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Keywords:

  • PRAME;
  • real-time PCR;
  • minimal residual disease;
  • tumour antigen;
  • cytotoxic T cell

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Forty-one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real-time PCR for a period of 10–37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr-abl gene. Therefore, quantitative monitoring of the PRAME gene using real-time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME-positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME-specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.

Despite recent progress, 30–40% of patients with leukaemia relapse after high-dose chemotherapy followed by stem cell transplantation (Lowenberg et al, 1999). Accordingly, new methods for early diagnosis and treatment need to be developed (Campana & Pui, 1995; Baer, 1998). In haematological malignancies, minimal residual disease (MRD) after treatment has been successfully monitored in some patients using leukaemia-specific gene rearrangements, including t(9;22)(q34;q11) (Cross et al, 1993; Lion et al, 1995; Lin et al, 1996; Preudhomme et al, 1997; Radich et al, 1997; Drobyski & Hessner, 1998; Moravcova et al, 1998), t(8;21)(q22;q22) (Muto et al, 1996; Tobal & Yin, 1996), and rearrangement of immunogloblin (Ig) (Roberts et al, 1997) and T-cell receptor (TCR) (Beishuizen et al, 1994; Cave et al, 1994; Seriu et al, 1995). Leukaemia specific markers, however, are not yet available for many patients, although the WT1 gene has been reported to be a useful leukaemia marker for these patients (Inoue et al, 1994, 1996).

PRAME (Preferentially expressed antigen of melanoma) has been identified as a tumour antigen recognized by cytotoxic T-cells (CTLs) that was originally established from a melanoma patient (Ikeda et al, 1997). It codes for a protein consisting of 509 amino acids and its function is unknown. It has been reported that this gene is expressed in a variety of cancer cells including leukaemic cells using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) (Neumann et al, 1998; van Baren et al, 1998, 1999; Pellat-Deceunynck et al, 2000).

In the present study, we evaluated whether quantitative measurement of PRAME using the real-time PCR method is useful for monitoring MRD in patients with leukaemia. By screening bone marrow (BM), peripheral blood (PB) or lymph node (LN) samples from 98 Japanese patients using semiquantitative RT-PCR, PRAME expression was detected in 42% of these samples. A series of samples from eight patients with leukaemia were then monitored for PRAME expression using real-time PCR for a period of 10–37 months. A decrease in PRAME expression after the induction of complete remission (CR) and the reincrease before cytological diagnosis of relapse were observed in some patients, suggesting that PRAME is a good marker for monitoring small numbers of leukaemic cells.

As PRAME has been identified as a tumour antigen recognized by CTLs, it may be used as a target antigen for T cells in patients with leukaemia and lymphoma. Several studies in animal models (Greenberg, 1991) and clinical trials in patients with melanoma have demonstrated that immunotherapies specific for tumour antigens are effective in some patients (Nestle et al, 1998; Rosenberg et al, 1998; Marchand et al, 1999). Immunotherapies were reported to also be effective for haematological malignancies. Various immunotherapies with idiotypes of antibody for B-cell lymphoma and multiple myeloma have been reported to be effective (Hsu et al, 1997; Bendandi et al, 1999; Massaia et al, 1999; Reichardt et al, 1999). Immunotherapy against Epstein–Barr virus (EBV) antigens has been reported to prevent the development of EBV-related lymphoma after bone marrow transplantation (BMT) (Heslop et al, 1996). In addition, allogeneic BMT and donor leucocyte infusion (DLI) have been reported to be effective for patients with various types of leukaemia, particularly chronic myelogenous leukaemia. In these cases, allogeneic antigens, including minor histocompatibility antigens, have been thought to be target antigens (Goulmy et al, 1996; Warren et al, 1998). A variety of other antigens expressed on leukaemic and lymphoma cells have recently been investigated as target antigens for immunotherapies. These include chimaeric proteins caused by chromosome translocation such as the bcr-abl protein (Bosch et al, 1996; Mannering et al, 1997; Nieda et al, 1998; Yotnda et al, 1998), tissue-specific proteins such as proteinase 3 (Molldrem et al, 1996) and overexpressed proteins in cancer cells such as WT1 (Ohminami et al, 2000). In this study, we also indicated the possibility that a peptide from PRAME is expressed on the cell surface of leukaemic cells and can be recognized by CTL.

These results demonstrate that quantitative monitoring of PRAME is useful for the development of new diagnostic and treatment methods for patients with leukaemia and lymphoma. Immunotherapy for patients with MRD is a particularly interesting strategy.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patient samples Samples from 98 Japanese patients with various haematological malignancies were collected with informed consent in several Japanese institutions from 1993 to 1999. Acute leukaemia was classified according to the criteria devised by the French–American–British Committee between 1992 and 1998. Chronic myelogenous leukaemia was classified into chronic phase (CP), accelerated phase (AP) or blastic crisis (BC) according to the criteria of the International Bone Marrow Transplant Registry (Speck et al, 1984). Diagnostic material from 98 patients was screened for PRAME expression using semiquantitative RT-PCR (Table I). PRAME-positive patients (n = 8) were monitored using real-time PCR for PRAME expression in BM samples during the follow-up period (Table II). Mononuclear cells (MNCs) were obtained from the PB or BM using Ficoll density centrifugation. Lymph nodes (LNs) were resected from patients with non-Hodgkin's lymphoma. MNC and LN samples were frozen and stored at −80°C for RNA extraction. MNCs from five BM, 20 PB and two cord blood (CB) samples of healthy donors were used as a control.

