Prognostic significance of HOXB4 in de novo acute myeloid leukemia

As research into hematopoiesis advances, new factors associated with hematopoietic stem cell (HSC) activity have been discovered, and the contribution of HSC factors to hematopoiesis is now actively being investigated. Since the involvement of stem cells is considered to be important in hematological malignancies as well as normal hematopoiesis, we examined the expression of newly defined HSC factors including HOXB4, TCFEC, HMGB-1, FOS, and SPI-1 in the bone marrow (BM) from de novo acute myeloid leukemia (AML) patients. Expression levels of mRNA extracted from frozen specimens of AML patients and normal controls were measured by real-time polymerase chain reaction (PCR). Among the HSC factors, HOXB4 exhibited significantly higher expression in the BM of AML as compared with normal controls. Immunostaining for HOXB4 revealed that the HOXB4-positive cell ratios correlated well with the expression levels of mRNA for HOXB4 in AML BM. Based on the HOXB4-positive cell ratio, AML patients were divided into two groups (cases with higher ratios and lower ratios). The group with higher HOXB4-positive cell ratios had better prognoses as compared with the group with lower ratios. Multivariate analysis confirmed the HOXB4-positivity as an independent predictor of overall survival of AML patients. Lastly, mRNA expression levels for HOXB4 were inversely correlated with the expression levels of at least two genes, including ABCB1, which is known to be a multidrug-resistance gene. Our study distinguishes a subgroup of AML with higher HOXB4 expression and better prognosis, and this might be reflected by relative drug sensitivity in leukemic cells.     

Prognostic significance of chromosome analysis in de novo acute myeloid leukemia (AML)

Summary Between 1981 and 1986 cytogenetic studies of bone marrow and/or blood cells in 139 patients with de novo acute myeloid leukemia (AML) were performed. The overall incidence of chromosomal aberrations was 53%, and this was not significantly influenced by sex, age nor the FAB classification. The aberrations most often found were: complex anomalies (n=13), t(8; 21) (n=10), trisomy 8 (n=9), monosomy 7 (n=6), monosomy 5, 5q-, trisomy 11, 12p- (n=4) and trisomy 6, 11q-, inv [16] (n=3). The prognostic significance of chromosomal findings was evaluated in 112 patients treated by combination chemotherapy. The chromosomal status NN, AN, AA did neither significantly influence complete remission rate (NN: 68%, AN: 71%, AA: 60%) nor mean survival (NN: 24, AN: 26.6, AA: 35.6 months). On the other hand, certain types of chromosomal anomalies were of prognostic value. CR was obtained in all 10 patients with t(8; 21) but only in 2 out of 9 patients with complex aberrations. Median duration of CR in patients with t(8; 21) was significantly longer than in patients with a normal karyotype (30 vs 7 months).     

Neurofibromatosis‐1 gene deletions and mutations in de novo adult acute myeloid leukemia

Germline heterozygous alterations of the tumor‐suppressor gene neurofibromatosis‐1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real‐time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1‐deleted patients. We defined a small ～0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1‐deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression. Am. J. Hematol. 88:306–311, 2013. © 2013 Wiley Periodicals, Inc.     

Anti‐DNA topoisomerase IIα autoantibodies in localized scleroderma

Objective

To determine the prevalence and clinical correlation of anti‐DNA topoisomerase IIα (anti–topo IIα) antibody in patients with localized scleroderma.

Methods

Anti–topo IIα antibodies or anti‐DNA topoisomerase I (topo I) antibodies were determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. Inhibition of topo IIα enzymatic activity by the antibodies was evaluated by decatenation assays using kinetoplast DNA as a substrate.

