Preferentially Expressed Antigen Of Melanoma Research Articles

The relevance of preferentially expressed antigen of melanoma (PRAME) as a marker of disease activity and prognosis in acute promyelocytic leukemia

The gene for preferentially expressed antigen of melanoma (PRAME) has been shown to be over-expressed in acute promyelocytic leukemia, but its actual incidence and clinical impact are still unknown. We studied PRAME expression at diagnosis using real-time quantitative polymerase chain reaction in 125 patients with acute promyelocytic leukemia enrolled in the Spanish PETHEMA-96 (n=45) and PETHEMA-99 (n=80) clinical trials. In addition, PRAME expression was evaluated as a marker of disease activity in 225 follow-up samples from 67 patients with acute promyelocytic leukemia. At diagnosis, PRAME expression in patients with acute promyelocytic leukemia was significantly higher (p<0.001) than in patients with non-M3 acute myeloid leukemia (n=213) and in healthy controls (n=10). Furthermore, patients with acute promyelocytic leukemia with high PRAME expression had a favorable outcome. Thus, the 5-year relapse-free survival was better in patients with >100-fold PRAME expression (86% vs. 74%; p=0.03), and this cut-off established two sub-groups with different relapse-free survival rates among patients with a white cell count <10(9)/L (5-year relapse-free survival 94% vs. 80%, p=0.01). This effect was similar in patients with a white cell count >10(9)/L, although differences were not statistically significant. In multivariate analysis, white cell count >10(9)/L (p<0.001), bone marrow blasts >90% (p=0.001), and PRAME expression <100-fold (p=0.009) were associated with short relapse-free survival. Samples at remission showed PRAME levels similar to those in normal controls while samples at relapse over-expressed PRAME again. Furthermore, 12/13 samples collected within the 6-month period preceding relapse showed a >10-fold increase in PRAME expression levels. Low PRAME expression defines a subgroup of patients with acute promyelocytic leukemia with a short relapse-free survival. This marker could be useful as a secondary marker for monitoring patients with acute promyelocytic leukemia.

Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia

AbstractThe cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02–restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNγ in response to PRAME peptides (between 113 ± 8 and 795 ± 23 spot forming cells/105 T cells) and lysed PRAME peptide–loaded cells (45 ± 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02+/PRAME+ tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high αβTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.

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.

Tumor associated antigen PRAME is a marker of favorable prognosis in childhood acute myeloid leukemia patients and modifies the expression of S100A4, Hsp 27, p21, IL-8 and IGFBP-2 in vitro and in vivo

Preferentially expressed antigen of melanoma (PRAME) is expressed in a wide variety of tumors, but in contrast with most other tumor associated antigens, it is also expressed in leukemias. In a previous study, we showed that overexpression of PRAME induced apoptosis, inhibited cell proliferation and reduced tumorigenicity of leukemic cells in vivo. We also demonstrated that PRAME overexpression induced the repression of three genes (Hsp27, S100A4 and p21) associated with an unfavorable prognosis in leukemia. Here, we further investigated the mechanisms of PRAME-induced tumor suppression in vitro and in vivo. We performed a gene profiling study by analysing PRAME shRNA-silenced leukemic cells on high-density micro-arrays (Affymetrix) and found that PRAME altered the expression of two additional genes potentially involved in cancerogenesis and cancer progression: IL-8 and IGFBP-2. In a series of 28 acute myeloid leukemia pediatric patients, we observed that PRAME expression was associated with an increased leukemia-free survival. Importantly, the correlation between PRAME expression in leukemic cell lines and the decreased expression of Hsp27, S100A4, p21, IL-8 and the increased expression of IGFBP-2 was also observed in vivo, in leukemic patients. Our results suggest that the favorable prognosis of PRAME could be mediated, at least in part, by the modified expression of those genes.

Preferentially Expressed Antigen of Melanoma (PRAME)-Specific Cytotoxic T-Lymphocytes (CTLs) and Transgenic T Cells To Target Chronic Myelogenous Leukemia (CML).

