Original Manuscript

Leukemia (2005) 19, 1338–1344. doi:10.1038/sj.leu.2403835; published online 23 June 2005

Acute Non-Lymphocytic Leukemias

DEK-CAN molecular monitoring of myeloid malignancies could aid therapeutic stratification

L Garçon1,6,7, M Libura1,7, E Delabesse1, F Valensi1, V Asnafi1, C Berger2, C Schmitt3, T Leblanc4, A Buzyn5 and E Macintyre1

  1. 1Faculté de Medecine, Université Paris-Descartes, INSERM EMI U210 and AP-HP Hématologie-biologique, Hôpital Necker- Enfants Malades, rue de Sèvres, Paris cedex, France
  2. 2Pédiatrie Oncologie A, CHU Hôpital Nord, Saint-Etienne, France
  3. 3Médecine Infantile II, Hôpitaux de Brabois - Hôpital d'Enfants, Vandoeuvre-les-Nancy, France
  4. 4Hématologie Pédiatrique, Hôpital St. Louis, Paris Cedex, France
  5. 5Hématologie Clinique, Hôpital Necker-Enfants Malades, rue de Sèvres, Paris cedex, France

Correspondence: Professor E Macintyre, Laboratoire d'Hématologie, Tour Pasteur, Hôpital Necker-Enfants Malades, 149-161, rue de Sèvres, 75743 Paris cedex 15, France. Fax: +33 1 44 38 17 45; E-mail: elizabeth.macintyre@nck.ap-hop-paris.fr

6Current address: Laboratoire d'Hématologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France

7These two authors have contributed equally to this work

Received 30 April 2005; Accepted 3 May 2005; Published online 23 June 2005.

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Abstract

The t(6;9)(p23;q34) is a recurrent chromosomal abnormality observed in 1% of acute myelogenous leukemia (AML), which generates a fusion transcript between DEK and CAN/NUP214 genes. We used a DEK-CAN real-time quantitative (RQ)-PCR strategy to analyze 79 retrospective and prospective samples from 12 patients. Five patients reached DEK-CAN negativity (sensitivity 10-5); all underwent early allogeneic hematopoietic stem cell transplantation (median 5.5 months from diagnosis) with some demonstrating molecular positivity at the time of allograft. All four cases in CCR with adequate follow-up (median 18.5 months, range 13–95) demonstrate persistent molecular negativity, whereas all seven patients with persistent DEK-CAN positivity died at a median of 12 months from diagnosis (range 7–27). We conclude that DEK-CAN molecular monitoring by RQ-PCR in t(6;9) malignancies is a useful tool for individual patient management and that molecular negativity is indispensable for survival, but should not be a prerequisite for allografting in this rare, poor prognosis, subset of AML.

Keywords:

acute leukemia, DEK-CAN transcript, minimal residual disease, real-time PCR

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Introduction

The t(6;9)(p23;q34) is a cytogenetic event occurring recurrently in acute myelogenous leukemia (AML) and myelodysplasia (MDS). Since its first description in 1976,1 more than 70 cases have been reported, allowing the identification of clinical and morphological characteristics: a young age at diagnosis, a predominance of AML-M2 according to the FAB classification, signs of multilineage dysplasia frequently with bone marrow (BM) basophilia, and a very poor prognosis with short disease-free survival (DFS) and overall survival (OS).2, 3, 4, 5

At the molecular level, this translocation generates a chimeric protein, resulting from the fusion between DEK and the 3'-terminus of the CAN gene, also known as NUP214.6 DEK, originally described as a proto-oncogene, is now known to be a major component of metazoan chromatin able to modify the structure of DNA by introducing supercoils.7, 8 Its level was found to be increased in AML BM samples.9 CAN is a nuclear pore complex protein with multiple FG-peptide sequence motifs and is implicated in nucleocytoplasmic transport.10, 11 The CAN gene is involved in several fusion transcripts described in acute leukemia: not only with DEK in t(6;9)(p23;q34) AML, but also with the SET gene and recently with ABL in T-cell acute lymphoblastic leukemia (ALL).12, 13 The size and structure of the DEK-CAN chimeric protein is the same in all cases, as DNA breakpoints within both genes are always located in the same intron.14

Quantitative reverse transcription-polymerase chain reaction (RQ-PCR) is currently used for the detection of minimal residual disease (MRD) in several AMLs.15 By providing information about the kinetics of clearance of the leukemic clone, reliable MRD quantification can have important predictive value for relapse and can identify groups of patients for whom more intensive treatment can be of benefit.16 Real-time quantitative (RQ)-PCR can provide useful information, as demonstrated in ALL as well as AML with PML-RARalpha, CBFbeta-MYH11, and AML1-ETO transcripts.17 Few studies of MRD have been performed for DEK-CAN+ AML, with most using only qualitative RT-PCR.18, 19, 20, 21 A limited number of patients have been analyzed by RQ-PCR, but little is known about the evolution of the DEK-CAN fusion transcript under therapy.22, 23

We therefore decided to quantify DEK-CAN fusion transcript in a series of 12 AML/MDS patients with the t(6;9) translocation and show that molecular evolution under therapy correlates with clinical outcome.

