Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10−1, and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.
Specific, reciprocal chromosomal translocations are recurrent features of hematological malignancies and sarcomas. Recent studies indicate that these specific chromosomal alterations may influence the diagnosis and choice of treatment for patients.1 These rearrangements have two main consequences: the activation of proto-oncogenes or, more frequently, the generation of a fusion gene (FG). To date, more than 200 different translocations specific to particular types of leukemia and lymphoma have been described and there is increasing evidence that specific rearrangements may be utilized for risk stratification and therapeutic option decisions.1, 2
Among these cancer-related chromosomal aberrations, a family of rearrangements involving the MLL gene (myeloid/lymphoid leukemia or mixed lineage leukemia, also called HRX, Htrx-1, ALL-1) on chromosome 11, band q23, is of particular interest.3 The MLL rearrangements include nonconstitutional or acquired deletions, duplications, amplifications and reciprocal translocations.4 The latter result in the replacement of the MLL coding sequences 3′ to the breakpoint by the translocation partner. These aberrations usually occur in tumors of specific hematological lineages, and suggest a crucial role for MLL in determining disease phenotype or tumor tropism.5 Most of the genomic breakpoints occur in a clustered region between exons 5 and 11 in an 8 kb genomic region, but some rearrangements have been recently observed outside this region in adult T-ALL.6 More than 30 different partners resulting in various MLL FG transcripts have been identified (Table 1).4, 7 These rearrangements account for 5–10% of acquired chromosomal rearrangements in pediatric and adult ALL (B- and T-ALL), acute myeloblastic leukemia (AML), and poorly differentiated or bi-phenotypic leukemia and myelodysplastic syndromes (MDS).2, 6 Strikingly, up to 80% of acute leukemia in neonates and infants, as well as a majority of secondary or treatment-related (ie topoisomerase II inhibitor induced) leukemia, involve a reciprocal clonal rearrangement of 11q23.8, 9, 10
Analyses of leukemia karyotypes by several groups have revealed a relative association between particular translocations and subtypes of leukemia.2, 11 For instance, MLL-AF9 is primarily found in AML, and translocation involving the AF4 gene occurs almost exclusively in B-cell lineage tumors.5 The most common partners are AF4 in pro-B ALL and AF9 in AML, mainly M5, accounting for 40 and 27% of the 11q23 translocations, respectively.11 19p13 is another frequently encountered rearrangement observed in approximately 12% of 11q23 translocations in both ALL and AML.11 Additionally, MLL gene rearrangements can occur in AML patients with +11, or without any evidence of chromosomal abnormality, resulting in translocation (MLL-LARG) or internal tandem duplication (DUP).12, 13
Overall, 11q23 rearrangements have been shown to be predictive of poor clinical outcome, independent of their association with other high-risk clinical features at presentation. Thus, despite the use of aggressive induction regimens and stem cell transplantation in first remission, patients with 11q23 rearrangements globally continue to have poor long-term survival.2, 14 However, improved survival has been observed for t(9;11) in pediatric and adult AML, suggesting that prognosis may depend on the translocation partner.2, 15 Likewise, whole-genome profiling studies demonstrated that leukemia bearing MLL translocation can specify a unique gene expression program,16 suggesting that precise and systematic determination of the MLL status of acute leukemia will become increasingly important in defining appropriate clinical treatment.17
Cytogenetics analysis is a routine part of diagnostic testing for newly diagnosed and relapsed patients, and has been reported to serve as an independent prognostic factor, providing a framework for stratified treatment approaches to these malignancies.14 However, analysis must be performed on metaphases spreads and a translocation can only be detected and identified if it causes a clear difference in chromosome banding pattern. Southern blot analysis can identify MLL translocations, but requires at least two probes and several enzymatic digestions, resulting in laborious and time-consuming experiments, unsuitable for routine clinical use. Assays based on fluorescence in situ hybridization (FISH) have been described for rapid detection of MLL rearrangements in acute leukemia.18, 19 Split signal FISH methods can detect all 11q23 rearrangements, with low false positivity.20, 21 However, these techniques cannot be used for precise identification of the involved partner gene. PCR-based methods can be utilized to detect chimerical RNA with comparable reliability to FISH,22 but can only detect MLL gene translocations with well-defined (sequenced) partner gene and is not practical for FG identification due to the large number of FGs and breakpoint variants.
