correspondence
Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia
I-7 had monosomy 7. The eldest son of patient I-7 (II-1, Fig 1) has thrombocytopenia. The history of familial thrombocytopenia coupled with development of AML suggested a diagnosis of FPD-AML, which was confirmed by Sanger sequencing identification of a heterozygous RUNX1 p.Arg166X mutation in the leukaemic blasts and constitutional buccal scrapes of both affected patients. Patient I-3 underwent a reduced intensity conditioning allogeneic stem cell transplant (ASCT) from RUNX1 wild type sibling donor I-5. Patient I-7 underwent a myeloblative ASCT from RUNX1 wild type sibling donor I-6. Both patients achieved 100% donor chimerism by day-100 post-ASCT. For NGS, amplicon libraries were generated from AML diagnostic bone marrow DNA of I-3 and I-7 using the Ion AmpliseqTM AML Panel (Thermo Fisher Scientific, Life Technologies, Paisley, UK), a four primer-pool panel that generates 237 amplicons to allow interrogation of 19 commonly mutated genes implicated in AML. Amplicons cover the entire coding region of DNMT3A, CEBPA, GATA2, TET2, TP53 and mutational hot spot regions of ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1 and WT1. Sequencing was performed on an Ion PGMTM with data analysed and reviewed using Torrent Browser and Ion Reporter 42 software (Thermo Fisher Scientific, Life Technologies). Criteria to allow confident calling of somatic mutations were a minimum target coverage of 500X, the presence of a mutation at >5% and a predicted change in amino acid sequence. Sanger sequencing was also performed of exon 8 of CDC25C, encompassing the mutation hotspot previously described (Yoshimi et al, 2014). In addition to confirmation of the heterozygous RUNX1 p.Arg166X mutation, targeted NGS demonstrated the presence of further mutations in the genes known to disrupt epigenetic (ASXL1, IDH1, TET2) and transcription factor (CEBPA, RUNX1) function in AML (Table I). Although
Familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) is a rare, autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities with a propensity to develop a myelodysplastic syndrome (MDS) or AML. FPD-AML kindred are defined by germ-line mutations of RUNX1 (Song et al, 1999), which encodes a transcription factor essential for definitive haematopoiesis and myeloid cell differentiation, commonly dysregulated by translocations, mutations or amplification in de novo and secondary MDS and acute leukaemias. Most germ line RUNX1 mutations are unique to the individual FPDAML pedigree with variability observed in the MDS or AML phenotype and the incidence of leukaemic transformation of affected individuals (Nickels et al, 2013). The spectrum of somatic genetic events associated with progression to MDS or AML have not been fully appreciated but acquisition of cytogenetic abnormalities, single gene defects that occur in de novo MDS and AML, and bi-allelic RUNX1 mutations have all been demonstrated (Minelli et al, 2004; Preudhomme et al, 2009; Shiba et al, 2012). More recently, mutations of CDC25C have been identified in approximately half of affected FPD-AML patients. CDC25C mutations appear to disrupt a critical cell cycle check point in pre-leukaemic clones, allowing subsequent acquisition of further sub-clonal mutations (Yoshimi et al, 2014). Emerging next-generation sequencing (NGS) technologies, platforms and diseasetargeted panels allow the simultaneous identification of numerous mutational events. Such a targeted NGS approach was applied to a known RUNX1 mutated FPD-AML kindred to identify additional molecular events that co-operate with the germ line RUNX1 mutation in driving leukaemic transformation. A 56-year-old male and a 45-year-old female sibling both presented with AML with myelodysplastic features (Fig 1, I– 3 and I–7 respectively). At diagnosis, I-3 had trisomy 8 and
