急性白血病患者WT1、PRAME、ERG、TRX-2基因表达的检测及其与疾病分型、疗效的关系

目的:研究急性白血病患者肾母细胞瘤基因(WT1)、黑色素瘤优先表达抗原(PRAME)、ETS相关基因(ERG)、硫氧还蛋白-2(TRX-2)基因表达及其与疾病分型、疗效的关系.方法:将本院收治的90例白血病患者纳入研究的白血病组,将同期体检健康者纳入健康组;进一步根据分型将白血病患者分为ALL组和AML组,根据化疗后疗效将白血病患者分为完全缓解组和复发组.采集患者的外周血,检测WT1、PRAME、ERG、TRX-2基因的mRNA含量.结果:白血病组血液中WT1、PRAME、ERG、TRX-2基因的mRNA含量高于对照组,差异有统计学意义(P＜0.05);AML组血液中WT1、PRAME、ERG、TRX-2基因的mRNA含量高于ALL组,差异有统计学意义(P＜0.05);复发组的血液中WT1、PRAME、ERG、TRX-2基因的mRNA含量高于完全缓解组,差异有统计学意义(P＜0.05).结论:WT1、PRAME、ERG、TRX-2基因异常高表达与白细胞的发生有关,也与疾病的分型、化疗的效果密切相关.     

PRAME基因在成人急性白血病的表达及其与预后的相关性

本研究探讨黑色素瘤特异性抗原（preferentially expressed antigen of melanoma，PRAME）基因在成人急性白血病中的表达及其与预后的相关性。应用逆转录一聚合酶链反应（RT—PCR）技术检测73例初发、3例复发成人急性白血病患者、7例特发性血小板减少性紫癜（ITP）患者和8例正常人骨髓单个核细胞（BMMNC）以及K562和U937二个白血病细胞株PRAME mRNA的表达水平，分析其在成人急性白血病中的表达规律及其在判断疗效及预后、监测微小残留病（MRO）和在特异性免疫治疗中的意义。结果表明：PRAME基因在成人急性白血病中的表达率为36．8％（n=28），急性髓系白血病（AML）为42．9％（n=24），急性淋巴细胞白血病（ALL）为20％（n=4）。AML的表达率显著高于ALL（P〈0．01）。在K562和U937细胞都有PRAME基因表达，它们的表达水平分别为0．57和0．46。在15例对照（正常人、ITP患者）未检出PRAME基因表达。AML各亚型之间（除去病例数为0例的M7，M1及M6各1例）、ALL各亚型（L1，L2）之间PRAME mRNA的表达水平无显著性差异。患者的年龄、初诊或者复发时白细胞计数、血红蛋白、血小板计数、骨髓中原始细胞、幼稚细胞比例以及脾脏厚度对PRAME mRNA的表达无相关性。完全缓解组PRAME mRNA的表达水平与部分缓解组和死亡组有显著性差异，并且随着PRAME mRNA的表达水平升高，治疗效果越好。对1例病人动态监测发现，在复发之前PRAMEmRNA的表达会升高。结论：PRAME基因在成人急性白血病中存在高表达，其表达水平与预后密切相关，并可以作为监测MRD的标志基因。PRAME在白血病特异性免疫治疗中是潜在的靶抗原。     

Expression of NY-ESO-1, MAGE-A3, PRAME and WT1 in different subgroups of breast cancer: An indication to immunotherapy?

ObjectivesCancer Testis Antigens are immunogenic tumor-specific proteins. We investigated NY-ESO-1, MAGE-A3 and PRAME, in addition to WT1 expression in different Breast Cancer (BC) subtypes. We then evaluated the expression rate of NY-ESO-1 in early Triple Negative breast cancer (TNBC), and investigated whether its expression would be maintained or lost in the metastatic setting to explore possible immunotherapy indication.Materials and methodsThree subgroups of BC patients were selected by the expression of ER, PgR and Her2. Tissue microarray was performed on a total of 92 Invasive BC. Sections were stained for NY-ESO-1, MAGE-A3, PRAME and WT1. The second cohort was composed by 26 metastatic TNBC patients from whom both the primary and secondary lesion tissues were available. Sections were stained for NY-ESO-1.ResultsNY-ESO-1 was the only differentially expressed antigen and was absent in ER+ and ER-PgR + tumors, as for an exclusive expression of either NY-ESO-1 or at least one hormonal receptor (HR+). NY-ESO-1 was particularly represented in TNBC. No correlation has been found between MAGE-A3 and PRAME expression and subtype WT1 had low expression, except in the Her2+ group. In the second cohort, NY-ESO-1 was expressed in 12 and 24% of primary and metastatic lesions respectively.ConclusionsThis study defines a distinction between HR+ and HR-tumors through NY-ESO-1 expression.TNBC subgroup has the highest frequency of NY-ESO-1+ cases, and it could be the candidate population for the development of anti-NY-ESO-1 vaccine, both in the adjuvant or metastatic setting, and for the selection of cases suitable for immunotherapy.     

