JMD Association for Molecular Pathology 2008 Annual Meeting
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kadkol, S. S.
Right arrow Articles by Ravandi, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kadkol, S. S.
Right arrow Articles by Ravandi, F.
JMD 2004, Vol. 6, No. 1
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

Comprehensive Analysis of CBFß-MYH11 Fusion Transcripts in Acute Myeloid Leukemia by RT-PCR Analysis

ShriHari S. Kadkol*, Annette Bruno*, Carol Dodge*, Valerie Lindgren* and Farhad Ravandi{dagger}

From the Departments of Pathology * and Medicine, {dagger} University of Illinois at Chicago, Chicago, Illinois


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CBFß-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype. Patients who express CBFß-MYH11 fusion transcripts respond favorably to high-dose chemotherapy and are generally spared allogeneic bone marrow transplantation. Hence it is important to identify this fusion in all patients with acute myeloid leukemia M4Eo leukemia. The fusion can be detected by cytogenetics, fluorescence in-situ hybridization (FISH), or by molecular analysis with RT-PCR. Multiple fusion transcripts arising as a result of various breakpoints in the CBFß and MYH11 have been identified. In this report we describe a comprehensive RT-PCR assay to identify all known fusion transcripts and provide an algorithm for molecular analysis of CBFß-MYH11 fusions from patient specimens. Further, identification of the fusion transcript by such an assay would help in the diagnosis and follow up of patients with cryptic inversion 16 translocations (such as patient 2 in this report) not detected by standard cytogenetics or FISH and for rational design of probes for quantitative analysis by real-time PCR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CBFß-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype and occur as a result of inversion 16 (p13q22) or t(16;16) chromosomal abnormalities.1, 2, 3, 4 These leukemias have a relatively favorable response to high-dose chemotherapy and such patients are generally spared allogeneic bone marrow transplantation.5, 6 Thus detection of CBFß-MYH11 fusion transcripts at diagnosis is important for making therapeutic decisions.

The fusion transcripts involve the CBFß gene on 16q22 and the MYH11 gene on 16p13. CBFß together with CBF{alpha} subunit forms the core binding factor complex. The DNA binding activity of CBF{alpha} is stabilized by dimerizing with CBFß.7 The resulting CBF{alpha}-CBFß complex has transcriptional activity and recognizes the core regulatory sequence TGTGGT, present in several cellular and viral promoters and enhancers as well as in genes expressed in hematopoietic cells.8 Three genes encode CBF{alpha} (RUNX2, RUNX1 and RUNX3), whereas a single gene codes for CBFß. CBF{alpha} expression is limited to hematopoietic system in adults whereas CBFß appears to be ubiquitously expressed.7 Knock-out and knock-in studies show that CBF{alpha} and CBFß expression is absolutely essential for normal hematopoiesis and differentiation.9, 10 MYH11 codes for the smooth muscle myosin heavy chain. The repeated coiled coil structure of the tail domain of MYH11 may result in dimerization of the CBFß-MYH11 fusion protein and lead to alterations in transcriptional regulation. The CBFß-MYH11 fusion protein is believed to bind and sequester CBF{alpha} resulting in dysfunctional transcription and leukemogenesis.7

Three different techniques are used to identify CBFß-MYH11 fusions: cytogenetics, fluorescence in-situ hybridization (FISH), and molecular analysis using RT-PCR. The breakpoints in the CBFß and MYH11 are variable and thus testing by all three methods may be necessary.8, 11 Some reports suggest that RT-PCR analysis is more sensitive than standard cytogenetic or FISH analysis to detect CBFß-MYH11 fusions.12, 13, 14 Standard cytogenetics and FISH generally have a sensitivity of 1 to 5%, depending on the number of cells examined and the type of probe used, respectively. In most cases cytogenetic and molecular analysis correlate with each other, but acute myeloid leukemia (AML) M4 Eo patients who are negative for inv(16) by standard cytogenetics but positive for fusion gene transcripts by RT-PCR have been described.12, 13, 14, 15, 16 Cytogenetic analysis is still required to obtain a complete karyotype in every patient with AML because of the well-described prognostic significance of the karyotype in this disease.17 In addition, cytogenetic analysis helps to follow evolutionary changes in karyotype that may be related to overall prognosis.