I
1
Fig 1. Pedigree of the familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) kindred. The index cases (I-3, I-7) are indicated as black symbols. II-1 (grey symbol) suffered from thrombocytopenia
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
2
3
4
5
6
II
7
1
8
2
3
doi: 10.1111/bjh.13838
Correspondence Table I. Mutations identified by targeted next-generation sequencing in the two affected siblings. Patient
Mutated gene
Nucleotide change
Amino acid change
Variant effect
% Mutant allele
COSMIC (Disease association)
I-3
RUNX1 ASXL1 IDH1 RUNX1 RUNX1 TET2 RUNX1 CEBPA
c.496C>T c.2083C>T c.394C>T c.608C>G c.496C>T c.5162T>G c.497G>A c.898C>T
p.Arg166X p.Gln695X p.Arg132Cys p.Pro203Arg p.Arg166X p.Leu1721Trp p.Arg166Gln p.Arg300Cys
nonsense nonsense missense missense nonsense missense missense missense
53% 26% 14% 6% 50% 50% 11% 5%
COSM24769 (AML, ETP-ALL) COSM1738037 (MDS) COSM28747 (AML, Glioma) – COSM24769 (AML, ETP-ALL) – COSM36055 (AML) –
I-7
COSMIC, Catalogue of somatic mutations in cancer (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/); AML, acute myeloid leukaemia; ETP-ALL, early T cell precursor-acute lymphoblastic leukaemia; MDS, myelodysplastic syndrome.
matched germ line material was not studied by NGS, the fact that several of these mutations have been previously described in haematological malignancies (Table I) and that they are present at allele frequencies less than 50% implies they are somatic and not heterozygous germ line in nature. No mutations of CDC25C exon 8 were detected in either patient. Proof of principle feasibility and utility of a targeted NGS approach are demonstrated in a FPD-AML kindred with a known germ line RUNX1 mutation. A targeted NGS approach using more comprehensive MDS- and AML-associated gene panels or whole exome sequencing is likely to offer several advantages over standard sequencing methodologies in familial AML: identification of the causative germ line mutation is possible where the phenotype may be uninformative (Obata et al, 2015); the simultaneous detection of co-operating mutations that may be clinically actionable, such as IDH and TET2 demonstrated in the FPD-AML kindred described herein (Falini et al, 2015); the ability to infer patterns of clonal evolution in patients with overt AML (Tawana et al, 2015); and affords the opportunity to monitor identified thrombocytopenic individuals from FPD-AML kindred to identify progression to MDS or AML, thus allowing earlier clinical intervention. While no CDC25C mutations were detected in the two FPD-AML affected patients described, the role of these abnormalities requires clarification in other FPD-AML kindred. If NGS technologies become integrated into routine practice, more cases of familial leukaemia are likely to be resolved (Churpek et al, 2013)
References Churpek, J.E., Lorenz, R., Nedumgottil, S., Onel, K., Olopade, O.I., Owen, C.J., Bertuch, A.A. & Godley, L.A. (2013) Proposal for the clinical detection and management of patients and their family members with familial myelodysplastic syndrome/acute leukemia predisposition syndromes. Leukemia & Lymphoma, 54, 28–35. Falini, B., Sportoletti, P., Brunetti, L. & Martelli, M.P. (2015) Perspectives for therapeutic targeting of gene mutations in acute myeloid leukae-
2
with this approach, possessing the potential to enhance genetic testing and improve clinical management.
Author contributions K.H. performed laboratory studies. K.H. and S.E.L. analysed the data. K.H., S.E.L. and E.V. conceived and designed the study. A.H. and E.C. provided patient care and clinical information. All authors contributed to manuscript preparation and gave final approval.
