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Clinical Cancer Research Vol. 12, 3043-3049, May 15, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

NOTCH1 Mutations in T-Cell Acute Lymphoblastic Leukemia: Prognostic Significance and Implication in Multifactorial Leukemogenesis

Yong-Mei Zhu1, Wei-Li Zhao1, Jian-Fei Fu1, Jing-Yi Shi1, Qin Pan1, Jiong Hu1, Xiao-Dong Gao1, Bing Chen1, Jun-Min Li1, Shu-Min Xiong1, Long-Jun Gu2, Jing-Yi Tang2, Hui Liang2, Hui Jiang3, Yong-Quan Xue4, Zhi-Xiang Shen1, Zhu Chen1 and Sai-Juan Chen1

Authors' Affiliations: 1 State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Shanghai Rui Jin Hospital, 2 Department of Hematology/Oncology, Shanghai Children's Medical Center and Shanghai Xin Hua Hospital, and 3 Department of Hematology, Shanghai Children's Hospital, Medical School of Shanghai Jiao Tong University, Shanghai, China; and 4 Jiangsu Institute of Hematology, Leukemia Research Unit, The First Hospital affiliated to Suzhou University, Medical School of Suzhou University, Suzhou, China

Requests for reprints: Wei-Li Zhao, Shanghai Institute of Hematology, Shanghai Rui-Jin Hospital, Medical School of Shanghai Jiao Tong University, 197 Rui Jin Er Road, Shanghai 200025, China. Phone: 86-21-6437-7859; Fax: 86-21-6474-3206; E-mail: weili_zhao_sih{at}yahoo.com.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients with T-ALL.

Experimental Design: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/biological data of the patients.

Results: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (≤18 years), possibly due to the relatively good overall response of childhood T-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11.

Conclusion: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant disease of thymocytes, accounting for 10% to 15% of pediatric and 25% of adult ALL cases. Although the outcome of T-ALL has improved dramatically with current therapy, significant challenges remain, including understanding of the factors that contribute to the malignant behavior of these leukemic cells and developing subsequently optimal biomarkers to predict prognosis in T-ALL patients (1).

Oncogenic transcription factors are often aberrantly expressed in T-ALL and related to leukemia development (2). Initially identified from chromosomal translocations where fusion genes are generated, disruption of normal expression control of transcription factors may present irrespectively to these rearrangements and lead to dysregulation of differentiation, self-renewal, and proliferation of hematopoietic stem cells.

NOTCH signaling controls T-cell fate decisions and regulates normal T-cell development (3). NOTCH1, as an important receptor of NOTCH family, was first discovered through its involvement in a t(7;9)(q34;q34) chromosomal translocation seen in T-ALL and was subsequently proved essential for T-cell leukemogenesis (4, 5). Mice reconstituted with hematopoietic progenitor cells expressing activated NOTCH1 allele develop T-cell leukemia (5, 6) whereas blockade of NOTCH pathway suppresses the growth and survival of NOTCH1-transformed T-ALL cells (7). Recently, gain-of-function NOTCH1 mutations were reported in T-ALL patients, confirming the pathogenetic role of NOTCH1 activation on T-ALL (8). However, clinical significance of NOTCH1 mutations, especially their effect on prognosis, remains to be investigated.

In this study, we evaluate the clinical significance and prognostic value of the NOTCH1 mutation in T-ALL patients. Moreover, because leukemia development involves multiple processes combining abnormality at both transcriptional regulation and cytoplasmic signaling levels, as already shown in acute and chronic myeloid leukemia models (9, 10), we raised the same question for NOTCH1 mutations and other oncogenic transcription factors in T-ALL. By reverse transcription-PCR, the expression of three additional oncogenes specifically involved in T-ALL, homeobox genes HOX11 and HOX11L2, as well as SIL-TAL1 fusion gene (11), were assessed to investigate their possible association with NOTCH1 mutation on disease outcome in T-ALL.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patients. Seventy-seven newly diagnosed T-ALL patients (61 male and 16 female) who achieved complete remission after treatment and subsequently possessed follow-up data were enrolled in this study, including 53 pediatric patients (≤18 years; median, 10 years) and 24 adult patients (>18 years; median, 24 years). The main characteristics of these patients are summarized in Table 1 . T-cell lineage was defined by the presence of the T-cell antigen CD3 either on the cell surface or in the cytoplasm, with at least two of the B-cell markers (CD19, CD20, CD22, CD79a) and three of the myeloid markers (CD11b, CD13, CD33, myeloperoxidase) tested negative.


