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Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response a
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a
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Yan-Yu Wang , Jie Hao , Zhan-Yun Liu , Xiang-Qin Weng , Yan Sheng , Chun-Lei Jiang , Yongb
b
b
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b
c
Mei Zhu , Bing Chen , Shu-Min Xiong , Jun-Min Li , Qiu-Sheng Chen , Hao-yue Chen , Chun d
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Qiao & Yu Chen a
Department of Hematology, Bei Zhan Hospital, Shanghai, China
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State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China c
Click for updates
Jingjiang People's Hospital, The Seventh Affiliated Hospital of Yangzhou University, Jingjiang, Jiangsu Province, China d
Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China Published online: 15 Apr 2015.
To cite this article: Yan-Yu Wang, Jie Hao, Zhan-Yun Liu, Xiang-Qin Weng, Yan Sheng, Chun-Lei Jiang, Yong-Mei Zhu, Bing Chen, Shu-Min Xiong, Jun-Min Li, Qiu-Sheng Chen, Hao-yue Chen, Chun Qiao & Yu Chen (2015): Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response, Leukemia & Lymphoma To link to this article: http://dx.doi.org/10.3109/10428194.2015.1007454
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Leukemia & Lymphoma, 2015; Early Online: 1–4 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2015.1007454
LETTER TO THE EDITOR
Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response Yan-Yu Wang1, Jie Hao1, Zhan-Yun Liu1, Xiang-Qin Weng2, Yan Sheng2, Chun-Lei Jiang2, Yong-Mei Zhu2, Bing Chen2, Shu-Min Xiong2, Jun-Min Li2, Qiu-Sheng Chen2, Hao-yue Chen3, Chun Qiao4 & Yu Chen2 1Department of Hematology, Bei Zhan Hospital, Shanghai, China, 2State Key Laboratory of Medical Genomics, Shanghai
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Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China, 3Jingjiang People’s Hospital, The Seventh Affiliated Hospital of Yangzhou University, Jingjiang, Jiangsu Province, China and 4Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China
A great majority of patients with acute promyelocytic leukemia (APL) have the specific translocation t(15;17) (q22;q21), leading to formation of the PML–RARA (promyelocytic leukemia–retinoic acid receptor alpha) fusion gene [1]. By degrading the PML–RARA fusion protein, targeted drugs such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) can effectively eliminate leukemic cells. Therefore, PML–RARA is the key molecular mechanism of APL leukemogenesis. In 2008, the World Health Organization (WHO) declared APL expressing the PML–RARA fusion gene as a subtype of acute myeloid leukemia (AML) with recurrent genetic abnormalities [2]. Moreover, other variant genes combined with the RARA gene have also been identified, such as promyelocytic leukemia zinc finger (PLZF, 11q23), nucleophosmin (NPM1, 5q35), nuclear mitotic apparatus (NuMA, 11q13), signal transducer and activator of transcription 5β (STAT5B, 17q21), protein kinase A regulatory subunit type (PRKAR1A, 17q24), FIP1-like 1 (FIP1L1, 4q12), BCL6 corepressor (BCOR, Xp11), oligonucleotide/ oligosaccharide-binding fold containing 2A (OBFC2A, 2q32) [3] and transducin β-like 1 X-linked receptor 1 (TBLR1, 3q26) [4] (Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/10428194.2015. 1007454). Unlike other forms, variant APL associated with STAT5B–RARA or PLZF–RARA is resistant to ATRA [5]. We herein report a case of APL associated with a novel STAT5B– RARA fusion gene transcript. A 22-year-old Chinese male patient was admitted to our hospital because of recurrent fever, and anemia for 3 months, on 18 January 2013. Physical examination revealed severe anemia without obvious skin petechiae. Routine blood test showed a white blood cell count (WBC) of 2.37 ⫻ 109/L, hemoglobin (Hb) of 50 g/L and platelet count (Plt) of 125 ⫻ 109/L, while the coagulation test showed that fibrinogen (Fg) was 0.6 g/L. The bone marrow (BM)
smear revealed markedly increased cellularity, characterized by 76.5% abnormal promyelocytes [Figure 1(A)], while cytochemical analysis demonstrated a strong positivity for peroxidase (POX) staining in abnormal promyelocytes [Figure 1(B)]. RHG (R-bands by heating using Giemsa) banding karyotype was 46,XY,inv(9)(p13q13) c[18]/44∼45,XY,inv(9)(p13q13)c[cp4]/[22] [Figure 1(C)]. Flow cytometric analysis of the leukemia cells showed low or moderate expression of CD45 with high side scatter (SSC) accounting for 66.2%, with strong expression of myeloperoxidase (MPO), CD117, CD13, CD33 and CD56, but not expression of CD34, CD11b, CD38, human leukocyte antigen (HLA)-DR and B- or T-lineage [Figure 1(E)]. However, reverse transcriptase-polymerase chain reaction (RT-PCR) did not detect the PML–RARA fusion transcript. FLT3-ITD, FLT3-TKD, NPM, N-RAS, CEBPA, DNMT3A and N-RAS were found to be wild type, while MLL–AF9, MLL–AF6, MLL–ENL, MLL–AF10, MLL–AF17 and MLL–ELL did not indicate any rearrangement abnormality (data not shown). The