Table I.  Relative expression of the PRAME gene in 98 Japanese patients with a haematological malignancy and healthy donors.
 Relative PRAME expressionNumber of samples(%)
Sample*4+3+2+1++/–> 2+
  • *

    Patient samples include BM, PB and LN.

  •   †Semi-quantitative measurement was performed with RT-PCR analysis by comparing ethidium bromide-stained band density with the standard K562 cDNA dilutions (4+: 1/28, 3+: 1/211, 2+: 1/214, 1+: 1/216, +/–: 1/218).

  •   ‡The PRAME expression higher than 2+ was defined as high expression, which corresponds to ‘positive’ previously reported by van Baren et al (1998, 1999).

AML M00010101/2
 M11003111/6
 M22210335/11 (45%)
 M32132006/8 (75%)
 M41100022/4
 M50001000/1
 M60011001/2
 M70000010/1
       17/35 (49%)
CML CP1025233/13 (23%)
 AP2000142/7 (29%)
 BC2123045/12 (42%)
       10/32 (31%)
ALL t(9;22)+0230015/6
t(9;22)–1212114/8
       9/14 (64%)
ATL1001001/2
NHL1113433/13 (23%)
MM0000020/2
Total      41/98 (42%)
Healthy donor
 PB00005150/20
 BM0000140/5
 CB0000110/2

Semi-quantitative RT-PCR Total RNA extraction, cDNA synthesis and RT-PCR were performed as previously described (Ikeda et al, 1997) with modifications. Briefly, total RNA was extracted from the MNCs using the guanidine–isothiocyanate procedure. Total RNA was extracted from lymph nodes using Isogen (NipponGene, Tokyo, Japan), in an alternated acid guanidium thiocyanate–phenol–chloroform extraction method. Reverse transcription was performed with 2 μg of total RNA in 20 μl of reverse transcriptase buffer (Gibco BRL, Gaithersburg, MD, USA), 1 μl of 20 mmol/l solution of dNTPs (Invitrogen, Groningern, The Netherlands), 1 μl of a 20 mmol/l solution of random hexamer (Worthington Biochemical, Lakewood, NJ, USA), 20 U of Rnasin (Promega Biotec, Madison,Wisconsin, USA), 2 μl of 0·1 mol/l dithiotreitol (Gibco BRL) and 200 U of MoMLV reverse transcriptase (Gibco BRL). The reaction was performed by incubation at 42°C for 60 min. The cDNA product (1 μl) was then supplemented with 2·5 μl of 10× PCR buffer (Takara Shuzo, Shiga, Japan), 2 μl of a 2·5 mmol/l solution of dNTP, 1 μl of 10 mmol/l solution of primers, 1 U of TakaraTaq polymerase (Takara) and water to a final volume of 25 μl. The PCR primers were OPC189 and OPC190 for PRAME amplification (OPC189, 5′-CTGTACTCATTTCCAGAGCCAGA-3′; OPC190, 5′-TATTGAGAGGGTTTCCAAGGGGTT-3′) and A-F, A-R for beta-actin (A-F, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′; A-R, 5′CGTCATACTCCTGCTGATCCACATCTGC-3′). The PCR conditions for PRAME were 5 min at 94°C, followed by 34 cycles of 1 min at 94°C, 2 min at 63°C, 3 min at 72°C. The PCR conditions for β-actin were 5 min at 94°C, followed by 25 cycles of 30 s at 94°C, 1 min at 68°C and 1 min at 72°C. The PCR products were examined after gel electrophoresis in ethidium bromide-stained 1·5% agarose gel. Each PCR run included five standard samples. The densities of the bands stained with ethidium bromide were measured using Molecular Imager (Bio-Rad Lab.Hercules, CA, USA) and compared with those of the standards. For the PRAME standard, cDNA synthesized from the PRAME-positive K562 cell line was diluted in twofold steps into water from 1:28 to 1:218. Standards were aliquoted and stored at −20°C. Precautions were taken in all aspects of sample handling and preparation to avoid contamination of PCRs, according to published recommendations (Kwok & Higuchi, 1989).

RT-PCR for other genetic markers Quantitative RT-PCR for the bcr-abl fusion transcript and the WT1 gene was performed as described previously (Inoue et al, 1996; Eder et al, 1999) with minor modifications. In brief, 2 μg of total RNA from leukaemic cells was converted into cDNA. Real-time PCR was performed for the bcr-abl gene and competitive PCR was performed for the WT1 gene. Semi-quantitative RT-PCR for the PML-RARα fusion transcripts was performed as described previously (Miller et al, 1992).

Southern blotting The specificity and sensitivity of the PCR products were confirmed by Southern blot analysis using specific probes (PRAME, 5′-GTAGACTCCTCCTCTCCCAC-3′; β-actin, 5′-ATCATGTTTGAGACCTTCAACACCCCAGCC-3′). The PCR products electrophoresed on an agarose gel were transferred onto a nylon membrane (Hybond N+ Amersham International, Buckinghamshire, UK) and then hybridized with the probe labelled using the ECL 3′ oligo labelling and detection kit (Amersham International). The primers and probes are sequence-specific primers, which allows discrimination between genomic DNA and cDNA.