To overcome this, multiplex protocols for routine screening of patients have been developed to characterize small groups of chromosomal translocations found in leukemia cells. However, additional analyses, such as split-out and modified post-PCR southern blot analysis are required for the detection and verification of an FG.23, 24, 25, 26 A recent publication describes a multiplex reaction followed by biochip detection to screen samples for the presence of translocations, but this technology is only applicable to well-characterized fusion events.27 Moreover, the continual identification of new breakpoints, especially for MLL partner genes, as well as the technical complexity of this kind of assay, increase the risk of false negative and false positive results due to cross-contamination.23, 24
To overcome the intrinsic difficulties in the analysis of complex molecular rearrangements of the MLL gene, we developed an assay, which combines the specific amplification of MLL fusion transcripts and identification of the amplified product by hybridization on a low-density biochip spotted with specific oligonucleotides. This design allows for rapid, accurate and comprehensive detection of the 32 well-characterized MLL gene rearrangements, and precise identification of the FG partner and breakpoint positions. Although the described assay is limited to 32 MLL FG partners, this device may be easily adapted to screen for additional partners. Here, we report the development and validation of this new diagnostic method, and demonstrate that biochip-based devices can be utilized to provide important molecular information that may contribute to improved patient care.
Materials and methods
Cell lines and patient samples
Assay performance was tested using cell lines known to express specific MLL fusion RNA: MV4-11, t(4;11); ML2, t(6;11); Karpas45 t(X;11) and only normal MLL (U937, K562). Cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) or ‘Deutsche Sammlung von Mikroorganismen und Zellkulturen’ (DSMZ, Braunschweig, Germany), and cultured according to provider recommendations.
Leukemia samples were obtained from peripheral blood or bone marrow cells from affected individuals at diagnosis and assessed by standard cytogenetics (±FISH). Assays were blindly performed on stored frozen samples and test results cannot be linked to a specific patient nor provide information that impact upon the patient's condition.
Total RNA from cell lines was isolated by various commercially available methods (TRIzol®, Gibco®, Life Technologies®; RNAqueous®-4PCRKit, Ambion® Ltd; Nucleospin®RNAII, Machery-Nagel Sarl; SVTotalRNA Isolation System, Promega® France; RNeasy® MiniKit, Qiagen® France SA; RNAble® Eurobio France) or by standard cesium chloride extraction, with no obvious differences in terms of quality or assay performance. Total RNA was re-suspended in RNase-free water and concentration and quality were determined by ultraviolet spectroscopy and analysis on an Agilent® Bioanalyser (RNA 6000 Nano Assay reagent set-Agilent®). Stock solutions containing 1.0 μg/μl RNA were stored at −80°C.
Gene specific, long (50-mers) oligonucleotides for MLL and its known potential partners were designed with the primer analysis software OLIGO 6.0 (Molecular Biology Insights Inc.) based on cDNA sequences deposited in GenBank (Table 1) and according to specific properties, such as melting temperature, GC content and free energy (including 3′ stability; duplex free; maximum length of dimer (2 bp); maximum acceptable homology; maximum number sequence repeat). The resulting selected sequences were systematically compared to the NCBI database (standard BLASTN 2.2.2, Dec-14-2001) and between each other to circumvent nonspecific hybridization. These gene-specific oligonucleotides (www.mllfusionchip.com) were purchased from Invitrogen. For each MLL partner, a set of oligonucleotides was selected according to the published splice-junction site known to be implicated in gene fusion events. When possible, at least one oligonucleotide upstream of the described breakpoint was chosen to serve as a negative control. In addition, the specific oligonucleotides corresponding to the other genes partners can be regarded as negative controls. The MLL specific oligonucleotides are arranged as an L shape from the 5′ (upper left) to the 3′ (lower right) region and the oligonucleotides for each FG partner arranged in line from 5′ (left) to 3′ (right) (Figure 1). The 12 different oligos representative of the MLL sequence are repeated several times, and each specific partner's oligo was spotted once. A nonrelevant unique biotinylated oligonucleotide is included on the chip and serves as an internal control for colorimetric reaction, and accurate test interpretation (inverted L shape). Oligonucleotides (50 μ M for all MLL and partner genes and 25 μ M for biotinylated controls) are spotted onto Vivid™ Gene Array Slides (Pall® Life Science) using a Microgrid II robot (Biorobotics Genomic Solutions) and fixed onto the membrane for 2 h at 80°C.
In our assay, the MLL specific primers represent sequences of the exon 4c of MLL.28 The anchored-random primer used for the RT step contains a random nonamer at its 3′ end, linked to a 47-mer anchor sequence at its 5′ end, allowing the use of two different reverse primers for consecutive rounds of amplification (Figure 2a and b).