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  		Table 1. Patient characteristics and NOTCH1 mutation

 
For adult patients, the induction course was VDCP regimen administered over a 4-week period (vincristine 1.5 mg/m2, days 1, 8, 15, and 22; daunorubicin 30 mg/m2 or idarubicin 9 mg/m2, days 1-3; cyclophosphamide 800 mg/m2, days 1 and 8; prednisone 60 mg/m2, days 1-28). A supplement dose of 6,000 IU/m2 L-asparaginase every other day was added on days 15 to 29 when bone marrow blasts persisted >5% on day 14. The patients then received a course combining cytorabine (1 g/m2 every 12 hours, days 1-4) with mitoxantrone (10 mg/m2, days 3-5).

For pediatric patients, the conventional protocol for remission induction was VDLP regimen (vincristine 1.5 mg/m2, days 1, 8, 15, and 22; daunorubicin 30 mg/m2 or idarubicin 9 mg/m2, days 8-10; L-asparaginase 6,000 IU/m2 every other day, days 9-23; prednisone 60 mg/m2 per day, days 1-28), followed by one cycle of cyclophosphamide (800 g/m2, day 1) + cytarabine (1 g/m2 every 12 hours, days 2-4) + 6-mercaptopurine (75 mg/m2, days 1-7) regimen.

The consolidation therapy of all the patients included three cycles of high-dose methotrexate regimen (methotrexate 3-5 g/m2 for 1 day every 10 days, three courses continuously), one cycle of VDLDex regimen (same dosage of vincristine and daunorubicin/idarubin on days 1 and 8, with L-asparaginase 6,000 IU/m2 every other day on days 2-12 and dexamethasone 6 mg/m2 per day on days 1-14), followed by one cycle of EA regimen (etoposide, 300 mg/m2, cytarabine, 300 mg/m2, days 1, 4, and 7). The maintenance therapy was 6-mercaptopurine 75 mg/m2 per day for 3 weeks and methotrexate 20 mg/m2 per week, thrice, combined with intermittent intensive chemotherapy using VDLDex, EA, or high-dose methotrexate regimen. The whole therapy was stopped at 3 years after complete remission.

Seventy patients with B-ALL and 102 healthy volunteers were referred as controls. Approval for these studies was obtained from the University and Institutional Review Boards. All patients gave accordingly their informed consent.

RNA extraction and reverse transcription-PCR analysis. For each patient, bone marrow sample was collected at diagnosis and mononuclear cells were enriched by density-gradient centrifugation with Ficoll solution. As assessed morphologically and cytochemically, all samples consisted of >85% leukemic blasts. Total RNA was extracted using TRIzol agent (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized from 1 µg total RNA using Superscript II reverse transcriptase (Invitrogen) and random hexamers according to the instructions of the manufacturer.

The transmembrane segment and intracellular region of NOTCH1 were amplified using standard protocol with seven pairs of primers: NOTCH1-1 (F: 5'-CCTGGAAGAACTGCACGCA-3' and R: 5'-CAATCTCCAGGTAGACGATGGAG-3') for the NH2-terminal region of the heterodimerization domain (HD-N); NOTCH1-2 (F: 5'-ATCTTCCCCTACTACGGCCG-3' and R: 5'-AAGAACAGAAGCACAAAGGCG-3') for the COOH-terminal region of the HD (HD-C); NOTCH1-3 (F: 5'-TGCACTTCATGTACGTGGCG-3' and R: 5'-CCTGGTAGATGAAGTCGGAGATG-3') for the RAM domain and the N-region of the ankyrin repeat (ANK) domain; NOTCH1-4 (F: 5'-CAACAGCGAGGAAGAGGAGG-3' and R: 5'-TGTCCCGGTTGGCAAAGTG-3') for the middle part of the ANK domain; NOTCH1-5 (F: 5'-CGCAGTTGTGCTCCTGAAGAAC-3' and R: 5'-ACGGACGGAGACTGCTGGAA-3') for the C-region of ANK domain and one part of the transcription activation domain (TAD); NOTCH1-6 (F: 5'-TGGAGTCACCCCATGGCTAC-3' and R: 5'-CTCGGCTCTCCACTCAGGAA-3') for the rest of the TAD domain and one part of the proline/glutamic acid/serine/threonine (PEST) domain; and NOTCH1-7 (F: 5'-GCTGCACAGTAGCCTTGCTG-3' and R: 5'-GCGCGCCGTTTACTTGAAG-3') for the rest of the PEST domain.

