Letters to the Editor 1075 References patients express virtually identical immunoglobulins. Blood 2004; 104: 2499–2504. 1 Duke VM, Gandini D, Sherrington PD, Lin K, Heelan B, 5 Widhopf II GF, Goldberg CJ, Toy TL, Rassenti LZ, Wierda WG, Amlot P et al. V(H) usage differs in germline and mutated Byrd JC et al. Non-stochastic pairing of immunoglobulin heavy B-cell chronic lymphocytic leukemia. Haematologica 2003; 88: and light chains expressed by chronic lymphocytic leukemia 1259–1271. B-cells is predicated on the heavy chain CDR3. Blood 2007 2 Matthews C, Catherwood MA, Morris TC, Alexander HD. V(H)3-48 [E-pub ahead of print]. and V(H)3-53, as well as V(H)3-21, gene rearrangements define 6 Stamatopoulos K, Belessi C, Hadzidimitriou A, Smilevska T, unique subgroups in CLL and are associated with biased lambda Kalagiakou E, Hatzi K et al. Immunoglobulin light chain repertoire light chain restriction, homologous LCDR3 sequences and poor in chronic lymphocytic leukemia. Blood 2005; 106: 3575–3583. prognosis. Leuk Res 2007; 31: 231–234. 7 Giudicelli V, Duroux P, Ginestoux C, Folch G, Jabado-Michaloud J, 3 Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A, Willander K Chaume D et al. IMGT/LIGM-DB, the IMGT comprehensive data- et al. Subsets with restricted immunoglobulin gene rearrange- base of immunoglobulin and T-cell receptor nucleotide sequences. ment features indicate a role for antigen selection in the develop- Nucleic Acids Res 2006; 34 (Database issue): D781–D784. ment of chronic lymphocytic leukemia. Blood 2004; 104: 8 Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL, Kolitz J 2879–2885. et al. Multiple distinct sets of stereotyped antigen receptors indicate 4 Widhopf II GF, Rassenti LZ, Toy TL, Gribben JG, Wierda WG, Kipps a role for antigen in promoting chronic lymphocytic leukemia. J Exp TJ. Chronic lymphocytic leukemia B-cells of more than 1% of Med 2004; 200: 519–525.

Characterization of with PTPN11 : the mutation is closely associated with NPM1 mutation but inversely related to FLT3/ITD

Leukemia (2008) 22, 1075–1078; doi:10.1038/sj.leu.2405005; were also found in patients with juvenile myelo- published online 1 November 2007 monocytic leukemia, , childhood AML and acute lymphoblastic leukemia. To the best of our knowl- Acute myeloid leukemia (AML) is a relentless hematological edge, however, the characteristics of adult AML with PTPN11 malignancy characterized by overproduction of hematopoietic mutation have not been comprehensively studied yet, and the precursor cells with impaired differentiation. Increasing evi- gene alteration that is cooperative with the PTPN11 mutation in dences suggest that the development of AML is a multistep the pathogenesis of AML remains largely unknown. In this study, process that requires collaboration of at least two classes of we investigated prevalence and clinical relevance of PTPN11 mutations: class I mutations activate signal-transduction path- mutations and their association with other genetic changes from way and confer a proliferation advantage to hematopoietic cells, 272 consecutive patients with primary AML. PTPN11 mutations and the class II mutations affect transcription factors and serve were detected by PCR and direct sequencing using the primers primarily to impair hematopoietic differentiation.1,2 covering exons 3 and 13.4 Mutational analyses of N-RAS, The PTPN11 gene encodes human SHP2, a nonreceptor K-RAS, NPM1, FLT3/ITD, FLT3/TKD, CEBPA, KIT, AML1 and tyrosine phosphatase on 12q24, which partici- MLL/PTD were performed as previously described.5,6 pates in signal events downstream of the receptors of growth The correlation of genetic mutations with clinical and factors and cytokines and plays a key role in the proliferation laboratory characteristics is summarized in Supplementary and survival of hematopoietic cells. Germ line PTPN11 Table 1. Among the 272 AML patients recruited, 165 were mutations in patients afflicted with Noonan syndrome were first males and 107 were females with a median age of 52.5 years reported by Tartaglia et al.3 Subsequently, somatic PTPN11 (range, 19–90). Overall, 15 PTPN11 mutations were detected in

