Letters to the Editor
1555 4
Institute of Pathology, Hannover Medical School, Hannover, Germany; 5 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany; 6 Outpatient Oncology Center Hannover, Hannover, Germany; 7 Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Medical Faculty, Duesseldorf, Germany and 8 Medical Department I, University Hospital Carl Gustav Carus, Dresden, Germany E-mail: [email protected] 9 These authors contributed equally to this work. 10 These authors share the senior authorship.
REFERENCES 1 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013; 369: 2379–2390. 2 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013; 369: 2391–2405. 3 Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor SM et al. Calreticulin: non-endoplasmic reticulum functions in physiology and disease. FASEB J 2010; 24: 665–683. 4 Chao MP, Majeti R, Weissman IL. Programmed cell removal: a new obstacle in the road to developing cancer. Nat Rev Cancer 2011; 12: 58–67. 5 Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood 2013; e-pub ahead of print 26 December 2013.
6 Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia 2014; 28: 1407–1413. 7 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472–1477. 8 Kroger N, Holler E, Kobbe G, Bornhauser M, Schwerdtfeger R, Baurmann H et al. Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Blood 2009; 114: 5264–5270. 9 Alchalby H, Yunus DR, Zabelina T, Kobbe G, Holler E, Bornhauser M et al. Risk models predicting survival after reduced-intensity transplantation for myelofibrosis. Br J Haematol 2012; 157: 75–85. 10 Alchalby H, Badbaran A, Zabelina T, Kobbe G, Hahn J, Wolff D et al. Impact of JAK2V617F mutation status, allele burden, and clearance after allogeneic stem cell transplantation for myelofibrosis. Blood 2010; 116: 3572–3581. 11 Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13: 54–61. 12 Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005; 123: 321–334. 13 Locher C, Rusakiewicz S, Tesniere A, Ghiringhelli F, Apetoh L, Kroemer G et al. Witch hunt against tumor cells enhanced by dendritic cells. Ann N Y Acad Sci 2009; 1174: 51–60. 14 Wemeau M, Kepp O, Tesniere A, Panaretakis T, Flament C, De Botton S et al. Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia. Cell Death Dis 2010; 1: e104. 15 Hussein K, Pardanani AD, Van Dyke DL, Hanson CA, Tefferi A. International Prognostic Scoring System-independent cytogenetic risk categorization in primary myelofibrosis. Blood 2010; 115: 496–499.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Calreticulin mutation was rarely detected in patients with myelodysplastic syndrome Leukemia (2014) 28, 1555–1557; doi:10.1038/leu.2014.71 Recently, somatic mutations in exon 9 of calreticulin (CALR) were detected in patients with myeloproliferative neoplasms (MPN) who
Table 1. UPN
had nonmutated JAK2 and MPL.1,2 Similar genetic alterations, such as mutations of ASXL1, IDH, TET2 and so on, have been identified in both myelodysplastic syndrome (MDS) and MPN,3–6 raising the possibility that CALR mutations may also occur in patients with MDS. In a report by Nangalia et al.,1 10 of the 120 patients with MDS had
Mutational studies in the three MDS patients who had CALR mutations at diagnosis and/or at follow-ups Interval (months)a
1
Age/sex
77/M 42.5
2 3
63/F 65/M 5.5 9.5 18.5
FABb
RARS RARS RA RAEBT RAEBT RAEBT AML, MDS
Karyotype
46,XY 46,XY 46,XX 46,XY ND 46,XY 46,XY
CALR mutation
Other accompanied gene mutations
Location
nt change
aa change
Exon9 Exon9 Exon9 Exon9 Exon9 Exon9 Exon9
c.1122_1125del c.1122_1125del c.1092_1143del — — — c.1092_1143del
K374NfsX55 K374NfsX55 L367TfsX46 —c —c —c L367TfsX46
DNMT3A, SF3B1 DNMT3A, SF3B1 — ASXL1, SF3B1 ASXL1, SF3B1 ASXL1, SF3B1 RUNX1, ASXL1, SF3B1
Abbreviations: aa, amino acid; AML, acute myeloid leukemia; F, female; MMDS, myelodysplastic syndrome.; ND, not done; RA, refractory anemia; RARS, refractory anemia with ring sideroblasts; RAEBT, refractory anemia with excess blasts in transformation; UPN, unique patient number. aInterval between the two successive studies. bMDS entity with bone marrow blasts of 20–29% was subclassified as RAEBT according to the FAB classification and that with bone marrow blasts 430% as AML, MDS. cThe CALR mutation could not be detected by TA cloning in this patient either (65, 43 and 54 clones were analyzed).