Following allogeneic hematopoietic stem cell (HSC) transplantation, a graft versus leukemia (GVL) effect mediated by donor T cells contributes to the maintenance of long-term remission. GVL is likely mediated not only by alloreactive T cells, but also by donor lymphocytes recognizing tumor associated antigens overexpressed by leukemic cells. Selective expansion of such tumor-specific CTLs could augment GVL without increasing the risk of graft versus host disease (GVHD). PRAME is a cancer testis antigen (CTA) that is over-expressed by many hematological malignancies, including CML. We therefore generated PRAME-specific CTLs and evaluated their activity against primary CML tumor cells. CD8+ cells from 9 HLA-A*02 healthy donors and 5 CML patients were primed with autologous CD40L-activated B blasts loaded with HLA-A*02 restricted PRAME-peptides in the presence of low concentrations of IL-12, IL-7 and IL-15. Cells were then re-stimulated once a week using an artificial antigen presenting cell line (aAPC), consisting of K562 cell line that was genetically modified to stably express HLA-A*02, CD80 and CD40L and loaded with the same PRAME-peptides. In 8 out of 9 HLA A*02 healthy donors, we expanded (22±7 fold) T cells specific for the ALY-PRAME peptide. Similarly, PRAME-specific CTLs could be expanded from 3 out of 5 CML pts. When assessed by Elispot assay, these CTLs released IFNγ in response to ALY-peptide (511±260 SFC/105 cells) compared to 30±10 IFNγ SFC/105 against an irrelevant peptide. They also lysed autologous-PHA blasts loaded with ALY (84%±12% specific 51Cr release at 20:1 E:T ratio), but not with an irrelevant peptide (<10%). Both IFNγ release and cytotoxic activity were blocked by MHC class I antibodies, indicating that these effects were MHC-restricted. PRAME-specific CTLs also recognized primary CML cells since they produced IFNγ (320±31 SFC/105) in response to CD33+ CML blasts overexpressing PRAME (as assessed by Real Time PCR) isolated from HLA-A*02+ CML patients. Since routine expansion of PRAME-specific CTLs for clinical trials of adoptive T-cell transfer may remain problematic, we next cloned the αβT cell receptor (TCR) from a CTL clone with high affinity for the ALY-peptide (621 IFNγ SCF/105 cells at 0.2 nM peptide). The α- and β-chains were cloned in the SFG retroviral vector using an IRES sequence, and the transgenic αβTCR was expressed in primary T lymphocytes. T cells expressing the transgenic PRAME-specific αβTCR produced IFNγ on exposure to PRAME+ CML blasts (448± 69 IFNγ SFC/105 cells) and killed PHA blasts loaded with the ALY-peptide (42%±7% at 20:1 E:T ratio). In conclusion, our data show that PRAME is a promising target antigen on CML blasts and that adoptive transfer of CTLs or of T cells expressing a transgenic αβTCR targeting this antigen may be of value for patients with the disease.

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.

A functional genetic screen identifies retinoic acid signaling as a target of histone deacetylase inhibitors.

Understanding the pathways that are targeted by cancer drugs is instrumental for their rational use in a clinical setting. Inhibitors of histone deacetylases (HDACI) selectively inhibit proliferation of malignant cells and are used for the treatment of cancer, but their cancer selectivity is understood poorly. We conducted a functional genetic screen to address the mechanism(s) of action of HDACI. We report here that ectopic expression of two genes that act on retinoic acid (RA) signaling can cause resistance to growth arrest and apoptosis induced by HDACI of different chemical classes: the retinoic acid receptor alpha (RARalpha) and preferentially expressed antigen of melanoma (PRAME), a repressor of RA signaling. Treatment of cells with HDACI induced RA signaling, which was inhibited by RARalpha or PRAME expression. Conversely, RAR-deficient cells and PRAME-knockdown cells show enhanced sensitivity to HDACI in vitro and in mouse xenograft models. Finally, a combination of RA and HDACI acted synergistically to activate RA signaling and inhibit tumor growth. These experiments identify the RA pathway as a rate-limiting target of HDACI and suggest strategies to enhance the therapeutic efficacy of HDACI.