Table 1 PTPN 11 mutations for 14 adult AML patients

Patient no. Age (years) Gender FAB Nucleotide Amino-acid Karyotype Domain Other gene substitution change mutation

411 39 Male M1 G214Aa Ala72Thr Normal N-SH2 F 449 60 Male M1 C218Ta Thr73Ile cplx, À5, del(7q) N-SH2 AML1 474 47 Male M1 G214Aa Ala72Thr Normal N-SH2 NPM1 696 78 Male M4 G226Aa Glu76Lys Normal N-SH2 F 702 47 Male M4 G226Aa Glu76Lys Normal N-SH2 F 709 72 Male M2 C215Ta Ala72Val Normal N-SH2 NPM1 735 64 Male M5b A227Ga Glu76Gly Normal N-SH2 MLL/PTD 791 52 Male M4 G214Aa Ala72Thr Normal N-SH2 NPM1, NRAS 811 64 Male M5b C215Ta Ala72Val No mitosis N-SH2 NPM1 815 75 Female M4 G226Ca Glu76Gly Normal N-SH2 NPM1 T220A Leu74Met N-SH2 859 71 Male M2 T211C Phe71Leu À7 N-SH2 AML1 860 50 Female M4 G205Aa Glu69Lys Normal N-SH2 NPM1 357 75 Male M2 G1508Ca Gly503Ala Normal PTP AML1 699 74 Male M0 G1508Ca Gly503Ala del (5q) PTP F Abbreviations: AML, acute myeloid leukemia; cplx, complex; MLL/PTD, partial tandem duplication of MLL. aThese gene mutations have been reported previously.7

Leukemia Letters to the Editor 1076

Figure 1 Representative sequence changes at initial diagnosis and subsequent complete remission for two patients (no. 859 and 860). (a) PTPN11 T211C mutation from patient no. 859 at diagnosis (upper panel); PTPN11 sequence at complete remission (lower panel). (b) PTPN11 G205A mutation from patient no. 860 at diagnosis (upper panel); PTPN11 sequence at complete remission (lower panel). Arrows indicate the location of the mutation.

Table 2 Comparison of other genetic alterations between AML patients with and without PTPN11 mutations

Gene Total patients PTPN11-mutated patients PTPN11-wild patients P-value

No. studied No. altered (%) No. studied No. altered (%) No. studied No. altered (%)

CEBPA 269 38 (14.1) 14 0 255 38 (14.9) 0.231 FLT3/ITD 271 67 (24.7) 14 0 257 67 (26.1) 0.025 FLT3/TKD 267 16 (6.0) 13 0 254 16 (6.3) 40.9999 AML1 268 36 (13.4) 14 3 (21.4) 254 33 (13.0) 0.412 NPM 266 57 (21.4) 13 6 (46.2) 253 51 (20.2) 0.037 NRAS 271 33 (12.2) 14 1 (7.1) 257 32 (12.5) 40.9999 KRAS 271 8 (3.0) 14 0 257 8 (3.1) 40.9999 KIT 270 4 (1.5) 14 0 256 4 (1.6) 40.9999 MLL/PTD 263 13 (4.9) 14 1 (7.1) 249 12 (4.8) 0.517 Abbreviations: AML, acute myeloid leukemia; FLT3/ITD, internal tandem duplication of FLT3; FLT3/TKD, activation loop of the second tyrosine kinase domain of FLT3; MLL/PTD, partial tandem duplication of MLL.