Accepted article preview online 17 February 2014; advance online publication, 11 March 2014
& 2014 Macmillan Publishers Limited
Leukemia (2014) 1543 – 1572
Letters to the Editor
1556 CALR mutations; however, Klampfl et al.2 showed none of the 73 patients with MDS and 64 patients with chronic myelomonocytic leukemia (CMML) harbored these abnormalities. To further characterize CALR mutations in MDS, we performed Sanger sequencing of CALR exon 9 in a much larger cohort of patients with de novo MDS, according to the World Health Organization (WHO) classification, as well as those with CMML and acute myeloid leukemia (AML) with blast percentage of 20–30%, that is refractory anemia with excess blasts in transformation (RAEBT) by the French-American-British (FAB) classification. Mutational analysis was performed by genomic DNA PCR followed by direct sequencing as previously reported.7 The primer sequences were as follows: human CALR exon 9 sense primer 50 -GGC AACGAGACGTGGGGCGT-30 and antisense primer 50 -CAGAGA CATTATTTGGCGCGGCC-30 . Abnormal sequencing results were
confirmed by at least two repeated analyses. Totally, 453 patients were enrolled, including 162 patients with refractory anemia (RA), 33 RA with ring sideroblasts (RARS), 160 RA with excess blasts (RAEB), 50 RAEBT and 48 CMML. Sequential studies were also performed in 312 samples from 108 patients, including 1 CALRmutated and 107 CALR-wild patients, during clinical follow-ups to investigate the role of CALR mutation in disease progression. This study was approved by the Institutional Review Board of the National Taiwan University Hospital. Totally, 2 (0.56%) of 355 WHO-defined MDS patients had CALR mutations at diagnosis (Table 1). Patient 1, who had initial presentation of anemia and thrombocytopenia, was diagnosed as having RARS. He had intermediate-1-risk MDS based on the International Prognostic Scoring System (IPSS) and low-risk MDS by the revised IPSS (IPSS-R). He also harbored DNMT3A and SF3B1
Figure 1. Sequential analyses of CALR mutations in patient 3 showing acquisition of a novel CALR mutation at leukemia transformation. Wildtype CALR at diagnosis (RAEBT, upper lane) and subsequent follow-ups (RAEBT, middle two lanes); CALR mutation with L367TfsX46 (lower lane) was acquired 33.5 months after diagnosis when the disease progressed from RAEBT to AML by the FAB classification. The arrow indicates the location of the mutation. Leukemia (2014) 1543 – 1572
& 2014 Macmillan Publishers Limited
Letters to the Editor
1557 mutations in addition to CALR mutation at diagnosis. He was still alive without disease progression at the time of this study, that is 57 months after diagnosis. Patient 2, diagnosed as having RA, had low-risk characteristics according to both the IPSS and the IPSS-R. She remained transfusion dependent during the follow-up period of 27.4 months. We did not find any CALR mutation in 98 patients with CMML or RAEBT at diagnosis. The variation of the incidence of CALR mutations in WHO-defined MDS (0–8.3%) in different studies1,2 and in this study may be due to bias in patient selection, ethnic and/or geographic differences, but more studies are needed to clarify it. Intriguingly, among the 107 CALR-wild patients who were sequentially studied, one (patient 3, Table 1) acquired CALR mutation 33.5 months after diagnosis when the disease progressed from RAEBT to AML by the FAB classification (Figure 1). A novel RUNX1 mutation was also acquired at the same time in this patient. The CALR of other 106 patients remained in germline during follow-ups. Because direct sequencing might not be sensitive enough to detect low level of CALR mutant, we therefore did TA cloning followed by direct sequencing7 for the three CALR-wild samples obtained at diagnosis and subsequent follow-ups from patient 3. We could not