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Materials and methods

Patients and samples

We analyzed a group of 10 patients with AML and two with MDS demonstrating a t(6;9)(p23;q34) by conventional cytogenetics. For all these patients, presence of the DEK-CAN fusion transcript was confirmed by RT-PCR. Patients were treated in one of the seven following French centers: Assistance Publique – Hôpitaux de Paris de la Pitié-Salpétrière, Trousseau, Hôtel-Dieu, Necker-Enfants Malades and Saint-Louis, Paris; Hôpital Nord, Saint-Etienne; Hôpital d'Enfants-Brabois, Nancy. All patients were treated and followed in their original institutions between 1996 and 2004. RT-PCR quantification of DEK-CAN fusion transcript at diagnosis or during follow up was performed in the laboratory of Biological Hematology at the Necker-Enfants Malades hospital, after informed consent according to the Declaration of Helsinki. The total number of samples analyzed was 79. For eight patients, diagnosis and follow up samples were analyzed. For the others, only follow up samples at different end points of treatment were available.

Analysis of DEK-CAN fusion transcript by RT-PCR and RQ-PCR

For cDNA analysis 1 mug of total RNA obtained from BM or peripheral blood (PB) mononuclear cells was reverse transcribed and cDNA quality was assessed relative to the ABL housekeeping gene as described24 on an ABI PRISM™ 7000 (Applied Biosystems, Foster City, CA, USA). Samples with CtABL values above 32 (fluorescent threshold 0.1) were considered degraded. Qualitative RT-PCR at diagnosis was performed in the presence of 200 muM dNTP, 2.5 mM MgCl2 and 200 nM of the following primers: DEK-primer C: gCCAAAAgAg AAAAACCTAAA; CAN-primer B: gCAAggATTT ggTgTgAgAT. Real-time quantitative-PCR was performed in a total volume of 25 mul in the presence of 300 nM primers, 1/10 of the cDNA, and 200 nM probe using conditions standardized within the Europe Against Cancer program.25 Each run included four nontemplate controls to exclude false-positive results. Results of RQ-PCR analysis were normalized according to ABL gene expression using the following formula: 2(CTABL-CTDEK-CAN). Sequence of primers and probes for the DEK-CAN fusion gene was designed using Primer Express (Applied Biosystems) DEK S: AAAgTTgAAgAAACCCCCTACAgA; CAN AS: CATCATTCACATCTTggACAgCA; DEK-CAN probe: FAM-CATACTgATgAAggCgCCgAATTTCCT-TAMRA. For DEK-CAN amplification, the t(6;9) negative U937 negative control cDNA was used with dilutions of BM cDNA from a positive patient (UPN 3063) for the standard curve. Figure 1 represents the DEK-CAN amplification plot of one representative experiment. All dilutions were carried out in duplicate.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Amplification plot of standard log dilutions of cDNA from a DEK-CAN+ patient into U937 cDNA. (a) Schematic representation of genomic rearrangement between the DEK and CAN genes on chromosome 6p23 and 9q34, respectively. Position of the ABL gene relative to the breakpoint on chromosome 9 is also represented. (b) Position of primers and probe used for DEK-CAN detection: Primers C (forward) and B (reverse) were used for qualitative RT-PCR. Primer F and R (forward and reverse, respectively) and probe P were used for RQ-PCR assays. (c) Quantification of DEK-CAN and ABL transcripts by real-time RQ-PCR was assayed in parallel with serial dilutions of bone marrow cDNA from UPN3063 used for the standard curve. Each dilution was made in duplicate. For each experiment, U937 cDNA was used as a negative control and water was used to verify the absence of contamination.

