Concurrent Somatic Mutations in Exon 14 of Janus KINASE2 (JAK2) and Exon 10 of Myeloprolifeative Leukemia Virus Oncogene (MPL) in Myeloproliferative Neoplasms (MPN) and Myelodysplastic Syndromes (MDS)/MPN

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5211-5211
Author(s):  
Krishna Gundabolu ◽  
Catalina C Amador ◽  
Allison M Cushman-Vokoun ◽  
Vijaya R. Bhatt ◽  
Lori J. Maness ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Refractory Anemia ◽  
Current Evidence ◽  
Frequent Allele ◽  
Exon 10 ◽  
Mutation Allele ◽  
Mutation Allele Frequency

Abstract INTRODUCTION: JAK2 p.V617F mutations are common in Polycythemia Vera (>90%), Essential Thrombocythemia (50%; ET) and Primary Myelofibrosis (30-50%; PMF). Somatic mutations of codons 515 or 505 in MPL Exon 10 are present in 5% of non-JAK2-mutated ET and 1% of PMF. MPL p.W515L mutation has also been identified in patients with Refractory Anemia with Ring Sideroblasts associated with Thrombocytosis (RARS-T). MPL mutation in Exon 10 codon 505 (S505N) was originally identified as a germ line mutation associated with hereditary thrombocytopenia, but was subsequently reported in ET and PMF. Most current evidence suggests that JAK2 and MPL mutations are mutually exclusive. Pardanani et al had reported 6 cases out of 1182 patients with concurrent JAK2 and MPL mutations, using assays of different sensitivities. In the reported cases the more frequent allele mutated is MPL and less frequent allele is JAK2 as a possible second hit. Here, we report 2 cases with concurrent JAK2 and MPL mutations. METHODS: DNA was extracted and subjected to library preparation using the 50 gene Ion AmpliSeq™ Cancer Hotspot Panel v2, clonal amplification and Ion Sphere™ Particle enrichment on the Ion One Touch™ 2 and One Touch™ ES, and Next Generation Sequencing (NGS) on the Ion Torrent PGM System™. Data was analyzed using Variant Caller Software v.4.0 and NextGENe software v.2.3.4. The original target region BED file for the Ion AmpliSeq™ Cancer Hotspot Panel v2 was modified to include only MPL exon 10 and JAK2 exon 14 amplicons. Assay coverage averaged approximately 3000X. RESULTS: From February 2015, JAK2/MPL NGS was performed on 105 specimens to determine somatic mutations in possible MPN and MDS/MPN cases. 2/105 (1.9%) of the specimens harbored concurrent JAK2 and MPL mutations compared to 6/1182 patients reported previously (0.5%). One patient had RARS-T and one had PMF. Mean age (69 and 74 years) was 71 years, hemoglobin (8.3 and 10.3 gm/dL) 9.3 gm/dL, hematocrit (25 and 26%) 25%, Mean Corpuscular Volume (92 and 92 fL) 92 fL, platelet count (171 and 1320 x 10E3/cmm) 745 x 10E3/cmm, White blood count-WBC (6.2 and 10.3 x 10E3/UL) 8.25 x10E3/cmm, monocytes (3-5%) 4%, Basophils (1 and 2%) 2%, Neutrophils (61 and 66%) 64%, Bands (3and 15%) 9%, Eosinophils (0 and 2%) 1%, peripheral blood blasts (0 and 4%) 2%, bone marrow fibrosis grade of 2 in a grading scale of 1-3. Both were males. Spleen was not palpable at diagnosis in either patient. The case with RARS-T had 4.5% MPL p.W515L mutation allele frequency and 22.7% JAK2 p.V617F mutation allele frequency. The case with PMF had 3.3% MPL p.S505N mutation allele frequency and 2.4% JAK2 p.V617F mutation allele frequency (detected at the follow up 6 months bone marrow in May 2015). They were treated with EPO analogues/Anagrelide and Ruxolitinib respectively. None had thrombosis or bleeding complications. At a mean follow-up of 366 days (113 and 620 day), both patients are alive. CONCLUSION: MPL (codons 505 and 515) and JAK2 p.V617F mutations are not always mutually exclusive. NGS panels with dual gene coverage and high sensitivity identify coexistent mutations that would not be identified by single gene assays. The clinical significance of concurrent mutations remains unclear. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5515-5515
Author(s):  
Yuko Mishima ◽  
Yuji Mishima ◽  
Masahiro Yokoyama ◽  
Noriko Nishimura ◽  
Yoshiharu Kusano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genomic Dna ◽  
Research Funding ◽  
Somatic Mutations ◽  
Clinical Course ◽  
Bone Marrow Aspiration ◽  
Long Term Follow Up

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA>50 ng/mL (72.9%) than the group with cfDNA<50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3702-3702
Author(s):  
Ken Ishiyama ◽  
Chiharu Sugimori ◽  
Hirohito Yamazaki ◽  
Akiyoshi Takami ◽  
Shinji Nakao
Keyword(s):  
Bone Marrow ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Failure ◽  
Bone Marrow Examination ◽  
Refractory Anemia ◽  
Clonal Population ◽  
Dividing Cells ◽  
Humara Assay

Abstract Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.

