we performed xenotransplantation of HPCs from several sAML-iPSC lines into sublethally irradiated NSG mice. HPCs from all tested sAML-iPSC lines engrafted at 13 to 15 weeks after transplant. HPCs from FLT3-ITD-positive sAML-iPSC lines (n=5) displayed a higher ability to engraft and induced more severe sAML-like disease in recipient mice than those from FLT3-wild-type (wt) sAML-iPSC lines (n=4) (median spleen weight, 145.4 mg vs 55.7 mg; P < .05). Survival of mice injected with FLT3-ITD-positive sAML-iPSC-derived HPCs was shorter than those injected with FLT3-wt sAML-iPSC-derived HPCs (194 days vs 361 days; P< .001). To confirm the transformation properties of the FLT3-ITDs in this MDS/sAML patient, we transduced FLT3-wt sAML-iPSC-derived HPCs with retrovirus expressing a FLT3-ITD and transplanted the HPCs into recipient mice. FLT3-ITD-transduced recipients developed an aggressive sAML-like disease in 100% of recipient mice with shorter latency around 6 to 10 weeks, as evidenced by marked splenomegaly and massive expansion of immature myeloid cells in bone marrow and spleen.Our data suggest that this iPSC-based system could be useful for analysing clonal architecture and biological feature of subclones of MDS/sAML to identify candidate genes responsible for initiation and progression of MDS. DisclosuresTakaori-Kondo:Shionogi: Research Funding; Toyama Chemical: Research Funding; Mochida Pharmaceutical: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Alexion Pharmaceuticals: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Yamanaka:iPS Academia Japan: Consultancy.

Frequency and Clinical Impact of Cytogenetic Clonal Evolution in Myelodysplastic Syndromes (MDS) with Isolated Del(5q)

▪Introduction: Inmyelodysplastic syndromes (MDS) the karyotype is one of the most important predictor of disease advancement, response to the treatment and patients' outcome. Clonal cytogenetic abnormalities are detected in the bone marrow cells in approximately 50% MDS patients. The interstitial deletion of the long arm of chromosome 5-del(5q)-is the most common finding, accounting for roughly 30% of abnormal karyotypes. According to IPSS-R, MDS with isolated del(5q) are associated with a favorable clinical course and are recognized as a specific subtype of MDS. However in some cases, acquisition of additional genetic aberrations may occur during the course of the disease and it has been proved it negatively influences outcome of MDS patients. The aim of the study was: (1) to evaluate the frequency of cytogenetic clonal evolution during the course of the disease in MDS patients with isolated del(5q), (2) to analyze the pattern of acquired cytogenetic abnormalities, and (3) to assess the impact of clonal evolution on transformation to acute myeloid leukemia (AML) and/or overall survival (OS).Patients and Methods: A detailed retrospective and/or prospective genome-wide analysis of fixed bone-marrow cells of 184 adults with del(5q), identified with conventional G-banding at the diagnosis of MDS, was performed during the course of their disease. The chromosomal aberrations were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods using the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with CytoChip Cancer SNP 180K (BlueGnome, Cambridge, UK) or with Illumina Human CytoSNP-12 arrays (Illumina, San Diego, CA). Amplicon deep sequencing of TP53 mutations (exons 4-11) was performed on a Roche 454 GS Junior system (Roche, Indianapolis, IN) using oligonucleotide primer plate assays validated according to the IRON-II (Interlaboratory Robustness Of Next generation sequencing).Results: Cytogenetic clonal evolution was observed in 21/184 MDS patients with isolated del(5q) (11.4%). Abnormalities most frequently acquired during the course of the disease were non-balanced rearrangements involving chromosomes 5, 7, 3, 17, 12 and 11. Deleted chromosome 5 was highly unstable and was often involved in different types of cryptic unbalanced rearrangements (translocations, insertions). Fragmentation of 5q was present as well.Clonal evolution was frequently associated with TP53 mutations and/or unbalanced aberrations of 17p.The median time between the first and the last evaluation was 23 months (range 2-83 months). Clonal evolution was detected between 2 and 83 months (median 14 months) after first cytogenetic evaluation. Median OS was 35 months (range 6-89 months), median survival from the first emergence of cytogenetic clonal evolution was 7.5 months (range 1-31 months). Progression to RAEB 1 or RAEB 2 was detected in 10 patients, 11 patients transformed into AML.Conclusions: Clonal evolution was detected in 11.4% of patients with MDS and isolated del(5q). The pattern of acquired abnormalities was similar to the changes previously described in high risk MDS at primary evaluation. The emergence of specific clones with additional genetic aberrations frequently correlated with altered clinical parameters. Cytogenetic clonal evolution was strongly associated with higher frequency of loss of heterozygosity (LOH) of 17p and/or TP53 mutations, shorter OS, rapid disease progression and transformation to AML. This data substantiate a need for regular molecular cytogenetic monitoring to guide clinical treatment decision in MDS with isolated del(5q).This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, PRVOUK-P27/LF1/1, and MHCR 00023736. DisclosuresNo relevant conflicts of interest to declare.