Disclosures All authors disclose no conflicts of interest. Karl Haslam1 Stephen E. Langabeer1 Amjad Hayat2 Eibhlin Conneally3 Elisabeth Vandenberghe1,3 1
Cancer Molecular Diagnostics, St. James’s Hospital, Dublin, 2Depart-
ment of Haematology, Galway University Hospital, Galway, and 3
Department of Haematology, St. James’s Hospital, Dublin, Ireland. E-mail: [email protected]
Keywords: FPD-AML, RUNX1, next-generation sequencing
mia with normal cytogenetics. British Journal of Haematology, 170, 305–322. Minelli, A., Maserati, E., Rossi, G., Bernardo, M.E., De Stefano, P., Cacchini, M.P., Valli, R., Albano, V., Pierani, P., Leszl, A., Sainati, L., Lo Curto, F., Danesino, C., Locatelli, F. & Pasquali, F. (2004) Familial platelet disorder with propensity to acute myelogenous leukemia: genetic heterogeneity and progression to leukemia via acquisition of clonal chromosomal anomalies. Genes Chromosomes & Cancer, 40, 165–171. Nickels, E.M., Soodalter, J., Churpek, J.E. & Godley, L.A. (2013) Recognizing familial myeloid
leukemia in adults. Therapeutic Advances in Hematology, 4, 254–269. Obata, M., Tsutsumi, S., Makino, S., Takahashi, K., Watanabe, N., Yoshida, T., Tamiya, G. & Kurachi, H. (2015) Whole exome sequencing confirmation of a novel heterozygous mutation in RUNX1 in a pregnant woman with platelet disorder. Platelets, 26, 364–369. Preudhomme, C., Renneville, A., Bourdon, V., Philippe, N., Roche-Lestienne, C., Boissel, N., Andre, J.M., Cornillet-Lefebvre, P., Baruchel, A., Mozziconacci, M.J. & Sobol, H. (2009) High frequency of RUNX1 biallelic alteration in acute
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
Correspondence myeloid leukemia secondary to familial platelet disorder. Blood, 113, 5583–5587. Shiba, N., Hasegawa, D., Park, M.J., Murata, C., Sato-Otsubo, A., Ogawa, C., Manabe, A., Arakawa, H., Ogawa, S. & Hayashi, Y. (2012) CBL mutation in chronic myelomonocytic leukemia secondary to familial platelet disorder with propensity to develop acute myeloid leukemia (FPD/AML). Blood, 119, 2612–2614. Song, W.J., Sullivan, M.G., Legare, R.D., Hutchings, S., Tan, X., Kufrin, D., Ratajczak, J., Resende, I.C., Haworth, C., Hock, R., Loh, M., Felix, C., Roy, D.C., Busque, L., Kurnit, D.,
Willman, C., Gerwitz, A.M., Speck, N.A., Bushweller, J.H., Li, F.P., Gardiner, K., Poncz, M., Maris, J.M. & Gilliland, D.G. (1999) Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukemia. Nature Genetics, 23, 166–175. Tawana, K., Wang, J., Renneville, A., B€ od€ or, C., Hills, R., Loveday, C., Savic, A., Van Delft, F.W., Treleaven, J., Georgiades, P., Uglow, E., Asou, N., Uike, N., Debeljak, M., Jazbec, J., Ancliff, P., Gale, R., Thomas, X., Mialou, V., D€ ohner, K., Bullinger, L., Mueller, B., Pabst, T., Stelljes, M., Schlegelberger, B., Wozniak, E.,
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
Iqbal, S., Okosun, J., Araf, S., Frank, A.K., Lauridsen, F.B., Porse, B., Nerlov, C., Owen, C., Dokal, I., Gribben, J., Smith, M., Preudhomme, C., Chelala, C., Cavenagh, J. & Fitzgibbon, J. (2015) Disease evolution and outcomes in familial AML with germline CEBPA mutations. Blood, 126, 1214–1223. Yoshimi, A., Toya, T., Kawazu, M., Ueno, T., Tsukamoto, A., Iizuka, H., Nakagawa, M., Nannya, Y., Arai, S., Harada, H., Usuki, K., Hayashi, Y., Ito, E., Kirito, K., Nakajima, H., Ichikawa, M., Mano, H. & Kurokawa, M. (2014) Recurrent CDC25C mutations drive malignant transformation in FPD/AML. Nature Communications, 5, 4770.