Real-time PCR Primers and probes (PRAME forward primer, 5′-TCTTCCTACATTTCCCCGGA-3′; PRAME reverse primer, 5′-GCACTGCAGACTGAGGAACTGA-3′; PRAME probe, 5′-(FAM)AAGGAAGAGCAGTATATCGCCCAGTTCACC-(TAMRA)-3′; β-actin forward primer, 5′-TCACCCACACTGTGCCCATCTACGA-3′; β-actin reverse primer, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′; β-actin probe, 5′-(FAM)ATGCCC-(TAMRA)-CCCCCATGCCATCCTGCGTp-3′) were designed using Primer-Express software (PE Biosystems, Foster city, CA, USA). Real-time PCR amplification and data analysis were performed using the ABI Prism 7700 Sequence Detector System (PE Biosystems). Each sample (1 μl) was mixed with 49 μl of Ma stermix [1× TaqMan Buffer, 3·5 mmol/l MgCl2, 300 nmol/l each primer, 200 mmol/l dATP, 200 mmol/l dCTP, 200 mmol/l dGTP, 200 mmol/l dUTP, 0·5 U AmpErase Uracil N-glycosylase (UNG) and 1·25 U of AmpliTaq Gold]. The PCR conditions were 2 min at 50°C, 10 min at 95°C, followed by 60 cycles of 15 s at 95°C and 1 min at a primer-specific annealing temperature. Experiments were performed in duplicate. Each PCR run included five standards. Precautions against cross contamination of genomic DNA were taken as follows. Reverse transcription reaction was performed without reverse transcriptase, then the resulting mixture was used as the template for real-time PCR. We confirmed that the amount of PRAME-specific PCR product with this template corresponded to 10−5−10−6 dilution of leukaemic cell line, which was under the quantitative detection level of this method.

For the PRAME standard, Hi2, a plasmid containing PRAME cDNA (Ikeda et al, 1997) was serially diluted in 10-fold steps into water from 1 pg/μl to 0·01 fg/μl for real-time PCR. For the beta-actin standard, the K562 cDNA was diluted in twofold steps into water from 1:1 to 1:16. Standards were aliquoted and stored at −20°C. PCR products equivalent to 1 fg/μl plasmid DNA, taking into account the expression level of the beta-actin gene, were defined as 1 U. Subsequently, the normalized PRAME PCR products were defined as follows:

Relative PRAME expression (U)   =  (PRAMEsample/PRAMEplasmid(1 fg/μl))/ (beta-actinsample/beta-actinK562cDNA)

Sensitivity To determine the sensitivity, total RNA from K562 was diluted in 10-fold steps into RNA from PRAME-negative PBMNCs from healthy donors; semiquantitative RT-PCR, Southern blotting and real-time PCR were then performed.

Cytotoxicity assay K562 was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma Chemical, St.Louis, MO, USA) containing 10% fetal calf serum (FCS). LB33-MEL.B was cultured in Iscove's modified Dulbecco's medium (IMDM; Sigma) containing 10% FCS, supplemented with l-arginine (116 mg/l), l-asparagine (36 mg/l) and l-glutamine (216 mg/l). Anti-LB33-E CTL (CTL17) was established as previously described (Ikeda et al, 1997). The cytotoxic activity of the CTL was evaluated using a standard 4-h 51Cr-release assay. Target cells were incubated in a culture medium supplemented with interferon (IFN)-γ (50 U/ml) for 48 h before the assay. Some assays were performed in the presence of the murine anti-HLA-B,C antibody, B1.23.2 (Ikeda et al, 1997). CTL17, LB33-MEL.B and B1.23.2 antibodies were kindly provided by Dr P. G. Coulie (Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium).

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Screening of PRAME expression in 98 haematological malignancies in Japan

The relative expression of the PRAME gene in 98 BM, PB or LN samples from Japanese patients with leukaemia and lymphoma, including 35 cases of acute myelogenous leukaemia (AML), 14 of acute lymphocytic leukaemia (ALL), two cases of adult T-cell leukaemia (ATL), 32 of chronic myelogenous leukaemia (CML), 13 of non-Hodgkin's lymphoma (NHL) and two cases of multiple myeloma (MM), was screened using semiquantitative RT-PCR with ethidium bromide staining, comparing the dilutions of the K562 cDNA standards (Table I). Forty-one out of 98 samples (42%) showed a higher expression than a 1/214 dilution of K562 (> 2+) which was previously defined to be PRAME-positive (van Baren et al, 1998). The PRAME expression was detected in high percentages in AML M2 (45%), AML M3 (75%), CML BC (42%) and ALL (64%), but in a relatively low percentage of lymphoma cases (23%). This expression pattern was almost the same as previously reported data in Caucasian patients, except the relatively high percentage in ALL. The percentages of leukaemic cells in samples that were determined morphologically ranged between 12·6% and 99·5% (mean: 70·0%) for highly PRAME-positive (> 2+) samples and between 3·4% and 97·0% (mean: 58·5%) for other patients. No correlation was observed between the levels of the PRAME expression and the percentage of affected leukaemic cells, suggesting that the PRAME expression in leukaemic cells differs among patients. As the WT1 gene is known to be expressed in a variety of leukaemia cells, we also examined the WT1 expression using quantitative RT-PCR in 28 patients and compared it with the PRAME expression (Table III). Four samples of ALL (patient 9), ATL (patient 23), AML (patient 24) and NHL (patient 27) were found to be PRAME-positive (> 2+) and WT1-negative (< 10 × 102 copies/μg RNA).

