Figures
Abstract
Background
Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.
Methods
We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.
Results
Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.
Citation: Chen Y-K, Hou H-A, Tang J-L, Jhuang J-Y, Lai Y-J, Lee M-C, et al. (2015) Clinical and Prognostic Implications of Roundabout 4 (Robo4) in Adult Patients with Acute Myeloid Leukemia. PLoS ONE 10(3): e0119831. https://doi.org/10.1371/journal.pone.0119831
Academic Editor: Halvard Böenig, German Red Cross Blood Service Frankfurt, GERMANY
Received: June 17, 2014; Accepted: January 16, 2015; Published: March 20, 2015
Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The Roundabout (Robo) family proteins are highly conserved transmembrane cell adhesion molecules that are expressed predominantly in neuronal tissues and play a role in the development of the nervous system. [1, 2] Roundabout 4 (Robo4), the fourth member of Robo family, was first identified by Huminiecki et al in 2002. [3] It is distinct from the other family members in that it is expressed specifically in endothelial cells [4,5] and hematopoietic stem/progenitor cells (HSPCs). [6,7] Robo4 acts as a receptor for Slit 2 and modulates vascular endothelial growth factor A (VEGF)–VEGF receptor signaling. [8,9] The Slit-Robo signaling is essential for angiogenesis, [6,10] especially tumor angiogenesis. [11] obo4 expression was found to be significantly up-regulated in tumor endothelia compared to their normal counterparts. [12] In addition to angiogenesis, Slit2-Robo4 signaling pathway also plays an essential role in BM homing and mobilization of HSPCs. [7,13] Smith-Berdan et al demonstrated that Robo4, in cooperation with CXCR4, acted as an HSPC-specific adhesion receptor to localize HSPC to BM niches. [7]
Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy regarding the pathogenesis, clinical features and treatment outcomes. In addition to intrinsic changes of leukemic cells including cytogenetic, genetic and epigenetic alterations, impaired hematopoietic microenvironment of the bone marrow (BM) may also contribute to the development of AML. [14] It has been shown that adhesion of AML cells to the BM stromal cells promotes their survival and proliferation. [15,16] Angiogenesis also plays an important role in AML. [17,18] Higher BM angiogenesis predicts a higher relapse rate and poorer prognosis in AML patients. [18,19]
Robo4 has been shown to be expressed on leukemic stem cells. [20] However, little is known about the expression of Robo4 and its clinical implications in AML. In the present study, we investigated the BM Robo4 expression by quantitative real-time polymerase chain reaction (RQ-PCR) in a cohort of 218 adults with de novo AML and correlated the results with clinical features, major molecular mutations and outcomes of the patients. To the best of our knowledge, this is the first report to address the prognostic implication of Robo4 expression in AML patients. We found that higher BM Robo4 expression was an independent unfavorable prognostic factor for overall survival (OS) in these patients, and the finding was also validated in an independent validation cohort.
Materials and Methods
Patients
The original cohort comprised 218 patients with newly diagnosed de novo AML at the National Taiwan University Hospital (NTUH) from April 1996 to December 2007 who had complete clinical data and enough cryopreserved BM samples for analysis. Twenty normal marrow donors were also enrolled for comparison. Diagnosis and classification of AML were made according to the French-American-British (FAB) Cooperative Group Criteria. Among the 218 patients, 148 (67.9%) received standard intensive chemotherapy. The 134 non-M3 patients received induction chemotherapy with idarubicin 12 mg/m2 per day on days 1–3 and cytarabine 100 mg/m2 per day on days 1–7 and then consolidation chemotherapy with 2–4 courses of high-dose cytarabine (2000 mg/m2 q12h days 1–4, total 8 doses), with or without an anthracycline (idarubicin or novatrone), after achieving complete remission (CR). [21,22] The 14 patients with acute promyelocytic leukemia (M3 subtype) received concurrent all-trans retinoic acid and chemotherapy (idarubicin 12 mg/m2 per day on days 1–2) as induction treatment. Consolidation chemotherapy with anthracycline-based regimen was given for 4 courses after CR was achieved. The validation cohort comprised 53 adult AML patients diagnosed at the NTUH from Jan 2008 to June 2009. (S4 Table) These patients were treated with the same regimens as those in the original cohort and were used to validate the prognostic effect of BM Robo4 expression in AML. This study was approved by the Institutional Review Board of the NTUH; and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.