PRAME mRNA levels in cases with acute leukemia: clinical importance and future prospects

The PRAME (preferentially expressed antigen of melanoma) gene has been shown to be expressed in high levels in some solid tumors and hemopoietic neoplasias but not or only weakly expressed in normal tissues. It encodes an antigen recognized by autologous cytolytic T lymphocytes. PRAME is a good candidate for tumor immunotherapy and is a useful marker gene for detection of minimal residual disease (MRD). In this study, PRAME mRNA using real-time RT-PCR was studied in 74 adult cases with acute leukemia-68 had de-novo acute leukemia, 3 had chronic myeloid leukemia-blastic crisis (CML-BC), and 3 had myelodysplastic/myeloproliferative syndrome-blastic transformation (MDS/MPD-BT)-and the results were compared with 30 age-matched healthy volunteers. Nineteen of 74 cases with leukemia expressed PRAME, while only 2 controls showed weak expression. The prevalence of PRAME expression in AML and ALL cases was 30% and 17%, respectively. We did not find any important correlation between PRAME expression and clinical characteristics, such as age, sex, organomegaly/lymphadenopathy, Hb, WBC count, platelet count, LDH level, alkaline phosphatase, albumin, cell-surface antigens, response to therapy, or progression-free and overall survival. PRAME was monitored in 15 cases during remission and/or relapse. There was a good correlation between PRAME mRNA and hematological remission and/or relapse. Interestingly, PRAME was very high in one case with AML but was not found 3 months after allogeneic transplantation. PRAME mRNA is observed in about one-third of AML cases; it may be a useful marker to detect MRD, and it may also be a good predictor for the timing of donor lymphocyte infusions (DLI) in the post-transplant period in cases of molecular relapse.     

PRAME基因在急性白血病中的表达及临床意义分析

本研究检测黑色素瘤优先表达抗原(PRAME)基因在不同类型急性白血病(acute leukemia,AL)中的表达及其与病情发展的相互关系.用建立的检测PRAME基因的SYBR Green Ⅰ荧光实时定量逆转录聚合酶链反应(RQ-RT-PCR)方法,对55例急性白血病患者(急性髓系白血病43例、急性淋巴细胞性白血病9例、其它类型的白血病3例)的骨髓样本进行PRAME基因表达情况的检测.同时选择7例非恶性血液疾病患者的骨髓样本和8例正常人外周血样本作为阴性对照,并用白血病细胞系K562作为阳性对照.结果表明35例急性白血病患者中检测出不同水平的PRAME基因表达,在其余20例急性白血病患者及15例对照样本中均未检测出PRAME基因,总阳性率64%,两组之间具有非常显著的差异(p＜0.005).在35例PRAME基因阳性表达的患者中,AML 28例,阳性检出率为65%,其中以M3、M4和M2亚型的阳性检出率较高,分别为90%、70%、67%;ALL 5例,阳性检出率为56%.在31例融合基因阳性患者中,PRAME基因阳性患者为23例,而在24例融合基因阴性患者中,PRAME基因阳性患者达到12例.比较各种临床因素与PRAME表达水平的相关性未发现明显相关.短期观察5例PRAME基因阳性表达的患者显示出经化疗达到完全缓解(CR)后,患者PRAME基因表达均转阴,下降水平为63-23921/106×GAPDH拷贝.长期观察1例M3患者显示化疗后PRAME基因水平逐渐下降并转阴,患者至今仍处于CR状态.结论PRAME基因在65%的急性白血病中呈阳性表达,其中以M3、M4和M2亚型最常见,在正常个体和非恶性血液病患者中不表达.不同白血病个体之间PRAME基因表达水平不同,随病情缓解其表达水平降低或转阴.PRAME基因可以成为微小残留病(MRD)检测的一种分子标志.     

Aberrant hypomethylation of the cancer–testis antigen PRAME correlates with <Emphasis Type="Italic">PRAME</Emphasis> expression in acute myeloid leukemia

PRAME is a tumor-associated antigen, which belongs to the family of cancer-testis antigens (CTA). The expression of CTA is mainly restricted to the testis and various tumors. In contrast to other CTA, PRAME expression is also frequently detected in acute and chronic leukemias. Due to this expression pattern, PRAME has attracted great interest as a prognostic tumor marker that can be used for the detection of minimal residual disease and as a potential target for immunotherapy. In acute myeloid leukemia (AML), PRAME expression has been observed in 30-64% of cases. To evaluate whether epigenetic mechanisms contribute to PRAME activation in AML, we studied DNA methylation of 15 CpG dinucleotides within a CpG-rich region located in the intron 1 of the PRAME gene. DNA methylation was determined by sequence analysis of cloned PCR products generated from bisulfite-treated genomic DNA. Methylation patterns were correlated with PRAME mRNA levels as determined by microarray analysis and real-time PCR. We found almost complete methylation in mononuclear blood cells from two healthy donors and in bone marrow cells of four PRAME-negative AML patients. In contrast, the degree of PRAME methylation was clearly reduced in four PRAME-positive AML bone marrow samples. In particular, these samples were characterized by the presence of clones, which were completely devoid of methylation. The significant inverse correlation between the degree of methylation and PRAME expression suggests a causal role of DNA methylation in PRAME regulation. Such a role is further supported by the observation that treatment of PRAME-negative cell lines U-937 and THP-1 with the demethylating agent 5'-Aza-2'dC resulted in a dose-related upregulation of PRAME expression.     