3
Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia
I-7 had monosomy 7. The eldest son of patient I-7 (II-1, Fig 1) has thrombocytopenia. The history of familial thrombocytopenia coupled with development of AML suggested a diagnosis of FPD-AML, which was confirmed by Sanger sequencing identification of a heterozygous RUNX1 p.Arg166X mutation in the leukaemic blasts and constitutional buccal scrapes of both affected patients. Patient I-3 underwent a reduced intensity conditioning allogeneic stem cell transplant (ASCT) from RUNX1 wild type sibling donor I-5. Patient I-7 underwent a myeloblative ASCT from RUNX1 wild type sibling donor I-6. Both patients achieved 100% donor chimerism by day-100 post-ASCT. For NGS, amplicon libraries were generated from AML diagnostic bone marrow DNA of I-3 and I-7 using the Ion AmpliseqTM AML Panel (Thermo Fisher Scientific, Life Technologies, Paisley, UK), a four primer-pool panel that generates 237 amplicons to allow interrogation of 19 commonly mutated genes implicated in AML. Amplicons cover the entire coding region of DNMT3A, CEBPA, GATA2, TET2, TP53 and mutational hot spot regions of ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1 and WT1. Sequencing was performed on an Ion PGMTM with data analysed and reviewed using Torrent Browser and Ion Reporter 42 software (Thermo Fisher Scientific, Life Technologies). Criteria to allow confident calling of somatic mutations were a minimum target coverage of 500X, the presence of a mutation at >5% and a predicted change in amino acid sequence. Sanger sequencing was also performed of exon 8 of CDC25C, encompassing the mutation hotspot previously described (Yoshimi et al, 2014). In addition to confirmation of the heterozygous RUNX1 p.Arg166X mutation, targeted NGS demonstrated the presence of further mutations in the genes known to disrupt epigenetic (ASXL1, IDH1, TET2) and transcription factor (CEBPA, RUNX1) function in AML (Table I). Although
Familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) is a rare, autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities with a propensity to develop a myelodysplastic syndrome (MDS) or AML. FPD-AML kindred are defined by germ-line mutations of RUNX1 (Song et al, 1999), which encodes a transcription factor essential for definitive haematopoiesis and myeloid cell differentiation, commonly dysregulated by translocations, mutations or amplification in de novo and secondary MDS and acute leukaemias. Most germ line RUNX1 mutations are unique to the individual FPDAML pedigree with variability observed in the MDS or AML phenotype and the incidence of leukaemic transformation of affected individuals (Nickels et al, 2013). The spectrum of somatic genetic events associated with progression to MDS or AML have not been fully appreciated but acquisition of cytogenetic abnormalities, single gene defects that occur in de novo MDS and AML, and bi-allelic RUNX1 mutations have all been demonstrated (Minelli et al, 2004; Preudhomme et al, 2009; Shiba et al, 2012). More recently, mutations of CDC25C have been identified in approximately half of affected FPD-AML patients. CDC25C mutations appear to disrupt a critical cell cycle check point in pre-leukaemic clones, allowing subsequent acquisition of further sub-clonal mutations (Yoshimi et al, 2014). Emerging next-generation sequencing (NGS) technologies, platforms and diseasetargeted panels allow the simultaneous identification of numerous mutational events. Such a targeted NGS approach was applied to a known RUNX1 mutated FPD-AML kindred to identify additional molecular events that co-operate with the germ line RUNX1 mutation in driving leukaemic transformation. A 56-year-old male and a 45-year-old female sibling both presented with AML with myelodysplastic features (Fig 1, I– 3 and I–7 respectively). At diagnosis, I-3 had trisomy 8 and
I
1
Fig 1. Pedigree of the familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) kindred. The index cases (I-3, I-7) are indicated as black symbols. II-1 (grey symbol) suffered from thrombocytopenia
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
2
3
4
5
6
II
7
1
8
2
3
doi: 10.1111/bjh.13838
Correspondence Table I. Mutations identified by targeted next-generation sequencing in the two affected siblings. Patient
Mutated gene
Nucleotide change
Amino acid change
Variant effect
% Mutant allele
COSMIC (Disease association)
I-3
RUNX1 ASXL1 IDH1 RUNX1 RUNX1 TET2 RUNX1 CEBPA
c.496C>T c.2083C>T c.394C>T c.608C>G c.496C>T c.5162T>G c.497G>A c.898C>T
p.Arg166X p.Gln695X p.Arg132Cys p.Pro203Arg p.Arg166X p.Leu1721Trp p.Arg166Gln p.Arg300Cys
nonsense nonsense missense missense nonsense missense missense missense
53% 26% 14% 6% 50% 50% 11% 5%
COSM24769 (AML, ETP-ALL) COSM1738037 (MDS) COSM28747 (AML, Glioma) – COSM24769 (AML, ETP-ALL) – COSM36055 (AML) –
I-7
COSMIC, Catalogue of somatic mutations in cancer (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/); AML, acute myeloid leukaemia; ETP-ALL, early T cell precursor-acute lymphoblastic leukaemia; MDS, myelodysplastic syndrome.