Quantitative real-time polymerase chain reaction (RQ-PCR)
BM mononuclear cells from 271 patients, including the original and validation cohorts, before chemotherapy and 20 healthy transplantation donors were isolated and cryopreserved until use. Total RNA was extracted and reverse transcribed. The gene expression level was quantified utilizing TaqMan technology on the Applied Biosystems 7500/7500 Fast Real-Time PCR System as previously described. [23] Gene-specific primers and probe of Robo4 were available as TaqMan Gene Expression Assay (Assay ID, Hs00219408_m1*, Applied Biosystems). Each sample was tested at least twice independently. The amount of the target gene was normalized to that of the housekeeping gene RPLP0. [17, 24, 25] The copies of target gene were quantified only after successful amplification of the internal control, using the standard curves derived from cloned plasmids. All data were presented as log ratio of the target gene/RPLP0.
Immunohistochemical staining (IHC) for Robo4 protein
We performed IHC to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the original cohort, 17 having higher Robo4 mRNA expression and another 13, lower Robo4 mRNA expression. The tissue section was prepared from formalin-fixed, paraffin-embedded tissue. After deparaffinization and rehydration as previously described, [23] the slides were boiled in 0.01 mol/L citrate buffer (pH = 6.1) for 5 minutes to retrieve the antigens. The endogenous peroxidase activity was blocked by incubation with 3% H2O2 in methanol for 30 minutes. Then the sections were blocked with 10% normal goat serum (VECTASTAIN Elite ABC kit; Vector Laboratories). The primary antibody we used was rabbit polyclonal anti-human Robo4 (PAB12049, Abnova) at a 1:250 dilution. A negative, no-antibody control was included for each staining. The slides were incubated with a biotinylated secondary antibody (1:200 dilution; goat anti-rabbit IgG; VECTASTAIN Elite ABC kit; Vector Laboratories) for 60 minutes at room temperature and AB reagent was applied based on the manufacturer's instructions (VECTASTAIN Elite ABC kit; Vector Laboratories). The antigen detection was conducted by a color reaction with 3,3'-diaminobenzidine (DAB peroxidase substrate kit; catalog number SK-4100; Vector Laboratories). Finally, the sections were counterstained with hematoxylin and mounted. Robo4 expression was assessed by staining intensity and frequency of positively stained cells. A score of 1–5 was calculated for each specimen according to the addition of the score of staining intensity (0 = none, 1 = weak, 2 = strong) and the score of percentage of myeloid cells positively stained (1 = 0~10%, 2 = 10~50%, 3>50%) by the pathologists who were blind to the results of Robo4 mRNA expression. [26–28] Erythroid cells were excluded from calculation.
Cytogenetics
BM mononuclear cells were harvested directly or after 1–3 day of unstimulated culture as described previously. [29] Metaphase chromosomes were banded by trypsin-Giemsa technique and karyotyped according to the International System for Human Cytogenetic Nomenclature.
Immunophenotype analysis
A panel of monoclonal antibodies to myeloid associated antigens, including CD13, CD33, CD11b, CD15, CD14, and CD41a, as well as lymphoid-associated antigens, including CD2, CD5, CD7, CD19, CD10, and CD20, and lineage nonspecific antigens HLA-DR, CD34, and CD56 were used to characterize the immunophenotypes of the leukemia cells as previously described. [21]
Mutation analysis
Mutation analyses of 17 relevant molecular alterations, including Class I mutations, such as FLT3/ITD and FLT3/TKD, [30] NRAS, [31] KRAS, [31] JAK2, [31] KIT [32] and PTPN11 [33] mutations and Class II mutations, such as MLL/PTD, [34] CEBPA [35] and RUNX1 [36] mutations, as well as mutations in NPM1, [37] WT1, [38] and those genes related to epigenetic modifications, such as ASXL1, [39] IDH1, [40] IDH2, [41] TET2 [42] and DNMT3A, [21] were performed as previously described. Abnormal sequencing results were confirmed by at least two repeated analyses.