find any CALR mutation in the 65, 43 and 54 clones analyzed using cloning technique in this patient (Table 1 and Figure 1). Nevertheless, we could not exclude the possibility that minor population of cells with CALR mutant existed initially but escaped the detection of direct sequencing or TA cloning technique. In this study, two different kinds of CALR mutations (L367TfsX46 and K374NfsX55, Table 1) were detected in three patients. Both of these frame-shift mutations are predicted to generate mutant proteins with a novel C-terminal, resulting in the loss of most of the C-terminal acidic domain and the Lys–Asp–Glu–Leu (KDEL) signal. The KDEL signal is responsible for retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi apparatus and maintaining these proteins in the ER.8 Therefore, these alterations are thought to compromise retrieval or retention of these proteins in the ER. To the best of our knowledge, this study recruited the largest cohort of MDS patients to clarify the clinical relevance of CALR mutations in MDS. We showed that CALR mutations were rarely detected in patients with MDS both at diagnosis and disease progression. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was partially sponsored by grants NSC 100-2314-B002-057-MY3, NSC 100-2314-B-002 -112 -MY3 and NSC 100-2628 -B-002-003-MY3 from the National
Science Council (Taiwan), MOHW103-TD-B-111-04 from the Ministry of Health and Welfare (Taiwan), and NTUH 102P06 and UN 102-015 from the Department of Medical Research, National Taiwan University Hospital.
AUTHOR CONTRIBUTIONS H-AH and Y-YK were responsible for study design, literature collection, data management and interpretation, statistical analysis and manuscript writing; P-HC and W-CC were responsible for mutation analysis and interpretation; H-FT planned, designed and coordinated the study over the entire period and wrote the manuscript.
H-A Hou1,2,5, Y-Y Kuo3,5, W-C Chou1,4, P-H Chen3 and H-F Tien1 Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 3 Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan and 4 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan E-mail: [email protected] 5 These authors contributed equally to this work.
1
REFERENCES 1 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. New Engl J Med 2013; 369: 2391–2405. 2 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. New Engl J Med 2013; 369: 2379–2390. 3 Solary E, Bernard OA, Tefferi A, Fuks F, Vainchenker W. The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases. Leukemia 2014; 28: 485–496. 4 Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014; 28: 241–247. 5 Itzykson R, Fenaux P. Epigenetics of myelodysplastic syndromes. Leukemia 2014; 28: 497–506. 6 Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010; 24: 1128–1138. 7 Hou HA, Kuo YY, Liu CY, Chou WC, Lee MC, Chen CY et al. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications. Blood 2012; 119: 559–568. 8 Teasdale RD, Jackson MR. Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu Rev Cell Dev Biol 1996; 12: 27–54.
Acute myeloid leukemia cells harboring MLL fusion genes or with the acute promyelocytic leukemia phenotype are sensitive to the Bcl-2-selective inhibitor ABT-199 Leukemia (2014) 28, 1557–1560; doi:10.1038/leu.2014.72 Acute myeloid leukemia (AML) is a malignant disease of cells arising from the myeloid lineage. As a heterogeneous disease, AML consists of many different subtypes, each with different
treatment sensitivities and prognoses. Despite advances in care, AML remains a difficult-to-treat disease, with overall survival being B25% and 65%, in the adult and pediatric populations, respectively.1,2 A major factor contributing to treatment failure in AML is the resistance to standard chemotherapy, consisting primarily of a combination of cytarabine and an anthracycline.