14 (5.1%) patients. These PTPN11 alterations were exclusively who had paired bone marrow samples for analysis (Figure 1). missense mutations (Table 1) affecting residues located within The mutation occurred more frequently in FAB M4 or M5 the N-SH2 (12 cases) and PTP domains (2 cases). The PTPN11 subtypes than in other subtypes (10.4 vs 3.4%, P ¼ 0.049). The mutation disappeared at complete remission in the four patients AML patients with PTPN11 mutations were older than those

Leukemia Letters to the Editor 1077 without this mutation (median 64 vs 51 years, P ¼ 0.036) and Total AML Patients had a trend of higher median white blood cell counts at 1.0 diagnosis than those without (51.2 Â 109 vs 19.4 Â 109 per liter, P ¼ 0.069). Apart from this, there were no differences in other clinical parameters, including hemoglobin, platelet count and 0.8 lactate dehydrogenase value between patients with and without mutations. 0.6 As best we are aware, there was a relative paucity of relevant cytogenetic data for AML patients with PTPN11 mutation in the PTPN11-wild 0.4 literature, such that the relationship between the PTPN11 mutation and certain cytogenetic change remains unknown. In PTPN11-mutated this study, the PTPN11 mutation was found to be closely Proportion Surviving 0.2 associated with a normal karyotype. Among those patients who P=0.284 exhibited a normal karyotype, 8.0% featured a PTPN11 0.0 mutation, compared with 2.1% of those individuals who demonstrated chromosomal abnormalities (P ¼ 0.044). No 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 PTPN11 mutation was seen for patients with t(15;17), t(8;21) Time (months) or inv(16). For immunophenotyping, the PTPN11 mutation was closely associated with expression of CD14, but not with AML patients without NPM1 mutation ¼ expression of other markers, on leukemia cells (P 0.026, data 1.0 not shown). This is the first report to correlate PTPN11 mutations with 0.8 other gene alterations among adult AML patients. While the overall incidence of the FLT3/ITD was 24.7% for our patients, 0.6 none of the 14 patients who demonstrated PTPN11 mutations PTPN11-wild revealed an FLT3/ITD (Table 2). In other words, the PTPN11 and 0.4 FLT3/ITD mutations were mutually exclusive (P ¼ 0.025). On the other hand, 6 (46.2%) of the 13 patients with PTPN11 PTPN11-mutated 0.2 mutations who had mutation analysis of NPM1 gene showed Proportion Surviving concurrent NPM1 mutations (Tables 1 and 2), including one (no. P=0.001 0.0 791) with an extra NRAS mutation, compared with 51 (20.2%) of the patients without PTPN11 mutations did so (P ¼ 0.037). In 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 other words, 6 (10.5%) of the 57 patients with NPM1 mutations Time (months) showed PTPN11 mutations. None of the 14 patients who expressed PTPN11 mutations revealed any simultaneous altera- AML patients with NPM1 mutation tion of KIT, CEBPA, KRAS or FLT3/TKD, although three of these patients revealed AML1 mutations and the other one had MLL/ 1.0 PTD (Tables 1 and 2). These findings are compatible with a two- PTPN11-mutated hit theory of leukemogenesis and suggest that PTPN11 mutants 0.8 (class I mutation) may cooperate with class II mutations or NPM1 mutations, but usually not with class I mutations in the 0.6 multistep pathogenesis of AML. 0.4 An important question here is whether mutant PTPN11 significantly affects the treatment response or survival of AML 0.2 PTPN11-wild patients. Tartaglia et al. revealed that the PTPN11 mutation Proportion Surviving constituted no prognostic significance for pediatric patients with P=0.738 0.0 AML. In this study, similar findings were found for adult AML patients. The complete remission rate (75 vs 62%), the overall 0.0 20.0 40.0 60.0 80.0 100.0 120.0 survival (median 13±8.95 vs 25.5±6.54 months, Figure 2a) Time (months) and relapse-free survival (19±3.27 vs 25±14.04 months) were similar between patients with and without PTPN11 mutations. Figure 2 Overall survival according to PTPN11 status. (a) There However, subgroup analysis did reveal that the PTPN11 was no survival difference in overall survival between adult acute mutation was a poor risk factor for overall survival of AML myeloid leukemia (AML) patients with and those without PTPN11 patients who did not have NPM1 mutations (P ¼ 0.001, mutations (P ¼ 0.284, as revealed by the logrank test). (b) Among the patients not featuring the NPM1 mutation, patients that did express Figure 2b). It is possible that a cooperative gene alteration, such PTPN11 mutations revealed a significantly shorter overall survival than as the AML1 mutation or MLL/PTD, may influence the overall those without PTPN11 mutations. (P ¼ 0.001 by the logrank test). outcome for such patients. No such difference was apparent (c) No survival difference was observed between patients with among patients without NPM1 mutations (P ¼ 0.738, Figure 2c). and without PTPN11 mutations among AML patients with the In short, we demonstrated that although PTPN11 mutations NPM1 mutation. (P ¼ 0.738 by the log rank test). were not a frequent molecular event in adult patients with primary AML, those patients with this gene mutation did reveal leukemia cells. It also implied that the PTPN11 mutation, in some distinct clinical and biological characteristics. Typically, cooperation with the NPM1 mutation or a class II mutation, may they were usually elderly persons and had higher incidence of play a role for leukemogenesis in a proportion of adult patients M4/M5 subtype, CD14 expression, a normal karyotype and afflicted with AML. Further comprehensive studies are needed to concurrent NPM1 mutation, but no alteration of the FLT3 in disclose the molecular mechanisms of the interactions between