Statistical analysis
Cut-off value of Robo4 expression level was determined by sequential cutting strategy and was validated by bootstrap resampling method. The discrete variables of patients with lower and higher Robo4 expression were compared using the Chi-square tests or Fisher’s exact test. We used Mann–Whitney U-test to compare continuous variables and medians of distributions. Whole patient population was included for analyses of the correlation between Robo4 expression and clinical characteristics, however, only those receiving conventional standard chemotherapy, as mentioned above, were included in analyses of survivals. OS was measured from the date of first diagnosis to death from any cause or the last follow-up, whereas relapse was defined as a reappearance of at least 5% leukemic blasts in a BM aspirate or new extramedullary leukemia in patients with a previously documented CR. Disease-free status indicated that the patient achieved CR and did not relapse by the end of this study, and disease-free survival (DFS) was defined as the time from recruitment to the first of three events: treatment failure, leukemia relapse or death from any cause. To exclude confounding influences of different treatment regimens, patients who received allogeneic HSCT were censored on the day of cell infusion. [21, 22] We adopted Kaplan–Meier estimation to plot survival curves, and used log-rank tests to examine the difference between groups. Relative risk (RR) and 95% confidence interval (CI) were estimated by Cox proportional hazards regression models to determine independent risk factors associated with survival in multivariate analyses. Two-sided P-values less than 0.05 were considered statistically significant. All statistical analyses were accomplished with the SPSS 17 (SPSS Inc., Chicago, IL, USA) and Statsdirect (Cheshire, England, UK).
Results
Correlation of BM Robo4 expression with clinical features and laboratory data in the original cohort
Robo4 expression was significantly higher in AML patients than in normal BM donors. (P = 0.0007, S1 Fig.) The patients were then divided into two groups: one with lower expression of Robo4 (n = 119) and the other with higher expression (n = 99), by using a cut-off point of 0.010 (Robo4/RPLP0). The comparison of clinical characteristics between patients with lower and higher BM Robo4 expression is shown in Table 1. Patients with higher BM Robo4 expression had lower white blood cell (WBC) and blast counts than those with lower expression. There was no difference in sex, age and initial hemoglobin levels, platelet counts and serum lactate dehydrogenase levels between the two groups of patients. Compared to patients with lower BM Robo4 expression, those with higher expression had less frequently FAB M3 subtype (P = 0.006, Table 1).
Higher BM Robo4 expression were positively associated with the expression of HLA-DR (P<0.0001), CD13 (P = 0.0417), CD34 (P = 0.0009) and CD56 (P = 0.039) on the leukemia cells (S1 Table). There was no difference in the expression of other antigens between the patients with higher and lower Robo4 expression.
Correlation between Robo4 RNA expression and protein expression in the original cohort
Robo4 protein expression measured by scoring IHC of BM biopsy specimens correlated with Robo4 mRNA expression in the 30 patients studied. The patients with higher scores measured by IHC had also higher Robo4 mRNA expression than those with lower scores (P = 0.018). Representative IHC of a sample with a high score and the other one with a low score are demonstrated in S2 Fig. In addition to vasculoendothelial cells, Robo4 protein staining could be clearly seen in leukemia cells in the BM with a high score. Because not every patient in this study received BM biopsy, we only did IHC in selected patients. The correlation between Robo4 expression and angiogenesis in the BM of AML patients could not be analyzed in these limited samples.
Correlation of BM Robo4 expression with karyotypes and molecular gene mutations in the original cohort
Chromosome data were available in 207 (95%) patients at diagnosis. The comparison of cytogenetic changes between patients with higher and lower BM Robo4 expression is shown in S2 Table. Higher Robo4 expression was closely associated with chromosomal abnormality t(8;21), but inversely correlated with t(15;17) (15.8% versus 1.8%, P = 0.0002 and 1.1% versus 12.5%, P = 0.0019, respectively). With regard to the AML associated genetic alterations (Table 2), higher BM Robo4 expression was closely associated with ASXL1 mutation (15.2% versus 6.7%, P = 0.0489) but negatively associated with CEBPA mutation (6.1% versus 18.5%, P = 0.0076). AML patients with higher Robo4 expression had a trend of higher probability of concurrent DNMT3A mutation and KIT mutation (P = 0.0707 and 0.0825, respectively).