Molecular analysis by RT-PCR is a rapid and sensitive method for analyzing the expression of CBFß-MYH11 fusion transcripts. Breakpoint locations in the CBFß and MYH11 can also be determined by molecular analysis. This information might have prognostic significance, although such significance has not yet been established. There are eight known breakpoints in the MYH11 and three known breakpoints in the CBFß (Figure 1)Go . The breakpoints in the MYH11 occur at nucleotide (nt) positions 994 (Type E), 1098 (Type H), 1201 (Type D or G), 1308 (Type X), 1528 (Type C), 1708 (Type B), 1921 (Type A), and 2134 (Type S) compared with MYH11 GenBank sequence D10667.1 GI:532875.8 The breakpoints in the CBFß occur at nt positions 495 [most common], 45518 or 3998, 11, 19 compared with GenBank sequence AF294326.1 GI:9885832. The most common fusion is between nt 495 of the CBFß and nt 1921 of the MYH11 sequence. This fusion accounts for up to 88% of all CBFB-MYH11 chimeric transcripts. Fusions between nt 495 of the CBFß sequence and nt 994 or 1201 of the MYH11 sequence account for about 10% of cases. The remaining breakpoints account for less than 2% of cases. However, one can never predict a particular breakpoint prospectively and hence there is a need for a comprehensive molecular assay to detect all known fusions from clinical specimens in a systematic manner. In addition, alternative splicing of CBFß-MYH11 fusion transcripts has been reported.4 Further, we are unaware of any published report of the presence of CBFß-MYH11 fusion transcripts in blood from healthy individuals by standard RT-PCR assays.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Primer locations in relation to known breakpoints in the CBFß and MYH11 cDNA.

 
To the best of our knowledge, published reports to date have used only a few primer sets to identify the breakpoints and fusions resulting from them. False-negative results can occur if the breakpoints lie outside the ability of detection with a particular primer set. The problem can be overcome by using multiple primer sets to target all of the known breakpoints in the two genes. A disadvantage to using multiple primer sets is the need to run multiple reactions on every sample, which may be difficult if the amount of sample RNA is limiting. Multiplexing different primers in the same reaction is an option, but the amplification products obtained by multiplexing primer sets may generate a complicated pattern of differently sized products that may be hard to interpret. Also, RNA degradation, a potential problem in clinical samples, will require that the amplicon sizes be short to avoid false negatives. In this report, we propose an optimized algorithm that uses multiple primer pairs in different reactions in a stepwise manner to detect all known breakpoints in both CBFß and MYH11. We further illustrate the utility of such an algorithm to detect CBFß-MYH11 fusion transcripts in two patients with AML M4Eo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples and RNA Extraction
RNA was extracted from blood or bone marrow samples and from the ME-1f2 cell line with the RNeasy Mini kit according to the manufacturer’s directions (Qiagen, Valencia, CA). Contaminating genomic DNA was eliminated by on-column digestion with DNase I (Qiagen). The ME-1f2 cell line was a kind gift from Dr. Ikulya Sakai, First Department of Internal Medicine, Ehime University, Ehime, Japan.20 ME-1f2 was maintained in RPMI 1640 with 10% fetal calf serum (FCS) and antibiotics in a humidified atmosphere containing 5% CO2. This cell line is a subline of ME-1, which was established from a patient with AML M4Eo. ME-1f2 harbors the inversion 16 chromosomal abnormality and expresses Type A CBFß-MYH11 fusion transcripts.

Primer Design
Primers were designed using GenBank sequences D10667.1 GI:532875 (CBFß) and AF294326.1 GI:9885832 (MYH11) as templates and were optimized using Oligo Version 6.0 software (Molecular Biology Insights, Inc., Cascade, CO). The primer sequences are listed in Table 1Go . The locations of the primers in relation to the breakpoints in CBFß and MYH11 are shown in Figure 1Go .