Leukemia Letters to the Editor 1078 PTPN11 mutants and other genetic alterations as regards the References pathogenesis of AML. 1 Frohling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies: pathogenetic and clinical implications. J Clin Oncol Acknowledgements 2005; 23: 6285–6295. 2 Kelly LM, Gilliland DG. Genetics of myeloid leukemias. Annu Rev This work was sponsored partially by grants from the National Genomics Hum Genet 2002; 3: 179–198. Science Council of Taiwan, NSC 95-2314-B-002-243-MY2 and 3 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H the Department of Medical Research at the National Taiwan et al. Mutations in PTPN11, encoding the tyrosine phosphatase University Hospital, Taiwan. SHP-2, cause Noonan syndrome. Nat Genet 2001; 29: 465–468. 4 Chen CY, Lin LI, Tang JL, Tsay W, Chang HH, Yeh YC et al. H-A Hou1,2, W-C Chou3, L-I Lin4, C-Y Chen2, J-L Tang2, Acquisition of JAK2, PTPN11, and RAS mutations during disease M-H Tseng2, C-F Huang2, R-J Chiou2, F-Y Lee3, M-C Liu3 and progression in primary myelodysplastic syndrome. Leukemia 2006; H-F Tien2 20: 1155–1158. 1Division of Hematology, Department of Internal Medicine, 5 Chou WC, Tang JL, Lin LI, Yao M, Tsay W, Chen CY et al. National Taiwan University Hospital Yun-Lin Branch, College Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolu- of Medicine, National Taiwan University, Douliou, Taiwan; 2 tion. Cancer Res 2006; 66: 3310–3316. Division of Hematology, Department of Internal Medicine, 6 Chen CY, Lin LI, Tang JL, Ko BS, Tsay W, Chou WC et al. RUNX1 National Taiwan University Hospital, College of Medicine, gene mutation in primary myelodysplastic syndromeFthe mutation National Taiwan University, Taipei, Taiwan; 3 can be detected early at diagnosis or acquired during disease Department of Laboratory Medicine, National Taiwan progression and is associated with poor outcome. Br J Haematol University Hospital, College of Medicine, National Taiwan 2007; 139: 405–414. University, Taipei, Taiwan and 7 Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, Cordeddu 4 Department of Clinical Laboratory Sciences and Medical V et al. Diversity and functional consequences of germline and Biotechnology, College of Medicine, National Taiwan somatic PTPN11 mutations in human disease. Am J Hum Genet University, Taipei, Taiwan 2006; 78: 279–290.