Sensitivities of the assays

The sensitivities of three detection methods, including RT-PCR detected with ethidium bromide staining, RT-PCR combined with Southern blotting and real-time PCR, were compared using mRNA from K562 cells diluted into mRNA from PRAME-negative normal PBMNCs. Minimal detectable amounts of PRAME were a 10−4 dilution for the RT-PCR detection with ethidium bromide staining (Fig 1A) and a 10−6 dilution for the RT-PCR with Southern blotting (Fig 1B). Thus, combination with Southern blotting made the RT-PCR assay more sensitive, however, measurement of band densities using the imager is still semiquantitative. In contrast, in real-time PCR using the same RNA samples, PRAME was quantitatively measured at the range from 10−4 to 100 of K562 dilution (Fig 1C). Therefore, real-time PCR was considered to be the most quantitative, reproducible and simple method, and we applied the real-time PCR to monitoring PRAME expression.

image

Figure 1. Comparison of sensitivities among different detection methods. RNA from K562 cell was serially diluted into RNA from normal PRAME-negative PBLs from 100 to 10−8, and RT-PCR was performed. Then relative amounts of PRAME were measured as described below. (A) PCR products visualized on ethidium bromide-stained agarose gels. (B) PCR products visualized by Southern blotting. (C) Real-time amplification plots and standard curve with linear correlation between the cycle threshold (CT) and the initial amount of template cDNA. H, healthy donor; W, water.

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PRAME expression in samples from healthy donors

For using PRAME as a tumour marker, it has to be expressed completely specific to tumour cells or the expression level in normal samples has to be low enough to be distinguishable from tumour cells. Therefore, the expression of the PRAME gene was examined in samples from healthy volunteers. Using semiquantitative RT-PCR, low levels of PRAME expression were observed in 7 out of 27 samples (Table I). To further evaluate this expression in a quantitative manner, real-time PCR was also performed. As shown in Fig 2, PRAME expression in the samples from healthy donors ranged from 10−1 U to 100 U. The mean level of expression was 0·2 ± 0·2 U with PBMNCs (n = 20) and 1·7 ± 1·8 U with BMMNCs (n = 5). In contrast, PRAME expression in fresh PRAME-positive leukaemic samples (higher than 2+) ranged from 101 U to 103 U with a mean level of 313·6 U (n = 10). Therefore, most of the PRAME-positive leukaemic cells could be distinguished from samples without leukaemic cells.

image

Figure 2. Quantification of the PRAME expression in BM samples from healthy donors and leukaemic patients. The PRAME-positive samples from healthy donors show low level expression at the range from 10−1 U to 100 U (open circles). The representative PRAME-positive samples from leukaemic patients showed higher expression at the range of 101 U to 103 U (closed circles). r, correlation coefficient.

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Serial monitoring of PRAME-positive patients with leukaemia using real-time PCR

BM samples from eight of these patients were analysed at more than three time-points using real-time PCR. Figure 3 shows the monitoring of PRAME expression using real-time PCR in patient 1, who was diagnosed with Ph1-positive ALL. The PRAME expression was 10·2 U at the time of relapse after the first allogeneic BMT, it then decreased and became almost negative in the third and fourth months after the second allogeneic BMT. It increased again to 14·9 U at the fifth month after BMT, when the level of leukaemic blasts in BM was still 1·2%. However, 1 month later, the blast count increased to 76% along with increased PRAME expression (48·8 U). The level of the WT-1 gene expression monitored by competitive PCR did not increase, even at the time the patient was diagnosed as being in relapse (data not shown), but the presence of bcr-abl rearrangement monitored by real-time PCR was detected similarly to the PRAME expression. Thus, the detection of PRAME expression would appear to be useful for the early diagnosis of leukaemia relapse. In patient 2, who was diagnosed with AML M3, the PRAME expression was 49·0 U at diagnosis and then decreased when the patient achieved CR after treatment with all-trans retinoic acid (ATRA) and conventional chemotherapy (data not shown). The patient then relapsed with positive PRAME expression (20·8 U) as well as a blast level of 54% in the BM (Fig 4A). After readministration of ATRA, PRAME expression continued to be high as 5·3 U, although the blast counts in the BM decreased to 1·6%. Additional treatments with chemotherapy and allogeneic BMT resulted in a significant reduction in PRAME expression (0·03 U). However, 3 months after the BMT, PRAME increased again without any clinical signs of relapse. Similar changes were observed in monitoring with the PML-RARα rearrangement using semiquantitative RT-PCR. As both PRAME and PML-RARα levels increased again, immunosupressive therapy with cyclosporin A for the prevention of graft-vs.-host disease (GVHD) was discontinued to evoke graft-vs.-leukamia (GVL) effects in this patient. After that, the levels of the PRAME and PML-RARα expression reduced to undetectable amounts. The patient is now being followed-up carefully and remains in CR. In patient 3, who was diagnosed with AML M4, high levels of PRAME expression were detected at diagnosis, which decreased slowly after allogeneic BMT and became almost negative 18 months later. However, the PRAME levels increased again 24 months after the BMT without any clinical sign of relapse (Fig 4B). The WT-1 expression in this patient also changed similarly. This patient is now being followed-up. In patient 4, who was diagnosed with chronic phase CML, the PRAME expression decreased after 10 months of treatment with IFN-α. After allogeneic BMT, it finally became almost negative. The presence of bcr-abl rearrangement changed in a similar fashion (Fig 4C). Figure 5 shows four additional patients (patients 5–8) who remained PRAME-negative after induction therapy and none of whom has relapsed. These results suggest that the monitoring of PRAME expression is useful for following-up patients with leukaemia.

image

Figure 3. Monitoring of PRAME expression in a Ph1-positive ALL patient. BM samples at different time-points were analysed for PRAME expression. Top: the changes of PRAME expression detected by real-time PCR (▪) and the change of bcr-abl expression level detected by real-time PCR (▴). Percentages of leukaemic cells in the samples are indicated in parenthesis. Bottom: the corresponding bands detected by RT-PCR combined with Southern blotting.