HOX11, HOX11L2, and SIL-TAL1 expressions were assessed using the corresponding primers by reverse transcription-PCR: HOX11 (F: 5'-TGGATGGAGAGTAACCGCAGAT-3' and R: 5'-AGGTACTTCTGGCGGTGGAA-3'); HOX11L2 (F: 5'-CGCCAAGTCCCTCAAAATGA-3' and R: 5'-CGGGAACCTTGGAACTATCCT-3'); and SIL-TAL1 (F: 5'-CCGCGACCCCAACGT-3' and R: 5'-CTCATTCTTGCTGAGCTTCTTGTC-3').

NOTCH1 mutation detection. The resultant PCR products were purified on Qiagen columns (Qiagen, Inc., Valencia, CA) and sequenced by NOTCH1 primers on ABI Prism 3700 DNA Analyzer using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

For further confirmation of the insertion and deletion mutations, the purified PCR products were ligated into pGEM-T Easy Vector Systems (Promega Corporation, Madison, WI) and used to transform DH5{alpha} E. coli cells. On agarose gel with ampicillin, individual colonies were screened by isopropyl-L-thio-ß-D-galactopyranoside and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, quantified on electrophoresis, and sequenced by T7 and SP6 primers following the same protocol.

Statistical analyses. Patient characteristics were compared using {chi}2 analysis and Fisher's exact test. Overall survival was measured from the date of diagnosis to the date of death or last follow-up through January 31, 2006. Relapse-free survival was calculated from the date of complete remission to the date of first relapse. Survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. Multivariate survival analysis was done using a Cox regression model. P < 0.05 was considered statistically significant. All statistical analyses were evaluated using SAS 8.2 software (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
NOTCH1 mutations in T-ALL. Through direct sequencing and elimination of a number of single-nucleotide polymorphisms by typing 102 healthy volunteers, 32 NOTCH1 mutations were revealed in 29 of the 77 patients, involving HD (23 cases, 29.9%), PEST (5 cases, 6.5%), TAD (3 cases, 3.9%), and ANK regions (1 case, 1.3%). None of these mutations was found in 70 patients with B-ALL and 102 healthy volunteers. Interestingly, mutation was absent from remission bone marrow sample obtained from one patient whose T-ALL harbored NOTCH1 mutations in HD sequence (Fig. 1A, I ).


Figure 1
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  		Fig. 1. NOTCH1 mutations in T-ALL. A, NOTCH1 mutation of HD-C (L1679P; I) presented at diagnosis became undetectable after complete remission. A point mutation (N1900I; II) was found in ANK domain. In TAD region, both point mutation (P2272S; III) and frameshift mutation (G2262Xfs; V) were observed. In PEST domain, a premature stop of NOTCH1 was induced either by point mutation (Q2407X; IV) or insertion (S2423X; VI). Black arrows, site of point mutation; asterisks, stop codons. B, schematic representation of human NOTCH1 showing the distribution of mutations in T-ALL. White arrows, mutations first described in our study; black arrows, previously reported mutations.

 
All the NOTCH1 mutations detected were listed in Table 2 and illustrated in Fig. 1B. The mutations occurring in HD included 13 point mutations, 5 insertions, 2 deletions, 2 deletion/insertions, and 1 insertion + duplication. These mostly fell in HD-N (16 cases) whereas mutations of HD-C were also identified (7 cases). The HD-N mutations were all clustered in a "hotspot" spanning residues 1,575 to 1,610 and included, more frequently, three L-to-P missense mutations on residue 1,586 (nos. 3-5) and four insertions from residue 1,607 (nos. 12-15). Overall, seven new types of mutation were found on the sites previously reported (ref. 8; nos. 6, 10, and 12-16). In HD-C, with the missense mutations on residue 1679 (nos. 18-20) most often observed, one new type of insertion from residue 1,677 (no. 17) and one insertion on an undescribed site from residue 1,741 (no. 23) were identified. NOTCH1 mutations within HD region coexisted with mutations of PEST domain (one case, no. 3) and TAD region (two cases, nos. 17 and 20).