PRAME expression is not associated with down-regulation of retinoic acid signaling in primary acute myeloid leukemia

The tumor antigen preferentially expressed antigen of melanoma (PRAME) is frequently overexpressed in a wide variety of malignant diseases, including acute myeloid leukemia (AML). It was recently shown that PRAME can function as a repressor of retinoic acid signaling, as indicated by down-regulation of retinoic acid receptor-β (RARB) and cyclin-dependent kinase inhibitor 1A (CDKN1A). Another study suggested that PRAME can induce caspase-independent apoptosis via down-regulation of heat shock 27-kDa protein 1 (HSPB1) and S100 calcium–binding protein A4 (S100A4). The transcriptional repression of PRAME depends on the formation of a complex with the enhancer of zeste homolog 2 (EZH2). To test whether these mechanisms play an important roll in AML, we analyzed the expression of PRAME, EZH2, RARB, HSPB1, S100A4, and CDKN1A by real-time polymerase chain reaction in primary leukemic cells from 52 children with AML. All genes were expressed in many patients, but the level of expression of the last four genes was not associated with either PRAME expression or PRAME and EZH2 co-expression. In conclusion, the above mechanisms do not seem to play a major role in the pathogenesis of AML; they could be neutralized by other pathways that affect the same targets.

Prevalence and prognostic and predictive relevance of PRAME in breast cancer

Preferentially expressed antigen of melanoma (PRAME) has been described as a potential candidate for immunotherapeutic targeting. However, the prognostic and predictive relevance of PRAME in breast cancer has never been investigated. PRAME gene expression was evaluated in 103 breast tumour biopsies, using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Normal breast tissue was also analysed for comparative purposes. All qRT-PCRs were performed in triplicate. Kaplan-Meier survival curves, Chi-squared and Cox Regression analyses were used to identify associations between PRAME expression and patients' clinicopathological and survival data. PRAME mRNA was detected in approximately 53% of tumour specimens and 37% of normal breast specimens. Kaplan-Meier analysis showed expression of PRAME to correlate significantly with unfavourable disease outcome for patients, in terms of both their disease-free survival (p = 0.0004) and overall survival (OS) (p = 0.0052) times from diagnosis. Multivariate analysis indicated PRAME expression to be an independent prognostic factor for shortened disease-free survival (p = 0.026) and OS (p = 0.02). Furthermore, for patients who received adjuvant chemotherapy, significantly (p = 0.0291) shorter relapse-free survival was achieved for those whose tumour expressed PRAME, compared to those that did not express this transcript. Our results suggest that PRAME mRNA expression may be a useful prognostic and predictive marker for breast cancer.

Hypomethylation of PRAME is responsible for its aberrant overexpression in human malignancies

The preferentially expressed antigen of melanoma (PRAME) is expressed at high levels in large fractions of human malignancies, e.g., acute myeloid leukemia. Therefore, PRAME is an important marker for diagnosis of various malignant diseases and a relevant parameter for monitoring minimal residual disease. It is supposed to be involved in tumorigenic processes. Because of these important aspects we investigated its transcriptional regulation in detail. Most relevant was a detailed DNA methylation analysis of the PRAME 5' region by genomic sequencing in correlation with PRAME expression in various human patient samples and cell lines. In combination with DNA-truncation/transfection experiments with respect to DNA methylation, we show that changes in the methylation pattern in defined parts of the regulatory regions of PRAME are sufficient for its upregulation in cells usually not expressing the gene.

A Causal Role for the Human Tumor Antigen Preferentially Expressed Antigen of Melanoma in Cancer

Tumor antigens are of interest as diagnostic and prognostic markers and potential therapeutic targets. The tumor antigen preferentially expressed antigen of melanoma (PRAME) is frequently overexpressed in a wide variety of cancers and is a prognostic marker for clinical outcome. It has been shown recently that PRAME functions as a repressor of retinoic acid signaling. Here, we discuss this novel insight in the context of the increasing interest in tumor antigens as targets for therapy.

