Preferentially Expressed Antigen Of Melanoma Gene Research Articles

Correlation of preferentially expressed antigen of melanoma (PRAME) gene expression with clinical characteristics in acute leukemia patients

BackgroundPreferentially expressed antigen of melanoma (PRAME) gene is regularly overexpressed in acute leukemia (AL) and other malignant diseases which are recognized by human leucocyte antigen (HLA-24) located in the human chromosome of 22q11 coded by 509 amino acids. To rule out the PRAME gene expression in AL patients and its correlation with clinical characteristics in the Indian population set up by RT-qPCR. ResultsA total of 42 samples collected, 29 (69.4%) were males, and 13 (30.95%) were females, with a mean and standard deviation for age were 39.07 ± 22.22 years. Of which AML were of 22 (52.38%) cases, ALL were of 14 (33.33%) cases, and 6 (14.2%) cases which included other forms of leukemia. PRAME gene expression was highly expressed in thirty-three 27 (64.28%) AL patients compared to the least expression in healthy individuals. No significant difference between the different forms of AL (p=0.3203) was observed. Cytogenetic analysis of normal karyotype (NK), abnormal karyotype (Ab. K), and culture failure (CF) displayed statistical non-significance (p=0.5801). Among cytogenetic abnormalities obtained, no significant differences between the groups were observed (p=0.8507). Chloride, potassium, and absolute lymphocyte count (ALC) was found to be statistically significant with p=0.0038**, p=0.0358*, and p=0.0216*, respectively, between all other clinical characteristics. There was no correlation between the PRAME gene expression and clinical parameters. ConclusionPRAME gene expression in AL patients was highly expressed, comparable to studies reported globally with significant cytogenetic results. PRAME gene could be used as a potential diagnostic marker for monitoring the malignancies and minimal residual disease in AL.

Clinical features and prognosis of childhood B-lineage acute lymphoblastic leukemia expressing the PRAME gene

To study the clinical and prognostic significance of the preferentially expressed antigen of melanoma (PRAME) gene in the absence of specific fusion gene expression in children with B-lineage acute lymphoblastic leukemia (B-ALL). A total of 167 children newly diagnosed with B-ALL were enrolled, among whom 70 were positive for the PRAME gene and 97 were negative. None of the children were positive for MLL-r, BCR/ABL, E2A/PBX1, or ETV6/RUNX1. The PRAME positive and negative groups were analyzed in terms of clinical features, prognosis, and related prognostic factors. Compared with the PRAME negative group, the PRAME positive group had a significantly higher proportion of children with the liver extending >6 cm below the costal margin (P<0.05). There was a significant reduction in the PRAME copy number after induction chemotherapy (P<0.05). In the minimal residual disease (MRD) positive group after induction chemotherapy, the PRAME copy number was not correlated with the MRD level (P>0.05). In the MRD negative group, there was also no correlation between them (P>0.05). The PRAME positive group had a significantly higher 4-year event-free survival rate than the PRAME negative group (87.5%±4.6% vs 73.5%±4.6%, P<0.05), while there was no significant difference between the two groups in the 4-year overall survival rate (88.0%±4.4% vs 85.3%±3.8%, P>0.05). The Cox proportional-hazards regression model analysis showed that positive PRAME expression was a protective factor for event-free survival rate in children with B-ALL (P<0.05). Although the PRAME gene cannot be monitored as MRD, overexpression of PRAME suggests a good prognosis in B-ALL.

Both Methylation and Copy Number Variation Participated in the Varied Expression of PRAME in Multiple Myeloma.