In brief, 1 μg of total RNA is retro-transcribed through a random anchored reaction. In all, 5 μl of the cDNA synthesis reaction are amplified in a first PCR reaction with MLL-Ext and Anc-Ext primers (Figure 2a). In all, 1 μl of the first PCR reaction is amplified in a second nested PCR amplification (labeling) containing MLL-Int and Anc-Int primers. Quality of the resulting biotinylated-complexes is analyzed by electrophoresis on an ethidium bromide stained 1% agarose gel. The detailed protocol is available at www.mllfusionchip.com.
Hybridization and detection
BioChips are prehybridized then hybridized for 2 h and for 18 h, respectively, at 65°C. Following hybridization, chips are washed 5 min at room temperature (RT), then 30 min at 65°C. Chips are then incubated three times 5 min at RT in a new washing buffer, followed by 1 h in blocking buffer at RT, then 1 h in renewed blocking buffer plus 3.6 U Streptavidine Alkaline Phosphatase at 37°C. Chips are washed successively for 5 min at RT, 15 min at RT and 15 min at 37°C, and then incubated 15 min in BCIP/NBT solution.
Results are visually interpreted. The inverted L shape on the right of the chip confirms the efficiency of the detection reaction and defines a grid to allow rapid and accurate interpretation of the test. Results are interpreted by comparing the processed chip with the template (Figure 1). Even if the MLL pattern is partial, when at least two consecutive spots appears for one of the partner gene line, the test is positive, that is, the tested RNA carries an abnormal MLL transcript. When the MLL pattern is complete, and no signal appears for any of the spotted partner genes, the test is negative, that is, the test RNA carries only normal MLL transcripts or an MLL fusion transcript with a partner gene not represented on the chip. Negative test results may also occur if the sample carries an MLL fusion with an alternative breakpoint located in the small 3′ region of a known partner gene not covered by the oligonucleotides spotted on the biochip (relative positioning of all oligonucleotides are available on www.mllfusionchip.com). When the MLL pattern is not complete and no signal appears for any of the partner genes, or when two different partners are positive, the test cannot be interpreted.
Results and discussion
Analytical performances of the biochip
Limit of detection
The limit of detection for the MLLFusionChip™ was determined on cell line data by testing dilutions of MLL positive RNAs (MV4-11 or Karpas45 total RNA) in the presence of MLL negative RNAs (K562 or U937 total RNA). Tested dilution ratios of the MLL positive RNA were 1, 1/2, 1/5, 1/10, 1/20 and 0 (MLL negative RNA only) with a constant total amount of 1 μg. The resulting mixed RNAs were tested in parallel by two different operators. The minimal dilution giving positive results for both operators was 10% (data not shown), and one operator (working on a dilution of Karpas45 within U937 RNA) had a positive MLL-AFX result for the 5% dilution (position 4.3 on the chip reading grid, data not shown). A limit of detection of 20% was consider as sufficient for the MLLFusionChip™ to provide clinically useful information if used at the time of diagnosis on acute leukemia patients.29 Although FISH and PCR techniques have been reported to exhibit higher limits of sensitivity (10−1 and 10−5 respectively), results from these tests must be confirmed by alternate techniques, and are directly dependent on the availability and choice of the probes. In contrast, the MLLFusionChip™ allows the detection of the 32 rearrangements (Figure 1) with the MLL specific primers described above.
Intra-assay precision was tested by one global assessment of a positive sample previously characterized by standard cytogenetics and PCR as bearing a t(11;19) translocation (total RNA extracted from leukemia clinical sample cells IPS-18). The RNA was processed for six reverse transcriptions (RT). Three out of six resulting cDNAs were divided and amplified in two parallel PCR I reactions and the remaining three cDNAs were amplified in one PCR I reaction each as specified in our standard protocol. Each resulting PCR I product (ie nine products from six different RT) was subsequently amplified in PCR II labeling reactions (two pooled reactions per PCR I, as specified in our protocol). These nine reactions were hybridized onto nine individual MLLFusionChips. The nine hybridizations yielded 100% identical results, positive for the MLL-ELL translocation (position 3.1 on the chip reading grid, data not shown).
