Accepted Manuscript Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies Emiliano Fabiani, Giulia Falconi, Luana Fianchi, Francesco Guidi, Silvia Bellesi, Maria Teresa Voso, Giuseppe Leone, Francesco D’Alò PII:
S0301-472X(14)00156-8
DOI:
10.1016/j.exphem.2014.04.011
Reference:
EXPHEM 3129
To appear in:
Experimental Hematology
Received Date: 26 March 2014 Revised Date:
24 April 2014
Accepted Date: 28 April 2014
Please cite this article as: Fabiani E, Falconi G, Fianchi L, Guidi F, Bellesi S, Voso MT, Leone G, D’Alò F, Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies, Experimental Hematology (2014), doi: 10.1016/j.exphem.2014.04.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Letter to the Editor
Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies Emiliano Fabiani, Giulia Falconi, Luana Fianchi, Francesco Guidi, Silvia Bellesi, Maria Teresa Voso, Giuseppe Leone and Francesco D’Alò
M AN U
E.F. and G.F. equally contributed to this study.
SC
Institute of Hematology, Università Cattolica del Sacro Cuore, Rome, Italy
Contact Information for correspondence: Francesco D’Alò, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome, Italy, email [email protected], phone +390630154180, fax +390635503777
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Microenvironment is actively involved of the in the pathophysiology of hematopoietic malignancies. Mesenchymal stromal cells (MSC) support hematopoiesis through the production and secretion of cytokines, cell-cell interactions and immunomodulating properties. These cells are defined plastic-adherent growing cells, are known to express CD105, CD73 and CD90, lack common hematopoietic antigens and differentiate to osteoblasts, adipocytes and chondroblasts in vitro [1]. Anomalies of multiple MSC features have been described in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). These include significantly reduced growth, proliferative and differentiating capacities, premature replicative senescence, abnormal expression of surface
1
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molecules and chemokines, and reduced ability to support hematopoietic stem and progenitor cell (HSPC) growth in long-term culture assays [2].
RI PT
The molecular basis of MSC dysfunction in MDS and AML are still under investigation. Previous studies have shown the occurrence of non-clonal chromosomal aberrations in bone marrow MSC isolated from patients with MDS and AML, which only very rarely correspond to the cytogenetic markers observed in the hematopoietic leukemic clone of the same individual [3].
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Somatic mutations of multiple genes have been described in myeloid malignancies and often concur to identify distinct leukemia subtypes or prognostic subgroups. Recently, deep sequencing approaches have identified new recurrent mutations of genes involved in epigenetic and spliceosome machineries in AML and MDS samples [4-6]. We investigated the frequency of recurrent mutations of epigenetic and spliceosomal genes, of FLT3 and NPM1 genes in matched bone marrow hematopoietic cells and MSC isolated from 41 patients with myeloid malignancies. The study population included 9 de novo AML, 9 MDS, 7 chronic myeloproliferatve neoplasms (MPN), 3 secondary AML (sAML, 2 evolved from MDS, 1 from MPN), and 13 therapy-related myeloid neoplasm (7 t-AML and 6 t-MDS).
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Bone marrow mononuclear cells (BM-MNC) were isolated at the time of diagnosis by Ficoll gradient centrifugation. MSCs were expanded using Mesencult medium (Stem Cell Technologies, Voden Medical Instruments spa, Milan, Italy) in plastic-adherent cultures up to the second passage. Flow cytometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive) in more than 99% of MSC population. DNA was extracted from BM-MNC and MSC using QIAamp DNA Mini Kit (Qiagen srl, Milan, Italy). The following hotspot mutations were studied on genomic DNA by Sanger sequencing (ABI PRISM 3100; Applied Biosystems/Life technologies, Milan, Italy): IDH1 R132, IDH2 R140 and R172, DNMT3A R882, U2AF1 S34 and R35, SF3B1 exons 13–14 and 15–16, and SRSF2 exon 1 [7]. In addition, FLT3
2
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and NPM1 mutations were analyzed in patients with de novo or therapyrelated AML. FLT3 Internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations were studied by RT-PCR and RFLP RT-PCR, respectively, while NPM1 exon 12 mutations were detected by RT-PCR with high resolution melting curve analysis (HRMCA), followed by Sanger sequencing of positive cases.