PurposeThe cancer-testis antigen, which is a preferentially expressed antigen of melanoma (PRAME), is an ideal target for immunotherapy and cancer vaccines. Since the expression of this antigen is relevant to therapy responses, the heterogeneity in its expression and the underlying mechanism need to be investigated.Patients and MethodsPlasma cell sorting was performed in 48 newly diagnosed multiple myeloma (MM) patients. Real-time quantitative PCR was performed to examine the PRAME transcript levels and gene copy numbers. Bisulfate clone sequencing of the PRAME promoter and exon 1b regions was performed in 4 patients. Quantitative methylation-specific PCR of the +287 CpG site was performed for all patients. The human MM cell lines RPMI8226, LP-1 and MOLP-2 were treated with 5-azacytidine.ResultsThe median PRAME transcript level was 3.1% (range: 0–298.3%) in the plasma cells sorted from the 48 MM patients. Eleven (22.9%) and 37 (77.1%) patients were individually categorized into the PRAME low- and high-expression groups according to the cut-off value of 0.05%. The methylation ratios of the promoter and the 3ʹ region of exon 1b region were both negatively related to the transcript levels. The degrees of methylation at the +287 CpG site were significantly negatively related to the transcript levels in all 48 patients (r=−0.44, P=0.0018), and those in the high-expression group (r=−0.69, P<0.0001) but not those in the low-expression group (r=−0.27, P=0.43). All 5 patients with homozygous deletions were categorized into the low-expression group. There were no significant differences in the PRAME transcript levels between the hemizygous deletion (n=8) and no deletion (n=35) groups (P=0.40). Furthermore, the PRAME transcript levels significantly increased in the MM cell lines after treatment with 5-azacytidine.ConclusionBoth methylation and copy number variation may participate in the regulation of PRAME expression in MM; in patients with no homozygous deletion, PRAME expression is mainly controlled by methylation, and a proportion of fairly low expression is caused by homozygous deletion.

PRAME Gene Copy Number Variation Is Related to Its Expression in Multiple Myeloma.

Multiple myeloma (MM) patients commonly present abnormal expression of cancer-testis antigens, which may serve as immunotherapeutic targets and prognostic factors. We previously reported that preferentially expressed antigen of melanoma (PRAME) overexpression in bone marrow mononuclear cells is related to progression in MM patients treated with non-bortezomib-containing regimens. The mechanism underlying variations in PRAME expression remains unknown. To investigate the impact of gene copy number variation (CNV) on PRAME expression, plasma cells were sorted from 50 newly diagnosed patients and 8 healthy volunteers to measure PRAME transcript levels and gene copy numbers by real-time quantitative polymerase chain reaction. A total of 14 (28.0%), 7 (14.0%), and 29 (58.0%) patients exhibited overexpression, expression within the normal range, and low expression, respectively. PRAME overexpression was significantly related to a lower 1-year progression-free survival rate compared with PRAME low expression (20.0% vs. 88.9%, p = 0.043). The mean PRAME gene copy number relative to albumin (ALB) in normal samples was ∼1.0, whereas 4.0%, 24.0%, 70.0%, and 2.0% of patients had PRAME gene relative copy numbers of approximately 0, 0.5, 1.0, and 2.0, respectively. Patients with PRAME gene deletion (relative copy number of 0 or 0.5) had significantly higher frequency of PRAME nonoverexpression and lambda light chain expression than those with no deletion (p = 0.011 and 0.003). Thus, PRAME gene CNV occurs in MM. Gene deletion may be one mechanism leading to PRAME nonoverexpression and related to immunoglobulin lambda light chain locus rearrangement. PRAME overexpression in plasma cells might be an adverse prognostic factor for progression in MM.

Methylation pattern of preferentially expressed antigen of melanoma in acute myeloid leukemia and myelodysplastic syndromes.

Preferentially expressed antigen of melanoma (PRAME), a tumor-associated antigen, is overexpressed in a variety of hematologic malignancies with a great variation in expression. The majority of patients with acute myeloid leukemia (AML) 1-eight-twenty one (ETO)+ AML and a certain number of myelodysplastic syndromes (MDS) have an abnormally high increase in PRAME expression level. The landscape of PRAME methylation requires evaluation in order to determine the most relevant sites and the exact association of its methylation with expression level and type of disease. In the present study, bone marrow samples collected from 8 AML1-ETO+ AML, 4 MDS, 3 AML1-ETO- AML and 2 normal volunteers underwent bisulfate sequencing to analyze the methylation status of all four 5'-C-phosphate-G-3' (CpG) regions within the entire PRAME gene. The median PRAME transcript level of 15 patients was 204.5% (range, 0.02-710.3%). PRAME transcript levels were inversely associated with the degree of methylation of the -389 to -146 CpG sites (r=-0.69; P=0.002) in the 3' part of the promoter region and the +132 to +363 CpG sites (r=-0.69; P=0.006) in the exon 1b region. However, not every sample strictly followed this correlation: Certain samples with high degrees of methylation demonstrated abnormally high expression levels, and vice versa. The methylation ratios of CpG sites in exon 1a were low for all samples (range, 0.0-13.8%), and those in exon 2 were similar in 16 samples (range, 72.4-93.4%), with the exception of one patient with high expression (425.2%) and significantly low degree of methylation in the PRAME gene (22.2%). MDS patients revealed similar methylation ratios in the 3' section of the promoter region, but tended to have lower methylation ratios in the exon 1b region (P=0.62 and P=0.09, respectively) compared with those observed in AML1-ETO+ patients with AML and similar degree of PRAME overexpression. Therefore, the hypomethylation of CpG sites in the 3' part of the promoter region and in exon 1b was typically found with PRAME overexpression in AML and MDS. Methylation of other CpG islands, epigenetic and genetic mechanisms, and type of disease may also be involved.