Results

IgG or IgM anti–topo IIα antibody was detected in 76% (35 of 46) of patients with localized scleroderma, and in 85% (11 of 13) of patients with generalized morphea, the severest form of localized scleroderma. This prevalence of the antibody in patients with localized scleroderma was much higher than that found in patients with systemic sclerosis (SSc) (5 of 37 [14%]), systemic lupus erythematosus (2 of 26 [8%]), dermatomyositis (2 of 20 [10%]), and in healthy controls (3 of 42 [7%]). Immunoblotting confirmed the presence of IgG anti–topo IIα antibody in sera from patients with localized scleroderma and showed no cross‐reactivity of anti–topo IIα antibody with topo I. Anti–topo I antibody was not detected by ELISA in any sera from patients with localized scleroderma. In addition, anti–topo I antibody from SSc patients did not cross‐react with topo IIα. The presence of anti–topo IIα antibody was associated with a greater total number of sclerotic lesions and number of plaque lesions in patients with localized scleroderma. Furthermore, anti–topo IIα antibody was able to inhibit topo IIα enzymatic activity.

Conclusion

The results of the present study indicate that anti–topo IIα is a major autoantibody in localized scleroderma, and is distinct from anti–topo I antibody in SSc.

     

Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China

The 2-hydroxyglutarate (2-HG) has been reported to result from mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes and to function as an “oncometabolite.” To evaluate the clinical significance of serum 2-HG levels in hematologic malignancies, acute myeloid leukemia (AML) in particular, we analyzed this metabolite in distinct types of human leukemia and lymphoma and established the range of serum 2-HG in appropriate normal control individuals by using gas chromatograph–time-of-flight mass spectrometry. Aberrant serum 2-HG pattern was detected in the multicenter group of AML, with 62 of 367 (17%) patients having 2-HG levels above the cutoff value (2.01, log₂-transformed from 4.03 μg/mL). IDH1/2 mutations occurred in 27 of 31 (87%) AML cases with very high 2-HG, but were observed only in 9 of 31 (29%) patients with moderately high 2-HG, suggesting other genetic or biochemical events may exist in causing 2-HG elevation. Indeed, glutamine-related metabolites exhibited a pattern in favor of 2-HG synthesis in the high 2-HG group. In AML patients with cytogenetically normal AML (n = 234), high 2-HG represented a negative prognostic factor in both overall survival and event-free survival. Univariate and multivariate analyses confirmed high serum 2-HG as a strong prognostic predictor independent of other clinical and molecular features. We also demonstrated distinct gene-expression/DNA methylation profiles in AML blasts with high 2-HG compared with those with normal ones, supporting a role that 2-HG plays in leukemogenesis.Acute myeloid leukemia (AML) represents a group of clonal hematopoietic progenitor malignancies with considerable diversities in clinical and biological features (1). In addition to clinical parameter, such as age and white blood cell counts (WBC), a variety of biomarkers have been shown to be predictive of outcome in AML patients, including cytogenetic characteristics and patterns of recurrent gene mutations in the blasts, as exemplified by nucleophosmin (NPM1), fms-related tyrosine kinase 3 (FLT3), and CCAAT/enhancer binding protein-alpha (CEBPA) (1). Even with the remarkable progress of genomic studies on AML (2, 3), clinically useful biomarkers with prognostic values are still needed in a part of cases for better clinical management, particularly in cytogenetically normal AML (CN-AML) patients. Of note, gene mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were reported recently not only in gliomas but also in AML (4, 5). The prognostic significance of these mutations appears controversial in AML (6–10), although a metaanalysis suggests a poor outcome in cases with IDH1 mutations (11).IDHs are key enzymes that participate in the tricarboxylic acid metabolic cycle. Three members (IDH1, IDH2, and IDH3) are encoded by the IDH gene family and their activities are NADP(+)/NAD(+)-dependent. IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Mutant enzymes form a heterodimer, which display reduced catalytic activity to produce α-KG but a newly acquired activity to convert α-KG to 2-hydroxyglutarate (2-HG) (12). Recent studies have suggested that 2-HG may be an “oncometabolite” and play a role in driving malignant phenotype (13–15). Interestingly, 2-HG has been shown to competitively inhibit α-KG–dependent dioxygenases, such as the ten-eleven-translocation 2 (TET2) enzyme, and disturb the epigenetic regulatory network, leading to genome-wide histone and DNA damages with hypermethylation (12–14). Notably, in patients with d-2-hydroxyglutaric aciduria, there is an elevated risk of brain tumors (15). 2-HG can be metabolized by the enzyme 2-HG dehydrogenase (D2HGDH), but until now the possible contribution of genetic variants of this enzyme to the turnover of 2-HG has not yet been addressed.Although IDH1/2 mutations are biomarkers in gliomas, the serum levels of 2-HG in these mutations are usually normal (16). In AML harboring IDH1/2 mutations, serum 2-HG concentrations were elevated in some cases (17–21). Very recent reports suggested that high 2-HG could predict IDH1/2 mutations in AML and be used as a marker for minimal residual disease during clinical remission (21, 22). However, the significance of serum 2-HG levels in prognosis remains obscure. In this work, we examined the levels of 2-HG as a part of the metabolomic study in a large series of AML cases, as well as other hematologic malignancies and healthy controls, and characterized their relevant clinical and molecular features. In particular, we attempted to address the potential prognostic value of 2-HG in AML.     