Reproducibility was blindly evaluated by three different operators performing the MLLFusionChip™ test in three independent experiments utilizing total RNA from cell lines (three positive, Karpas45, MV4-11, and ML2, and one negative, K562) and a clinical leukemia sample (IPS-11). The positive clinical sample was previously characterized by standard cytogenetics and PCR as bearing a t(4;11) translocation. The mean operator-to-operator, and day-to-day reproducibility was 93 and 98%, respectively (Table 2). The only nonreproducible result was an MLL-AF6 translocation detected in addition to the expected MLL-AF4 translocation for the positive RNA IPS-11. This result is almost certainly due to a cross-contamination during the amplification steps since the positive cell line RNA ML2 was amplified in the same experiment, and is probably responsible for the weak AF6 signal detected on the positive RNA IPS-11. This sample was tested more than eight times in a series of experiments without false positive results. Hence, it is highly unlikely that this single spurious assay results from lack of probe specificity. Nevertheless, this inconsistent result demonstrates that the two consecutive nested amplifications must be performed with extreme caution, following strict GLP procedure for PCR, to prevent mRNA, cDNA or PCR I product carryover contamination.
It is noteworthy to correlate the ML2 results we observed with the characteristics of this cell line, derived from a patient with AML that developed after complete remission of T-cell lymphoma.30 ML2 cells have retained an 11q23–24 deletion from the lymphoma stage and have acquired t(6;11) with development of AML. Consequently, the ML-2 cells have no normal MLL gene on Southern blot analysis. On our MLLFusionChip™, the MLL positive spots for the ML2 RNA corresponded to positions 3688 and 3968 (MLL-3688 & MLL-3968). No signal was observed for the following MLL spots (MLL-4124 and further), confirming that the MLL normal transcript is not expressed in ML2 (Figure 3).
Studies on leukemia cells comparison with existing methods
Results obtained on the MLLFusionChip™ were compared to existing technologies for cross validation. Experiments were conducted in a retrospective study on leukemia cells from 31 patients, 25 of whom had been previously diagnosed as bearing a MLL rearrangement (MLL R) either by karyotype, FISH, Southern blot or RT-PCR analysis. The remaining seven patients were diagnosed as bearing either the germline MLL gene (MLL G) or a genetic alteration of chromosome 11 (deletion or trisomy) without any evidence of rearrangement. In all cases, results obtained with the MLLFusionChip™ were fully concordant with results from existing methods (Table 3). Of note, although most tested samples were collected at the time of diagnosis with a high percentage of blasts cells (ca 90%), we also tested a sample with 35% of blasts (IPS-18), and obtained the correct positive result for the MLL-ELL fusion transcript. Importantly, this result is consistent with the limit of detection studied on RNA extracted from cell lines.
Five clinical samples were positive for the MLL-AF9 FG (PSL-01, PSL-02, PSL-06, IPS-16 and IPS-17); the positive spots on the biochip for four out of these five samples started at position 4, indicating that the breakpoint on AF9 was upstream of exon 7 (Figure 4). These cases correspond to the most frequent AF9 breakpoint located at nucleotide 1321 of the mRNA sequence L13744 and named site A.31 Interestingly, the positive AF9 spots on the sample PSL-06 started at position 2, indicating that the breakpoint on AF9 was upstream of exon 5. This alternative site corresponds to the additional breakpoint described at nucleotide 616 and named site C.31
Three clinical samples were positive for the MLL-AF4 FG (IPS-10, IPS-11 and IPS-12); the positive spots on the biochip for samples IPS-11 and IPS-12 started at position 2, indicating that AF4 exon 5 was present in the chimerical transcript (Figure 5). Furthermore, signal for the spot at position 3 for the same samples indicated that the sequence specific to exon 10 was also present. The positive AF4 spots for sample IPS-10 started at position 4, indicating that at least the first 10 AF4 exons were absent in the chimerical transcript, and that breakpoint on AF4 was located upstream of exon 11 (Figure 5).
Alternative results for a given partner gene was not observed among the remaining fusion transcripts described in this study: five MLL-ENL and two MLL-ELL fusion transcripts were detected and all started from position 1 on the corresponding partner line. Four MLL-AF6 fusion transcripts were detected from the oligo at position 2, indicating that the breakpoint was upstream of exon 4 on AF6, as previously described.30 Positive results for MSF (IPC-02), AF17 (IPS-09), AF10 (IPS-08) and AF1q (IPS-21, IPS-22) partner genes were also observed. It should be noted that, even if the development of the device was carried out using cell lines RNA corresponding to three different fusion transcripts, this study enables us to extend the validation of this technology to the detection of seven other partner genes in clinical samples.