View this table:
[in this window]
[in a new window]
  		Table 1. Primer Sequences

 
RT-PCR Analysis
Molecular analysis was performed by reverse transcription followed by nested PCR amplification. All amplifications were performed in a MJ Research PTC 200 DNA Engine thermocycler (MJ Research, Watertown, MA). For the first-round reaction, 1 µg of RNA was reverse transcribed and amplified using the One Step RT-PCR kit (Qiagen). Each 50 µl reaction contained 1X RT-PCR buffer with 2.5 mmol/L MgCl2, 0.4 mmol/L of each dNTP, 30 pmols of forward and reverse primers, 1X Q solution, 10U RNase inhibitor (Promega, WI), and 2 µl of an enzyme mix that contained reverse transcriptase and HotStarTaq DNA polymerase. Reverse transcription was at 48° for 1 hour, followed by 95° for 15 minutes to inactivate reverse transcriptase and simultaneously activate HotStarTaq DNA polymerase. This was followed by 40 cycles of PCR amplification. Each cycle was 92° for 30 seconds, 61° for 30 seconds, and 72° for 35 seconds. A final extension step was performed at 72° for 10 minutes.

Nested PCR amplification was performed with the HotStarTaq DNA polymerase kit (Qiagen). Each 50 µl reaction contained 1X PCR buffer containing 1.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP (Sigma, St. Louis, MO), 20 pmols of forward and reverse primers and 0.5 µl (2.5U) of HotStarTaq DNA polymerase enzyme. One µl of a 1:10 dilution of the first-round product was used as template. Each reaction was heated at 95° for 15 minutes to activate the HotStarTaq enzyme and to denature the template and this step was followed by 33 cycles of PCR amplification. Each cycle was 92° for 30 seconds, 64° for 28 seconds, and 72° for 30 seconds. A final extension step was at 72° for 10 minutes.

Gel Electrophoresis
Ten µl of the nested PCR product was electrophoresed on a 2% agarose gel (E-Gel, Invitrogen, Carlsbad, CA). The gel was photographed under UV transillumination with the AlphaImager system (AlphaInnotech, San Leandro, CA).

Cloning and Sequencing
Nested PCR products were cloned into pCRII TOPO vector (Invitrogen) for good quality sequencing results and to generate positive-control plasmids and RNA for further use in the laboratory. TOP10 cells were transformed and plated on to LB/amp plates with blue/white screening. Representative clones were sequenced by the DNA Sequencing Core Facility at the University of Illinois at Chicago. Sequence analysis was performed with the BLAST program (www.ncbi.nih.gov).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT-PCR Analysis
The algorithm in FigureGo 2was applied to RNA samples from two patients with AML M4Eo and the cell line ME1-f2. In the initial step, first-round amplification was performed with primers 1/2 followed by nested reactions with primers 3/4, 3/11, or 3/12. Patient 1 showed products of 892 bp, 1134 bp, and 504 bp with nested primers 3/4, 3/11, or 3/12, respectively (Figure 3AGo 3BGo 3CGo , lane 1). Patient 2 and ME-1f2 cells showed 268-bp and 510-bp products with nested primers 3/4 (Figure 3AGo , lanes 2 and 3) and 3/11, respectively (Figure 3BGo , lanes 2 and 3). No product was obtained from patient 2 and ME1-f2 cells when the nested reaction was performed with primers 3/12 (Figure 3CGo , lanes 2 and 3).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. First-round RT-PCR with primers 1/2 followed by nested PCR with primers 3/4 (A), 3/11 (B) or 3/12 (C). M-1Kb Plus molecular weight marker (Invitrogen). Lane 1, patient 1; lane 2, patient 2; lane 3, ME-1f2 cells; lane 4, HL-60; lane 5, water control.

 
In the next step, a separate first-round amplification was performed with primers 5/6 followed by nested reactions with primers 7/8 or 7/9. Patient 1 showed a 197-bp product with nested primers 7/8 (Figure 4AGo , lane 1). No product was obtained from patient 1 with primers 7/9 (Figure 4BGo , lane 1). Also, no products were obtained from patient 2 and ME-1f2 cells with primers 7/8 or 7/9 (Figure 4Go , A and B, lanes 2 and 3).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. First-round RT-PCR with primers 5/6 followed by nested PCR with primers 7/8 (A) or 7/9 (B). M-1Kb Plus molecular weight marker (Invitrogen). A: Lane 1, patient 1; lane 2, patient 2; lane 3, ME-1f2 cells; lane 4, HL-60; lane 5, water control. B: Lane 1, patient 1; lane 2, patient 2; lane 3, ME-1f2 cells; lane 4, water.