Impact of BM Robo4 expression on clinical outcome in the original cohort
Of the 148 AML patients receiving conventional intensive induction chemotherapy, 107 (72.3%) patients achieved a CR (Table 1). The patients with higher Robo4 expression had a trend of a lower CR rate than those with lower Robo4 expression, albeit the difference was not statistically significant (63.9% versus 78.2%, P = 0.0643). Similar trend was also found in the subgroup of 99 patients with intermediate-risk cytogenetics (CR rate, 59.5% versus 75.8%, P = 0.114) (S3 Table). With a median follow-up of 37.2 months (ranges, 1.0–160), patients with higher Robo4 expression had significantly poorer OS (Fig. 1) and DFS (Fig. 2) than those with lower Robo4 expression (median, 17.0 months versus 95.0 months, P = 0.023, and medium, 5.0 months versus 15.0 months, P = 0.024, respectively). In the subgroup of patients with intermediate-risk cytogenetics, the differences between the two groups in OS (S3 Fig.) (median, 13.5 months versus 95.0 months, P = 0.007) and DFS (S4 Fig.) (median, 4.0 months versus 10.0 months, P = 0.025) were even bigger.
Among a total of 148 patients with AML who received conventional intensive chemotherapy, patients with higher Robo4 expression had shorter overall than those with lower Robo4 expression.
Among a total of 148 patients with AML who received conventional intensive chemotherapy, patients with higher Robo4 expression had shorter OS (A) and DFS (B) than those with lower Robo4 expression.
In multivariate analysis (Table 3), the independent poor risk factors for OS were older age > 50 years, higher WBC count >50,000/μL, unfavorable karyotype, and higher Robo4 expression (RR 1.779, 95% CI 1.005–3.149, P = 0.048). On the other hand, CEBPAdouble-mutation and NPM1+/FLT3-ITD- (mutant NPM1 in the absence of FLT3-ITD) were independent favorable prognostic factors. The independent poor risk factors for DFS included older age > 50 years, higher WBC count >50,000/μL, unfavorable karyotype, and higher Robo4 expression (RR 1.600, 95% CI 1.026–2.495, P = 0.038). CEBPAdouble-mutation and NPM1+/FLT3-ITD- were independent favourable factors for DFS.
The prognostic impact of BM Robo4 expression in the validation cohort
The cut-off point in the original cohort was used to define lower- and higher-expression groups in the validation cohort (n = 53). After a median follow-up time of 39.2 months (range 0.7 to 51.3), higher BM Robo4 expression remained to be a significant unfavorable prognostic factor for OS in total patients (Fig. 3) and those with intermediate-risk cytogenetics in the validation cohort (Fig. 4) (median 13.4 months versus not reached, P = 0.014; median 14.2 months versus not reached, P = 0.017, respectively).
The patients with higher BM Robo4 expression had a shorter OS than those with lower expression both among a total of 53 patients. The median value of BM Robo4 expression in the original cohort of 148 patients was used as the cutoff point to define lower- and higher-expression groups.
The patients with higher BM Robo4 expression had a shorter OS than those with lower expression both among 25 patients with intermediate-risk cytogenetics. The median value of BM Robo4 expression in the original cohort of 148 patients was used as the cutoff point to define lower- and higher-expression groups.
Discussion
Robo4, a receptor for Slit2, is highly expressed in solid tumors, including brain, lung, urinary bladder and colorectal cancers and its expression is restricted to tumor vasculature but down-regulated in mature vasculature. [3, 43, 44] Interestingly, up-regulation of Robo4 was noticed in cancerous tissues but not in corresponding noncancerous part using paired samples in 50 patients with colorectal cancers. [43] Robo4 is thus an excellent tumor endothelial markers and a potential target for anti-angiogenesis therapy. [11,45] There have been few studies concerning the clinical relevance of Robo4 expression in cancers. Gorn et al demonstrated that elevated serum levels of Robo4 before treatment predicted poor prognosis in patients with non-small cell lung cancer, but it did not reach statistical significance in a univariate Cox regression model. [44] To the best of our knowledge, the current study was the first to evaluate Robo4 expression and its clinical implications in AML patients. We demonstrated that Robo4 expression was significantly elevated in AML patients compared to normal controls. Furthermore, higher BM Robo4 expression predicted poorer DFS and OS in AML patients, especially those with intermediate-risk cytogenetics. The prognostic impact of higher BM Robo4 expression was further validated in an independent validation cohort.