patient was initially treated with ATRA (25 mg/m2/ day, days 1–28). After 10 days, the patient manifested a slight increase in WBC and Fg, but the BM aspirate after 13 days did not show obvious transformation of promyelocytes to myelocytes [Figure 1(D)]. As seen from Supplementary Figure 1 to be found online at http://informahealthcare. com/doi/abs/10.3109/10428194.2015.1007454, the WBC and Fg at day 23 reached 4.9 ⫻ 109/L and 1.7 g/L, respectively. After obtaining informed consent, the patient was started on ATO (10 mg/day, days 11–18) and the IA regimen (idarubicin 10 mg/m2/day, days 25–27 and cytarabine 100 mg/m2/ day, days 25–32). BM aspiration on days 35 and 75 revealed 59% and 30% of leukemia cells, respectively (Supplementary Table II to be found online at http://informahealthcare. com/doi/abs/10.3109/10428194.2015.1007454). Nevertheless, the patient refused to undergo further chemotherapy,
Correspondence: Yu Chen, State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Tel: ⫹ 86-1264370045. Fax: ⫹ 86-2164085875. E-mail: [email protected] Received 19 August 2014; revised 1 January 2015; accepted 10 January 2015
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2 Y.-Y. Wang et al.
Figure 1. Characterization of leukemia cells: (A) BM examination showed 76.5% abnormal promyelocytes with Auer rods before treatment (Wright’s staining, original magnification ⫻ 1000); (B) promyelocytes showed strong positivity for POX staining (⫻ 1000); (C) Cytogenetic analysis of R-banding was 46,XY,inv(9)(p13q13)c[18]/44∼45,XY,inv(9)(p13q13)c[cp4]/[22]. Arrows indicate breakpoint of inv(9); (D) transformation of promyelocytes to myelocytes was not obvious after ATRA treatment; (E) flow cytometric analysis: leukemia cells showed low or moderate expression of CD45 with high side scatter (SSC) accounting for 66.2%, strong positive reaction for CD13 (99.2%), CD33 (95.3%), CD117 (71.9%), MPO (98.5%) and CD56 (84.2%), negative for HLA-DR, CD34, CD38, B- or T-lineage antigen; (F) interphase FISH analysis with PML–RARA dualcolor dual-fusion translocation probe showed two separated signals (red and green), but no fusion signal in cells; (G) gel image of STAT5B–RARA fusion transcript with nested RT-PCR analysis in this patient (lane M, marker; lane 1, patient sample (402 bp); lane 2, housekeeping gene (146 bp); lane 3, no template); (H) RT-PCR sequencing results show GCT codon in STAT5B coding for alanine (Ala) and ACC codon in RARA coding for threonine (Thr), eventually forming GCC chimeric sequence at joining site which still codes for alanine (Ala); (I) sequencing analysis for STAT5B– RARA transcript. Base sequence to the right of the dotted separator line is that of exon 16 of STAT5B and to the left is that of exon 3 of RARA, and this cDNA sequencing analysis for nucleotides shows that the fusion event occurred between exon 16 of STAT5B and exon 3 of RARA; (J) schematic representation of STAT5B–RARA fusion protein shows amino acids encoded by exon 15 located between G593 and Q636, covering the first half of the SH2 domain. Amino acids encoded by exon 16 are located between Q636 and A693, covering the second half of the SH2 domain. Coiled-coil, coiled-coil domain; DBD, DNA binding domain; Linker, linker region; SH2, Src homology 2 domain; DBD, LBD, ligand binding domain.
and died of disease progression 9 months after diagnosis. For identification of the variant translocation, fluorescence in situ hybridization (FISH) was performed using the commercially available Vysis LSI PML–RARA dual-color dualfusion translocation probe according to the manufacturer’s protocols. The probe showed two RARA (green) signals on chromosome 17 and two PML (red) signals on chromosome
15, indicating that no PML–RARA fusion signal (yellow) was detected [Figure 1(F)]. Four of the variant fusion partners of RARA, namely PLZF–RARA, NPM–RARA, NuMA–RARA and STAT5B–RARA fusion genes, were tested as previously reported [6]. Interestingly, the results revealed only the presence of STAT5B–RARA fusion gene transcripts. Primers for the STAT5B–RARA first-
M/32 F/29 M/26 M/17 M/22 5 6 7 8 9
*These data have been modified from Strehl et al. [13]. APL, acute promyelocytic leukemia; M, male; F, female; FAB, French–American–British classification; DIC, disseminated intravascular coagulation: ⫺ none, ⫹ moderate, ⫹⫹ severe; WBC, white blood cell count; Hb, hemoglobin; PLT, platelet count; ATRA, all-trans retinoic acid; ATO, arsenic trioxide; NR, no response; NA, not available; DA, daunorubicin and cytarabine; CR, complete morphological remission; IA, idarubicin and cytarabine; MTN, mitoxantrone; FLAG, fludarabine, cytarabine and recombinant human granulocyte colony stimulating factor (G-CSF); CAG, cytarabine, G-CSF and aclarubicin; VP-16, etoposide; DFS, disease-free survival measured as time from disease diagnosis to treatment failure such as relapse, refractory disease, death or alive in CR at last follow-up (censored); OS, overall survival measured as time from date of disease diagnosis to death (failure) or alive at last follow-up (censored); PS, present study.