 
RNA from HL-60 cells did not amplify with any primer set. Unidirectional workflow in the laboratory was strictly followed and great care was taken to prevent contamination. Water-only reactions did not show any amplification products, diminishing the likelihood of cross-contamination. Further, peripheral blood or bone marrow-derived RNA from 32 cases of unrelated leukemia and 20 healthy individuals did not show amplification products with any of the primer sets.

Two previous RNA samples from patient 1 (Figure 5AGo , lanes 1 and 2) did not show any specific amplification product with primers 1/2 followed by a nested reaction with primers 3/4. A few non-specific products of incorrect size were seen in the sample analyzed in lane 1. However, the same two RNA samples amplified with primers 5/6 followed by a nested reaction with primers 7/8 (Figure 5BGo , lanes 1 and 2). The presence of amplifiable RNA was confirmed by prior analysis of tyrosine kinase mRNA (ABL) expression. The ABL primers amplified a product of 315 bp (data not shown) in the two samples. Sequencing results suggested that partial RNA degradation in the previous samples from patient 1 was the likely cause of amplification failure with primer sets 1/2 and 3/4. In this patient, first-round primers 1/2 are expected to amplify a 1327-bp product in contrast to a 498-bp product from primers 5/6. Thus if RNA degradation reduced the size of intact transcripts, primers 5/6 would have had a better chance of generating an amplified product compared to primers 1/2. Partial RNA degradation would also explain the positive ABL result (315-bp product) and a negative result with primer sets 1/2 and 3/4. Hence false-negative results occurred with primers 1/2 and 3/4 in the two previous samples from patient 1. These samples were in fact positive for fusion transcripts as detected by primers 5/6 and 7/8. The built-in redundancy in the assay assures against false-negative results and represents a significant advantage of this analysis protocol.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 5. Effect of RNA degradation on amplification. A: Analysis with primers 1/2 followed by primers 3/4: lanes 1 and 2, two previous RNA samples from patient 1; lane 3, most recent sample from patient 1 (same as in lane 1, Figure 3Go A); lane 4, ME-1f2 cells; lane 5, HL-60; lanes 6 and 7, water controls. B: Analysis with primers 5/6 followed by primers 7/8. Lanes 1 and 2, two previous samples from patient 1; lane 3, most recent sample from patient 1 (same as lane 1, Figure 4AGo ); lane 4, water control.

 
Sequence Analysis
The pattern of bands in patient 1 suggested a proximal 5' breakpoint in MYH11. The 197-bp product (generated by primers 5/6 and 7/8) that was sequenced showed that the breakpoint in CBFß was at nt 399 and the breakpoint in MYH11 was at nt 1201. This represents a "Type D" breakpoint in MYH11 (reference 11 and Figure 1Go ). Individual breakpoints at nt 399 in CBFß and at nt 1201 in MYH11 have been reported before.8, 11, 17 However, to the best of our knowledge, there is only one previously published report of a single case of a fusion between nt 399 of CBFß and nt 1201 of MYH11.8 Patient 1 in our report is thus the second case of CBFß-MYH11 fusion occurring as a result of a breakpoint at nt 399 in CBFß and nt 1201 in MYH11. Sequence analysis of the 268-bp product from patient 2 and ME-1f2 cells (generated by primers 1/2 and 3/4) showed that the breakpoint in CBFß was at nt 495 and in MYH11 was at nt 1921. This represents a typical "Type A" fusion transcript that is seen in almost 85 to 88% of cases with CBFß-MYH11 fusions. Interestingly, cytogenetic and FISH analysis failed to detect inversion 16 or t(16;16) inpatient 2 where as ME-1f2 cells and leukemic cells from patient 1 showed the inversion 16 abnormality by cytogenetics. Thus, the only evidence of CBFß-MYH11 fusion inpatient 2 was from molecular analysis by RT-PCR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we describe a comprehensive assay to identify CBFß-MYH11 fusion transcripts. The assay utilizes two first-round RT-PCR reactions and five nested reactions and can be completed within 24 hours after RNA extraction. Because only two first-round RT-PCR reactions are performed, the assay can be performed with as little as 2 µg of total RNA (1 µg/reaction). Our assay utilizes a nested format with a larger number of primer sets and has distinct advantages over previously published reports.8, 11 First, the primer sets cover all currently known breakpoints in the CBFß and MYH11 genes.11 Expected product sizes generated with the primer sets from all potential fusions are shown in Table 2Go . Hence, accurate identification of the breakpoints in CBFß and MYH11 genes is possible. The protocol does use multiple primer sets in separate reactions, making the assay seem a bit tedious. It is certainly possible to assay the most common fusion transcript first (Type A, primers 1/2, and nested primers 3/11) and then use the other primer sets if the patient is negative for Type A transcripts. However, the purpose of this report is to provide a comprehensive assay to detect all known fusion transcripts arising from multiple breakpoints in AML M4 Eo cases. The strength of the comprehensive format is that more than one nested reaction performed concurrently produces a product from the same fusion transcript (albeit of a different size, Table 2Go ). This approach significantly improves specificity and sensitivity and confers a high level of confidence in interpreting results without the need for further confirmation by probe hybridization and detection. Such an approach also improves turn-around times for clinical samples because all reactions are performed simultaneously. Multiplexing different primer pairs was considered but such an approach resulted in primer-dimer formation and decreased the efficiency of specific amplification.