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image

Figure 4. Monitoring of expression of PRAME and other marker genes in leukaemia patients. The serial changes in expression of PRAME and other marker genes during disease course of three patients with leukaemia are shown. ●, PRAME expression detected by real-time PCR; ▪, other gene markers. Percentages of leukaemic cells in the samples are indicated in parenthesis. Short arrows indicate administration of chemotherapy. (A) AML M3 patient treated by ATRA, chemotherapy and allogeneic BMT. PML-RARα expression was detected using semiquantitative PCR. (B) AML M4 patient treated by chemotherapy and allogeneic BMT. WT-1 expression was detected using competitive RT-PCR. (C) CML CP patient treated by IFN-α and allogeneic BMT. Expression of major bcr-abl was detected by real-time PCR.

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image

Figure 5. Monitoring of patients with PRAME-positive leukaemia using real-time PCR. The serial changes of PRAME expression level in samples from eight follow-up patients were evaluated using real-time PCR. Black bar, more than 5% blast cells in bone marrow; white bar, haematological complete remission; black circles, PRAME expression more than mean normal level (1·7 U); white circles, PRAME expression less than mean normal level (1·7 U).

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Recognition of leukaemia cells by anti-PRAME CTL

To evaluate whether leukaemic cells also present the PRAME peptide on the cell surface and are sensitive to recognition and lysis by T cells, as demonstrated in melanoma, the lysis of leukaemic cells by the PRAME-specific CTLs established from a melanoma patient was examined. The HLA-A24-restricted PRAME-specific CTL, CTL clone 17, recognizes the 9mer PRAME peptide LYVDSLFFL presented by HLA-A24 (Ikeda et al, 1997). As CTL17 expresses killer inhibitory receptors (KIRs) that bind to HLA-Cw7, it is not able to lyse target cells expressing HLA-Cw7. It can lyse either HLA-A24-positive, HLA-Cw7-negative and PRAME-positive target cells, or HLA-A24-positive and PRAME-positive cells, in the presence of an anti-HLA-Cw7 blocking antibody such as the anti-HLA-B,C monoclonal antibody, B1.23.2 (Ikeda et al, 1997). CTL17 did not lyse the HLA-negative, PRAME-positive erythroleukaemia cell line K562, but could lyse K562-A24 that was stably transfected with HLA-A24 cDNA (unpublished observations) (Fig 6A). This CTL also lysed freshly isolated, PRAME-positive AML-M2 leukaemic cells in the presence of the anti-HLA-B,C antibody (Fig 6B). Thus, there is a possibility that fresh leukaemic cells are also able to process the PRAME protein and present its peptide on their cell surface by HLA-A24 to be lysed by cytotoxic T cells.

image

Figure 6. Cytolysis by PRAME-specific CTLs of PRAME-positive K562 leukaemic cell line and leukaemic cells from an AML-M2 patient. Lytic activities of CTL17 against PRAME-positive leukaemic cell line (A) and leukaemic cells from AML-M2 patient (B) were measured by Cr51 release assay. Right: the expression of the PRAME gene in target cells assessed using semiquantitative RT-PCR.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Detection of MRD has been successfully performed using PCR for altered genes in leukaemic cells as tumour-specific markers, such as bcr-abl and PML-RARα fusion genes. These techniques allow for the early detection of MRD in patients with CR defined by conventional morphological study. However, these tumour-specific markers have still not been identified for many haematological malignancies, except the WT-1 gene, which is expressed in many AML cases. PRAME was originally isolated as a melanoma antigen recognized by T cells and has since been found to be expressed in various cancers, including leukaemia. In the present study, we have attempted to develop a quantitative and sensitive method for the detection of MRD using a novel leukaemia marker, PRAME, which is highly expressed in both AML and ALL.

We have screened PRAME expression in 98 Japanese patients as well as in healthy individuals. Overall, 42% of the samples from Japanese patients expressed the PRAME gene at high levels, which is consistent with the data of Caucasian patients. However, frequent expression of the PRAME gene was also observed in ALL (64%) in Japanese patients, which is not shown in other studies. Pre-B ALL may express PRAME, as 12 out of 14 ALL samples in our study and all 14 PRAME-positive ALL samples in other studies were pre-B ALL.

A correlation between the Ph1 chromosome and PRAME expression has previously been suggested (van Baren et al, 1998) and PRAME has been reported to be induced in a leukaemic cell line by transfection of the bcr-abl fusion gene (Watari et al, 2000). In this study, the PRAME expression was particularly detected in five out of six Ph1-positive ALL patients, however, only 31% of 32 CML samples expressed PRAME. Therefore, the relationship between t(9;22) and PRAME expression is still unclear.

The real-time PCR method has recently been used for monitoring MRD using leukaemia/lymphoma-specific genetic markers, including bcr-abl, and the Ig idiotype (Gerard et al, 1998; Luthra et al, 1998; Marcucci et al, 1998; Mensink et al, 1998; Pongers-Willemse et al, 1998). In this study, we established the real-time PCR method for the detection of PRAME-positive cells. This method is quantitative and less labour-intensive. We monitored PRAME expression in eight PRAME-positive patients with leukaemia using real-time PCR. In all of the patients, PRAME expression significantly decreased after induction therapy. However, two of them relapsed with increased PRAME expression and one of them relapsed 1 month after the detection of PRAME expression. Among these patients, one Ph1-positive ALL patient (patient 1), one APL patient (patient 2) and one CML patient (patient 4) were also followed by quantitative RT-PCR with bcr-abl or PML-RARα leukaemia-specific rearrangement markers, and PRAME expression was correlated with the expression of these markers. Although further study with larger numbers of patients with leukaemia and lymphoma would be required, this pilot study indicated that quantitative monitoring of PRAME expression using real-time PCR is a sensitive tool for the detection of MRD, especially in leukaemia without known genetic markers.