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In BM-MNC, FLT3 ITD and FLT3 TKD mutations were found in two patient (one patient with de novo AML and one patient with a t-AML respectively), while NPM1 exon 12 mutations were present in six AML patients (4 de novo, 3 COSM158604 and 1 COSM1319219; 1 sAML, COSM158604; 1 tAML, COSM28937). No FLT3 and NPM1 mutations were found in MSC compartment in all studied patients. IDH1 R132 mutations were found in BM-MNC isolated from one patient with de novo AML (R132C) and in two t-AML patients (one R132H and one R132L). One t-AML patient presented IDH2 R140Q mutation in BMMNC, while no mutations were detected at the codon R172. Neither IDH1 nor IDH2 mutations occurred in matched MSC.
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DNMT3A R882 mutations were found in BM-MNC from one de novo AML (R882H), one t-AML (R882H) and one sAML evolved from Polycythemia Vera (R882C), but not in corresponding MSC.
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One U2AF1 S34Y mutation was found in BM-MNC isolated from one patient with intermediate-risk MDS, while a SF3B1 K666N mutation was detected in BM-MNC from one patient with AML following a primary MDS. SRFS2 P95 mutations were found in BM-MNC isolated from two patients with de novo AML (P95L and P95R), three patients with MDS (P95L, P95H and P95 frameshift mutation) and one patient with MPN (P95H). Again, U2AF1, SF3B1 and SRFS2 mutation were absent in MSC. All described mutations described were heterozygous. Results of mutational analysis in hematopoietic BM-MNC are reported in Figure 1.
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We found no mutations for any of the studied genes in the MSC compartment, both in carriers of mutations in the hematopoietic compartment and in wild-type patients.
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The absence of NPM1 and FLT3 mutations in MSC from AML patients has been previously reported [3]. So far, the prevalence of somatic mutations of epigenetic and spliceosomal genes in MSC compartment in patients with myeloid malignancies is not known. Mutations of DNMT3A and IDH1/2 have been described in 22% and 16% of AML patients and in 8% and 12% of MDS patients, respectively [4,8-9]. These mutations have been associated to deregulated DNA methylation and poor prognosis. Abnormal DNA methylation has been advocated as responsible of the premature senescence and reduced growth of MSC in MDS. Specific patterns of DNA methylation in MSC have been reported in MDS subtypes compared to healthy donors [2]. According to our results, abnormal methylation profiles described in MSC are unlikely to be related to mutations of epigenetic regulatory genes, even though a wider mutational analysis, including also ASXL1 and TET2 genes, could be more informative.
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Abnormal splicing patterns of multiple genes have been described in myeloid neoplasms due to mutations of spliceosome machinery genes [10]. This phenomenon is considered to play a significant role in myeloid leukemogenesis due to selective missplicing of tumor-associated genes. The contribution of missplicing to MSC dysfunction in myeloid neoplasms is still a matter of investigation. The absence of recurrent spliceosome gene mutations in MSC contrasts with the hypothesis that these mutations may play a significant role.
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We conclude that common mutations of genes involved in epigenetic regulation and spliceosome machinery are absent in the mesenchymal compartment of leukemic bone marrows and are restricted only to the malignant myeloid clone, in agreement with the distinct origin from hematopoietic cells.
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Acknowledgments: This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.)
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References
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1. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315-7.
SC
2. Geyh S, Oz S, Cadeddu RP, et al. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells. Leukemia. 2013;27:1841-51.
M AN U
3. Blau O, Baldus CD, Hofmann WK, et al. Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts. Blood. 2011;118:5583-92. 4. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424-33.
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5. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361:1058-66. 6. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478:64-9.
EP
7. Voso MT, Fabiani E, Fianchi L, et al. Mutations of epigenetic regulators and of the spliceosome machinery in therapy-related myeloid neoplasms and in acute leukemias evolved from chronic myeloproliferative diseases. Leukemia. 2013;27:982-5.
AC C
8. Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia. 2011;25:1153-8.
6
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9. Patnaik MM, Hanson CA, Hodnefield JM, et al. Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients. Leukemia. 2012;26:101-5.
AC C
EP
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M AN U
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10. Przychodzen B, Jerez A, Guinta K, et al. Patterns of missplicing due to somatic U2AF1 mutations in myeloid neoplasms. Blood. 2013;122:9991006.
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Figure 1: Distribution of IDH1, IDH2, DNMT3A, U2AF1, SF3B1, SRSF2,
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NPM1 and FLT3 mutations in the hematopoietic compartment of the study population. No mutations were found in mesenchymal stem cell
compartment. Each column represents a single patient. Black boxes represent mutation. FLT3 and NPM1 mutational analysis was not
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performed in MDS, MPN and t-MDS patients (white boxes).