Expression of tumor-associated antigens in breast cancer subtypes

ObjectivesTumor-associated antigens (TAAs) are frequently overexpressed in several cancer types. The aim of this study was to investigate the expression of TAAs in breast cancer.Material and methodsA total of 250 selected invasive breast cancers including 50 estrogen receptor (ER)-positive (Luminal B like), 50 triple-negative (TN), 50 ER-positive lobular type, 50 ER- and progesterone receptor (PgR)-positive (Luminal A like) and 50 cerbB2-positive breast cancers, were assessed for New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Wilms tumor antigen (WT-1) and PReferentially expressed Antigen of MElanoma (PRAME) antigen expression by immunohistochemistry (IHC).ResultsA significantly higher expression of cancer testis (CT)-antigens NY-ESO-1 and WT-1 antigen was detected in TN breast cancers compared with ER-positive tumors. NY-ESO-1 overexpression (score 2 + and 3+) assessed by monoclonal and polyclonal antibodies was detected in 9 (18%) TN cancers as compared to 2 (4%) ER-positive tumors (p = 0.002). WT1 over-expression (score 2 + and 3+) was confirmed in 27 (54%) TN tumor samples as compared to 6 (12%) ER-positive (p < 0.0001). PRAME over-expression (score 2 + and 3+) was detected in 8 (16%) HER2 positive tumor samples as compared to no TN and ER-positive cancers (p = 0.0021).ConclusionsNY-ESO-1 and WT1 antigens are overexpressed in TN breast cancers. Because of the limited therapeutic options for this patient subgroup, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.     

PRAME基因在血液恶性肿瘤中的表达及相关研究

目的:研究PRAME基因在慢性粒细胞白血病、淋巴瘤和多发性骨髓瘤中的表达及临床意义,探讨PRAME基因在血液恶性肿瘤微小残留病(MRD)监测中的意义。方法:应用半定量逆转录-聚合酶链反应(RT-PCR)技术,检测76例血液肿瘤病人,其中36例慢性粒细胞白血病(CML)、20例淋巴瘤(NHL18例,HD2例)、20例多发性骨髓瘤(MM),10例正常人骨髓单个核细胞(BMMNC)以及K562细胞株PRAME mRNA的表达水平;同时检测CML患者BCR/ABL融合基因的表达并与PRAME的表达结果相比较。结果:10例正常人骨髓PRAME基因表达均呈阴性。PRAME在慢性粒细胞白血病中的表达率为22.2%(n=8),慢粒慢性期(CML-CP)表达阴性,慢粒加速期(CML-AP)表达率为16.7%(n=1),慢粒急变期(CML-BC)表达率为46.7%(n=7),慢粒急变期的表达率显著高于慢性期和加速期(P<0.05)。36例CML患者BCR/ABL融合基因表达率为97.2%(n=35),与PRAME基因表达无相关性(Fisher精确概率法,P>0.05)。3例经历了慢性期、加速期和急变期的CML患者均在急变期检测到PRAME基因的表达。淋巴瘤中PRAME表达率为10%(n=2),其中2例阳性均为弥漫大B细胞淋巴瘤。PRAME在多发性骨髓瘤中的表达率为20%(n=4),其中4例阳性均是在MMⅢ期(28.6%),与I、II期的表达无明显差异(P>0.05)。PRAME mRNA表达水平与性别、年龄、初诊或复发时白细胞计数、血红蛋白、血小板计数、骨髓中原幼细胞比例无明显相关性(P>0.05)。结论:PRAME基因在多种血液恶性肿瘤中表达,与慢性粒细胞白血病病情进展密切相关,与CML患者BCR/ABL基因的表达无相关性,可作为PRAME阳性血液恶性肿瘤MRD监测的标志基因。     

Differential gene expression in liposarcoma, lipoma, and adipose tissue

Malignant transformation is thought to be associated with changes in the expression of a number of genes, and this alteration in gene expression is felt to be critical to the development of the malignant phenotype. Sarcomas represent a diverse group of tumors derived from cells of mesenchymal origin. Marked heterogeneity exists in the biological behavior of sarcomas, even within histologic subtypes of sarcomas. In an effort to better understand the biology of liposarcomas, gene expression in normal adipose tissue, lipomas, and liposarcomas was examined using the Affymetrix® microarray technology. Differences in gene expression were quantified as the fold change in gene expression among the sample sets. Differences in gene expression among normal adipose tissue, lipomas, and liposarcomas were observed. In addition, genes expressed uniquely in liposarcoma among these and 18 other tissue sample sets were identified. Gene sets were devised that allowed the separation of liposarcomas from other samples, and most normal adipose tissue from most lipomas using the Eisen clustering software “Cluster.” We conclude that differences in gene expression can be identified among different tumors derived from the adipocyte series. Such differences in gene expression may help differentiate among subtypes of sarcomas, and may also yield clues to the pathophysiology of this heterogeneous group of tumors.     

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.