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Childhood T-cell non-Hodgkin’s lymphoma, colorectal carcinoma and brain tumor in association with cafe´-au-lait spots caused by a novel homozygous PMS2 mutation

Leukemia (2008) 22, 1078–1080; doi:10.1038/sj.leu.2405008; MLH1, MSH2, MSH6 or PMS2, cause Lynch syndrome, a well- published online 15 November 2007 characterized dominant cancer syndrome associated with hereditary non-polyposis colorectal cancer, and other malig- nancies (reviewed in Peltomaki3). Tumors arising in these Mismatch repair (MMR) is an essential process that maintains individuals result from somatic loss of the remaining wild type genome integrity. The likelihood of replication errors in MLH1, MSH2, MSH6 or PMS2 allele, which leads to impaired newly synthesized DNA is dramatically decreased by the MMR and accumulation of somatic mutations. action of a highly conserved MMR system. Mismatches are In contrast to individuals with Lynch syndrome who harbor a recognized by one of two heterodimers, MSH2 Á MSH6 (MutSa) heterozygous mutant MMR gene allele, rare cases with biallelic or MSH2 Á MSH3 (MutSb), that are believed to travel with the germline mutations have been reported. Biallelic mutations of replication fork. MutSa is involved in the repair of base/base MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood mismatches and misalignments of one or two nucleotides, while cancer syndrome characterized by cafe´-au-lait spots and other the less abundant MutSb recognizes larger mismatches. MutSa signs of neurofibromatosis type 1, childhood brain tumors, (or MutSb) recruits a second heterodimer, MLH1 Á PMS2 leukemia, lymphoma, colorectal cancer and other malignancies (referred to as MutLa), that possesses a probable endonuclease (reviewed in Felton et al.4). This syndrome is referred to as 5 active site, which has been localized to a PMS2 DQHA(X)2- childhood cancer syndrome, mismatch repair deficiency 6 7 E(X)4E motif. This endonuclease active site enables MutLa to (MMR-D) syndrome or Lynch III syndrome. To date, 14 introduce random nicks at sites spanning the mismatch. families with a biallelic PMS2 lesion have been described,5,8–17 Subsequent loading of EXO1 at the 50 side of the mismatch and PMS2 deficient mice have been shown to be at high tumor leads to activation of its 50-to-30 exonuclease activity, resulting (mainly lymphoma) risk.18 in removal of the error-containing DNA fragment. The repara- Herein, we report on a 6-year-old Turkish girl with multiple tion process is completed by polymerase d and its cofactors, cafe´-au-lait spots presenting with severe respiratory distress. proliferation cell nuclear antigen and replication factor C Radiologic examination revealed a large mediastinal mass, that fill in the single-stranded gap. Finally, ligase I seals the pleural effusions and bilateral infiltration of both kidneys. The remaining nick (Figure 1; reviewed in Jiricny1). In addition to diagnosis of T-cell lymphoblastic lymphoma was established by DNA repair activity, the MMR system is also involved in morphologic examination of pleural fluid showing lymphoblasts apoptotic response to a variety of DNA damaging agents and flow cytometry revealing 490% of cells positive for CD2, (reviewed in Jiricny2). CD3, CD5, CD7, CD8 and TdT with coexpression of CD13. Heterozygous germline loss-of-function mutations of the Cytogenetic analysis of these cells showed a normal karyotype. encoding the crucial components of this MMR system, Analyses of bone marrow cells did not uncover blast infiltration.

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