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  		Table 2. NOTCH1 mutations in T-ALL patients

 
The PEST mutations were less frequently detected, including three point mutations, one insertion, and one deletion/insertion, all of which were newly identified. They induced either a substitution in reading frame (no. 27) or premature stop codons (nos. 3, 26, 28, and 29; Fig. 1A, IV and VI).

In addition to HD and PEST sequence, mutation detection was done on RAM, ANK, and TAD regions. One N-to-I mutation on residue 1,900 was found in ANK domain (no. 24; Fig. 1A, II). Two frameshift mutations on residues 2,262 and 2,330 (nos. 20 and 17) inducing premature stop codons, as well as one point mutation (P-to-S on residue 2,272, no. 25) of NOTCH1, were found in TAD region (Fig. 1A, III and V). These mutations were not reported previously.

Clinical and prognostic significance associated with NOTCH1 mutation. As shown in Table 1, among nine clinical and biological variables, NOTCH1 mutation was more frequent in the patients with WBC count >10 x 109/L than those with normal level [26 of 59 (44.1%) versus 3 of 18 (16.7%), P = 0.0357]. When the cutoff was elevated to 50 x 109/L or 100 x 109/L, although a similar increase of NOTCH1 mutation was found [18 of 41 (43.9%) versus 11 of 36 (30.6%) and 14 of 31 (45.2%) versus 15 of 46 (32.6%), respectively], they failed to reach statistical significance (P = 0.2278 and P = 0.2649, respectively).

Of the 77 patients, the 3-year relapse-free survival and overall survival rate (± SE percentage) were respectively 47.2% (±6.4%) and 54.6% (±6.7%), with median relapse-free survival at 24 months and median overall survival not reached at the time of last follow-up. Poor relapse-free survival and overall survival rate were correlated with NOTCH1 mutation. The 3-year relapse-free survival and overall survival rate (± SE percentage) for patients with NOTCH1 mutation were 28.8% (±8.8%) and 31.8% (±9.5%), significantly shorter than patients without mutation [59.8% (±8.4%) and 71.7% (±7.9%); P = 0.0053 (Fig. 2A ) and P = 0.0026, respectively].


Figure 2
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  		Fig. 2. Kaplan-Meier estimates of relapse-free survival according to NOTCH1 mutation. Notable difference of relapse-free survival existed between patients positive (bold black line) and negative for NOTCH1 mutation (black line; A). According to the age at diagnosis, this statistically significant difference was not observed in patients ≤18 years (B) but was observed in patients >18 years (C).

 
Multivariate analysis revealed that NOTCH1 mutation independently correlated with inferior relapse-free survival [P = 0.0084; hazard ratio, 2.4; 95% confidence interval (95% CI), 1.3-4.6] and overall survival (P = 0.0065; hazard ratio, 2.9; 95% CI, 1.3-6.1) in T-ALL patients. Other variables entered with NOTCH1 mutation in the Cox regression model were age and WBC count.

Because age was also an independent negative indicator both on relapse-free survival (P = 0.0369; hazard ratio, 2.1; 95% CI, 1.0-4.0) and overall survival (P = 0.0221; hazard ratio, 2.4; 95% CI, 1.1-5.1), we further divided the 77 patients into two groups according to the age at diagnosis. In the pediatric group, although decreased survival did present in patients displaying NOTCH1 mutation, no significant difference was obtained [P = 0.2763 for relapse-free survival (Fig. 2B); P = 0.1712 for overall survival]. In the adult group, patients positive for NOTCH1 mutation had a clearly worse prognosis than those negative for mutation [P = 0.0015 for relapse-free survival (Fig. 2C); P = 0.0041 for overall survival]. Moreover, NOTCH1 mutation was still an independent adverse prognostic factor in this group both for relapse-free survival (P = 0.0049; hazard ratio, 5.4; 95% CI, 1.7-17.5) and for overall survival (P = 0.0106; hazard ratio, 5.5; 95% CI, 1.5-15.3).