Mutation Analysis of TET2 Reveals the Clonal Nature of Refractory Anemia with Ring Sideroblasts

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1862-1862 ◽  
Author(s):  
Luca Malcovati ◽  
Angela Brisci ◽  
Anna Gallì ◽  
Francesca Bruno ◽  
Erica Travaglino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mutation Analysis ◽  
X Chromosome ◽  
Somatic Mutations ◽  
Refractory Anemia ◽  
Hematopoietic Stem ◽  
Myeloid Neoplasms ◽  
Mutation Burden ◽  
Ring Sideroblasts ◽  
Clonal Nature

Abstract Abstract 1862 Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated anemia, erythroid dysplasia only, less than 5% blasts and 15% or more ring sideroblasts in the bone marrow (2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues). The natural history of RARS is characterized by an initial phase of erythroid hyperplasia and ineffective erythropoiesis, which is usually stable for many years but in a proportion of patients may be followed by a phase of marrow failure, with or without the later emergence of leukemic blasts. Overall, RARS is a benign condition with a median survival of about 9 years (J Clin Oncol. 2005;23:7594-603). Since the vast majority of these patients have no cytogenetic abnormalities, the clonal nature of RARS has been questioned. However, a few studies of X-chromosome inactivation patterns performed in female patients have suggested that RARS derives from clonal proliferation of a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation. Somatic mutations of TET2 have been recently found in myeloid neoplasms including MDS, where they appear occur early during disease evolution (Nat Genet. 2009;41:838-42), and are currently considered as a reliable clonal marker of these disorders. In this study, we therefore performed a mutation analysis of TET2 in patients with myeloid neoplasms associated with ring sideroblasts. Using direct sequencing, we studied 33 patients with RARS and 28 patients with refractory cytopenia with multilineage dysplasia (RCMD) having 15% or more ring sideroblasts in the bone marrow. Somatic mutations of TET2 were detected in circulating granulocytes from 10 out of 33 (30%) patients with RARS and 10 out of 28 (36%) patients with RCMD and ring sideroblasts. Most of these mutations were novel at the time of this writing. Fourteen patients had a single somatic mutation, and the mutation burden ranged from 10 to 80%. In 9 of these 14 cases, the mutation burden was approximately 50%, consistent with a fully clonal hematopoiesis characterized a single dominant clone that was heterozygous for the mutation. In a female patient with 10% mutant alleles, however, granulocytes carrying mutant TET2 represented only one tenth of clonal granulocytes as determined by X-chromosome inactivation patterns, suggesting the existence of alternative genetic events preceding the TET2 mutation and sustaining clonal dominance. Six patients had multiple somatic mutations of TET2: two mutations in 3 cases, three mutations in 2 cases, and four mutations in the last case. Quantitative evaluation of mutation burden showed concordant values (about 50%) for the multiple mutations in two patients (one with 4 and the other one with 3 somatic mutations of TET2), indicating the existence of a single dominant clone with multiple mutations. In the remaining 4 patients, discordant mutation loads were detected: the dominant mutation was present in about 50% alleles, while the remaining one(s) involved a lower proportion (10-35%) of alleles. These findings are consistent with the initial emergence of a clone of hematopoietic cells carrying a single mutation of TET2 and the subsequent development of subclones that carry additional TET2 mutations and become dominant with time. We also compared gene expression profiles of CD34-positive cells from patients with and without somatic mutations of TET2. While these 2 patient groups both had up-regulation of ALAS2 and down-regulation of ABCB7, distinctive “sideroblastic” features at the molecular level (Blood. 2006;108:337-45), no differentially expressed gene was identified between the 2 groups. These data indicate that somatic mutations of TET2 are unlikely to have a major impact on metabolic pathways at the CD34-positive cell level, and are more consistent with an epigenetic regulation function of TET2. In summary, this study shows that about one third of patients with RARS carry somatic mutations of TET2 in circulating granulocytes, clearly indicating that RARS is a true clonal disorder of hematopoiesis despite it presents as a benign erythroid disorder. In most cases, TET2 mutations appear to cause clonal dominance of hematopoietic stem cells, thus initiating the myelodysplastic process. During the clinical course of the disease subclonal evolution may occur through the acquisition of additional somatic mutations of TET2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow-Derived Cell Therapies to Heal Long-Bone Nonunions: A Systematic Review and Meta-Analysis—Which Is the Best Available Treatment?