matched germ line material was not studied by NGS, the fact that several of these mutations have been previously described in haematological malignancies (Table I) and that they are present at allele frequencies less than 50% implies they are somatic and not heterozygous germ line in nature. No mutations of CDC25C exon 8 were detected in either patient. Proof of principle feasibility and utility of a targeted NGS approach are demonstrated in a FPD-AML kindred with a known germ line RUNX1 mutation. A targeted NGS approach using more comprehensive MDS- and AML-associated gene panels or whole exome sequencing is likely to offer several advantages over standard sequencing methodologies in familial AML: identification of the causative germ line mutation is possible where the phenotype may be uninformative (Obata et al, 2015); the simultaneous detection of co-operating mutations that may be clinically actionable, such as IDH and TET2 demonstrated in the FPD-AML kindred described herein (Falini et al, 2015); the ability to infer patterns of clonal evolution in patients with overt AML (Tawana et al, 2015); and affords the opportunity to monitor identified thrombocytopenic individuals from FPD-AML kindred to identify progression to MDS or AML, thus allowing earlier clinical intervention. While no CDC25C mutations were detected in the two FPD-AML affected patients described, the role of these abnormalities requires clarification in other FPD-AML kindred. If NGS technologies become integrated into routine practice, more cases of familial leukaemia are likely to be resolved (Churpek et al, 2013)
References Churpek, J.E., Lorenz, R., Nedumgottil, S., Onel, K., Olopade, O.I., Owen, C.J., Bertuch, A.A. & Godley, L.A. (2013) Proposal for the clinical detection and management of patients and their family members with familial myelodysplastic syndrome/acute leukemia predisposition syndromes. Leukemia & Lymphoma, 54, 28–35. Falini, B., Sportoletti, P., Brunetti, L. & Martelli, M.P. (2015) Perspectives for therapeutic targeting of gene mutations in acute myeloid leukae-
2
with this approach, possessing the potential to enhance genetic testing and improve clinical management.
Author contributions K.H. performed laboratory studies. K.H. and S.E.L. analysed the data. K.H., S.E.L. and E.V. conceived and designed the study. A.H. and E.C. provided patient care and clinical information. All authors contributed to manuscript preparation and gave final approval.