Tumor mRNA-Transfected Dendritic Cells Stimulate the Generation of CTL That Recognize Neuroblastoma-Associated Antigens, Kill Tumor Cells: Immunotherapeutic Implications

Several observations suggest a potential role of T-cell- mediated immunity in the control of neuroblastoma (NB). However, the generation of NB-specific cytotoxic T lymphocytes (CTL) on T-cell priming with tumor mRNA-transfected dendritic cells (DC) has never been investigated before. In the present study, the feasibility of this strategy has been analyzed, both in healthy donors, in NB patients. Monocyte-derived DC were raised from three human leukocyte antigen (HLA) A2+ NB patients, seven HLA-A1+ or HLA-A2+ healthy donors transfected with mRNA from four NB cell lines, cocultured with autologous CD8+ lymphocytes. Expanded CTL expressed an effector/memory phenotype, a T cytotoxic 1-like profile of cytokine secretion. CTL specificity was demonstrated by interferon-; release on incubation with HLA-matched NB cell lines. The latter cell lines, but not autologous T-cell blasts, were lysed by CTL in an HLA-restricted manner. Cytotoxicity was found to involve the release of granzyme B. When tested for reactivity against NB-associated antigens, CTL from normal individuals recognized anaplastic lymphoma-associated kinase (ALK), preferentially expressed antigen of melanoma (PRAME) peptides only, whereas patients' CTL reacted also to survivin, telomerase, tyrosine hydroxylase peptides. This study demonstrates that DC transfected with NB mRNA induce the generation of patients' CTL specific for different NB-associated antigens, supporting the feasibility of NB T-cell immunotherapy.

Childhood acute myelogenous leukaemia: Association between PRAME, apoptosis- and MDR-related gene expression

The gene PRAME (preferentially expressed antigen of melanoma) encodes an antigen recognised by autologous cytolytic T lymphocytes. The mRNA level of PRAME is used as a tumour marker due to its overexpression in various malignancies. Furthermore, it is known that the overexpression of genes encoding antiapoptotic proteins leads to the survival of leukaemic cells via exclusion of apoptosis. On the other hand, overexpression of genes encoding ABC transporters may lead to multi drug resistance (MDR). Therefore, we investigated whether there is a relationship between PRAME overexpression and the expression of apoptosis- and MDR-related genes in childhood de novo acute myelogenous leukaemia (AML) patient samples and, furthermore, whether this is a general or an AML-subtype specific event. Microarray analysis and real time quantitative PCR revealed that clinical samples showing PRAME upregulation are associated with a decreasing expression of genes coding for apoptotic proteins and an overexpression of genes encoding ABC transporters. Our results indicate that patients showing PRAME upregulation may have an increased risk of MDR induction.

PRAME mRNA levels in cases with chronic leukemia: Clinical importance and review of the literature

Preferentially expressed antigen of melanoma (PRAME) is a tumor antigen expressed in various malignant tumors including solid tumors and hemopoietic neoplasias but no or weak expression in normal tissues. The aim of this study is to determine the frequency and the clinical importance of PRAME expression in chronic myeloid leukemia (CML)/chronic myeloproliferative disorders (CMPD) and chronic lymphocytic leukemia (CLL). PRAME mRNA was measured by real time RT-PCR in 88 cases with chronic leukemia (CL) and 42 controls. Seventy cases had CML/CMPD (56 had chronic phase (CP)-14 had accelerated/blastic phase disease (AP/BP) and 18 cases had CLL (11 had early stage (Rai 0–I–II) and 7 had late stage (Rai III–IV). Mann Whitney U, Wilcoxon and Kruskal Wallis tests were used for statistical analyses. Twenty-four of 70 (34%) cases with CML/CMPD and 5 of 18 (28%) cases with CLL showed PRAME expression. Totally 29 of 88 cases showed PRAME expression (33%). However, only two controls showed weak PRAME expression. The difference for PRAME mRNA between the CLs and controls was significant ( p = 0.000). PRAME (+) and PRAME (−) cases were not different for age, Hb, Hct, WBC count, platelet count, stage of the disease and response to therapy. PRAME was monitorised in eight cases during follow-up: in three cases PRAME was negative at CP and expression developed at the AP/BP disease. PRAME was positive at the beginning in five cases (4 CML-1CLL) and expression disappeared after chemotherapy. In conclusion, PRAME is detected in one third of the cases with CLs. PRAME mRNA changes may be detected during the progression of these disorders and/or after therapy. PRAME mRNA may be a useful marker to detect the minimal residual disease (MRD) and to determine the response to therapy in CLs.