Robo4, in cooperation with CXCR4, plays a role in BM homing and mobilization of HSPCs. [7,13] It acts as an HSPC-specific adhesion receptor to localize HSPC to BM niches and its expression is dramatically down-regulated in mobilized HSPCs. [7] It has been shown that mobilization of AML cells by CXCR4 antagonist could enhance chemosensitivity of AML, which is translated into significant survival advantage. [46] Compatible with this finding, AML patients with lower Robo4 expression in the present study were more sensitive to induction chemotherapy and had a trend of higher CR rates.
In the comprehensive analyses of the 17 genetic alterations and cytogenetic changes in the original cohort, we found that higher BM Robo4 expression was closely associated with ASXL1 mutation and t(8;21), but negatively associated with CEBPA mutation and t(15;17). The translocation t(8;21) is a frequent cytogenetic abnormality in AML; however, the presence of the resultant RUNX1-RUNXT1 fusion protein alone is not sufficient per se to induce leukemia. [47] In the present study, 15 of the 17 patients with t(8;21) showed higher Robo4 expression (S2 Table). The higher Robo4 expression, which may lead to dysregulation of angiogenesis or microenvironment, may be another hit in patients with this chromosomal abnormality. The significance of close association of higher Robo4 expression and ASXL1 mutation in the pathogenesis of AML needs further exploration.
Conclusions
In conclusion, the present study provides evidences that higher BM Robo4 expression is closely associated with distinct clinical and biologic characteristics in AML patients. Further, higher BM Robo4 expression is an independent poor prognostic factor for OS. The unfavorable prognostic impact of higher BM Robo4 expression was also validated in an independent validation cohort. Higher BM Robo4 expression may serve as a new biomarker for foreseeing the clinical outcome of AML patients and may be a potential target for the treatment of AML patients with higher expression of this protein.
Supporting Information
S1 Fig. Comparison of BM Robo4 expressions between AML patients in the original cohort and normal controls.
The level was calculated as the log value of Robo4 mRNA expression normalized to the housekeeping gene RPLP0. The P value was calculated using the Mann-Whitney U test.
https://doi.org/10.1371/journal.pone.0119831.s001
(DOCX)
S2 Fig. Representative immunohistochemical staining of Robo4 protein in BM biopsy specimens from two patients.
One specimen from a patient with higher BM Robo4 mRNA expression showed strong staining of leukemic cells (A & B) and another one from a patient with lower BM Robo4 mRNA expression showed weak staining (C & D). (Magnification 100X and 400X, respectively)
https://doi.org/10.1371/journal.pone.0119831.s002
(DOCX)
S3 Fig. Kaplan–Meier survival curves for overall survival of AML patients with intermediate-risk cytogenetics stratified by BM Robo4 mRNA expression in the original cohort.
Among a total of 99 patients with intermediate-risk cytogenetics who received conventional intensive chemotherapy, patients with higher Robo4 expression had shorter overall survival than those with lower expression.
https://doi.org/10.1371/journal.pone.0119831.s003
(DOCX)
S4 Fig. Kaplan–Meier survival curves for disease-free survival of AML patients with intermediate-risk cytogenetics stratified by BM Robo4 mRNA expression in the original cohort.
Among a total of 99 patients with intermediate-risk cytogenetics who received conventional intensive chemotherapy, patients with higher Robo4 expression had shorter disease-free survival than those with lower expression.
https://doi.org/10.1371/journal.pone.0119831.s004
(DOCX)
S1 Table. Comparison of immune-phenotypes of leukemia cells between AML patients with higher and lower BM Robo4 expression.
https://doi.org/10.1371/journal.pone.0119831.s005
(DOCX)
S2 Table. Association of Robo4 expression level with cytogenetic abnormalities*.
https://doi.org/10.1371/journal.pone.0119831.s006
(DOCX)
S3 Table. Comparison of clinical manifestations between patients with higher and lower BM Robo4 expression in AML patients with intermediate cytogenetics.
https://doi.org/10.1371/journal.pone.0119831.s007
(DOCX)
S4 Table. Clinical characteristics of validation cohort.
https://doi.org/10.1371/journal.pone.0119831.s008
(DOCX)
Author Contributions
Conceived and designed the experiments: YKC HAH JLT HFT. Performed the experiments: YKC JYJ YJL M-C Lee YYK C-W Lin MHT CFH Y-C Chiang FYL M-C Liu C-W Liu. Analyzed the data: YKC HAH CYL SCC JYJ C-W Lin YJL. Contributed reagents/materials/analysis tools: WCC CYC JLT MY SYH BSK SCH SJW WT Y-C Chen. Wrote the paper: YKC HAH JLT HFT.