[10] [11] [12] [13] PS 46 15 6 53 9 52 9 0 24 0 (1) MTN, (2) FLAG/CR IA/CR CAG/NR, IA/NR IA ⫹ VP-16/CR IA/NR NR NR/NA NR NR NR 282 NA 94 NA 121 83 NA 73 NA 62 3.8 5.6 6.6 2.8 2.4 ⫹ ⫹ ⫹ ⫹ ⫹
M/67 M/57 M/42 M/41 1 2 3 4
M3 M3 NA M3 M3
45,X,⫺ Y,add(17)(q?) [40] 46,XY,t(10;11)(q22;q25),i(17)(q10) 46,X,⫺ Y,⫹ 11[9]/46,XY[11] 47,XY,del(9)(q?),add(17) (q12),⫹ mar1[13]/48,XY, idem,⫹ mar1[17] 46,XY 46,XX,t(3;17)(q26;q21) 46,XY 46,XY,?der(13),?der(17q)[14]/46,XY[6] 46,XY,inv(9)(p13q13)c[18]/44∼45,XY,inv(9) (p13q13)c[cp4]/[22]
⫹ ⫹⫹ ⫹⫹ ⫹
Ref.
[5] [7] [8] [9] NA 18 76 17 NA 9 75 3 NA DA/CR DA/CR IA/CR NR/NA NR/NA NR NR/NA 112 NA 64 51 118 NA 125 95 M1/M3v M3 M3 M3
6.7 23.8 3.6 77.8
OS (months) DFS (months) Induction therapy/ response ATRA/ATO PLT (⫻ 109/L) Hb (g/L) WBC (⫻ 109/L) DIC FAB Karyotype Sex/age (years) Case no.
step PCR were F1 5¢-GGTGCCATTTGCCGTGCCT-3¢ and R1 5¢-CAGCCCTCACAGGCGCTGAC-3¢. Nested PCR was then performed using the primers F2 5¢-GTTTGACGGTGTGATG GAAGTG-3¢ and R2 5¢-CTGGGCACTATCTCTTCAGAACT-3¢ with 1 μL of the first-step PCR product. Subsequently the sequencing analysis indicated that the 402 PCR fragment was the product of the STAT5B–RARA transcript [Figure 1(G)]. The GCT codon in STAT5B coding for alanine (Ala) and the ACC codon in RARA coding for threonine (Thr) eventually formed the GCC chimeric sequence at the joining site, which, however, still coded for alanine (Ala) [Figure 1(H)], hence indicating that the STAT5B–RARA fusion event is in-frame, occurring between the 16th exon of STAT5B and the third exon of RARA [Figure 1(I)]. APL is characterized by generation of the PML–RARA fusion transcript as a result of a reciprocal chromosomal translocation t(15;17)(q22;q21). There are a few reported cases harboring the typical morphological and immune-phenotypic features of APL while lacking the PML–RARA fusion transcript [5,7–13]. In these cases, RARA fused with variant alternative partner genes, and STAT5B was one of those variant partner genes. The STAT5B gene, located at 17q21.1-21.2, combines with the RARA gene to form the STAT5B–RARA fusion gene, which is the fifth fusion protein identified in APL [5]. The STAT5B–RARA fusion event is a result of a deletion of 1.9 Mb on chromosome 17q21.1-21.2 and gene rearrangement that most likely includes an inversion. Since small deletion or complex gene rearrangements are not easily seen by conventional large typing, the abnormality in chromosome 17 was not detected in our case, as well as three of other reported cases [8,10,12]. Previous reports stated that the breaking point of STAT5B was located between the 15th and 16th exons [5,7– 13], but in our case, we determined that the breaking point of STAT5B was located between the 16th and 17th exons, which, however, included not only 15 exons but also an additional 16th exon. More importantly, amino acids encoded by exon 15 are located between G593 and Q636, covering the first half of the Src homology 2 (SH2) domain, while amino acids encoded by exon 16 are located between Q636 and A693, covering the second half of the SH2 domain [Figure 1(J)]. Hence, the amino acids encoded by the novel STAT5B–RARA fusion transcript almost cover the whole SH2 domain (however, lacking three amino acids to cover the whole domain). The STAT5b protein is a member of the STAT family of transcription factors. SH2 plays an important role in signal transduction via tyrosine protein kinase. STAT5b protein can homodimerize in the cytoplasm via its phosphorylated SH2 domain, migrate to the nucleus and then bind to a gamma activated site (GAS) element. Meanwhile, the resulting fusion protein retains the RARA protein, allowing reaction via dimerization with the retinoid X receptor protein (RXRA) and eventually causing inhibition of the transcriptional activity of wild-type RARA [14]. Our patient received standard dose therapy but the outcome was ineffective. The correlation between the prognosis and the presence of the almost whole SH2 domain in the novel fusion protein is still unknown: the whole SH2 might have strengthened the combination between STAT5b –RARA protein and wild-type RARA, thereby largely inhibiting the transcriptional activity of wild-type RARA.