Real-time RT-PCR quantification of PRAME gene expression for monitoring minimal residual disease in acute myeloblastic leukaemia

Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. PRAME transcripts were quantified by real-time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME-positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME. The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME-positive leukaemia.

Duplication and positive selection among hominin-specific PRAME genes

BackgroundThe physiological and phenotypic differences between human and chimpanzee are largely specified by our genomic differences. We have been particularly interested in recent duplications in the human genome as examples of relatively large-scale changes to our genome. We performed an in-depth evolutionary analysis of a region of chromosome 1, which is copy number polymorphic among humans, and that contains at least 32 PRAME (Preferentially expressed antigen of melanoma) genes and pseudogenes. PRAME-like genes are expressed in the testis and in a large number of tumours, and are thought to possess roles in spermatogenesis and oogenesis.ResultsUsing nucleotide substitution rate estimates for exons and introns, we show that two large segmental duplications, of six and seven human PRAME genes respectively, occurred in the last 3 million years. These duplicated genes are thus hominin-specific, having arisen in our genome since the divergence from chimpanzee. This cluster of PRAME genes appears to have arisen initially from a translocation approximately 95–85 million years ago. We identified multiple sites within human or mouse PRAME sequences which exhibit strong evidence of positive selection. These form a pronounced cluster on one face of the predicted PRAME protein structure.ConclusionWe predict that PRAME genes evolved adaptively due to strong competition between rapidly-dividing cells during spermatogenesis and oogenesis. We suggest that as PRAME gene copy number is polymorphic among individuals, positive selection of PRAME alleles may still prevail within the human population.

PRAME-specific CD8+ T cells are spontaneously present in a large proportion of healthy donors and leukemic HLA-A0201 patients

The tumor antigen PRAME (preferentially expressed antigen of melanoma) is expressed in up to 50% of patients with AML. Four HLA-A*0201-restricted epitopes in the PRAME protein have been identified: P100-108 (P100), P142-151 (P142), P300-309 (P300) and P425-433 (P425). To detect very low frequencies of PRAME-specific CD8+ T cells, we used quantitative real-time reverse transcription polymerase chain reaction (qPCR) to measure interferon-g mRNA (IFN-g) production by PRAME peptide pulsed CD8+ T cells from 11 healthy donors and 10 HLA-A*0201+ patients with AML, one of whom had received an allogeneic stem cell transplant (SCT). After isolation, 1 x10 6 CD8+ T cells were stimulated in vitro with C1R-A2 cells (an MHC class I-defective LCL expressing HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM, to determine functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3h coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated the IFN-g copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/10 4 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/10 4 CD8 copies in peptide pulsed cultures/unpulsed cultures. Using this sensitive technique, we found responses in 8/11 HLA-A2-positive healthy donors and 7/10 AML patients. Four of eight healthy donors and 5/7 AML responders recognized 2 or 3 peptide epitopes. The mean response against each of the four epitopes was greater in leukemic patients compared with healthy donors (3597–9371 versus 172–1288 IFN-g mRNA copies/10 4 CD8 copies). PRAME peptide mediated responses, particularly to P300, were also detected using an optimized ELISPOT assay in 2/4 AML patients and 5/8 normal donors tested. In cross-comparison of 8 qPCR positive donors, 6 also generated IFN-g ELISPOTS confirming IFN-g mRNA transcription was also associated with protein secretion. Of note, the most immunogenic epitope in both donors and patients by both methods was P300. Six qPCR and ELISPOT assays were concordant, but there were 2 ELISPOT negative, qPCR positive patients, and 1 ELISPOT positive qPCR negative patient. In 2/5 samples tested (1 donor and 1 patient), peptide-specific ELISPOT responses expanded from 99 to 1627 and 280 to 758 spots per million plated PBMC respectively, after a single 7 day peptide pulse. Samples from a stem cell donor and the recipient pre and post SCT were also studied. The donor had CD8 + T-cell reactivity to P142, P300 and P425. The patient had no PRAME response prior to SCT but after SCT developed significant responses to P142 and P425 and P100, suggesting the transfer and expansion of PRAME-specific CD8 + T cells from donor to recipient. These results provide the first evidence for spontaneous T-cell reactivity against PRAME in healthy donors and AML patients. They support the immunogenicity of PRAME and its potential application in immunotherapy of leukemia.