Clinical Importance of PRAME Gene Expression in Acute Myeloid Leukemia

Background: The PRAME (preferentially expressed antigen of melanoma) gene is frequently over expressed in a wide variety of malignant diseases, including acute myeloid leukemia (AML). Objective: To investigate the expression of PRAME gene in AML and its importance in the prognosis. Patients and methodsThe transcript level of PRAME gene was detected in peripheral blood specimens from 72 de novo AML patients and 40 control subjects by using real-time quantitative PCR and the prognostic value of PRAME was determined through treatment outcome and survival analysis. Results: PRAME gene was highly expressed in 74% of de novo AML cases while low expression was detected among healthy controls, the mean value of PRAME gene expression was highest among M3 AML subtype and lowest among M5b AML subtype. No significant correlation could be observed between PRAME gene expression and different CBC parameters, age, gender or cytogenetics, but there was a significance positive correlation between PRAME gene expression with treatment outcome, disease free survival (DFS) and overall survival. Conclusion: PRAME gene over expression might be a good prognostic factor of AML and could be a useful target for immunotherapy.

Preferentially Expressed Antigen of Melanoma (PRAME) and Wilms' Tumor 1 (WT 1) Genes Expression in Childhood Acute Lymphoblastic Leukemia, Prognostic Role and Correlation with Survival.

BACKGROUND:Acute lymphocytic leukemia (ALL) is the most common hematologic malignancy in children. In young children it is also largely curable, with more than 90% of afflicted children achieving long-term remission. PRAME (Preferentially expressed antigen of melanoma) gene belongs to Group 3 class I HLA-restricted widely expressed antigens in which genes encoding widely expressed tumor antigens have been detected in many normal tissues as well as in histologically different types of tumors with no preferential expression on a certain type of cancer. It has been found to be expressed in a variety of cancer cells as leukemia & lymphoma. PRAME monitoring can be useful for detection of minimal residual disease and subsequent relapses particularly those leukemias in which specific tumor markers are unavailable. Wilms’ tumor1 (WT1) gene was identified as a gene that plays an important role in normal kidney development and inactivation of its function was shown to result in the development of Wilms’ tumors in paediatric patients. Disruption of WT1 function has been implicated in the formation of many different tumor types.AIM:to study how PRAME & WT 1 genes expression patterns influence cancer susceptibility & prognosis.PATIENTS & METHODS:50 patients with denovo childhood acute lymphoblastic leukemia, as well as 50 age and sex matched apparently healthy volunteers were genotyped for PRAME and WT1 genes expression by reverse transcription polymerase chain reaction (RT-PCR).RESULTS:PRAME gene was expressed in 34 of the patients (68%) and WT1 gene was expressed in 26 of the patients (52%). Expression of both genes was significantly higher compared to controls (P < 0.0001). Analysis of relapse free survival among our patients revealed that patients expressing PRAME gene or WT1 gene had better relapse free survival (p value=0.02 and 0.01 respectively). Relapse free survival increased significantly among patients coexpressing PRAME and WT 1(p value =0.001).CONCLUSION:It is concluded that the expression of PRAME and WT1 genes are indicators of favorable prognosis and can be useful tools for monitoring minimal residual disease (MRD) in acute leukemia especially in patients without known genetic markers. Differential expression between acute leukemia patients and healthy volunteers suggests that the immunogenic antigens (PRAME and WT1) are potential candidates for immunotherapy in childhood acute leukemia.