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Silvia Palombella ◽  
Silvia Lopa ◽  
Silvia Gianola ◽  
Luigi Zagra ◽  
Matteo Moretti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Healing ◽  
Healing Rate ◽  
Meta Analysis ◽  
Long Bone ◽  
Current Evidence ◽  
Positive Trend ◽  
Case Control Studies ◽  
Cell Therapies

Nonunions represent one of the major indications for clinical settings with stem cell-based therapies. The objective of this research was to systematically assess the current evidence for the efficacy of bone marrow-derived cell-based approaches associated or not with bone scaffolds for the treatment of nonunions. We searched MEDLINE (PubMed) and CENTRAL up to July 2019 for clinical studies focused on the use of cell-based therapies and bone marrow derivatives to treat bone nonunions. Three investigators independently extracted the data and appraised the risk of bias. We analysed 27 studies including a total number of 347 participants exposed to four interventions: bone marrow concentrate (BMAC), BMAC combined with scaffold (BMAC/Scaffold), bone marrow-derived mesenchymal stromal cells (BMSCs), and BMSC combined with scaffold (BMSC/Scaffold). Two controlled studies showed a positive trend in bone healing in favour of BMAC/Scaffold or BMSC/Scaffold treatment against bone autograft, although the difference was not statistically significant (RR 0.11, 95% CI -0.05; 0.28). Among single cohort studies, the highest mean pooled proportion of healing rate was reported for BMAC (77%; 95% CI 63%-89%; 107 cases, n=8) and BMAC/Scaffold treatments with (71%; 95% CI 50%-89%; 117 cases, n=8) at 6 months of follow-up. At 12 months of follow-up, an increasing proportion of bone healing was observed in all the treatment groups, ranging from 81% to 100%. These results indicate that BMAC or BMAC/Scaffold might be considered as the primary choice to treat nonunions with a successful healing rate at a midterm follow-up. Moreover, this meta-analysis highlighted that the presence of a scaffold positively influences the healing rate at a long-term follow-up. More case-control studies are still needed to support the clinical improvement of cell-based therapies against autografts, up to now considered as the gold standard for the treatment of nonunions.

A Survey on Clinical and Biological Characteristic and Therapy Management of an Italian Series of 455 Adult Patients with Systemic Mastocytosis on Behalf of Italian Registry of Mastocytosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3188-3188
Author(s):  
Lisa Pieri ◽  
Patrizia Bonadonna ◽  
Chiara Elena ◽  
Cristina Papayannidis ◽  
Federica Irene Grifoni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Systemic Mastocytosis ◽  
Myeloproliferative Neoplasm ◽  
Large Series ◽  
Refractory Anemia