Coexistence of NOTCH1 mutations with oncogenic transcription factors and their combined effect on disease outcome. All the patients were studied for HOX11, HOX11L2 expression, and SIL-TAL1 fusion gene. The NOTCH1 mutations were seen associated with expression of HOX11 (8 of 22 cases, 36.4%), HOX11L2 (6 of 14, 42.9%), and SIL-TAL1 (3 of 10, 30.0%). However, no significant relation was observed between NOTCH1 mutation and incidence of HOX11, HOX11L2, and SIL-TAL1 expression (Table 1).

In all, HOX11L2 expression indicated shorter relapse-free survival (P = 0.0052) but no difference in overall survival (P = 0.0649). When combining NOTCH1 mutation with HOX11L2 expression, significantly worse relapse-free survival and overall survival were observed in the patients positive for NOTCH1 mutation and HOX11L2 expression, compared with those negative for both factors [P < 0.0001 for relapse-free survival (Fig. 3A ); P = 0.0102 for overall survival, respectively].


Figure 3
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  		Fig. 3. Kaplan-Meier estimates of relapse-free survival according to NOTCH1 mutation and oncogenic transcription factors expression. Compared with NOTCH1 mutation (–) HOX11L2 (–) cases (black line), significantly reduced relapse-free survival was seen in NOTCH1 mutation (+) HOX11L2 (+) patients (bold black line; A). In contrast, NOTCH1 mutation (+) patients showed a different disease outcome according to HOX11 expression (B): NOTCH1 mutation (+) HOX11 (–) cases (black line) presented with poor prognosis whereas NOTCH1 mutation (+) HOX11 (+) patients (bold black line) presented with good prognosis.

 
HOX11 expression was associated with longer relapse-free survival and overall survival (P = 0.0497 and P = 0.0246). Distinguished according to NOTCH1 mutation and HOX11 expression, as shown in Fig. 3B, patients with NOTCH1 mutation were stratified into two outcomes depending on the status of HOX11 expression. Favorable prognosis was noted in patients additionally positive for HOX11, in contrast to those negative for HOX11 who presented with short survival (P = 0.0121 for relapse-free survival (Fig. 3B); P = 0.0163 for overall survival).

These prognostic effects of the genetic subgroup were not significant when further divided according to the age distribution, probably because of the limited number of patients included in each group.


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
NOTCH1 expression has been implicated in promoting T lineage choice from common lymphoid progenitors and its mutations are frequently present in T-ALLs (3, 8). In our study, using reverse transcription-PCR amplification and direct sequencing, we showed NOTCH1 mutations both in pediatric and adult T-ALL patients. These mutations were not observed in patients with B-ALL, indicating that they are restricted to T-ALL (8). Although the incidence of NOTCH1 mutation is relatively lower than that previously reported (8, 12), by the same technique, the confirmation of previously described mutations with the discovery of new ones suggested that racial difference might exist between Asian and Western countries (13). This needs further study on a larger series of patients. Of note, its disappearance after remission revealed that NOTCH1 mutation acquired within the malignant clones could thus be served as a biomarker for disease surveillance.

The NOTCH1 mutations were mainly observed in the HD and PEST domains. Responsible for maintaining stable association between extracellular and transmembrane subunit (14), the mutation within HD-N may destabilize the subunit interaction, resulting in constitutive NOTCH1 activation and subsequent cell transformation (15, 16). Moreover, mutations detected in the HD-C sequence could affect the proteolytic sites of furin-like convertase from residue 1,675 to 1,682 and S3 cleavage site from residue 1,743 to 1,744, and thus perturb the processing of the NOTCH1 receptor (17–20). The PEST region regulates protein turnover by targeting proteins to the ubiquitin-proteosome complex for subsequent degradation (21). The deletions of its COOH-terminal sequences enhance NOTCH1 intracellular signaling and have been previously reported in murine T-ALL models, presaging the detection of PEST mutations having the similar structural consequences in human T-ALL (16, 22).

In addition to HD and PEST domains, mutations of TAD and ANK domains were identified for the first time in this series of T-ALL. Functionally, ANK interacts with downstream transcription factors, displacing corepressors, and the TAD serves to recruit coactivator molecules (23). Consistent with a previous report in mice model that both domains are required for T-cell leukemogenesis (24), these mutations could also play a role in T-ALL development.