Full figure and legend (135K)

PCR detection of FLT3-ITD and FLT3-exon 17 D835 mutations

Polymerase chain reaction was performed as previously described.26 Briefly, PCR was performed from either genomic DNA (500 ng) or cDNA (0.2 mug RNA equivalent) in 50 mul with 0.3 muM primers, 5 mM of MgCl2, 0.75 mM dNTP, 1.25 U AmpliTaq GOLD (Applera™) at 94°C for 8 min, followed by 35 cycles at 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, and a final elongation step at 72°C for 10 min. PCR products were analyzed on 3% agarose gel or by Genescan analysis using a 5'HEX- Flt3Q labeled primer, analyzed on an ABI PRISM™ 310. Mutations within FLT3 exon 17 were detected as previously described.26 Briefly, 20 mul of PCR product was digested with 5 U EcoRV (NewEngland Biolabs, Frankfurt, Germany) for 1 h at 37°C and mutations involving D835 detected by PAGE of digested PCR products.

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Results

Patient characteristics

All 12 patients included in our study presented with a myeloid malignancy associated with translocation t(6;9)(p23;q34) by conventional cytogenetics. This translocation was isolated in 11 patients; for one patient, an additional t(7;7) translocation was associated with the t(6;9) (UPN1185, data not shown). The presence of a DEK-CAN fusion transcript was confirmed by qualitative RT-PCR in all cases (data not shown). Clinical and biological data of the patients are summarized in Table 1. The majority of patients were young adults (median age 18 years, range 3–44 years). The gender ratio was four males and eight females. OMS/FAB subtype distribution was mainly AML with maturation/M2 (1 AML without maturation/M1, 8 AML with maturation/M2, 1 AML/M4 and 2 RAEB-type MDS). The blood count was characterized by moderate hyperleucocytosis (median WBC of 15 800/mm3, range 4600–67 800). Median hemoglobin level and platelet count at diagnosis were, respectively, 7.2 g/dl (range 5.5–9.7) and 35 000/mm3 (range 6000–198 000). BM basophilia was noted for six patients. Multilineage dysplasia at diagnosis was noted in all patients for whom these data were available. Immunophenotype was available for seven patients (data not shown): CD13 and/or CD33 were positive in all patients and most of them expressed immature markers, such as CD34 (four out of seven), HLA-DR (five out of seven) and c-kit (three out of five).


FLT3 internal tandem duplication (FLT3-ITD) has been described to be frequently associated with DEK-CAN myeloid malignancies.27 In our series, this duplication was also frequent, being found in five out of the eight patients tested. Identical FLT3-ITD profiles were found in all four patients analyzed at diagnosis and relapse, including two initially FLT3-ITD positive cases (UPN2592 and UPN2804). All seven diagnosis samples tested for FLT3-D835/I836 point mutations were negative (Table 1).

Clinical outcome

The median follow-up of the cohort was 14 months (range 7–95 months). Chemotherapeutic protocols and response to treatment are summarized in Table 2. All but one patient received at least one course of induction chemotherapy (CT). Induction regimens were heterogeneous but classical, including anthracycline in association with cytarabine. Complete remission (CR) was obtained after one or two courses for nine patients among the 11 who received induction CT; partial remission (PR) was reached for one, and one patient was considered to be primary refractory to CT. After achieving CR, patients underwent different consolidation strategies. Six underwent allogeneic hematopoietic stem cell transplantation (HSCT) in CR1, PR1 or for refractory disease, one underwent autologous PB stem cell transplantation after high-dose CT, and five were treated by CT alone (either because there was no donor available or because of an early relapse). Seven patients relapsed with a median of 7 months (range 5–14 months) after diagnosis. Seven patients died with a median of 12 months after diagnosis (range 7–27 months). Five patients are alive (median follow-up 15 months, range 7–95 months), four in CR1 and one in CR2.


DEK-CAN monitoring by quantitative real-time RT-PCR

We used an RQ-PCR assay in order to follow the DEK-CAN transcript level response to therapy. Position of DNA breakpoints on chromosome 6p23 and 9q34, and location of primers and probe used for the PCR assays are represented in Figure 1a and b, respectively. Log dilutions of DEK-CAN+ cDNA (UPN3063) into DEK-CAN- U937 cell line cDNA demonstrated a reproducible sensitivity of at least 10-4 (Figure 1c). Samples were normalized using ABL expression and quantified relatively to the UPN3065 dilution series. Eight diagnosis and 71 follow-up samples from 12 patients were then analyzed by RQ-PCR (Figure 2). Median number of samples per patient was 5.5 (range 2–18).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic representation of DEK-CAN molecular monitoring by RQ-PCR: Circles: bone marrow; triangle: peripheral blood; filled: positive samples; empty: negative samples. ALLO: allogeneic; HSCT: hematopoietic stem cell transplantation; CT: chemotherapy; CR: complete response; NR: no response; PR: partial response; R: relapse; D: death; A: alive; CB: cord blood; SD: sibling donor; HP: haplo-identical; MUD: match unrelated donor; auto-PBSCT: autologous peripheral blood stem cells transplantation; RIC: reduced intensity conditioning regimen; DLI: donor lymphocyte infusion.