Clonal evolution in myelodysplastic syndromes.

During the past ten years, genome-wide analysis of genetic alterations in myelodysplastic syndromes (MDS) has improved our understanding of their pathogenesis. Especially, single nucleotide polymorphism array karyotyping and next-generation sequencing technologies (NGS) have unveiled frequent genetic changes in novel functional pathways, including DNA methylation, RNA splicing and cohesin complex formation, in MDS. Moreover, NGS shed light on the clonal evolution which occurs during the development and progression of MDS, pre-cancerous lesions of MDS, and the effects of germline mutations in MDS.

Myeloid Cell Maturation Is Disrupted By Monosomy 7 or By Gain of Additional Genetic Aberrations during Clonal Evolution in Myelodysplastic Syndromes.

Background: Myelodysplastic syndromes (MDS) are associated with cytogenetic clones. To follow the maturation sequence of original clones and evolved subclones with additional cytogenetic abnormalities, progenitor cells, immature and mature myeloid cells were sorted by flow cytometry and analyzed separately by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).Methods: Flow cytometry sorted cell fractions from bone marrows for sixteen patients with MDS-associated cytogenetic abnormalities were evaluated by FISH. Flow cytometric cell sorting was based on CD34+/low side scatter (SS) for progenitors, CD13+/CD16-/high SS for immature myeloid cells, and CD13+/CD16+/high SS for mature myeloid cells. Customized color labeling (spectrum orange, green and aqua) of FISH probe combinations were designed to detect and to analyze clonal evolution for each patient based on their known cytogenetic abnormalities and clonal evolution patterns. Three marrow aspirates were sorted for analysis by SNP/CGH microarray.Results: The 16 bone marrow specimens evaluated by FISH were categorized into three groups: (1) eight patients with a single, good-to-intermediate cytogenetic abnormality; (2) four patients with monosomy 7; and (3) four patients with more than one chromosome abnormality with evidence of clonal evolution by conventional cytogenetic analysis, excluding monosomy 7.The Group-1 abnormalities included deletion 20q (n=4), trisomy 8 (n=2), deletion 5q (n=1), and trisomy 11 (n=1). All specimens from this group showed FISH abnormalities in equal proportions in myeloid progenitors, immature and mature myeloid cells.Group-2 had four patients with monosomy 7. All four had monosomy 7 concentrated in the progenitor cells (45-79%) compared to immature and mature myeloid compartments (less than 9-36%).For Group-3, known original clones with single cytogenetic abnormalities (deletion 20q, monosomy 3, deletion 7q or 5q) were monitored by FISH analysis. Using customized FISH panels, the presence of subclones with additional cytogenetic aberrations (trisomy 8 in three patients and gain of a marker chromosome characterized by the centromere of chromosome 4 in a fourth patient) was assessed using single-cell resolution. The progenitor and immature myeloid compartments had the original founding clones containing a single cytogenetic abnormality at 15-34% and the subclones with the additional aberrations at 23-76%. In contrast, the mature myeloid cells were comprised of the original clone at 20-40%, but the subclones with additional aberrations were absent in the mature myeloid compartment in three patients and reduced by more than half in a fourth.For three patients sorted bone-marrow fractions were analyzed by SNP/CGH microarray. In one patient with trisomy 8 as the sole abnormality, the same aberration was observed in both the immature and mature myeloid compartments. In two other patients, additional abnormalities not seen in the mature myeloid cells were detected in the progenitor and immature myeloid compartments.Conclusions: These data show two main patterns for the distribution of clonal cytogenetic abnormalities among myeloid cells in MDS: 1) A continuous distribution at all stages of maturation was found for single aberrations with good-to-intermediate prognostic associations (Group 1) and 2) A disrupted distribution pattern with accumulation of the cytogenetic aberrations in the progenitor compartment as compared to the immature and mature myeloid compartments for specimens characterized by monosomy 7 (Group 2). Similarly, subclones characterized by additional cytogenetic abnormalities (Group 3) were sequestered in the progenitor and immature myeloid compartments while the original founding clone was evenly distributed throughout maturation.These data demonstrate that specific cytogenetic abnormalities associated with poor prognosis (e.g. monosomy 7) as well as acquired cytogenetic abnormalities as a result of clonal evolution can cause disruption of myeloid cell maturation in MDS. DisclosuresZehentner:HematoLogics Inc.: Employment, Equity Ownership. Hartmann:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Chapman:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:Hematologics: Employment, Equity Ownership.