Disclosures All authors disclose no conflicts of interest. Karl Haslam1 Stephen E. Langabeer1 Amjad Hayat2 Eibhlin Conneally3 Elisabeth Vandenberghe1,3 1
Cancer Molecular Diagnostics, St. James’s Hospital, Dublin, 2Depart-
ment of Haematology, Galway University Hospital, Galway, and 3
Department of Haematology, St. James’s Hospital, Dublin, Ireland. E-mail: [email protected]
Keywords: FPD-AML, RUNX1, next-generation sequencing
mia with normal cytogenetics. British Journal of Haematology, 170, 305–322. Minelli, A., Maserati, E., Rossi, G., Bernardo, M.E., De Stefano, P., Cacchini, M.P., Valli, R., Albano, V., Pierani, P., Leszl, A., Sainati, L., Lo Curto, F., Danesino, C., Locatelli, F. & Pasquali, F. (2004) Familial platelet disorder with propensity to acute myelogenous leukemia: genetic heterogeneity and progression to leukemia via acquisition of clonal chromosomal anomalies. Genes Chromosomes & Cancer, 40, 165–171. Nickels, E.M., Soodalter, J., Churpek, J.E. & Godley, L.A. (2013) Recognizing familial myeloid
leukemia in adults. Therapeutic Advances in Hematology, 4, 254–269. Obata, M., Tsutsumi, S., Makino, S., Takahashi, K., Watanabe, N., Yoshida, T., Tamiya, G. & Kurachi, H. (2015) Whole exome sequencing confirmation of a novel heterozygous mutation in RUNX1 in a pregnant woman with platelet disorder. Platelets, 26, 364–369. Preudhomme, C., Renneville, A., Bourdon, V., Philippe, N., Roche-Lestienne, C., Boissel, N., Andre, J.M., Cornillet-Lefebvre, P., Baruchel, A., Mozziconacci, M.J. & Sobol, H. (2009) High frequency of RUNX1 biallelic alteration in acute
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
Correspondence myeloid leukemia secondary to familial platelet disorder. Blood, 113, 5583–5587. Shiba, N., Hasegawa, D., Park, M.J., Murata, C., Sato-Otsubo, A., Ogawa, C., Manabe, A., Arakawa, H., Ogawa, S. & Hayashi, Y. (2012) CBL mutation in chronic myelomonocytic leukemia secondary to familial platelet disorder with propensity to develop acute myeloid leukemia (FPD/AML). Blood, 119, 2612–2614. Song, W.J., Sullivan, M.G., Legare, R.D., Hutchings, S., Tan, X., Kufrin, D., Ratajczak, J., Resende, I.C., Haworth, C., Hock, R., Loh, M., Felix, C., Roy, D.C., Busque, L., Kurnit, D.,
Willman, C., Gerwitz, A.M., Speck, N.A., Bushweller, J.H., Li, F.P., Gardiner, K., Poncz, M., Maris, J.M. & Gilliland, D.G. (1999) Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukemia. Nature Genetics, 23, 166–175. Tawana, K., Wang, J., Renneville, A., B€ od€ or, C., Hills, R., Loveday, C., Savic, A., Van Delft, F.W., Treleaven, J., Georgiades, P., Uglow, E., Asou, N., Uike, N., Debeljak, M., Jazbec, J., Ancliff, P., Gale, R., Thomas, X., Mialou, V., D€ ohner, K., Bullinger, L., Mueller, B., Pabst, T., Stelljes, M., Schlegelberger, B., Wozniak, E.,
ª 2015 John Wiley & Sons Ltd, British Journal of Haematology
Iqbal, S., Okosun, J., Araf, S., Frank, A.K., Lauridsen, F.B., Porse, B., Nerlov, C., Owen, C., Dokal, I., Gribben, J., Smith, M., Preudhomme, C., Chelala, C., Cavenagh, J. & Fitzgibbon, J. (2015) Disease evolution and outcomes in familial AML with germline CEBPA mutations. Blood, 126, 1214–1223. Yoshimi, A., Toya, T., Kawazu, M., Ueno, T., Tsukamoto, A., Iizuka, H., Nakagawa, M., Nannya, Y., Arai, S., Harada, H., Usuki, K., Hayashi, Y., Ito, E., Kirito, K., Nakajima, H., Ichikawa, M., Mano, H. & Kurokawa, M. (2014) Recurrent CDC25C mutations drive malignant transformation in FPD/AML. Nature Communications, 5, 4770.
3