References
- 1. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, et al. Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell. 1998;92(2):205–15. Epub 1998/02/11. pmid:9458045
- 2. Long H, Sabatier C, Ma L, Plump A, Yuan W, Ornitz DM, et al. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron. 2004;42(2):213–23. Epub 2004/04/20. pmid:15091338
- 3. Huminiecki L, Gorn M, Suchting S, Poulsom R, Bicknell R. Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis. Genomics. 2002;79(4):547–52. Epub 2002/04/12. pmid:11944987
- 4. Huminiecki L, Bicknell R. In silico cloning of novel endothelial-specific genes. Genome Res. 2000;10(11):1796–806. Epub 2000/11/15. pmid:11076864
- 5. Park KW, Morrison CM, Sorensen LK, Jones CA, Rao Y, Chien CB, et al. Robo4 is a vascular-specific receptor that inhibits endothelial migration. Developmental biology. 2003;261(1):251–67. Epub 2003/08/28. pmid:12941633
- 6. Shibata F, Goto-Koshino Y, Morikawa Y, Komori T, Ito M, Fukuchi Y, et al. Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype. Stem Cells. 2009;27(1):183–90. Epub 2008/10/18. pmid:18927479
- 7. Smith-Berdan S, Nguyen A, Hassanein D, Zimmer M, Ugarte F, Ciriza J, et al. Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches. Cell Stem Cell. 2011;8(1):72–83. Epub 2011/01/08. pmid:21211783
- 8. Jones CA, Nishiya N, London NR, Zhu W, Sorensen LK, Chan AC, et al. Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity. Nature cell biology. 2009;11(11):1325–31. Epub 2009/10/27. pmid:19855388
- 9. Jones CA, London NR, Chen H, Park KW, Sauvaget D, Stockton RA, et al. Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nature medicine. 2008;14(4):448–53. Epub 2008/03/18. pmid:18345009
- 10. Bedell VM, Yeo SY, Park KW, Chung J, Seth P, Shivalingappa V, et al. roundabout4 is essential for angiogenesis in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(18):6373–8. Epub 2005/04/26. pmid:15849270
- 11. Legg JA, Herbert JM, Clissold P, Bicknell R. Slits and Roundabouts in cancer, tumour angiogenesis and endothelial cell migration. Angiogenesis. 2008;11(1):13–21. Epub 2008/02/12. pmid:18264786
- 12. Seth P, Lin Y, Hanai J, Shivalingappa V, Duyao MP, Sukhatme VP. Magic roundabout, a tumor endothelial marker: expression and signaling. Biochemical and biophysical research communications. 2005;332(2):533–41. Epub 2005/05/17. pmid:15894287
- 13. Goto-Koshino Y, Fukuchi Y, Shibata F, Abe D, Kuroda K, Okamoto S, et al. Robo4 plays a role in bone marrow homing and mobilization, but is not essential in the long-term repopulating capacity of hematopoietic stem cells. PloS one. 2012;7(11):e50849. Epub 2012/12/12. pmid:23226403
- 14. Konopleva MY, Jordan CT. Leukemia stem cells and microenvironment: biology and therapeutic targeting. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2011;29(5):591–9. Epub 2011/01/12.