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p = 0.0009) than the mutation not detectable (mutation-) group. Multivariate analysis showed that the p53 mutation was an independent factor (p = 0.005) for short overall survival as well as 60 yr or older (p = 0.001) and unfavorable karyotypes (p = 0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti-p53 monoclonal antibody (DO-7). All samples carrying missense mutations (N = 6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation-/accumulation+ group (N = 8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.     

Prognostic significance of dysplastic features of hematopoiesis in patients with de novo acute myelogenous leukemia

 The detection of dysplastic features of hematopoiesis in de novo acute myeloid leukemia (AML) by light microscopy is defined as AML with trilineage myelodysplasia (AML/TLMD). The prognostic relevance of these dysplastic features for patients with de novo AML remains unclear. In order to evaluate the role of dysplasia in de novo AML, bone marrow aspirates from 69 patients were analyzed prospectively and investigated separately for erythropoiesis, granulopoiesis and megakaryopoiesis by three independent investigators. The overall complete remission (CR) rate was 48.8% and partial remission (PR) or nonresponders consituted 52.2% of the patients investigated. The median overall survival time was 5 months with a disease-free interval of 3.5 months for all patients. Dysgranulopoiesis (DysG) was observed in 30.4%, dysmegakaryopoiesis (DysM) in 50.7%, and dyserythropoiesis (DysE) in 43.5%. Of all patients, 26.0% showed trilineage dysplastic features and were thus classified as AML/TLMD. A significantly worse prognosis (Kaplan-Meyer plot, Student's t–test) was calculated for those patients with detection of only DysG (p=0.002), DysM (p=0.02), DysE (p=0.04) as compared with patients without any dysplastic signs. An unfavorable karyotype was correlated with patients showing DysG (P=0.02) and DysM (P=0.04). For these patients with an unfavorable karyotype, the occurrence of any dysplastic features had no additional prognostic impact. Dysplastic features (DysG, DysM, DysE) seem to be an important prognostic factor in de novo AML correlating with short overall survival. DysG and DysM correlated well with the appearance of unfavorable chromosomal abnormalities. It may be reasonable to assume that patients with dysplastic features should be considered for more aggressive treatment schedules at the time of diagnosis. Received: 25 March 1997 / Accepted: 10 July 1997     

Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia

Cytofluorimetric detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at the time of diagnosis in 158 patients with acute myeloid leukemia using the C219 monoclonal antibody (MoAb). In 108 of these cases the JSB1 MoAb was also tested. An improved histogram subtraction analysis, based on curve fitting and statistical test was applied to distinguish antigen-positive from antigen-negative cells. A marker was considered positive when more than 20% of the cells were stained. At onset, P-170 was detected in 43% of cases with C219 and in 73% of cases with JSB1. There was a strict correlation between C219 and JSB1 positivity, as all C219+ cases were also positive for JSB1 MoAb (P < .001). No relationship was found between sex, age, organomegaly, and MDR phenotype. Significant correlation was found between CD7 and both C219 and JSB1 expression (P < .001 and .001, respectively). C219-negative phenotype was more often associated with a normal karyotype (24 of 55 with P = .030). Rhodamine 123 (Rh123) staining and flow cytometry analysis showed a significantly decreased mean fluorescence in 51 C219+ and 38 JSB1+ patients compared to 42 MDR negative ones (P < .001). The rate of first complete remission (CR) differed both between C219+ and C219- cases and between JSB+ and JSB- ones (30.9% v 71.1% and 35.4% v 93.1%, respectively, P < .001). Of the 21 C219+ patients who had yielded a first CR, 19 (90.4%) relapsed, compared with 28 of 64 (43.7%) C219- patients (P < .001). Of the 28 JSB1+ patients in first CR, 17 (60.7%) relapsed relative to 8 (29.6%) of 27 JSBI- ones (P = .021). A higher rate of relapses among MDR+ compared with MDR- patients was observed both for C219 and JSB1 MoAbs taken separately (C219 80% v 44%; JSB1 52% v 27%), with no relationship to age. The survival rates (Kaplan-Meyer method) were significantly shorter both in C219+ patients and in JSB1+ cases (P < .001). Disease-free survival curves followed this same trend. The combination (C219- JSB1+) identified a subset of patients with an intermediate outcome compared to C219 positive cases. The prognostic value of both markers (C219 and JSB1) was confirmed in multivariate analysis. These results suggest that the assessment of MDR phenotype by flow cytometry may be an important predictor of treatment outcome.     

Chromosome aberrations in de novo acute myeloid leukemia patients in Kuwait

Cytogenetic analysis was successfully performed at the time of diagnosis in 45 patients with de novo acute myeloid leukemia, including 10 children and 35 adults. In approximately 73% of AML patients (35 patients) clonal chromosome abnormalities were detected at the time of diagnosis. Twelve patients (22.8%) had apparently normal karyotypes. Recurring aberrations found in 22 of patients with abnormal karyotypes included t(15;17)(q22;q11), t(8;21)(q22;q22), inv(16)(p13q22), trisomy 8, monosomy 7 and del(5q). The highest frequency of chromosome changes was observed in AML-M3. The occurrence of the classical cytogenetic abnormalities was not a ubiquitous phenomenon. In 11 patients previously not described miscellaneous clonal chromosomal abnormalities were detected. Clonal chromosomal abnormalities detected in AML have shown correlations between specific recurrent chromosomal abnormalities and clinico-biological characteristics of the patients, therefore have been repeatedly shown to constitute markers of diagnostic and prognostic significance. Moreover, ongoing cytogenetic analysis can identify new nonrandom chromosome aberrations in AML and contribute to the identification of novel genes involved in the development of cancer, which can lead to better understanding of the disease pathogenesis.     

Prognostic significance of CD20 expression in adults with de novo precursor B-lineage acute lymphoblastic leukemia

Immunophenotypic classification of acute lymphoblastic leukemia (ALL) has well-recognized prognostic implications. The significance of CD20 expression has been evaluated in childhood precursor B-lineage ALL with conflicting results. We retrospectively analyzed the influence of CD20 expression on outcome in 253 adults with de novo precursor B-lineage ALL treated with either conventional (VAD/CVAD) or intensive (hyper-CVAD) frontline chemotherapy regimens in the pre-rituximab era. Overall, CD20 positivity of at least 20% was associated with lower 3-year rates of complete remission duration (CRD; 20% vs 55%, P < .001) and overall survival (OS; 27% vs 40%, p = .03). In the CD20 negative subset, the 3-year rates for CRD (58% vs 42%, p = .04) and OS (60% vs 28%, P < .001) were superior for hyper-CVAD compared with VAD/CVAD; rates were particularly favorable for the CD20 negative younger age group (68% and 85%, respectively). In contrast, 3-year CRD and OS rates were uniformly poor for the CD20-positive group regardless of therapy (27% or less). Multivariate analysis for event-free survival identified older age, leukocyte count higher than 30 x 10(9)/L, presence of Philadelphia chromosome, high systemic risk classification, and CD20 positivity as independent predictors of worse outcome. In conclusion, CD20 expression in de novo adult precursor B-lineage ALL appears to be associated with a poor prognosis. Incorporation of monoclonal antibodies directed against CD20 into frontline chemotherapy regimens warrants investigation.     