These results collectively demonstrate the potential utility of this biochip assay, which allows the detection of an FG partner of MLL regardless of breakpoint location, and provides valuable information to facilitate primer design to follow the expression of the identified partner by standard PCR.32
MLL internal tandem duplications (MLL-DUP)
A positive result for MLL-DUP (spot position 1.2. on the biochip) is not always correlated with a genomic internal duplication detected by RT-PCR. Previous studies show that splicing of MLL is extremely complex, and several abnormally spliced MLL transcripts have been described in both malignant and normal tissues.33, 34 In these mis-spliced products, MLL exons are joined in aberrant genomic orientation (exon scrambling) and are not the result of exon duplication. For example, we observed DUP positive spots in several cases in addition to positive spots for another partner gene for the same patient sample. Further, MLL duplications were detected in patients without any evidence of genomic rearrangement and variable results were obtained for the same patient. For these reasons, confirmation of MLL-DUP using the MLLFusionChip™ should be performed using standard or long range PCR on genomic DNA. However, since scrambled transcripts are indistinguishable from transcripts resulting from genomic rearrangements, this precludes the use of nested RT-PCR as a screening method for the detection of internal tandem duplication of MLL.
The combination of RACE-PCR with hybridization on specific probes spotted on a biochip allows a more accurate identification of patients with molecular genetic lesions affecting the MLL gene. The use of nonradioactive detection allows for easy integration of this screening tool into the clinical laboratory setting for the diagnosis and management of leukemia. Additionally, the microarray approach provides unambiguous and definitive results for products of different chromosome translocations, as well as localization of breakpoints and splice variants for specific chromosomal translocations.
These results collectively demonstrate that the performance and clinical utility of the MLLFusionChip™ device is, at least equal to, or superior to FISH, RQ-PCR and Southern blot assays. FISH is commonly used for detecting chromosome translocations, but requires prior identification of the translocation, to select the appropriate DNA probe for detection.35 Dual-color FISH analysis can determine the partner chromosome, but cannot be used to identify the precise FG partner.20, 21 Nevertheless, the FISH methods have usually a very high sensitivity and enable the detection of unknown fusion as well as 3′ MLL gene deletions. Likewise, RQ-PCR assays are sensitive and quantitative, but lack multiplexing capabilities beyond two to four samples due to signal crossover and complexity when multiple fluorescent signals are used.36 Further, molecular target identification by amplicon size is insufficient and individual amplification and sequencing of FG partners are time consuming and impractical with these conventional approaches.
Designing clinically useful molecular diagnostics technologies have proven challenging because of the complexity of sample processing. However, the described method is robust and shown to be feasible in the clinical setting in a global multicenter trial (manuscript in preparation). A method to systematically screen for MLL partner genes may become increasingly important for patient stratification (as demonstrated in childhood and adult MLL-AF9 positive AML29, 37, 38), and all karyotype positive patients for MLL gene rearrangement may be screened9, 39 to identify partner genes and breakpoint location. This type of molecular information may be pivotal during patient follow-up to facilitate monitoring of minimal residual disease by RQ-PCR (ie with TaqMan® technology using patient-specific plasmid standards, and the MLL universal forward primer and probe combined to a partner specific reverse primer33). Recent work report that chromosomal translocations and their FG transcripts may be important molecular markers for leukemia and lymphoma subtyping and disease stratification.14, 16, 17, 26 These fusion proteins and/or downstream gene products are potential targets of intervention in leukemia patient treatment, and novel molecular-based therapeutic technologies for targeting chromosomal translocation products have been proposed.39 Elucidation of tumor-specific chromosomal translocations is a prerequisite for the development of targeted therapies and high-throughput technologies for identifying patient-specific chromosomal lesions may be integral to optimizing clinical use of these molecular target-based cancer therapeutics and contribute to improved care of patients with MLL rearrangements.
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We thank Dr C Bilhou Nabera (CHU, Bordeaux, France) and Dr N Dastugue (Hôpital Purpan, Toulouse, France) for providing samples, Dr D Birnbaum (IPC, Marsèille, France) for critical review of the manuscript, and Dr P Ravassard (LGN, Paris, France) for valuable discussion. We also gratefully acknowledge Ipsogen's Bioinformatics and Production Teams for their contributions. This work was partially supported by grants from PHRC97 (JG), the Agence Nationale de la Valorisation de la Recherche (ANVAR) and the Directions Régionales de l'Industrie de la Recherche et de l'Environnement (DRIRE).
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Maroc, N., Morel, A., Beillard, E. et al. A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia. Leukemia 18, 1522–1530 (2004). https://doi.org/10.1038/sj.leu.2403439
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