The WT1 gene is known to be a genetic marker for a wide variety of haematological malignancies. The detection sensitivities of WT1 and PRAME are almost the same, 10−3−10−4 dilution of leukaemic cell line for WT1 and 10−4 dilution for PRAME. However, WT1 expression is usually not so high in ALL or NHL as in AML. Thus, we compared the expression levels of both genes in the samples of ALL, ATL, AML and NHL patients. We found that WT1 was not expressed or was expressed at low levels in the range of 101−102 copies/μg RNA in samples from one APL, one ALL, one ATL and one NHL patient with high PRAME expression at diagnosis. Therefore, the use of both genetic markers may cover a broad range of patients with leukaemia.

We also demonstrated the possibility that PRAME is processed and presented by HLA on the cell surface of leukaemic cells and is lysed by CTL in a similar way to that observed in melanoma. Thus, PRAME may be a target antigen useful for immunotherapy in haematological malignancy. A variety of immunotherapies specific for tumour antigens have recently been developed, and tumour regression has been observed in some clinical trials for melanoma and haematological malignancy, including immunization with idiotypic proteins for B-cell lymphoma. We are now attempting to generate CTLs specific for PRAME from PBMNCs of patients with leukaemia. As immunotherapy may be more effective in patients with a low tumour burden, it may be an interesting strategy to use PRAME-specific immunotherapy in MRD patients with PRAME-positive leukaemia. For example, PRAME-specific CTLs prepared from PBMNCs of patients before the relapse of leukaemia may be administered to patients whose PRAME expression increases without increased blast counts. PRAME may be expressed in some normal blood cells, however, the expression is about three log lower than in tumour cells. It has previously shown that tumour antigens expressed at this level could not be recognized by CTLs (Lethe et al, 1997), thus, normal cells would be resistant to CTL lysis, even if they express PRAME.

In conclusion, the present study has demonstrated that PRAME is a good tumour marker for monitoring MRD in patients with leukaemia, particularly for malignancies without known genetic markers. In addition, PRAME may also be useful as a target antigen for immunotherapy of haematological malignancies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Dr Y. Hara (Keiyu Hospital, Kanagawa), Dr Kikuchi (Saiseikai-chuo Hospital, Tokyo), Dr Kimura (Tokyo Medical College, Tokyo), Dr S. Kuwabara (Urawa Municipal Hospital, Saitama), Dr Takahashi (Tokyo University, Tokyo), Dr J. Ueyama (Toranomon Hospital, Tokyo) and Dr S. Watanabe (Department of Laboratory Medicine, Keio University Hospital) for kindly providing leukaemic samples. We also thank Otsuka Assay Laboratories for measuring bcr-abl gene and WT1 gene expression.