- 15. Scholzel C, Lowenberg B. Stimulation of proliferation and differentiation of acute myeloid leukemia cells on a bone marrow stroma in culture. Experimental hematology. 1985;13(7):664–9. Epub 1985/08/01. pmid:3861327
- 16. Bendall LJ, Daniel A, Kortlepel K, Gottlieb DJ. Bone marrow adherent layers inhibit apoptosis of acute myeloid leukemia cells. Experimental hematology. 1994;22(13):1252–60. Epub 1994/12/01. pmid:7957711
- 17. Hou HA, Chou WC, Lin LI, Tang JL, Tseng MH, Huang CF, et al. Expression of angiopoietins and vascular endothelial growth factors and their clinical significance in acute myeloid leukemia. Leuk Res. 2008;32(6):904–12. Epub 2007/10/02. pmid:17904634
- 18. Shih TT, Hou HA, Liu CY, Chen BB, Tang JL, Chen HY, et al. Bone marrow angiogenesis magnetic resonance imaging in patients with acute myeloid leukemia: peak enhancement ratio is an independent predictor for overall survival. Blood. 2009;113(14):3161–7. Epub 2008/11/08. pmid:18988863
- 19. Hou HA, Shih TT, Liu CY, Chen BB, Tang JL, Yao M, et al. Changes in magnetic resonance bone marrow angiogenesis on day 7 after induction chemotherapy can predict outcome of acute myeloid leukemia. Haematologica. 2010;95(8):1420–4. Epub 2010/03/12. pmid:20220062
- 20. Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM, Weissman IL. Differential expression of novel potential regulators in hematopoietic stem cells. PLoS genetics. 2005;1(3):e28. Epub 2005/09/10. pmid:16151515
- 21. 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(2):559–68. Epub 2011/11/15. pmid:22077061
- 22. Hou HA, Lin CC, Chou WC, Liu CY, Chen CY, Tang JL, et al. Integration of cytogenetic and molecular alterations in risk stratification of 318 patients with de novo non-M3 acute myeloid leukemia. Leukemia. 2014;28(1):50–8. pmid:23929217
- 23. Cheng CL, Hou HA, Jhuang JY, Lin CW, Chen CY, Tang JL, et al. High bone marrow angiopoietin-1 expression is an independent poor prognostic factor for survival in patients with myelodysplastic syndromes. British journal of cancer. 2011;105(7):975–82. Epub 2011/09/01. pmid:21878936
- 24. Cheng WC, Chang CW, Chen CR, Tsai ML, Shu WY, Li CY, et al. Identification of reference genes across physiological states for qRT-PCR through microarray meta-analysis. PloS one. 2011;6(2):e17347. Epub 2011/03/11. pmid:21390309
- 25. Braig M, Pallmann N, Preukschas M, Steinemann D, Hofmann W, Gompf A, et al. A 'telomere-associated secretory phenotype' cooperates with BCR-ABL to drive malignant proliferation of leukemic cells. Leukemia. 2014. Epub 2014/03/08.
- 26. Atkin G, Barber PR, Vojnovic B, Daley FM, Glynne-Jones R, Wilson GD. Correlation of spectral imaging and visual grading for the quantification of thymidylate synthase protein expression in rectal cancer. Human pathology. 2005;36(12):1302–8. Epub 2005/11/29. pmid:16311124
- 27. Giles FJ, Bekele BN, O'Brien S, Cortes JE, Verstovsek S, Balerdi M, et al. A prognostic model for survival in chronic lymphocytic leukaemia based on p53 expression. British journal of haematology. 2003;121(4):578–85. Epub 2003/05/20. pmid:12752098
- 28. Edelman MJ, Watson D, Wang X, Morrison C, Kratzke RA, Jewell S, et al. Eicosanoid modulation in advanced lung cancer: cyclooxygenase-2 expression is a positive predictive factor for celecoxib + chemotherapy—Cancer and Leukemia Group B Trial 30203. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2008;26(6):848–55. Epub 2008/02/19.
- 29. Tien HF, Wang CH, Lin MT, Lee FY, Liu MC, Chuang SM, et al. Correlation of cytogenetic results with immunophenotype, genotype, clinical features, and ras mutation in acute myeloid leukemia. A study of 235 Chinese patients in Taiwan. Cancer Genet Cytogenet. 1995;84(1):60–8. pmid:7497445
- 30. Chou WC, Tang JL, Lin LI, Yao M, Tsay W, Chen CY, et al. Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolution. Cancer Res. 2006;66(6):3310–6. pmid:16540685
- 31. Chen CY, Lin LI, Tang JL, Tsay W, Chang HH, Yeh YC, et al. Acquisition of JAK2, PTPN11, and RAS mutations during disease progression in primary myelodysplastic syndrome. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2006;20(6):1155–8.