Abstract Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by proliferation and hyperactivation of clonal mast cells. Clinical manifestations are heterogeneous and encompass cutaneous lesions, gastrointestinal alterations, osteoporosis, anaphylaxis and involvement of bone marrow and other organs due to neoplastic mast cells (MC) infiltration. As consequence, diagnosis may be difficult and patients (pts) are often evaluated by different specialists before the disease is recognized. To date, only few studies (Lim 2009, Escribano 2009, Cohen 2014) described relatively large series of pts with SM. We performed a multicentre retrospective study to evaluate clinical and biological features and therapeutic management in a large series of pts from 10 Italian centres experienced in management of SM and organized in multidisciplinary groups of specialists. We collected 455 pts diagnosed with SM according to WHO criteria. Additionally 26 pts with mastocytosis in the skin (MIS) evaluated with BM examination did not fulfil criteria for SM, leading to diagnosis of Cutaneous Mastocytosis (CM); however 2/26 pts with CM had both cKITD816V mutation and CD2/CD25 expression on MC in BM, additional 3 showed either cKITD816V or CD2/CD25. Moreover, we found 22 pts without MIS but with features of monoclonal mast cell activation syndrome. Of the 455 pts with WHO-SM (male 56%), 252 (55%) had MIS: median age at MIS diagnosis (dg) was 37 years (y) (range 0-79), while at SM dg it was 46.5 (range 18-82). Time from onset of MIS to dg of SM was 9 y (range 0-43). In 18/252 pts (7%) MIS occurred before age of 18 y (median 9, range 0-17) and persisted over childhood. Median age at dg of SM without MIS (203/455 pts, 45%) was older: 54 y, range 19-79 (p<0.0001). First manifestations of SM were MIS in 46.5% of pts, anaphylaxis in 47.8%, mediator related symptoms in 6.4%, osteoporosis/bone lesions in 5.2%, organomegaly in 3.4%, hematologic alterations in 0.4%. Anaphylaxis was observed in 76/252 (30.1%) pts with MIS, of which 23 had tryptase <20 mcg/L. BM multifocal infiltrates of MC were present in 61.6% of pts, while in 38.4% dg was performed only by using minor criteria. cKIT D816V mutation was detected in BM of 394/432 analysed pts (91.2%). Three D816V-negative pts had different cKIT mutations: M541L, D816H and K546K. In peripheral blood cKIT D816V was evaluated in 165 pts and found in 49.7%. CD2 and/or CD25 expressing MC were found in 99% of pts of the 426 evaluated. Mean tryptase value at dg was 75.9±263 mcg/L. SM subtypes were indolent SM (ISM) 402/455 (88.4%), of which 140 isolated bone marrow mastocytosis (BMM) (34.5% of ISM) and 34 smoldering SM (SSM) (8.4% of ISM), aggressive SM (ASM) 32/455 (7%), SM associated with hematological non mast cell disorders (SM-AHNMD) 20/455 (4.4%), mast cell leukemia (MCL) 1/455 (0.2%). In SM-AHNMD the associated hematologic disease was chronic myelomonocytic leukemia (6/20, 30%), non-Hodgkin lymphoma and refractory anemia with ring sideroblasts and thrombocytosis (3/20 each, 15%), essential thrombocythemia and not otherwise characterized myeloproliferative neoplasm (2/20 each, 10%), myelodysplastic syndrome, myelofibrosis, multiple myeloma and acute myeloid leukemia (AML) (1/20 each, 5%). Median follow up was 23 months (mo), range 2-289. At last follow up, 27/455 pts died (5.9%). 52% of pts had ASM, 18.5% SM-AHNMD, 14.7% SSM, 7.4% ISM and 3.7% each MCL and BMM. Causes of death were disease progression in 21/27 pts (77.8%), other solid neoplasms in 3/27 (11.1%), arterial thrombosis in 2/27 (9.5%), cerebral haemorrhage in 1/27 (3.7%). Disease progression consisted in evolution to AML in 6 pts with ASM, 1 pts with SSM and 1 pts with SM-AHNMD; median time to progression to AML was 30 mo (range 13-149); 2 pts developed other AHNMD: chronic myeloid leukemia and myelofibrosis. 178/455 pts (39%) were treated with anti MC mediators therapies. Of the 60/455 (13%) treated with cytostatic therapy 47% had ASM, 12% SM-AHNMD, 2% MCL, 35% ISM of which 38% were SSM. The ISM cohort was treated mainly due to severe osteoporosis with vertebral fractures not C-findings or disease evolution. First line therapy was interferon (28.3%), hydroxyurea (20%), midostaurine (18.3%), imatinib (13.3%), cladribin (8.3%), dasatinib (6.7%) and masitinib (5%). This is one of the largest series reporting pts with SM that may provide useful information for clinical management of pts with this probably underestimated “rare” disease. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

scholarly journals Mutational Profiling of Peripheral Blood and Bone Marrow Samples Discriminates Reactive Monocytosis from Chronic Myelomonocytic Leukaemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3181-3181
Author(s):  
Catherine Cargo ◽  
Matt Cullen ◽  
Jan Taylor ◽  
Mike Short ◽  
Paul Evans ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Patient Group ◽  
Peripheral Blood ◽  
Somatic Mutations ◽  
Bone Marrow Sample ◽  
Myeloid Malignancy ◽  
Myeloid Malignancies ◽  
Paired Samples ◽  
Chronic Myelomonocytic Leukaemia