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ACCEPTED MANUSCRIPT
S0301-472X(14)00156-8
DOI:
10.1016/j.exphem.2014.04.011
Reference:
EXPHEM 3129
To appear in:
Experimental Hematology
Received Date: 26 March 2014 Revised Date:
24 April 2014
Accepted Date: 28 April 2014
Please cite this article as: Fabiani E, Falconi G, Fianchi L, Guidi F, Bellesi S, Voso MT, Leone G, D’Alò F, Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies, Experimental Hematology (2014), doi: 10.1016/j.exphem.2014.04.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
RI PT
Letter to the Editor
Mutational analysis of bone marrow mesenchymal stromal cells in myeloid malignancies Emiliano Fabiani, Giulia Falconi, Luana Fianchi, Francesco Guidi, Silvia Bellesi, Maria Teresa Voso, Giuseppe Leone and Francesco D’Alò
M AN U
E.F. and G.F. equally contributed to this study.
SC
Institute of Hematology, Università Cattolica del Sacro Cuore, Rome, Italy
Contact Information for correspondence: Francesco D’Alò, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome, Italy, email [email protected], phone +390630154180, fax +390635503777
TE D
Word count: 1000
AC C
EP
Microenvironment is actively involved of the in the pathophysiology of hematopoietic malignancies. Mesenchymal stromal cells (MSC) support hematopoiesis through the production and secretion of cytokines, cell-cell interactions and immunomodulating properties. These cells are defined plastic-adherent growing cells, are known to express CD105, CD73 and CD90, lack common hematopoietic antigens and differentiate to osteoblasts, adipocytes and chondroblasts in vitro [1]. Anomalies of multiple MSC features have been described in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). These include significantly reduced growth, proliferative and differentiating capacities, premature replicative senescence, abnormal expression of surface
1
ACCEPTED MANUSCRIPT
molecules and chemokines, and reduced ability to support hematopoietic stem and progenitor cell (HSPC) growth in long-term culture assays [2].
RI PT
The molecular basis of MSC dysfunction in MDS and AML are still under investigation. Previous studies have shown the occurrence of non-clonal chromosomal aberrations in bone marrow MSC isolated from patients with MDS and AML, which only very rarely correspond to the cytogenetic markers observed in the hematopoietic leukemic clone of the same individual [3].
TE D
M AN U
SC
Somatic mutations of multiple genes have been described in myeloid malignancies and often concur to identify distinct leukemia subtypes or prognostic subgroups. Recently, deep sequencing approaches have identified new recurrent mutations of genes involved in epigenetic and spliceosome machineries in AML and MDS samples [4-6]. We investigated the frequency of recurrent mutations of epigenetic and spliceosomal genes, of FLT3 and NPM1 genes in matched bone marrow hematopoietic cells and MSC isolated from 41 patients with myeloid malignancies. The study population included 9 de novo AML, 9 MDS, 7 chronic myeloproliferatve neoplasms (MPN), 3 secondary AML (sAML, 2 evolved from MDS, 1 from MPN), and 13 therapy-related myeloid neoplasm (7 t-AML and 6 t-MDS).
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EP
Bone marrow mononuclear cells (BM-MNC) were isolated at the time of diagnosis by Ficoll gradient centrifugation. MSCs were expanded using Mesencult medium (Stem Cell Technologies, Voden Medical Instruments spa, Milan, Italy) in plastic-adherent cultures up to the second passage. Flow cytometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive) in more than 99% of MSC population. DNA was extracted from BM-MNC and MSC using QIAamp DNA Mini Kit (Qiagen srl, Milan, Italy). The following hotspot mutations were studied on genomic DNA by Sanger sequencing (ABI PRISM 3100; Applied Biosystems/Life technologies, Milan, Italy): IDH1 R132, IDH2 R140 and R172, DNMT3A R882, U2AF1 S34 and R35, SF3B1 exons 13–14 and 15–16, and SRSF2 exon 1 [7]. In addition, FLT3
2
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RI PT
and NPM1 mutations were analyzed in patients with de novo or therapyrelated AML. FLT3 Internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations were studied by RT-PCR and RFLP RT-PCR, respectively, while NPM1 exon 12 mutations were detected by RT-PCR with high resolution melting curve analysis (HRMCA), followed by Sanger sequencing of positive cases.