Expression and prognostic significance of survivin in de novo acute myeloid leukaemia

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients with de novo acute myeloid leukaemia (AML) was investigated. By immunofluorescence of bone marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50-90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) (P = 0.008 by the Mann-Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2; P = 0.007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC (P = 0.03 by the Mann-Whitney test) and favourable/intermediate cytogenetics (P= 0.03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin-positive and survivin-negative AML patients (P = 0.15 by the log-rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2.8 and P = 0.026, by the likelihood-ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor of de novo AML and suggest a role for apoptosis inhibition in influencing disease outcome.     

Expression of klotho and β‐catenin in esophageal squamous cell carcinoma,and their clinicopathological and prognostic significance

Esophageal carcinoma is one of the most common types of cancers in the world; the molecular mechanism underlying its tumorigenesis is still not well understood. This study was aimed at investigating the expression of klotho and β‐catenin in patients with esophageal squamous cell carcinoma (ESCC) and analyzing their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho and β‐catenin were determined by tissue microarray and immunohistochemical technique in ESCC and normal tissues, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. The serum klotho levels in 40 ESCC patients and controls were measured by sandwich enzyme‐linked immunosorbent assay system (ELISA). The expression level of klotho was significantly lower in ESCC than in the adjacent noncancerous tissues (30 vs. 50%, P < 0.000), and the protein level was negative correlated with clinical staging, histological grade, lymph node metastasis, and invasion depth (P < 0.05). Whereas, the expression of β‐catenin was much higher in ESCC than their corresponding normal mucosa tissues (78.3 vs. 11.5%, P < 0.000), and the level of protein correlated only with histological grade and invasion depth (P < 0.05). Correlation analysis showed the expression level of klotho inversely correlated with that of β‐catenin (r = ?0.214, P < 0.01). Patients with klotho‐positive tumors had longer survival than those with klotho‐negative tumors (P < 0.01). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (hazard ratio, 0.371; 95% confidence interval, 0.201–0.685; P < 0.01). ELISA showed that the level of serum klotho was markedly higher (461.50 ± 43.30 pg/mL) than control group (239.37 ± 20.65 pg/mL) (P < 0.001). Receiver operating characteristic analysis gave a cut‐off value of 327.031 of serum klotho with a sensitivity of 81.3% and specificity of 81.2% (P < 0.000). Our present study demonstrated for the first time that klotho might be a novel biomarker candidate for predicting progression and prognosis in patients with ESCC.     

Prognostic significance of CD56 antigen expression in acute myeloid leukemia

BACKGROUND AND OBJECTIVES. CD56 antigen expression has been reported in several hematologic malignancies. In acute myeloid leukemia (AML)M2 with t(8;21) and acute promyelocytic leukemia (APL) it has been found to be consistently associated with an unfavorable prognosis, whereas in other AML subtypes its role remains uncertain. We investigated CD56 expression in a cohort of AML patients in order to assess its frequency and prognostic relevance. DESIGN AND METHODS. Immunophenotypic analysis including that of CD56 antigen was available for 171 consecutive AML patients (139 with AML and 32 with APL), enrolled between December 1995 and December 1999 at a single institution. A sample of fresh bone marrow cells taken at diagnosis was recorded as positive when at least 20% of the cells double-stained with specific monoclonal antibodies against CD56 and CD33 antigens. RESULTS. CD56 positivity was demonstrated in 37 cases (21.6%). Its frequency was lower in M4 (6%) and higher in M5 (37%). The median percentage for CD56+ blasts was 56% (range 21-99%). CD56 positivity did not correlate with age, sex, blast count, favorable or unfavorable cytogenetics at diagnosis, nor did it influence the outcome in terms of complete remission (CR) duration (606 vs. 417 days, p=n.s.) or overall survival (OS) (210 vs. 277 days, p= n.s.). In the APL subgroup a significant difference in relapse rate was found at 3 years (71.4% in the CD56 positive group vs. 12% in the CD56 negative group, p=0.005). INTERPRETATION AND CONCLUSIONS. Our data confirm that CD56 positivity in APL patients at diagnosis is associated with a worse prognosis, suggesting that close molecular monitoring is necessary in CD56 positive APL patients. In contrast, the prognostic role of CD56 remains uncertain in the other AML subtypes.