Abstract Background Chronic myelomonocytic leukaemia (CMML) presents a diagnostic challenge to the haematologist. Distinguishing between a reactive monocytosis and clonal expansion is difficult, and current diagnostic criteria allow for a diagnosis of CMML even in the absence of a clonal marker of disease as long as the monocytosis is persistent. This fails to correctly identify patients with prolonged reactive changes, increasing mis-diagnoses. More recently, large sequencing studies have identified somatic mutations in >90% of patients with CMML, providing potential objective evidence to support a diagnosis. To investigate the utility of high throughput sequencing to discriminate clonal disease and therefore improve diagnosis of CMML, we performed mutational analysis on all samples referred for investigation of a monocytosis to the Haematological Malignancy Diagnostic Service (HMDS) over a 2 year period. The aim of this study was to determine the frequency of mutations in this patient group and whether the presence of mutations can predict disease and outcome. Methods Samples from all patients (initial and follow-up) referred to HMDS with a monocytosis or suspected CMML between July 2014-July 2016 were included in the study. Those with a previous history of a myeloid malignancy diagnosed before July 2014 were excluded. 377 samples from 297 patients were processed and reported according to using current gold standard techniques, with targeted sequencing of 27 recurrently mutated genes in myeloid malignancies performed in parallel. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Detected variants were reported down to a minimum variant allele fraction of 5% and coverage of 100X. Low level variants were confirmed by repeat sequencing and SRSF2 regions were infilled using Sanger sequencing. Data from the literature as well as public online databases (dbSNP, COSMIC, ClinVar) and Alamut Visual were evaluated to annotate likely pathogenic variants. Results Of the164 patients who presented with an initial bone marrow sample, 95 had a confirmed diagnosis of CMML, of which 93 had a demonstrable mutation (98%). The spectrum of mutations in this group reflects that reported in the literature with TET2, SRSF2 and ASXL1 being most frequently mutated. A further 15 patients, all with mutations, were diagnosed with an alternative myeloid malignancy. In those without a confirmed diagnosis by conventional means, a somatic mutation was detected in 62% (39/54) of cases. Importantly, those with a mutation had both phenotypic and genotypic features indistinguishable from the CMML group. In particular CD56 overexpression by immunophenotyping was found almost exclusively in patients with a mutation whether a diagnosis was confirmed or not. To date, a follow-up sample has been received from 7 mutation-positive patients, of which 5 were diagnostic. All 5 cases had identical mutational profiles in the paired samples. In 133 patients, a peripheral blood sample was received as the initial specimen and 71% (94/133) of these harboured a mutation. As yet, 94 patients have not had a subsequent bone marrow biopsy so a conventional diagnosis has not been made. In those with a follow-up bone marrow sample (n=39), the mutational profile between paired samples was found to be highly concordant (98%). In addition, the detection of a mutation in the peripheral blood was strongly predictive of a myeloid malignancy in the bone marrow. Of the 30 patients with a detectable mutation, 29 had a confirmed diagnosis, including 20 patients with CMML, 5 with AML and 4 with other chronic myeloid malignancies. The remaining 9 patients without a mutation showed no morphological evidence of disease in the bone marrow (sensitivity 96.7%; CI 82.8%-99.9%, specificity 100%; CI 66.4%-100%). Conclusion This study has shown that patients investigated for a monocytosis commonly harbour somatic mutations which can be detected in the peripheral blood at a high frequency and with high confidence. The presence of a mutation correlates strongly with a CMML phenotype and detection in the peripheral blood is strongly predictive of a bone marrow diagnosis. Screening of peripheral blood samples using a targeted gene panel provides a highly effective tool to diagnose CMML. Follow-up of this patient group is ongoing and updated results will be available for presentation at the meeting. Disclosures Cargo: Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Molecular and clinical features of refractory anemia with ringed sideroblasts associated with marked thrombocytosis

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3538-3545 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Daniela Pietra ◽  
Emanuela Boveri ◽  
Andrea Pellagatti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Somatic Mutations ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Refractory Anemia ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasm ◽  
Normal Platelet ◽  
Cd34 Cells ◽  
Patient Groups

Abstract We studied patients with myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis. The combination of ringed sideroblasts 15% or greater and platelet count of 450 × 109/L or greater was found in 19 subjects fulfilling the diagnostic criteria for refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T), and in 3 patients with primary myelofibrosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells, but not in T lymphocytes, from 11 of 19 patients with RARS-T. Three patients with RARS, who initially had low to normal platelet counts, progressed to RARS-T, and 2 of them acquired JAK2 (V617F) at this time. In female patients with RARS-T, granulocytes carrying JAK2 (V617F) represented only a fraction of clonal granulocytes as determined by X-chromosome inactivation patterns. RARS and RARS-T patient groups both consistently showed up-regulation of ALAS2 and down-regulation of ABCB7 in CD34+ cells, but several other genes were differentially expressed, including PSIP1 (LEDGF), CXCR4, and CDC2L5. These observations suggest that RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical levels and that it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL, or other as-yet-unknown genes.

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