- 32. Chen CY, Lin LI, Tang JL, Ko BS, Tsay W, Chou WC, et al. RUNX1 gene mutation in primary myelodysplastic syndrome—the mutation can be detected early at diagnosis or acquired during disease progression and is associated with poor outcome. British journal of haematology. 2007;139(3):405–14. Epub 2007/10/04. pmid:17910630
- 33. Hou HA, Chou WC, Lin LI, Chen CY, Tang JL, Tseng MH, et al. Characterization of acute myeloid leukemia with PTPN11 mutation: the mutation is closely associated with NPM1 mutation but inversely related to FLT3/ITD. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2008;22(5):1075–8. Epub 2007/11/02.
- 34. Shiah HS, Kuo YY, Tang JL, Huang SY, Yao M, Tsay W, et al. Clinical and biological implications of partial tandem duplication of the MLL gene in acute myeloid leukemia without chromosomal abnormalities at 11q23. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2002;16(2):196–202.
- 35. Hou HA, Lin LI, Chen CY, Tien HF. Reply to 'Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favorable prognosis'. British journal of cancer. 2009;101(4):738–40. Epub 2009/07/23. pmid:19623175
- 36. Tang JL, Hou HA, Chen CY, Liu CY, Chou WC, Tseng MH, et al. AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations. Blood. 2009;114(26):5352–61. Epub 2009/10/08. pmid:19808697
- 37. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005;352(3):254–66. pmid:15659725
- 38. Hou HA, Huang TC, Lin LI, Liu CY, Chen CY, Chou WC, et al. WT1 mutation in 470 adult patients with acute myeloid leukemia: stability during disease evolution and implication of its incorporation into a survival scoring system. Blood. 2010;115(25):5222–31. Epub 2010/04/07. pmid:20368469
- 39. Chen TC, Hou HA, Chou WC, Tang JL, Kuo YY, Chen CY, et al. Dynamics of ASXL1 mutation and other associated genetic alterations during disease progression in patients with primary myelodysplastic syndrome. Blood cancer journal. 2014;4:e177. Epub 2014/01/21. pmid:24442206
- 40. Lin CC, Hou HA, Chou WC, Kuo YY, Liu CY, Chen CY, et al. IDH mutations are closely associated with mutations of DNMT3A, ASXL1 and SRSF2 in patients with myelodysplastic syndromes and are stable during disease evolution. American journal of hematology. 2013. Epub 2013/10/12.
- 41. Chou WC, Lei WC, Ko BS, Hou HA, Chen CY, Tang JL, et al. The prognostic impact and stability of Isocitrate dehydrogenase 2 mutation in adult patients with acute myeloid leukemia. Leukemia. 2011;25(2):246–53. Epub 2010/11/17. pmid:21079611
- 42. Chou WC, Chou SC, Liu CY, Chen CY, Hou HA, Kuo YY, et al. TET2 mutation is an unfavorable prognostic factor in acute myeloid leukemia patients with intermediate-risk cytogenetics. Blood. 2011. Epub 2011/08/11.
- 43. Grone J, Doebler O, Loddenkemper C, Hotz B, Buhr HJ, Bhargava S. Robo1/Robo4: differential expression of angiogenic markers in colorectal cancer. Oncology reports. 2006;15(6):1437–43. Epub 2006/05/11. pmid:16685377
- 44. Gorn M, Anige M, Burkholder I, Muller B, Scheffler A, Edler L, et al. Serum levels of Magic Roundabout protein in patients with advanced non-small cell lung cancer (NSCLC). Lung Cancer. 2005;49(1):71–6. Epub 2005/06/14. pmid:15949592
- 45. Yoshikawa M, Mukai Y, Okada Y, Tsumori Y, Tsunoda S, Tsutsumi Y, et al. Robo4 is an effective tumor endothelial marker for antibody-drug conjugates based on the rapid isolation of the anti-Robo4 cell-internalizing antibody. Blood. 2013;121(14):2804–13. Epub 2013/02/01. pmid:23365463
- 46. Nervi B, Ramirez P, Rettig MP, Uy GL, Holt MS, Ritchey JK, et al. Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100. Blood. 2009;113(24):6206–14. Epub 2008/12/04. pmid:19050309
- 47. Marcucci G, Caligiuri MA, Bloomfield CD. Molecular and clinical advances in core binding factor primary acute myeloid leukemia: a paradigm for translational research in malignant hematology. Cancer investigation. 2000;18(8):768–80. Epub 2000/12/07. pmid:11107447