Frequently occurring in T-ALL, NOTCH1 mutation relates to clinical variable and translates into differences in disease outcome in T-ALL patients. Constitutive NOTCH signaling was reported to be important to maintain T-ALL growth and survival in vitro (7). NOTCH1 is overexpressed in T cell–derived tumor cells of anaplastic large cell lymphoma and its activation accelerates the growth and inhibits the apoptosis of the lymphoma cells (25). This may explain that patients positive for NOTCH1 mutation displayed increased WBC count. However, it failed to reflect an extremely high proliferative status of the leukemic clone, which could possibly need a different and/or additional aberration. In clinical settings, a direct link has been found in multiple myeloma and lymphoma between activation of NOTCH1 and protection of tumor cells from apoptosis induced by chemotherapeutic agents or arsenite (25, 26), which could be related to poor disease outcome. In solid tumors, a high level of NOTCH1 was associated with advanced stage, metastasis, and poor prognosis in breast cancer (27). The fact that NOTCH1 mutations were correlated with decreased survival time in T-ALL patients further indicates the importance of NOTCH signaling abnormality in disease progression.

Interestingly, the adverse prognostic effect of NOTCH1 mutation relied on the age of the patients. Because pediatric patients respond well to current therapy, the significance of molecular biomarker could be weakened by the relatively good prognosis in pediatric group and no significant superiority of survival time was observed in patients without NOTCH1 mutation. However, in adult patients, who were known to be less frequently cured (1), NOTCH1 mutation was independently correlated with poor disease outcome and could be considered as a negative prognostic factor.

NOTCH1 mutations participate in T-ALL formation in E2A-PBX1 or C-MYC transgenic mice (16, 22). This capacity of NOTCH1 to cooperate with other genes in mice models is reminiscent of the association of NOTCH1 mutations with many molecular subtypes of human T-ALL. Point mutations, as well as insertions and deletions described above, could occur in multipotent hematopoietic progenitors, which normally express NOTCH1 (28). These aberrations would be predicted to induce daughter cells to adopt a T-cell fate and thereby increase the pool of cells at risk for additional leukemogenic events, including the overexpression of other critical transcription factors.

NOTCH signaling controls the generation and differentiation of early T lineage progenitors (29), which oncogenic transcription factor HOX11 could possibly act on (30). HOX11L2 is an orphan homeobox factor very similar to HOX11 and its ectopic expression reinforces the role of homeobox transcription factors on T-cell leukemogenesis (31). Clinically, HOX11L2 expression confers a worse response to treatment (30, 32) whereas HOX11 activation is significantly related to a favorable prognosis in T-ALL (11, 33). In our study, patients with NOTCH1 mutations had dual outcome in accordance with their expression for HOX11L2 and HOX11: patients additionally positive for HOX11L2 expression deteriorated quickly whereas those with HOX11 expression showed prolonged survival. This may reflect that NOTCH1-induced transformation of hematopoietic lineages requires the collaborative action of additional cellular oncogenes.

In conclusion, NOTCH1 mutations were present in patients with T-ALL and indicated poor prognosis. Cooperatively with other oncogenic transcription factors, NOTCH1 mutations participate in the pathogenesis of T-ALL, providing a promising rationale for targeted therapies that interfere with NOTCH signaling pathway.


   Acknowledgments
 
We thank Xiang-Qin Wen, Min Zhang, and Li-Quan Fan for technical assistance, and Yu Liu and Zhao-Jun Wen for illustrations.


   Footnotes
 
Grant support: Chinese National Key program for Basic Research (973:2004CB518600), the Chinese National High Tech Program (863:2002BA711A04), the National Natural Science Foundation of China, the Key Discipline Program of Shanghai Municipal Education Commission (Y0201), the Shanghai Commission of Science and Technology, the Shanghai Rising Star Program (05QMX1429), and the Samuel Waxman Cancer Research Foundation Laboratory, the Co-PI Program of Shanghai Rui Jin Hospital/Medical School of Shanghai Jiao Tong University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/26/05; revised 2/22/06; accepted 3/ 1/06.


   References
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 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

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