M AN U
SC
In BM-MNC, FLT3 ITD and FLT3 TKD mutations were found in two patient (one patient with de novo AML and one patient with a t-AML respectively), while NPM1 exon 12 mutations were present in six AML patients (4 de novo, 3 COSM158604 and 1 COSM1319219; 1 sAML, COSM158604; 1 tAML, COSM28937). No FLT3 and NPM1 mutations were found in MSC compartment in all studied patients. IDH1 R132 mutations were found in BM-MNC isolated from one patient with de novo AML (R132C) and in two t-AML patients (one R132H and one R132L). One t-AML patient presented IDH2 R140Q mutation in BMMNC, while no mutations were detected at the codon R172. Neither IDH1 nor IDH2 mutations occurred in matched MSC.
TE D
DNMT3A R882 mutations were found in BM-MNC from one de novo AML (R882H), one t-AML (R882H) and one sAML evolved from Polycythemia Vera (R882C), but not in corresponding MSC.
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EP
One U2AF1 S34Y mutation was found in BM-MNC isolated from one patient with intermediate-risk MDS, while a SF3B1 K666N mutation was detected in BM-MNC from one patient with AML following a primary MDS. SRFS2 P95 mutations were found in BM-MNC isolated from two patients with de novo AML (P95L and P95R), three patients with MDS (P95L, P95H and P95 frameshift mutation) and one patient with MPN (P95H). Again, U2AF1, SF3B1 and SRFS2 mutation were absent in MSC. All described mutations described were heterozygous. Results of mutational analysis in hematopoietic BM-MNC are reported in Figure 1.
3
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RI PT
We found no mutations for any of the studied genes in the MSC compartment, both in carriers of mutations in the hematopoietic compartment and in wild-type patients.
M AN U
SC
The absence of NPM1 and FLT3 mutations in MSC from AML patients has been previously reported [3]. So far, the prevalence of somatic mutations of epigenetic and spliceosomal genes in MSC compartment in patients with myeloid malignancies is not known. Mutations of DNMT3A and IDH1/2 have been described in 22% and 16% of AML patients and in 8% and 12% of MDS patients, respectively [4,8-9]. These mutations have been associated to deregulated DNA methylation and poor prognosis. Abnormal DNA methylation has been advocated as responsible of the premature senescence and reduced growth of MSC in MDS. Specific patterns of DNA methylation in MSC have been reported in MDS subtypes compared to healthy donors [2]. According to our results, abnormal methylation profiles described in MSC are unlikely to be related to mutations of epigenetic regulatory genes, even though a wider mutational analysis, including also ASXL1 and TET2 genes, could be more informative.
EP
TE D
Abnormal splicing patterns of multiple genes have been described in myeloid neoplasms due to mutations of spliceosome machinery genes [10]. This phenomenon is considered to play a significant role in myeloid leukemogenesis due to selective missplicing of tumor-associated genes. The contribution of missplicing to MSC dysfunction in myeloid neoplasms is still a matter of investigation. The absence of recurrent spliceosome gene mutations in MSC contrasts with the hypothesis that these mutations may play a significant role.
AC C
We conclude that common mutations of genes involved in epigenetic regulation and spliceosome machinery are absent in the mesenchymal compartment of leukemic bone marrows and are restricted only to the malignant myeloid clone, in agreement with the distinct origin from hematopoietic cells.
4
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Acknowledgments: This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.)
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References
RI PT
1. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315-7.
SC
2. Geyh S, Oz S, Cadeddu RP, et al. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells. Leukemia. 2013;27:1841-51.
M AN U
3. Blau O, Baldus CD, Hofmann WK, et al. Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts. Blood. 2011;118:5583-92. 4. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424-33.
TE D
5. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361:1058-66. 6. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478:64-9.
EP
7. Voso MT, Fabiani E, Fianchi L, et al. Mutations of epigenetic regulators and of the spliceosome machinery in therapy-related myeloid neoplasms and in acute leukemias evolved from chronic myeloproliferative diseases. Leukemia. 2013;27:982-5.
AC C
8. Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia. 2011;25:1153-8.
6
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9. Patnaik MM, Hanson CA, Hodnefield JM, et al. Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients. Leukemia. 2012;26:101-5.
AC C
EP
TE D
M AN U
SC
10. Przychodzen B, Jerez A, Guinta K, et al. Patterns of missplicing due to somatic U2AF1 mutations in myeloid neoplasms. Blood. 2013;122:9991006.
7
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Figure 1: Distribution of IDH1, IDH2, DNMT3A, U2AF1, SF3B1, SRSF2,
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NPM1 and FLT3 mutations in the hematopoietic compartment of the study population. No mutations were found in mesenchymal stem cell
compartment. Each column represents a single patient. Black boxes represent mutation. FLT3 and NPM1 mutational analysis was not
AC C
EP
TE D
M AN U
SC
performed in MDS, MPN and t-MDS patients (white boxes).
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