Accepted article preview online 17 February 2014; advance online publication, 7 March 2014
& 2014 Macmillan Publishers Limited
Leukemia (2014) 1543 – 1572
1555 4
Institute of Pathology, Hannover Medical School, Hannover, Germany; 5 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany; 6 Outpatient Oncology Center Hannover, Hannover, Germany; 7 Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Medical Faculty, Duesseldorf, Germany and 8 Medical Department I, University Hospital Carl Gustav Carus, Dresden, Germany E-mail: [email protected] 9 These authors contributed equally to this work. 10 These authors share the senior authorship.
REFERENCES 1 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013; 369: 2379–2390. 2 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013; 369: 2391–2405. 3 Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor SM et al. Calreticulin: non-endoplasmic reticulum functions in physiology and disease. FASEB J 2010; 24: 665–683. 4 Chao MP, Majeti R, Weissman IL. Programmed cell removal: a new obstacle in the road to developing cancer. Nat Rev Cancer 2011; 12: 58–67. 5 Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood 2013; e-pub ahead of print 26 December 2013.
6 Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia 2014; 28: 1407–1413. 7 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472–1477. 8 Kroger N, Holler E, Kobbe G, Bornhauser M, Schwerdtfeger R, Baurmann H et al. Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Blood 2009; 114: 5264–5270. 9 Alchalby H, Yunus DR, Zabelina T, Kobbe G, Holler E, Bornhauser M et al. Risk models predicting survival after reduced-intensity transplantation for myelofibrosis. Br J Haematol 2012; 157: 75–85. 10 Alchalby H, Badbaran A, Zabelina T, Kobbe G, Hahn J, Wolff D et al. Impact of JAK2V617F mutation status, allele burden, and clearance after allogeneic stem cell transplantation for myelofibrosis. Blood 2010; 116: 3572–3581. 11 Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13: 54–61. 12 Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005; 123: 321–334. 13 Locher C, Rusakiewicz S, Tesniere A, Ghiringhelli F, Apetoh L, Kroemer G et al. Witch hunt against tumor cells enhanced by dendritic cells. Ann N Y Acad Sci 2009; 1174: 51–60. 14 Wemeau M, Kepp O, Tesniere A, Panaretakis T, Flament C, De Botton S et al. Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia. Cell Death Dis 2010; 1: e104. 15 Hussein K, Pardanani AD, Van Dyke DL, Hanson CA, Tefferi A. International Prognostic Scoring System-independent cytogenetic risk categorization in primary myelofibrosis. Blood 2010; 115: 496–499.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Calreticulin mutation was rarely detected in patients with myelodysplastic syndrome Leukemia (2014) 28, 1555–1557; doi:10.1038/leu.2014.71 Recently, somatic mutations in exon 9 of calreticulin (CALR) were detected in patients with myeloproliferative neoplasms (MPN) who
Table 1. UPN
had nonmutated JAK2 and MPL.1,2 Similar genetic alterations, such as mutations of ASXL1, IDH, TET2 and so on, have been identified in both myelodysplastic syndrome (MDS) and MPN,3–6 raising the possibility that CALR mutations may also occur in patients with MDS. In a report by Nangalia et al.,1 10 of the 120 patients with MDS had
Mutational studies in the three MDS patients who had CALR mutations at diagnosis and/or at follow-ups Interval (months)a
1
Age/sex
77/M 42.5
2 3
63/F 65/M 5.5 9.5 18.5
FABb
RARS RARS RA RAEBT RAEBT RAEBT AML, MDS
Karyotype
46,XY 46,XY 46,XX 46,XY ND 46,XY 46,XY
CALR mutation
Other accompanied gene mutations
Location
nt change
aa change
Exon9 Exon9 Exon9 Exon9 Exon9 Exon9 Exon9
c.1122_1125del c.1122_1125del c.1092_1143del — — — c.1092_1143del
K374NfsX55 K374NfsX55 L367TfsX46 —c —c —c L367TfsX46
DNMT3A, SF3B1 DNMT3A, SF3B1 — ASXL1, SF3B1 ASXL1, SF3B1 ASXL1, SF3B1 RUNX1, ASXL1, SF3B1
Abbreviations: aa, amino acid; AML, acute myeloid leukemia; F, female; MMDS, myelodysplastic syndrome.; ND, not done; RA, refractory anemia; RARS, refractory anemia with ring sideroblasts; RAEBT, refractory anemia with excess blasts in transformation; UPN, unique patient number. aInterval between the two successive studies. bMDS entity with bone marrow blasts of 20–29% was subclassified as RAEBT according to the FAB classification and that with bone marrow blasts 430% as AML, MDS. cThe CALR mutation could not be detected by TA cloning in this patient either (65, 43 and 54 clones were analyzed).