This study was supported by grants from the Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Keio University Grant-in-Aid for Encouragement of Young Medical Scientists 1998, Research Fund of Mitsukoshi Health and Welfare Foundation 1998 and Keio Health Counselling Centre Foundation 1999.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • Baer, M.R. (1998) Assessment of minimal residual disease in patients with acute leukemia. Current Opinion in Oncology, 10, 1722.
  • Beishuizen, A., Verhoeven, M.A., Van Wering, E.R., Hahlen, K., Hooijkaas, H., Van Dongen, J.J. (1994) Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis. Blood, 83, 22382247.
  • Bendandi, M., Gocke, C.D., Kobrin, C.B., Benko, F.A., Sternas, L.A., Pennington, R., Watson, T.M., Reynolds, C.W., Gause, B.L., Duffey, P.L., Jaffe, E.S., Creekmore, S.P., Longo, D.L., Kwak, L.W. (1999) Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nature Medicine, 5, 11711177.DOI: 10.1038/13928
  • Bosch, G.J., Joosten, A.M., Kessler, J.H., Melief, C.J., Leeksma, O.C. (1996) Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide. Blood, 88, 35223527.
  • Campana, D. & Pui, C.H. (1995) Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood, 85, 14161434.
  • Cave, H., Guidal, C., Rohrlich, P., Delfau, M.H., Broyart, A., Lescoeur, B., Rahimy, C., Fenneteau, O., Monplaisir, N., D'Auriol, L., Elion, J., Vilmer, E., Grandchamp, B. (1994) Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes. Blood, 83, 18921902.
  • Cross, N.C., Feng, L., Chase, A., Bungey, J., Hughes, T.P., Goldman, J.M. (1993) Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood, 82, 19291936.
  • Drobyski, W.R. & Hessner, M.J. (1998) The use of the polymerase chain reaction to predict for subsequent relapse in unrelated marrow transplantation for chronic myelogenous leukemia. Leukemia and Lymphoma, 31, 317323.
  • Eder, M., Battmer, K., Kafert, S., Stucki, A., Ganser, A., Hertenstein, B. (1999) Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation. Leukemia, 13, 13831389.
  • Gerard, C.J., Olsson, K., Ramanathan, R., Reading, C., Hanania, E.G. (1998) Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards. Cancer Research, 58, 39573964.
  • Goulmy, E., Schipper, R., Pool, J., Blokland, E., Falkenburg, J.H., Vossen, J., Grathwohl, A., Vogelsang, G.B., Van Houwelingen, H.C., Van Rood, J.J. (1996) Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. New England Journal of Medicine, 334, 281285.
  • Greenberg, P.D. (1991) Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells. Advances in Immunology, 49, 281355.
  • Heslop, H.E., Ng, C.Y., Li, C., Smith, C.A., Loftin, S.K., Krance, R.A., Brenner, M.K., Rooney, C.M. (1996) Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nature Medicine, 2, 551555.
  • Hsu, F.J., Caspar, C.B., Czerwinski, D., Kwak, L.W., Liles, T.M., Syrengelas, A., Taidi-Laskowski, B., Levy, R. (1997) Tumor-specific idiotype vaccines in the treatment of patients with B- cell lymphoma – long-term results of a clinical trial. Blood, 89, 31293135.
  • Ikeda, H., Lethe, B., Lehmann, F., Van Baren, N., Baurain, J.F., De Smet, C., Chambost, H., Vitale, M., Moretta, A., Boon, T., Coulie, P.G. (1997) Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity, 6, 199208.
  • Inoue, K., Sugiyama, H., Ogawa, H., Nakagawa, M., Yamagami, T., Miwa, H., Kita, K., Hiraoka, A., Masaoka, T., Nasu, K., Kyo, T., Dohy, H., Nakauchi, H., Ishidate, T., Akiyama, T., Kishimoto, T. (1994) WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia. Blood, 84, 30713079.
  • Inoue, K., Ogawa, H., Yamagami, T., Soma, T., Tani, Y., Tatekawa, T., Oji, Y., Tamaki, H., Kyo, T., Dohy, H., Hiraoka, A., Masaoka, T., Kishimoto, T., Sugiyama, H. (1996) Long-term follow-up of minimal residual disease in leukemia patients by monitoring WT1 (Wilms tumor gene) expression levels. Blood, 88, 22672278.
  • Kwok, S. & Higuchi, R. (1989) Avoiding false positives with PCR. Nature, 339, 237238.
  • Lethe, B., Van Der Bruggen, P., Brasseur, F., Boon, T. (1997) MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte. Melanoma Research, 7, S83S88.
  • Lin, F., Van Rhee, F., Goldman, J.M., Cross, N.C. (1996) Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood, 87, 44734478.
  • Lion, T., Gaiger, A., Henn, T., Horth, E., Haas, O.A., Geissler, K., Gadner, H. (1995) Use of quantitative polymerase chain reaction to monitor residual disease in chronic myelogenous leukemia during treatment with interferon. Leukemia, 9, 13531360.
  • Lowenberg, B., Downing, J.R., Burnett, A. (1999) Acute myeloid leukemia. New England Journal of Medicine, 341, 10511062.
  • Luthra, R., McBride, J.A., Cabanillas, F., Sarris, A. (1998) Novel 5′ exonuclease-based real-time PCR assay for the detection of t(14;18) (q32;q21) in patients with follicular lymphoma. American Journal of Pathology, 153, 6368.
  • Mannering, S.I., McKenzie, J.L., Fearnley, D.B., Hart, D.N. (1997) HLA-DR1-restricted bcr-abl (b3a2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates. Blood, 90, 290297.
  • Marchand, M., Van Baren, N., Weynants, P., Brichard, V., Dreno, B., Tessier, M.H., Rankin, E., Parmiani, G., Arienti, F., Humblet, Y., Bourlond, A., Vanwijck, R., Lienard, D., Beauduin, M., Dietrich, P.Y., Russo, V., Kerger, J., Masucci, G., Jager, E., De Greve, J., Atzpodien, J., Brasseur, F., Coulie, P.G., Van Der Bruggen, P., Boon, T. (1999) Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA- A1. International Journal of Cancer, 80, 219230.DOI: 10.1002/(sici)1097-0215(19990118)80:2<219::aid-ijc10>3.3.co;2-j
  • Marcucci, G., Livak, K.J., Bi, W., Strout, M.P., Bloomfield, C.D., Caligiuri, M.A. (1998) Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia, 12, 14821489.
  • Massaia, M., Borrione, P., Battaglio, S., Mariani, S., Beggiato, E., Napoli, P., Voena, C., Bianchi, A., Coscia, M., Besostri, B., Peola, S., Stiefel, T., Even, J., Novero, D., Boccadoro, M., Pileri, A. (1999) Idiotype vaccination in human myeloma: generation of tumor-specific immune responses after high-dose chemotherapy. Blood, 94, 673683.