Table I. Characteristics of patients with APL harboring the STAT5B–RARA fusion gene transcript*.
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Letter to the Editor 3
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4 Y.-Y. Wang et al. Including our case, there are currently nine reported cases of STAT5B–RARA positive APL (Table I). The median age of the nine cases was 32 years (range 17–67 years), including one female and eight male patients. Our patient was also a male, further conforming to the male gender-biased sex ratio in STAT5B–RARA positive APL [13]. His treatment included standard doses of ATRA and ATO combined with the IA regimen. However, the patient failed to show any sign of remission. Analyzing the follow-up data of cases 3, 5, 7 and 8, the authors of case 8 (Strehl et al.) stated that the four patients were also treated with ATRA and ATO, but also did not show any response. These observations indicate that patients with STAT5B–RARA positive APL are resistant to ATRA and ATO [13]. There has not been any specific report about case 1. Case 2 attained a complete remission (CR) after one course of the daunorubicin–cytarabine (DA) regimen, but succumbed to transplant-related complications (TRCs) 18 months after diagnosis. Case 3 achieved a CR after poly-chemotherapy, but died of pulmonary hemorrhage 76 months after diagnosis. Case 4 achieved a CR with the IA regimen but died of disease progression 17 months after diagnosis. Case 5 attained a CR after two cycles of induction chemotherapy, underwent allogeneic stem cell transplant (allo-SCT) and maintained a molecular CR 46 months after diagnosis, but died of disease progression 52 months after diagnosis. Case 6 obtained a CR after a course of the IA regimen, but the disease rapidly relapsed and the patient died due to TRCs of allogeneic bone marrow transplant (allo-BMT) 15 months after diagnosis. Case 7 failed to achieve a CR even after treatment with ATRA and ATO as well as chemotherapy, and died of disease progression 6 months after diagnosis. Case 8 achieved a CR after one course of chemotherapy but succumbed to severe pulmonary infection. The estimated median disease-free survival (DFS) of the nine patients was 9 months (95% confidence interval [CI]: 0.94–17.05), while the estimated median overall survival (OS) of the nine patients was 17 months (95% CI: 12.84–21.15). Therefore, patients with STAT5B–RARA positive APL tend to have earlier disease progression and could be classified as having high-risk AML [15]. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
Supplementary material available online Tables and Figures showing further data.
References [1] de The H, Lavau C, Marchio A , et al. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 1991;66:675–684. [2] Borowitz MJ, Bene MC, Harris NL, et al. Acute leukemias of ambiguous lineage. In Swerdlow SH, Campo E, Harris NL, et al., editors. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2008:150–155. [3] De Braekeleer E, Douet-Guilbert N, De Braekeleer M, et al. RARa fusion genes in acute promyelocytic leukemia:a review. Expert Rev Hematol 2014;7:347–357. [4] Chen Y, Li S, Zhou C, et al. TBLR1 fuses to retinoid acid recepror α in a variant (3;17)(q26;q21) translocation of acute promyelocytic leukemia. Blood 2014;124:936–934. [5] Arnould C, Philippe C, Bourdon V, et al. The signal transducer and activator of transcription STAT5b gene is a new partner of retinoic acid receptor alpha in acute promyelocytic-like leukaemia. Hum Mol Genet 1999;8:1741–1749. [6] Shen Y, Zhu YM, Fan X, et al. Gene mutation patterns and their prognostic impact in a cohort of 1185 patients with acute myeloid leukemia. Blood 2011;118:5593–5603. [7] Gallagher RE, Mak S, Paietta E, et al. Identification of a second acute promyelocytic leukemia (APL) patient with the STAT5b-RARa fusion gene among PML-RARa-negative Eastern Cooperative Oncology Group (ECOG) APL Protocol Registrants. Blood 2004;104(Suppl. 