Prognostic Significance of WT1 and PRAME Gene Expression in Bone Marrow Samples of MDS and AML Patients Treated with Azacytidine

Introduction: The Wilms’ tumor gene (WT1) and preferentially expressed antigen of melanoma (PRAME) gene are frequently overexpressed in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) Combined quantitation of PRAME and WT1 transcript levels could provide a powerful molecular marker in newly diagnosed MDS patients, enabling the early identification of relapse through minimal residual disease monitoring and also to enable alternate treatment strategies (Qin et al, 2012).In this study, we investigated the prognostic impact of WT1 and PRAME gene expression on MDS and AML patients treated with azacytidine.Methods:In this ethically approved, retrospective study, we studied WT1 and PRAME gene expression in the bone marrow of 20 patients with MDS and AML (unsuitable for more intensive chemotherapy) prior to starting epigenetic therapy with azacytidine (100mg/M2 sc for 5 days every 28 days). 20 patients with lymphoma not involving the bone marrow were used as a control group. Quantitative assessment of WT1 and PRAME transcript levels was performed on formalin fixed paraffin-embedded trephine biopsies, using the WT1 Ipsogen ProfileQuant and a ‘homebrew’ kit respectively. A control GADPH was used. Overall survival was extimated using the Kaplan-Meier method and compared using the log-rank test. The logistic regression model was used to correlate IPSS-R with WT-1 and PRAME gene expression levels.Results: The median age of the patient group was 71. 16 patients had MDS (RCMD: 3; RARS: 2; RAEB1: 2; RAEB2: 5; CMML: 4) and 4 had AML. Median age of the control group was 61. Median number of cycles of azacytidine was 4 (range 1-21). In 11 of the patient group and in all of the controls, neither PRAME nor WT1 gene expression was detected.. PRAME gene expression without WT1 gene expression was detected in 5 patients, whilst, in the remaining 4 patients, both PRAME and WT1 gene expression was detected. There was no significant difference in mean percentage of bone marrow blasts between patients with detectable PRAME +/- WT1 gene expression (16.67%) and those without (12.64%).Median gene expression levels of PRAME +/- WT1 significantly correlated with IPSS-R (p=0.003).Overall survival (OS) was significantly inferior in patients who were positive for PRAME +/- WT1 (p=0.0103). Median OS for patients positive for both PRAME and WT1 was 270 days and for patients who were PRAME positive and WT1 negative was 300 days, whilst patients negative for both had a median OS of 630 days (figure 1).Conclusions: Our study results show that patients who have detectable WT1 and PRAME expression in their bone marrow prior to azacytidine therapy have more advanced disease in terms of IPSS-R score and have a much inferior OS compared to patients without detectable WT1 and PRAME. Such patients should be considered for either additional or alternate therapy to single agent azacytidine. DisclosuresNo relevant conflicts of interest to declare.

PRAME Gene Expression in Childhood Acute Lymphoblastic Leukemia: Impact on Prognosis

The PRAME (preferentially expressed antigen of melanoma) gene is frequently overexpressed in a wide variety of malignant diseases, including acute myeloid leukemia (AML) and acute B-cell malignancies. To study the expression of PRAME gene and clarify its prognostic impact on disease outcome. Screening for PRAME gene expression was assessed using real-time reverse transcriptase polymerase chain reaction in 55 pretreated ALL bone marrow samples. PRAME positivity was found in 14 (31.3%) of 45 patients. No significant correlation could be observed between PRAME expression and clinical characteristics. Positive PRAME expressers had a statistically higher CR (p = 0.001), lower relapse (p = 0.02), lower mortality (p < 0.001), a trend towards lower Refractory disease (p = 0.10), and a statistically longer DFS and OS (p < 0.001, < 0.001, respectively) in comparison to negative PRAME expressers. Our results suggested that PRAME was a predictor for better outcome, could be a useful target for immunotherapy, and might represent a candidate marker for the monitoring of minimal residual disease.