  • Mensink, E., Van De Locht, A., Schattenberg, A., Linders, E., Schaap, N., Geurts van Kessel, A., De Witte, T. (1998) Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. British Journal of Haematology, 102, 768774.DOI: 10.1046/j.1365-2141.1998.00823.x
  • Miller, Jr, W.H., Kakizuka, A., Frankel, S.R., Warrell, Jr, R.P., DeBlasio, A., Levine, K., Evans, R.M., Dmitrovsky, E. (1992) Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America, 89, 26942698.
  • Molldrem, J., Dermime, S., Parker, K., Jiang, Y.Z., Mavroudis, D., Hensel, N., Fukushima, P., Barrett, A.J. (1996) Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells. Blood, 88, 24502457.
  • Moravcova, J., Lukasova, M., Stary, J., Haskovec, C. (1998) Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation. Leukemia, 12, 13031312.
  • Muto, A., Mori, S., Matsushita, H., Awaya, N., Ueno, H., Takayama, N., Okamoto, S., Kizaki, M., Ikeda, Y. (1996) Serial quantification of minimal residual disease of t(8;21) acute myelogenous leukaemia with RT-competitive PCR assay. British Journal of Haematology, 95, 8594.
  • Nestle, F.O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., Schadendorf, D. (1998) Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nature Medicine, 4, 328332.
  • Neumann, E., Engelsberg, A., Decker, J., Storkel, S., Jaeger, E., Huber, C., Seliger, B. (1998) Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma: candidates for T-cell-based immunotherapies? Cancer Research, 58, 40904095.
  • Nieda, M., Nicol, A., Kikuchi, A., Kashiwase, K., Taylor, K., Suzuki, K., Tadokoro, K., Juji, T. (1998) Dendritic cells stimulate the expansion of bcr-abl specific CD8+ T cells with cytotoxic activity against leukemic cells from patients with chronic myeloid leukemia. Blood, 91, 977983.
  • Ohminami, H., Yasukawa, M., Fujita, S. (2000) HLA class I-restricted lysis of leukemia cells by a CD8 (+) cytotoxic T- lymphocyte clone specific for WT1 peptide. Blood, 95, 286293.
  • Pellat-Deceunynck, C., Mellerin, M.P., Labarriere, N., Jego, G., Moreau-Aubry, A., Harousseau, J.L., Jotereau, F., Bataille, R. (2000) The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells. European Journal of Immunology, 30, 803809.DOI: 10.1002/(sici)1521-4141(200003)30:03<803::aid-immu803>3.0.co;2-e
  • Pongers-Willemse, M.J., Verhagen, O.J., Tibbe, G.J., Wijkhuijs, A.J., De Haas, V., Roovers, E., Van Der Schoot, C.E., Van Dongen, J.J. (1998) Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia, 12, 20062014.
  • Preudhomme, C., Henic, N., Cazin, B., Lai, J.L., Bertheas, M.F., Vanrumbeke, M., Lemoine, F., Jouet, J.P., Deconninck, E., Nelken, B., Cosson, A., Fenaux, P. (1997) Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL). Leukemia, 11, 294298.
  • Radich, J., Gehly, G., Lee, A., Avery, R., Bryant, E., Edmands, S., Gooley, T., Kessler, P., Kirk, J., Ladne, P., Thomas, E.D., Appelbaum, F.R. (1997) Detection of bcr-abl transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia after marrow transplantation. Blood, 89, 26022609.
  • Reichardt, V.L., Okada, C.Y., Liso, A., Benike, C.J., Stockerl-Goldstein, K.E., Engleman, E.G., Blume, K.G., Levy, R. (1999) Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma – a feasibility study. Blood, 93, 24112419.
  • Roberts, W.M., Estrov, Z., Ouspenskaia, M.V., Johnston, D.A., McClain, K.L., Zipf, T.F. (1997) Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. New England Journal of Medicine, 336, 317323.
  • Rosenberg, S.A., Yang, J.C., Schwartzentruber, D.J., Hwu, P., Marincola, F.M., Topalian, S.L., Restifo, N.P., Dudley, M.E., Schwarz, S.L., Spiess, P.J., Wunderlich, J.R., Parkhurst, M.R., Kawakami, Y., Seipp, C.A., Einhorn, J.H., White, D.E. (1998) Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nature Medicine, 4, 321327.
  • Seriu, T., Hansen-Hagge, T.E., Erz, D.H., Bartram, C.R. (1995) Improved detection of minimal residual leukemia through modifications of polymerase chain reaction analyses based on clonospecific T cell receptor junctions. Leukemia, 9, 316320.
  • Speck, B., Bortin, M.M., Champlin, R., Goldman, J.M., Herzig, R.H., McGlave, P.B., Messner, H.A., Weiner, R.S., Rimm, A.A. (1984) Allogeneic bone-marrow transplantation for chronic myelogenous leukaemia. Lancet, 1, 665668.
  • Tobal, K. & Yin, J.A. (1996) Monitoring of minimal residual disease by quantitative reverse transcriptase-polymerase chain reaction for AML1-MTG8 transcripts in AML-M2 with t(8;21). Blood, 88, 37043709.
  • Van Baren, N., Chambost, H., Ferrant, A., Michaux, L., Ikeda, H., Millard, I., Olive, D., Boon, T., Coulie, P.G. (1998) PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukaemia cells. British Journal of Haematology, 102, 13761379.
  • Van Baren, N., Brasseur, F., Godelaine, D., Hames, G., Ferrant, A., Lehmann, F., Andre, M., Ravoet, C., Doyen, C., Spagnoli, G.C., Bakkus, M., Thielemans, K., Boon, T. (1999) Genes encoding tumor-specific antigens are expressed in human myeloma cells. Blood, 94, 11561164.
  • Warren, E.H., Greenberg, P.D., Riddell, S.R. (1998) Cytotoxic T-lymphocyte-defined human minor histocompatibility antigens with a restricted tissue distribution. Blood, 91, 21972207.
  • Watari, K., Tojo, A., Nagamura-Inoue, T., Nagamura, F., Takeshita, A., Fukushima, T., Motoji, T., Tani, K., Asano, S. (2000) Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene. FEBS Letters, 466, 367371.DOI: 10.1016/s0014-5793(00)01112-1
  • Yotnda, P., Firat, H., Garcia-Pons, F., Garcia, Z., Gourru, G., Vernant, J.P., Lemonnier, F.A., Leblond, V., Langlade-Demoyen, P. (1998) Cytotoxic T cell response against the chimeric p210 bcR-ABL protein in patients with chronic myelogenous leukemia. Journal of Clinical Investigation, 101, 22902296.