1): Abstract 3005. [8] Kusakabe M, Suzukawa K, Nanmoku T, et al. Detection of the STAT5B-RARA fusion transcript in acute promyelocytic leukemia with the normal chromosome 17 on G-banding. Eur J Haematol 2008;80:444–447. [9] Iwanaga E, Nakamura M, Nanri T, et al. Acute promyelocytic leukemia harboring a STAT5B-RARA fusion gene and a G596V missense mutation in the STAT5B SH2 domain of the STAT5B-RARA . Eur J Haematol 2009;83:499–501. [10] Qiao C, Zhang SJ, Chen LJ, et al. Identification of the STAT5BRARalpha fusion transcript in an acute promyelocytic leukemia patient without FLT3, NPM1, c-Kit and C/EBPalpha mutation. Eur J Haematol 2011;86:442–446. [11] Jovanovic JV, Rennie K, Culligan D, et al. Development of realtime quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia. Front Oncol 2011;1:35. [12] Chen H, Pan J, Yao L, et al. Acute promyelocytic leukemia with a STAT5b-RARalpha fusion transcript defined by array-CGH, FISH, and RT-PCR. Cancer Genet 2012;205:327–331. [13] Strehl S, Konig M, Boztug H, et al. All-trans retinoic acid and arsenic trioxide resistance of acute promyelocytic leukemia with the variant STAT5B-RARA fusion gene. Leukemia 2013;27: 1606–1610. [14] Zelent A , Guidez F, Melnick A , et al. Translocations of the RARa gene in acute promyelocytic leukemia. Oncogene 2001; 20:7186–7203. [15] Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010;115:453–474.
Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response a
a
a
b
b
b
Yan-Yu Wang , Jie Hao , Zhan-Yun Liu , Xiang-Qin Weng , Yan Sheng , Chun-Lei Jiang , Yongb
b
b
b
b
c
Mei Zhu , Bing Chen , Shu-Min Xiong , Jun-Min Li , Qiu-Sheng Chen , Hao-yue Chen , Chun d
b
Qiao & Yu Chen a
Department of Hematology, Bei Zhan Hospital, Shanghai, China
b
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China c
Click for updates
Jingjiang People's Hospital, The Seventh Affiliated Hospital of Yangzhou University, Jingjiang, Jiangsu Province, China d
Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China Published online: 15 Apr 2015.
To cite this article: Yan-Yu Wang, Jie Hao, Zhan-Yun Liu, Xiang-Qin Weng, Yan Sheng, Chun-Lei Jiang, Yong-Mei Zhu, Bing Chen, Shu-Min Xiong, Jun-Min Li, Qiu-Sheng Chen, Hao-yue Chen, Chun Qiao & Yu Chen (2015): Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response, Leukemia & Lymphoma To link to this article: http://dx.doi.org/10.3109/10428194.2015.1007454
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Leukemia & Lymphoma, 2015; Early Online: 1–4 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2015.1007454
LETTER TO THE EDITOR
Novel STAT5B–RARA fusion transcript in acute promyelocytic leukemia: identification and treatment response Yan-Yu Wang1, Jie Hao1, Zhan-Yun Liu1, Xiang-Qin Weng2, Yan Sheng2, Chun-Lei Jiang2, Yong-Mei Zhu2, Bing Chen2, Shu-Min Xiong2, Jun-Min Li2, Qiu-Sheng Chen2, Hao-yue Chen3, Chun Qiao4 & Yu Chen2 1Department of Hematology, Bei Zhan Hospital, Shanghai, China, 2State Key Laboratory of Medical Genomics, Shanghai
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Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China, 3Jingjiang People’s Hospital, The Seventh Affiliated Hospital of Yangzhou University, Jingjiang, Jiangsu Province, China and 4Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China
A great majority of patients with acute promyelocytic leukemia (APL) have the specific translocation t(15;17) (q22;q21), leading to formation of the PML–RARA (promyelocytic leukemia–retinoic acid receptor alpha) fusion gene [1]. By degrading the PML–RARA fusion protein, targeted drugs such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) can effectively eliminate leukemic cells. Therefore, PML–RARA is the key molecular mechanism of APL leukemogenesis. In 2008, the World Health Organization (WHO) declared APL expressing the PML–RARA fusion gene as a subtype of acute myeloid leukemia (AML) with recurrent genetic abnormalities [2]. Moreover, other variant genes combined with the RARA gene have also been identified, such as promyelocytic leukemia zinc finger (PLZF, 11q23), nucleophosmin (NPM1, 5q35), nuclear mitotic apparatus (NuMA, 11q13), signal transducer and activator of transcription 5β (STAT5B, 17q21), protein kinase A regulatory subunit type (PRKAR1A, 17q24), FIP1-like 1 (FIP1L1, 4q12), BCL6 corepressor (BCOR, Xp11), oligonucleotide/ oligosaccharide-binding fold containing 2A (OBFC2A, 2q32) [3] and transducin β-like 1 X-linked receptor 1 (TBLR1, 3q26) [4] (Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/10428194.2015. 1007454). Unlike other forms, variant APL associated with STAT5B–RARA or PLZF–RARA is resistant to ATRA [5]. We herein report a case of APL associated with a novel STAT5B– RARA fusion gene transcript. A 22-year-old Chinese male patient was admitted to our hospital because of recurrent fever, and anemia for 3 months, on 18 January 2013. Physical examination revealed severe anemia without obvious skin petechiae. Routine blood test showed a white blood cell count (WBC) of 2.37 ⫻ 109/L, hemoglobin (Hb) of 50 g/L and platelet count (Plt) of 125 ⫻ 109/L, while the coagulation test showed that fibrinogen (Fg) was 0.6 g/L. The bone marrow (BM)