Correlation between Preferentially Expressed Antigen of Melanoma and Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand Gene Expression in Different Types of Leukaemia Patients

Introduction: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) down-regulation by preferentially expressed antigen of melanoma (PRAME) is a general phenomenon in different types of solid tumours, but research on the correlation between PRAME and TRAIL gene expression in leukaemia patients is rare. Method: PRAME and TRAIL expression was detected in bone marrow samples from 80 newly diagnosed acute leukaemia (AL) patients and 40 chronic myeloid leukaemia (CML) patients using TaqMan-based real-time quantitative PCR methods, and a linear correlation analysis was performed on their levels of expression. A total of 15 normal bone marrow samples from individuals with non-malignant haematological diseases served as normal controls. Results: PRAME expression was higher in both AL and CML patients compared to controls (both p < 0.001). CML patients in both blast crisis (BC) and the accelerated phase (AP) had significantly higher PRAME levels than CML patients in the chronic phase (CP) (p = 0.006 and 0.0461, respectively). TRAIL expression was higher in both the acute myeloid leukaemia (AML) group and the acute lymphoblastic leukaemia (ALL) group than in the controls (p = 0.039 and 0.047, respectively). In contrast, CML patients had lower TRAIL levels than controls (p = 0.043), and TRAIL expression in CML patients in the advanced phases (BC and AP) was significantly lower than in CML-CP patients (p = 0.006). In CML patients, there was a significant inverse correlation (Spearman's R = -0.6669, p < 0.0001) between PRAME and TRAIL gene expression, while a greater significant inverse correlation was found in patients in the advanced phases (BC and AP) (R = -0.6764). In addition, no correlation was observed in AML and ALL patients. Conclusion: The simultaneous detection of PRAME and TRAIL gene expression may be helpful to monitor condition changes in leukaemia patients and evaluate therapeutic effects in clinical practice, particularly in CML patients.

The Cancer Testis Antigen PRAME As a Biomarker for Solid Tumor Cancer Management

Among the best-characterized tumor antigens are the cancer-testis antigens (CTAs), or germ cell antigens. These are a category of nonmutated genes that are restricted in expression to male germ cells and fetal tissues, but are detected in a variety of cancers and the cells derived from them. CTA gene families, including MAGE, BAGE, GAGE, LAGE/NY-ESO-1 and PRAME (preferentially expressed antigen of melanoma), encode antigenic peptides recognized by T lymphocytes. Numerous members of the CTA gene families are under intense study as targets for immunotherapy and as biomarkers for diagnosis and/or prognosis. Unlike other CTA genes, the PRAME gene (also known as MAPE or OIP4 ) has been shown to be expressed at low levels in ovarian, endometrial and adrenal tissues [1]. Originally characterized as a tumor antigen in a melanoma patient, PRAME is one of the best-characterized CTA family members because of its prevalence in both acute and chronic leukemias, and Hodgkin’s lymphoma [2]. Significant association of PRAME expression has been reported in acute myelogenous leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloma and chronic myelogenous leukemia (CML). In CML, expression has been shown to correlate with disease progression [2,3]. PRAME has been reported to be an independent prognostic factor in AML, being associated with increased overall survival in patients with a favorable or poor prognoses based on karyotype [2,3]. The other area of promising clinical utility is disease stratification and the detection of residual disease. Although it varies according to disease type, the level of PRAME expression can indicate probable resistance to chemotherapy in lymphomas and AML [4]. PRAME expression has been observed to significantly decrease in

PRAME Gene Expression in Acute Leukemia and Its Clinical Significance.

ObjectiveTo investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance.MethodsThe level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed.ResultsThe PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M3, 33.3% in M2, and 28.6% in M5. Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype, but not with age, gender, white blood count or percentage of blast cells.ConclusionsThe PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia.

PRAME (Preferentially Expressed Antigen of Melanoma) Is a Novel Marker for Differentiating Serous Carcinoma From Malignant Mesothelioma

The PRAME (preferentially expressed antigen of melanoma) gene was previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared with malignant mesothelioma using gene expression arrays. The objective of this study was to validate this finding at the messenger RNA (mRNA) and protein levels. Quantitative real-time polymerase chain reaction analysis of 126 müllerian carcinomas and 23 malignant mesotheliomas showed significantly higher PRAME mRNA expression in the former tumor (P < .001; test sensitivity and specificity, 89% and 91%, respectively). PRAME protein was expressed in 41 of 50 müllerian carcinomas and 0 of 30 mesotheliomas using Western blotting (P < .001; test sensitivity and specificity, 82% and 100%, respectively). PRAME levels in müllerian carcinoma were unrelated to survival; however, PRAME protein expression was up-regulated in solid metastases compared with primary carcinoma and effusions (P < .001). Our data confirm that PRAME effectively differentiates müllerian carcinoma from malignant mesothelioma at the mRNA and protein levels, suggesting a role in the diagnostic workup of serosal cancers.