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-Specific CD8+ T Cells Are Spontaneously Present in a Large Proportion of Healthy Donors and Leukemic HLA-A0201 Patients.

The tumor antigen PRAME (preferentially expressed antigen of melanoma) is expressed in up to 50% of patients with AML. Four HLA-A*0201-restricted epitopes in the PRAME protein have been identified: P100-108 (P100), P142-151 (P142), P300-309 (P300) and P425-433 (P425). To detect very low frequencies of PRAME-specific CD8+ T cells, we used quantitative real-time reverse transcription polymerase chain reaction (qPCR) to measure interferon-g mRNA (IFN-g) production by PRAME peptide pulsed CD8+ T cells from 11 healthy donors and 10 HLA-A*0201+ patients with AML, one of whom had received an allogeneic stem cell transplant (SCT). After isolation, 1 x106 CD8+ T cells were stimulated in vitro with C1R-A2 cells (an MHC class I-defective LCL expressing HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM, to determine functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3h coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated the IFN-g copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed cultures/unpulsed cultures. Using this sensitive technique, we found responses in 8/11 HLA-A2-positive healthy donors and 7/10 AML patients. Four of eight healthy donors and 5/7 AML responders recognized 2 or 3 peptide epitopes. The mean response against each of the four epitopes was greater in leukemic patients compared with healthy donors (3597–9371 versus 172–1288 IFN-g mRNA copies/104 CD8 copies). PRAME peptide mediated responses, particularly to P300, were also detected using an optimized ELISPOT assay in 2/4 AML patients and 5/8 normal donors tested. In cross-comparison of 8 qPCR positive donors, 6 also generated IFN-g ELISPOTS confirming IFN-g mRNA transcription was also associated with protein secretion. Of note, the most immunogenic epitope in both donors and patients by both methods was P300. Six qPCR and ELISPOT assays were concordant, but there were 2 ELISPOT negative, qPCR positive patients, and 1 ELISPOT positive qPCR negative patient. In 2/5 samples tested (1 donor and 1 patient), peptide-specific ELISPOT responses expanded from 99 to 1627 and 280 to 758 spots per million plated PBMC respectively, after a single 7 day peptide pulse. Samples from a stem cell donor and the recipient pre and post SCT were also studied. The donor had CD8+ T-cell reactivity to P142, P300 and P425. The patient had no PRAME response prior to SCT but after SCT developed significant responses to P142 and P425 and P100, suggesting the transfer and expansion of PRAME-specific CD8+ T cells from donor to recipient. These results provide the first evidence for spontaneous T-cell reactivity against PRAME in healthy donors and AML patients. They support the immunogenicity of PRAME and its potential application in immunotherapy of leukemia.

Preferentially Expressed Antigen of Melanoma (PRAME) in the Development of Diagnostic and Therapeutic Methods for Hematological Malignancies

PRAME (Preferentially expressed antigen of melanoma), highly expressed in various solid tumor cells and normal testis, was first isolated as a human melanoma antigen recognized by cytotoxic T cells (CTL). This gene was also expressed in some of the hematological malignancies, including acute myelogenous leukemia (AML) and multiple myeloma. We and others have extensively evaluated the PRAME expression in various hematological malignancies and demonstrated high expression of the PRAME gene in subsets of AML, chronic myelogenous leukemia, acute lymphocytic leukemia, lymphoma and multiple myeloma. In addition, we have demonstrated that PRAME was a useful marker for detection of minimal residual disease (MRD) in patients with leukemia, particularly those leukemias in which tumor specific markers are currently unavailable. Since PRAME was first identified as a tumor antigen recognized by T cells, the possibility that PRAME is a leukemia antigen recognized by T cells was evaluated, and it was found that PRAME-positive leukemia cell lines and fresh leukemia cells were susceptible to lysis by the PRAME-specific CTL. Five CTL epitopes associated with either HLA-A * 0201 or HLA-A * 2402 have recently been identified. It is, therefore, an attractive strategy to apply PRAME specific immunotherapy on patients with PRAME positive leukemia in MRD condition.