smear revealed markedly increased cellularity, characterized by 76.5% abnormal promyelocytes [Figure 1(A)], while cytochemical analysis demonstrated a strong positivity for peroxidase (POX) staining in abnormal promyelocytes [Figure 1(B)]. RHG (R-bands by heating using Giemsa) banding karyotype was 46,XY,inv(9)(p13q13) c[18]/44∼45,XY,inv(9)(p13q13)c[cp4]/[22] [Figure 1(C)]. Flow cytometric analysis of the leukemia cells showed low or moderate expression of CD45 with high side scatter (SSC) accounting for 66.2%, with strong expression of myeloperoxidase (MPO), CD117, CD13, CD33 and CD56, but not expression of CD34, CD11b, CD38, human leukocyte antigen (HLA)-DR and B- or T-lineage [Figure 1(E)]. However, reverse transcriptase-polymerase chain reaction (RT-PCR) did not detect the PML–RARA fusion transcript. FLT3-ITD, FLT3-TKD, NPM, N-RAS, CEBPA, DNMT3A and N-RAS were found to be wild type, while MLL–AF9, MLL–AF6, MLL–ENL, MLL–AF10, MLL–AF17 and MLL–ELL did not indicate any rearrangement abnormality (data not shown). The patient was initially treated with ATRA (25 mg/m2/ day, days 1–28). After 10 days, the patient manifested a slight increase in WBC and Fg, but the BM aspirate after 13 days did not show obvious transformation of promyelocytes to myelocytes [Figure 1(D)]. As seen from Supplementary Figure 1 to be found online at http://informahealthcare. com/doi/abs/10.3109/10428194.2015.1007454, the WBC and Fg at day 23 reached 4.9 ⫻ 109/L and 1.7 g/L, respectively. After obtaining informed consent, the patient was started on ATO (10 mg/day, days 11–18) and the IA regimen (idarubicin 10 mg/m2/day, days 25–27 and cytarabine 100 mg/m2/ day, days 25–32). BM aspiration on days 35 and 75 revealed 59% and 30% of leukemia cells, respectively (Supplementary Table II to be found online at http://informahealthcare. com/doi/abs/10.3109/10428194.2015.1007454). Nevertheless, the patient refused to undergo further chemotherapy,
Correspondence: Yu Chen, State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Tel: ⫹ 86-1264370045. Fax: ⫹ 86-2164085875. E-mail: [email protected] Received 19 August 2014; revised 1 January 2015; accepted 10 January 2015
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Figure 1. Characterization of leukemia cells: (A) BM examination showed 76.5% abnormal promyelocytes with Auer rods before treatment (Wright’s staining, original magnification ⫻ 1000); (B) promyelocytes showed strong positivity for POX staining (⫻ 1000); (C) Cytogenetic analysis of R-banding was 46,XY,inv(9)(p13q13)c[18]/44∼45,XY,inv(9)(p13q13)c[cp4]/[22]. Arrows indicate breakpoint of inv(9); (D) transformation of promyelocytes to myelocytes was not obvious after ATRA treatment; (E) flow cytometric analysis: leukemia cells showed low or moderate expression of CD45 with high side scatter (SSC) accounting for 66.2%, strong positive reaction for CD13 (99.2%), CD33 (95.3%), CD117 (71.9%), MPO (98.5%) and CD56 (84.2%), negative for HLA-DR, CD34, CD38, B- or T-lineage antigen; (F) interphase FISH analysis with PML–RARA dualcolor dual-fusion translocation probe showed two separated signals (red and green), but no fusion signal in cells; (G) gel image of STAT5B–RARA fusion transcript with nested RT-PCR analysis in this patient (lane M, marker; lane 1, patient sample (402 bp); lane 2, housekeeping gene (146 bp); lane 3, no template); (H) RT-PCR sequencing results show GCT codon in STAT5B coding for alanine (Ala) and ACC codon in RARA coding for threonine (Thr), eventually forming GCC chimeric sequence at joining site which still codes for alanine (Ala); (I) sequencing analysis for STAT5B– RARA transcript. Base sequence to the right of the dotted separator line is that of exon 16 of STAT5B and to the left is that of exon 3 of RARA, and this cDNA sequencing analysis for nucleotides shows that the fusion event occurred between exon 16 of STAT5B and exon 3 of RARA; (J) schematic representation of STAT5B–RARA fusion protein shows amino acids encoded by exon 15 located between G593 and Q636, covering the first half of the SH2 domain. Amino acids encoded by exon 16 are located between Q636 and A693, covering the second half of the SH2 domain. Coiled-coil, coiled-coil domain; DBD, DNA binding domain; Linker, linker region; SH2, Src homology 2 domain; DBD, LBD, ligand binding domain.