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

AbstractAbstract 1695 Introduction:The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods:The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results:A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions:Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures:No relevant conflicts of interest to declare.

Effect of 5-Azacytidine (5-AzaC) In the Expression of PRAME In Acute Myeloid Leukemia (AML)

AbstractAbstract 3615 Introduction:Preferentially expressed antigen of melanoma (PRAME) was first isolated as a human melanoma antigen by cDNA expression cloning using melanoma-reactive cytotoxic Tcells (CTL). PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies and solid tumors. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). In addition, several authors have suggested that PRAME could be used as a target for anticancer T-cell therapy. PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies. Methods:We analyzed PRAME mRNA expression of blast cells from AML patients at diagnosis versus CD34+ stem cells from healthy donors by RT-PCR without treatment or after exposure to 1mM 5-azaC during the four days of culture and correlated the expression of PRAME with the methylation status of the promoter. Results:PRAME is significantly over-expressed in blasts from AML patients (n=11) compared with normal CD34+ cells (n=8) ((700±1102 vs. 1.8±2.5 p=0.002). Interestingly, we found an inverse correlation between PRAME expression and the degree of methylation in the promoter among both AML samples and healthy donors (r=-0.77 p=0.010). In order to evaluate the effect of 5-azaC on PRAME gene expression, we treated blast cells and CD34+ cells from healthy donors with the drug and we observed that the exposure to the drug induced a decrease in the percentage of methylation in the promoter and subsequently increased the expression of PRAME but, interestingly, the higher the basal methylation of the promoter the more intense the effect of the drug among AML cells. By contrast, CD34+ cells from healthy donors were resistant to the effect of the drug so that no significant changes were observed neither in terms of methylation status of the promoter nor in the expression of PRAME prior to or after exposure to the drug among healthy donors. Conclusions:The promoter region is highly methylated in normal CD34+ cells compared to AML cells and this pattern correlates with a higher expression of PRAME in blasts. Furthermore, the level of PRAME methylation was reduced in AML patients after exposure to 5-azaC which correlated with an increase in the expression of PRAME. By contrast, the effect of 5-azaC on the methylation pattern of the promoter was significantly lower in CD34+ cells from healthy donors. Disclosures:Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is to increase PRAME expression in blasts from AML patients and generate CTL CD8+ specific response against tumor cells.

Development of PRAME in the diagnosis and treatment of children acute leukemia

The gene of preferentially expressed antigen of melanoma(PRAME) is a gene marker of leukemia,which can be used to monitor the minimal residual disease,and can make us to comprehend the curative effect,prognosis,the grade of risk and can predict relapse.In addition,PRAME as a human tumour antigen,can be recognized by autologous cytolytic T lymphocytes,is a ideal target of immunotherapy in leukemia,and investigate the methylation level of PRAME gene CpG island,which can help us comprehend the relation of the mechanism of PRAME and leukemia. Key words: PRAME; Minimal residual disease; Immunotherapy; Methylation

Quantification of the PRAME transcripts in patients with acute myeloid leukemia

To analyze the expression level and clinical significance of the preferentially expressed antigen of melanoma (PRAME) transcripts in patients with acute myeloid leukemia (AML). Real-time quantitative polymerase chain reaction with EvaGreen dye was established to detect the expression level of PRAME transcripts in the bone marrow mononuclear cells of 56 AML cases and 20 controls. The clinical association of PRAME transcripts was analyzed. The PRAME transcripts were 0-1.46% (median 0.18%) and 0-21 618.09% (median 9.79%) in controls and AML cases, respectively (P< 0.01). Among the FAB subtypes, those with M1, M2, M3 and M4 had significantly higher level of PRAME transcripts than controls. However, those with M5 had similar level of PRAME transcripts as controls. There was a significantly negative correlation between the PRAME transcripts and cytogenetic risk groups (r= -0.438, P= 0.001). Cases in low risk had significantly higher level of PRAME transcripts than those in intermediate and high risk. Among cases with AML-M2, those with t(8;21) had significantly higher level of PRAME transcripts (135.06% -21 618.09%, median 2201.88%) than those without t(8;21)(0.14% -1696.30%, median 17.97%)(P= 0.002). In a patient with sequential samples, PRAME transcripts significantly decreased after induction therapy and significantly increased after relapse. The PRAME transcript was highly expressed in AML patients and was a favorable marker of prognosis. Quantification of PRAME transcript can be used in monitoring disease status of AML.