Full figure and legend (66K)

The median DEK-CAN level at diagnosis was 152plusminus70% of UPN3065 (Figure 3a). Achievement of hematological CR1 correlated with a one to 4-log reduction of DEK-CAN levels between the initial and the lowest level (median 0.00014, s.e.m. 0.013). Only one patient (UPN5171) reached DEK-CAN negativity with CT alone before allografting. For all patients treated by CT alone as first-line therapy, neither the kinetics of initial response nor the nadir reached (down to 10-4–10-5) predicted subsequent relapse or outcome. Molecular data at the moment of relapse were available for five patients: median levels were 40plusminus142%, with a 1–4 log increase between the lowest level and the level at relapse (Figure 3a).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Evolution of DEK-CAN transcript levels under therapy. (a) Distribution of DEK-CAN transcript level at diagnosis, the nadir reached after induction/consolidation and at time of relapse: median DEK-CAN levels at diagnosis, during complete remission and at relapse were 1.52 (range 0.27–3.03), 0.00014 (range 0–0.0018), and 0.4 (range 0.0042–4.29), respectively. (b) Patients alive at the end of the study (n=5). All patients were in molecular remission with a DEK-CAN level below the threshold of detection. (c) Deceased patients (n=7). None of these patients reached DEK-CAN negativity at any point of follow up.

Full figure and legend (26K)

Follow-up samples from the five patients who are still alive were constantly negative for DEK-CAN by RQ-PCR (Figure 3b). Follow up was short for one patient (UPN3912), who underwent allogeneic HSCT in CR2, with PCR negativity only being found in the last sample. Since this patient was then lost to follow up it was excluded from further analysis. The four others demonstrated a DFS of more than 12 months, with persistent molecular remission. Median follow-up was 18.5 months (range 13–95). These four patients underwent allogeneic HSCT early in the course of the disease, either in CR1 (n=2, follow-up 13 and 22 months), PR1 (n=1, follow-up 15 months) or without prior CT (n=1, follow-up 95 months). Molecular negativity was achieved within 4–9 months after diagnosis in all four patients with adequate early sample points. Median time from diagnosis to allogeneic HSCT in these four patients was 5.5 months (3, 5, 6 and 14 months).

In contrast, DEK-CAN expression within the group of seven patients who died revealed that none achieved molecular negativity (Figure 3c). Two patients (UPN3255 and UPN2869) was treated by CT alone, relapsed and died from blastic disease. Four underwent allogeneic HSCT during refractory disease (UPN4216) or in CR2 after relapse and one died from relapse 9 months after allogeneic HSCT in CR1 (UPN2592). The five allografts were performed at a median of 8.5 months from diagnosis (range 4–17 months). Median survival was 12 months (range 7–27) in this group of patients.

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Discussion

The rarity of myeloid malignancies with t(6;9)(p23;q34) complicates analysis of their clinico-biological characteristics and response to treatment. We have analyzed 12 such cases by RQ-PCR quantification of DEK-CAN and demonstrate here that molecular follow-up is a useful tool to predict outcome and should be used to guide treatment.

The 12 patients demonstrated the characteristic features of t(6;9) myeloid malignancies: a relatively young age at diagnosis, a predominance of AML FAB M2 and frequent multilineage dysplasia and BM basophilia.2, 3, 4, 28, 29 We also confirmed the high incidence of FLT3-ITD: 5/8 cases compared to nine out of 10 published cases.27 It has been suggested that genomic instability in DEK-CAN+ blasts induces FLT3-ITD.27 The fact that all four cases analyzed here retained a stable FLT3-ITD phenotype between diagnosis and relapse argues against this, at least for these loci.

Poor prognosis is well recognized in t(6;9)(p23;q34) AML (2-5), which is consequently classified as high risk in the majority of therapeutic protocols.30, 31 Hematological CR was obtained after one or two induction courses in nine of the 11 patients undergoing induction. This CR1 rate is higher than prior series,2, 4, 30 possibly due to more intensive induction protocols. Despite this, seven patients subsequently died, with six demonstrating refractory disease despite intensive treatment, including allografting. Achievement of morphological CR1 does not therefore predict a favorable outcome in DEK-CAN AML.