and died of disease progression 9 months after diagnosis. For identification of the variant translocation, fluorescence in situ hybridization (FISH) was performed using the commercially available Vysis LSI PML–RARA dual-color dualfusion translocation probe according to the manufacturer’s protocols. The probe showed two RARA (green) signals on chromosome 17 and two PML (red) signals on chromosome
15, indicating that no PML–RARA fusion signal (yellow) was detected [Figure 1(F)]. Four of the variant fusion partners of RARA, namely PLZF–RARA, NPM–RARA, NuMA–RARA and STAT5B–RARA fusion genes, were tested as previously reported [6]. Interestingly, the results revealed only the presence of STAT5B–RARA fusion gene transcripts. Primers for the STAT5B–RARA first-
M/32 F/29 M/26 M/17 M/22 5 6 7 8 9
*These data have been modified from Strehl et al. [13]. APL, acute promyelocytic leukemia; M, male; F, female; FAB, French–American–British classification; DIC, disseminated intravascular coagulation: ⫺ none, ⫹ moderate, ⫹⫹ severe; WBC, white blood cell count; Hb, hemoglobin; PLT, platelet count; ATRA, all-trans retinoic acid; ATO, arsenic trioxide; NR, no response; NA, not available; DA, daunorubicin and cytarabine; CR, complete morphological remission; IA, idarubicin and cytarabine; MTN, mitoxantrone; FLAG, fludarabine, cytarabine and recombinant human granulocyte colony stimulating factor (G-CSF); CAG, cytarabine, G-CSF and aclarubicin; VP-16, etoposide; DFS, disease-free survival measured as time from disease diagnosis to treatment failure such as relapse, refractory disease, death or alive in CR at last follow-up (censored); OS, overall survival measured as time from date of disease diagnosis to death (failure) or alive at last follow-up (censored); PS, present study.
[10] [11] [12] [13] PS 46 15 6 53 9 52 9 0 24 0 (1) MTN, (2) FLAG/CR IA/CR CAG/NR, IA/NR IA ⫹ VP-16/CR IA/NR NR NR/NA NR NR NR 282 NA 94 NA 121 83 NA 73 NA 62 3.8 5.6 6.6 2.8 2.4 ⫹ ⫹ ⫹ ⫹ ⫹
M/67 M/57 M/42 M/41 1 2 3 4
M3 M3 NA M3 M3
45,X,⫺ Y,add(17)(q?) [40] 46,XY,t(10;11)(q22;q25),i(17)(q10) 46,X,⫺ Y,⫹ 11[9]/46,XY[11] 47,XY,del(9)(q?),add(17) (q12),⫹ mar1[13]/48,XY, idem,⫹ mar1[17] 46,XY 46,XX,t(3;17)(q26;q21) 46,XY 46,XY,?der(13),?der(17q)[14]/46,XY[6] 46,XY,inv(9)(p13q13)c[18]/44∼45,XY,inv(9) (p13q13)c[cp4]/[22]
⫹ ⫹⫹ ⫹⫹ ⫹
Ref.
[5] [7] [8] [9] NA 18 76 17 NA 9 75 3 NA DA/CR DA/CR IA/CR NR/NA NR/NA NR NR/NA 112 NA 64 51 118 NA 125 95 M1/M3v M3 M3 M3
6.7 23.8 3.6 77.8
OS (months) DFS (months) Induction therapy/ response ATRA/ATO PLT (⫻ 109/L) Hb (g/L) WBC (⫻ 109/L) DIC FAB Karyotype Sex/age (years) Case no.