Accepted article preview online 17 February 2014; advance online publication, 11 March 2014
& 2014 Macmillan Publishers Limited
Leukemia (2014) 1543 – 1572
Letters to the Editor
1556 CALR mutations; however, Klampfl et al.2 showed none of the 73 patients with MDS and 64 patients with chronic myelomonocytic leukemia (CMML) harbored these abnormalities. To further characterize CALR mutations in MDS, we performed Sanger sequencing of CALR exon 9 in a much larger cohort of patients with de novo MDS, according to the World Health Organization (WHO) classification, as well as those with CMML and acute myeloid leukemia (AML) with blast percentage of 20–30%, that is refractory anemia with excess blasts in transformation (RAEBT) by the French-American-British (FAB) classification. Mutational analysis was performed by genomic DNA PCR followed by direct sequencing as previously reported.7 The primer sequences were as follows: human CALR exon 9 sense primer 50 -GGC AACGAGACGTGGGGCGT-30 and antisense primer 50 -CAGAGA CATTATTTGGCGCGGCC-30 . Abnormal sequencing results were
confirmed by at least two repeated analyses. Totally, 453 patients were enrolled, including 162 patients with refractory anemia (RA), 33 RA with ring sideroblasts (RARS), 160 RA with excess blasts (RAEB), 50 RAEBT and 48 CMML. Sequential studies were also performed in 312 samples from 108 patients, including 1 CALRmutated and 107 CALR-wild patients, during clinical follow-ups to investigate the role of CALR mutation in disease progression. This study was approved by the Institutional Review Board of the National Taiwan University Hospital. Totally, 2 (0.56%) of 355 WHO-defined MDS patients had CALR mutations at diagnosis (Table 1). Patient 1, who had initial presentation of anemia and thrombocytopenia, was diagnosed as having RARS. He had intermediate-1-risk MDS based on the International Prognostic Scoring System (IPSS) and low-risk MDS by the revised IPSS (IPSS-R). He also harbored DNMT3A and SF3B1
Figure 1. Sequential analyses of CALR mutations in patient 3 showing acquisition of a novel CALR mutation at leukemia transformation. Wildtype CALR at diagnosis (RAEBT, upper lane) and subsequent follow-ups (RAEBT, middle two lanes); CALR mutation with L367TfsX46 (lower lane) was acquired 33.5 months after diagnosis when the disease progressed from RAEBT to AML by the FAB classification. The arrow indicates the location of the mutation. Leukemia (2014) 1543 – 1572
& 2014 Macmillan Publishers Limited
Letters to the Editor
1557 mutations in addition to CALR mutation at diagnosis. He was still alive without disease progression at the time of this study, that is 57 months after diagnosis. Patient 2, diagnosed as having RA, had low-risk characteristics according to both the IPSS and the IPSS-R. She remained transfusion dependent during the follow-up period of 27.4 months. We did not find any CALR mutation in 98 patients with CMML or RAEBT at diagnosis. The variation of the incidence of CALR mutations in WHO-defined MDS (0–8.3%) in different studies1,2 and in this study may be due to bias in patient selection, ethnic and/or geographic differences, but more studies are needed to clarify it. Intriguingly, among the 107 CALR-wild patients who were sequentially studied, one (patient 3, Table 1) acquired CALR mutation 33.5 months after diagnosis when the disease progressed from RAEBT to AML by the FAB classification (Figure 1). A novel RUNX1 mutation was also acquired at the same time in this patient. The CALR of other 106 patients remained in germline during follow-ups. Because direct sequencing might not be sensitive enough to detect low level of CALR mutant, we therefore did TA cloning followed by direct sequencing7 for the three CALR-wild samples obtained at diagnosis and subsequent follow-ups from patient 3. We could not find any CALR mutation in the 65, 43 and 54 clones analyzed using cloning technique