Quantification of specific oncogenic fusion transcripts is widely used for follow up of patients with leukemia. The DEK-CAN rearrangement lends itself well to RQ-PCR quantification, since the breakpoints are always located in the same introns,12, 14 thus allowing detection and quantification with at least theoretically comparable efficacy, using the same primers and probe for all patients. Systematic DEK-CAN detection by RQ-PCR in AMLs at diagnosis is probably not justified, since we screened 95 samples of de novo adult AML patients but found them all to be negative (unpublished data). It seems reasonable to use molecular analysis in DEK-CAN as confirmation of cytogenetic results and as a baseline for molecular follow-up. At diagnosis, DEK-CAN quantification showed relatively little inter-individual variation, particularly if variation in the degree of BM involvement is taken into consideration. DEK-CAN levels diminished by several logs following induction, correlating well with the hematological response, but rarely reached molecular negativity with CT alone. Even a 4-log decrease of DEK-CAN levels with CT was associated with subsequent relapse, confirming previous data showing relapse in two patients despite DEK-CAN RQ-PCR levels between 10-4 and 10-5 after treatment.23

Patient survival correlated strongly with persistent DEK-CAN RQ-PCR negativity. All five patients alive at the end of our study were in molecular remission, whereas all seven patients with persistent DEK-CAN positivity died during the study period (one from toxicity, all the others from refractory disease despite intensive treatment). Since the median follow-up time (18.5 months) for surviving patients all of which were in molecular remission exceeds the median time to death (12 months) in the PCR positive group, it is likely that molecular monitoring does identify patients with different prognoses, although this should obviously be tested in a prospective, protocol-based setting. It should also be noted that the negative follow-up points included blood and BM samples. Use of blood samples for MRD monitoring in AML is controversial; a strong correlation between BM and PB samples was found in PML-RARalpha AML;32 AML1-ETO monitoring by RQ-PCR was shown to be comparable in PB and BM although others report discordances by nested or single step PCR.33, 34 In our series, five follow-up points were available for both PB and BM, with no discrepancies in the four positive samples and the single negative one.

To our knowledge, the data presented here represent the first reported patients with prolonged RQ-PCR negativity in DEK-CAN myeloid malignancies. Three recently described patients remained positive after CT or autologous transplantation and all relapsed.23 All surviving DEK-CAN- patients in the present series had undergone allogeneic HSCT. Better results were obtained for patients who underwent allograft early in the course of the disease, in CR1/PR1 rather than after relapse. It is noteworthy that four of the five surviving patients were DEK-CAN+ in the last BM sample prior to allografting, with the majority being tested within 1 month. This suggests both that it may not be necessary to achieve RQ-PCR negativity before allograft, and also a potential GVL effect against the leukemic clone in this disease, in keeping with in-vitro data.35, 36 Allogeneic HSCT has a positive impact on OS in AMLs with unfavorable cytogenetics, including t(6;9).31 Based on a review of the literature, only a few other DEK-CAN+ patients obtained a DFS longer than 2 years, of which three had undergone allogeneic HSCT.2, 3, 4, 18 The only patient tested by nested, qualitative RT-PCR was negative up to 25 months after transplantation.18 Taken together, these data suggest that DEK-CAN AML should be allografted when possible, without waiting for molecular negativity, and that subsequent attainment of such negativity can be associated with relatively long-term survival in this poor prognosis group. Conversely, failure to obtain molecular negativity could represent an indication for DLI, since all patients with persistent positivity succumbed to their disease, including after allogeneic HSCT.

In conclusion, we demonstrate on a relatively large series of DEK-CAN myeloid malignancies that molecular quantification can help individual patient management, since persistence of DEK-CAN transcript under therapy is correlated with a bad prognosis, whereas prolonged molecular remission can be achieved after allogeneic HSCT, and seems to be crucial for survival. Further prospective studies are clearly justified in order to confirm this clinical relevance.

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Acknowledgements

We greatly acknowledge all the physicians and biologists for providing clinical and biological data and samples for DEK-CAN monitoring. We especially thank Dr Vasselon (Hôpital Nord, St-Etienne, France), Pr Leblond and Pr Merle-Béral (Hôpital de la Pitié-Salpétrière, Paris, France), Pr Leverger and Dr Adam (Hôpital Trousseau, Paris, France), Pr Rio (Hôtel Dieu, Paris, France), Dr Noguerra and Pr Baruchel (Hôpital St-Louis, Paris, France). Marta Libura is a recipient of a scholarship from the Postgraduate School of Molecular Medicine at the Medical University of Warsaw. This work was supported by la Ligue Nationale Contre le Cancer and l'Association de la Recherche sur le Cancer (ARC).

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