Landscape Of Genetic Lesions In 944 Patients With Myelodysplastic Syndromes

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. MethodsA total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). ResultsAfter excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p<0.01). Only 6 genes (TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1) were mutated in >10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4.Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P<0.05), and only SF3B1mutations were associated with a significantly better clinical outcome. Next, to evaluate the combined effect of these multiple gene mutations/deletions, together with common clinical/cytogenetic variables used for IPSS-R, OS was modeled by a conventional Cox regression.A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P<0.001). These results demonstrated that the mutation/deletion status of a set of genes could be used as variables independent of clinical parameters to build a clinically relevant prognostic score. When applied to the validation cohort, the novel molecular model was even shown to outperform the IPSS-R. ConclusionsLarge-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abrupt evolution of Philadelphia chromosome‐positive acute myeloid leukemia in myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a clonal disorder arising from an alteration in multipotent stem cells, which lose the ability of normal proliferation and differentiation. Disease progression occurs in approximately 30% MDS cases. Specific chromosomal alterations seem responsible for each step in the evolution of acute myeloid leukemia (AML). Multiple genetic aberrations occur during the clonal evolution of MDS; however, few studies report the presence of the Philadelphia (Ph) chromosome. We report a rare case of Ph-positive AML, which evolved during the course of low-risk MDS. The patient, a 76-year-old man with mild leukocytopenia, was diagnosed with MDS, refractory neutropenia (RN). After 1.5 yr, his peripheral blood and bone marrow were suddenly occupied by immature basophils and myeloblasts, indicating the onset of AML. A bone marrow smear showed multilineage dysplasia, consistent with MDS evolution. Chromosomal analysis showed an additional t(9;22)(q34;q11) translocation. Because progression occurred concurrently with emergence of the Ph chromosome, we diagnosed this case as Ph-positive AML with basophilia arising from the clonal evolution of MDS. The patient was initially treated with nilotinib. A hematological response was soon achieved with disappearance of the Ph chromosome in the bone marrow. Emergence of Ph-positive AML in the course of low-risk MDS has rarely been reported. We report this case as a rare clinical course of MDS.

Immune alterations in untreated and treated myelodysplastic syndrome

Introduction: Current literature suggests a variety of immune abnormalities are present in myelodysplastic syndrome (MDS) patients. However, they have not been comprehensively characterized or systematized to date. As a result, their clinical implications remain largely unknown. In addition, an increased incidence of various autoimmune conditions has been documented in MDS patients.Areas covered: The authors offer a comprehensive review of the existing literature on immune mechanisms involved in the pathogenesis of MDS, various immune abnormalities documented in its course, their link with the clonal evolution of MDS as well as immune alterations induced by the existing MDS therapies.Expert opinion: The course of MDS is accompanied by complex immune alterations, including T-cell and NK-cell defects, decreased functional abilities of neutrophils and antigen-presenting cells, altered antibody and cytokine production. While contemporary MDS therapies are shown to possess some immunomodulatory properties, authentic autoimmune conditions have also been documented with lenalidomide and recombinant erythropoietin. Caution should be exerted with the use of these agents in MDS patients with evidence of autoimmune disorders, as exacerbation of autoimmune phenomena can be anticipated. While the heterogeneity of MDS represents an inherent limitation, integration of emerging information from molecular biology, genetics, immunology research and clinical practice could translate into improved outcomes of this disease spectrum.

Clonal evolution in myelodysplastic syndromes with isolated del(5q): the importance of genetic monitoring

Myelodysplastic syndromes (MDS) with isolated deletion of chromosome arm 5q (del(5q)) have a good prognosis with low risk of transformation to acute myeloid leukemia (AML). Laboratory investigations typically demonstrate macrocytic anemia with preserved or increased platelet counts, and