step PCR were F1 5¢-GGTGCCATTTGCCGTGCCT-3¢ and R1 5¢-CAGCCCTCACAGGCGCTGAC-3¢. Nested PCR was then performed using the primers F2 5¢-GTTTGACGGTGTGATG GAAGTG-3¢ and R2 5¢-CTGGGCACTATCTCTTCAGAACT-3¢ with 1 μL of the first-step PCR product. Subsequently the sequencing analysis indicated that the 402 PCR fragment was the product of the STAT5B–RARA transcript [Figure 1(G)]. The GCT codon in STAT5B coding for alanine (Ala) and the ACC codon in RARA coding for threonine (Thr) eventually formed the GCC chimeric sequence at the joining site, which, however, still coded for alanine (Ala) [Figure 1(H)], hence indicating that the STAT5B–RARA fusion event is in-frame, occurring between the 16th exon of STAT5B and the third exon of RARA [Figure 1(I)]. APL is characterized by generation of the PML–RARA fusion transcript as a result of a reciprocal chromosomal translocation t(15;17)(q22;q21). There are a few reported cases harboring the typical morphological and immune-phenotypic features of APL while lacking the PML–RARA fusion transcript [5,7–13]. In these cases, RARA fused with variant alternative partner genes, and STAT5B was one of those variant partner genes. The STAT5B gene, located at 17q21.1-21.2, combines with the RARA gene to form the STAT5B–RARA fusion gene, which is the fifth fusion protein identified in APL [5]. The STAT5B–RARA fusion event is a result of a deletion of 1.9 Mb on chromosome 17q21.1-21.2 and gene rearrangement that most likely includes an inversion. Since small deletion or complex gene rearrangements are not easily seen by conventional large typing, the abnormality in chromosome 17 was not detected in our case, as well as three of other reported cases [8,10,12]. Previous reports stated that the breaking point of STAT5B was located between the 15th and 16th exons [5,7– 13], but in our case, we determined that the breaking point of STAT5B was located between the 16th and 17th exons, which, however, included not only 15 exons but also an additional 16th exon. More importantly, amino acids encoded by exon 15 are located between G593 and Q636, covering the first half of the Src homology 2 (SH2) domain, while amino acids encoded by exon 16 are located between Q636 and A693, covering the second half of the SH2 domain [Figure 1(J)]. Hence, the amino acids encoded by the novel STAT5B–RARA fusion transcript almost cover the whole SH2 domain (however, lacking three amino acids to cover the whole domain). The STAT5b protein is a member of the STAT family of transcription factors. SH2 plays an important role in signal transduction via tyrosine protein kinase. STAT5b protein can homodimerize in the cytoplasm via its phosphorylated SH2 domain, migrate to the nucleus and then bind to a gamma activated site (GAS) element. Meanwhile, the resulting fusion protein retains the RARA protein, allowing reaction via dimerization with the retinoid X receptor protein (RXRA) and eventually causing inhibition of the transcriptional activity of wild-type RARA [14]. Our patient received standard dose therapy but the outcome was ineffective. The correlation between the prognosis and the presence of the almost whole SH2 domain in the novel fusion protein is still unknown: the whole SH2 might have strengthened the combination between STAT5b –RARA protein and wild-type RARA, thereby largely inhibiting the transcriptional activity of wild-type RARA.
Table I. Characteristics of patients with APL harboring the STAT5B–RARA fusion gene transcript*.
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Letter to the Editor 3
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4 Y.-Y. Wang et al. Including our case, there are currently nine reported cases of STAT5B–RARA positive APL (Table I). The median age of the nine cases was 32 years (range 17–67 years), including one female and eight male patients. Our patient was also a male, further conforming to the male gender-biased sex ratio in STAT5B–RARA positive APL [13]. His treatment included standard doses of ATRA and ATO combined with the IA regimen. However, the patient failed to show any sign of remission. Analyzing the follow-up data of cases 3, 5, 7 and 8, the authors of case 8 (Strehl et al.) stated that the four patients were also treated with ATRA and ATO, but also did not show any response. These observations indicate that patients with STAT5B–RARA positive APL are resistant to ATRA and ATO [13]. There has not been any specific report about case 1. Case 2 attained a complete remission (CR) after one course of the daunorubicin–cytarabine (DA) regimen, but succumbed to transplant-related complications (TRCs) 18 months after diagnosis. Case 3 achieved a CR after poly-chemotherapy, but died of pulmonary hemorrhage 76 months after diagnosis. Case 4 achieved a CR with the IA regimen but died of disease progression 17 months after diagnosis. Case 5 attained a CR after two cycles of induction chemotherapy, underwent allogeneic stem cell transplant (allo-SCT) and maintained a molecular CR 46 months after diagnosis, but died of disease progression 52 months after diagnosis. Case 6 obtained a CR after a course of the IA regimen, but the disease rapidly relapsed and the patient died due to TRCs of allogeneic bone marrow transplant (allo-BMT) 15 months after diagnosis. Case 7 failed to achieve a CR even after treatment with ATRA and ATO as well as chemotherapy, and died of disease progression 6 months after diagnosis. Case 8 achieved a CR after one course of chemotherapy but succumbed to severe pulmonary infection. The estimated median disease-free survival (DFS) of the nine patients was 9 months (95% confidence interval [CI]: 0.94–17.05), while the estimated median overall survival (OS) of the nine patients was 17 months (95% CI: 12.84–21.15). Therefore, patients with STAT5B–RARA positive APL tend to have earlier disease progression and could be classified as having high-risk AML [15]. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
Supplementary material available online Tables and Figures showing further data.
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