Oncogene Associated cDNA Microarray Analysis Shows PRAME Gene Expression is a Marker for Response to Anthracycline Containing Chemotherapy in Patients with Diffuse Large B-cell Lymphoma

CHOP (cyclophosphamide, adriamycin, vincristine, and prednisolone) therapy achieves a response in more than 60% patients with diffuse large B-cell lymphomas (DLBCLs). However, DLBCL shows a heterogeneous response to chemotherapy, and some patients are refractory to CHOP therapy. This difference in response to therapy is most likely due to differences in biological characteristics. We used cDNA microarray analysis to identify genes differentially expressed in anthracycline containing chemotherapy-resistant DLBCLs (7 patients) compared with anthracycline containing chemotherapy-sensitive DLBCLs (6 patients). Nine genes on the cDNA chip showed increased expression in anthracycline containing chemotherapy-resistant patients. We chose the preferentially expressed antigen of melanoma (PRAME) gene because it showed the highest expression in anthracycline containing chemotherapy-resistant DLBCLs on the cDNA chip, and it has been linked to prognosis of hematological malignancies. We also examined the relationship between PRAME gene expression and progression-free survival (PFS) in 45 patients with DLBCL. The progression-free survival of PRAME-positive patients (n=12) was significantly worse than that of PRAME-negative patients (n=33) (p=0.0373). Our results therefore indicate that PRAME expression in DLBCL correlates with response to anthracycline containing chemotherapy.

PRAME expression and clinical outcome of breast cancer

The tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is expressed in a variety of malignancies, including breast cancer. We have analysed PRAME gene expression in relation to clinical outcome for 295 primary breast cancer patients. Kaplan–Meier survival curves show a correlation of PRAME expression levels with increased rates of distant metastases and decreased overall patient survival. This correlation existed both for the entire patient group (n=295) and for the subgroup of patients (n=185) who did not receive adjuvant chemotherapy. Multivariable analysis indicated that PRAME is an independent marker of shortened metastasis-free interval in patients who did not receive adjuvant chemotherapy. PRAME expression was associated with tumour grade and negative oestrogen receptor status. We conclude that PRAME expression is a prognostic marker for clinical outcome of breast cancer, independent of traditional clinicopathological markers.

The Expression and Clinical Significance of PRAME Gene in Acute Leukemia.

Objective To investigate the expression of PRAME (preferentially expressed antigen of melanoma) gene in acute leukemia and its clinical significance in monitoring prognosis, detecting minimal residual disease (MRD) and gene immunotherapy.Methods The expression of PRAME gene mRNA in bone marrow mononuclear cells is measured by reverse transcriptase polymerase chain reaction in 34 patients with acute leukemia and 12 bone marrow samples of health donors. The relationships between PRAME gene expressions and some clinical data, such as gender, age, white blood count, leukemic immunophenotype, the percentage of blast cells, and the karyotype of chromosome, were also estimated.Results PRAME gene was expressed in 38.2% of all the patients, 40.7% of all the AML patients, which was higher than the 28.6% of ALL patients (p >0.05). There was no expression of PRAME gene in healthy donors. In all the sub phenotypes of AML, the expressive rate of PRAME gene in M3 patients is 80%, which is higher than that in M2 (33.3%) and in M5 (28.6%). The expressive rate of PRAME gene was also positively correlated with the expression of CD15, CD33, and the abnormality in the karyotype of chromosome, but not correlated with age, gender, white blood count and percentage of blast cell in bone marrow.Conclusion PRAME gene is highly expressed in acute leukemia, and could be regarded as a useful tool for monitoring MRD. Differential expression in acute leukemia patients vs. healthy donors suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in acute leukemia.