in this patient (Table 1 and Figure 1). Nevertheless, we could not exclude the possibility that minor population of cells with CALR mutant existed initially but escaped the detection of direct sequencing or TA cloning technique. In this study, two different kinds of CALR mutations (L367TfsX46 and K374NfsX55, Table 1) were detected in three patients. Both of these frame-shift mutations are predicted to generate mutant proteins with a novel C-terminal, resulting in the loss of most of the C-terminal acidic domain and the Lys–Asp–Glu–Leu (KDEL) signal. The KDEL signal is responsible for retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi apparatus and maintaining these proteins in the ER.8 Therefore, these alterations are thought to compromise retrieval or retention of these proteins in the ER. To the best of our knowledge, this study recruited the largest cohort of MDS patients to clarify the clinical relevance of CALR mutations in MDS. We showed that CALR mutations were rarely detected in patients with MDS both at diagnosis and disease progression. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was partially sponsored by grants NSC 100-2314-B002-057-MY3, NSC 100-2314-B-002 -112 -MY3 and NSC 100-2628 -B-002-003-MY3 from the National
Science Council (Taiwan), MOHW103-TD-B-111-04 from the Ministry of Health and Welfare (Taiwan), and NTUH 102P06 and UN 102-015 from the Department of Medical Research, National Taiwan University Hospital.
AUTHOR CONTRIBUTIONS H-AH and Y-YK were responsible for study design, literature collection, data management and interpretation, statistical analysis and manuscript writing; P-HC and W-CC were responsible for mutation analysis and interpretation; H-FT planned, designed and coordinated the study over the entire period and wrote the manuscript.
H-A Hou1,2,5, Y-Y Kuo3,5, W-C Chou1,4, P-H Chen3 and H-F Tien1 Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 3 Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan and 4 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan E-mail: [email protected] 5 These authors contributed equally to this work.
1
REFERENCES 1 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. New Engl J Med 2013; 369: 2391–2405. 2 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. New Engl J Med 2013; 369: 2379–2390. 3 Solary E, Bernard OA, Tefferi A, Fuks F, Vainchenker W. The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases. Leukemia 2014; 28: 485–496. 4 Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014; 28: 241–247. 5 Itzykson R, Fenaux P. Epigenetics of myelodysplastic syndromes. Leukemia 2014; 28: 497–506. 6 Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010; 24: 1128–1138. 7 Hou HA, Kuo YY, Liu CY, Chou WC, Lee MC, Chen CY et al. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications. Blood 2012; 119: 559–568. 8 Teasdale RD, Jackson MR. Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu Rev Cell Dev Biol 1996; 12: 27–54.
Acute myeloid leukemia cells harboring MLL fusion genes or with the acute promyelocytic leukemia phenotype are sensitive to the Bcl-2-selective inhibitor ABT-199 Leukemia (2014) 28, 1557–1560; doi:10.1038/leu.2014.72 Acute myeloid leukemia (AML) is a malignant disease of cells arising from the myeloid lineage. As a heterogeneous disease, AML consists of many different subtypes, each with different
treatment sensitivities and prognoses. Despite advances in care, AML remains a difficult-to-treat disease, with overall survival being B25% and 65%, in the adult and pediatric populations, respectively.1,2 A major factor contributing to treatment failure in AML is the resistance to standard chemotherapy, consisting primarily of a combination of cytarabine and an anthracycline.
Accepted article preview online 17 February 2014; advance online publication, 7 March 2014
& 2014 Macmillan Publishers Limited
Leukemia (2014) 1543 – 1572