View this table:
[in this window]
[in a new window]
  		Table 2. Expected Amplicon Sizes Generated by the Respective Primer Pairs*

 
Second, the product sizes generated from the primer sets are such that reasonably preserved RNA can still be analyzed and false-negative results can be avoided. This was illustrated by results obtained from two previous samples from patient 1 (Figure 5)Go . RNA can degrade if sufficient care is not taken to preserve RNA integrity. The two previous samples from patient 1 were negative for fusion transcripts when analyzed with primers 1/2 and 3/4. However the same samples were positive when re-analyzed with primers 5/6 and 7/8. These results suggest that RNA degradation led to false-negative results with primers 1/2 and 3/4 as discussed in the Results section. Thus the assay is capable of analyzing accurately, even suboptimal RNA samples, although completely degraded RNA would be unsuitable for any analysis. In our opinion, the built-in redundancy of the assay represents a distinct advantage.

Third, some previous reports have used post-PCR steps such as oligonucleotide hybridization after a single round of RT-PCR to confirm identity of the amplicons.8 The algorithm presented in this report utilizes more than one set of nested amplification reactions to identify fusion transcripts in every case thereby ensuring a high degree of specificity, obviating the routine need for confirmation by additional probe hybridization. The assay can reliably detect CBFß-MYH11 fusion transcripts in as little as 10 pg of ME-1f2 cells when diluted into 1 µg of negative HL-60 RNA and hence can be used to monitor minimal residual disease qualitatively. This sensitivity was achieved only with a nested protocol. The sensitivity of a single round of amplification was about 100-fold lower than the nested protocol. Thus the protocol ensures adequate specificity and increased sensitivity without the need for post-PCR confirmatory testing. However, sequence confirmation may still be necessary when amplicons of unexpected sizes are found either from fusion transcripts arising from hitherto undescribed breakpoints or by alternative splicing. More extensive studies are necessary before the frequency of the need for sequencing can be determined.

Although only two cases were presented in this report, we believe that the assay is robust enough to comprehensively identify all known CBFß-MYH11 fusion transcripts. Further, in patient 2, routine cytogenetic analysis and FISH failed to reveal the inversion 16 abnormality and molecular analysis by RT-PCR was the only method that detected CBFß-MYH11 fusion transcripts.16 Such cases have been reported previously and probably arise from small insertions that are below the detection ability of standard cytogenetics and FISH.12, 13, 14, 15 We have also not observed any false-positive results with unrelated RNA specimens from patients with leukemias other than AML M4Eo.

Although the protocol outlined in this report can be used for qualitative detection of minimal residual disease in patients with AML and inv(16), quantitative analysis of fusion transcripts is necessary for determining the kinetics of minimal residual disease.21 However, before quantitative analysis can be performed, it is critical to determine the type of fusion transcript by qualitative analysis so that appropriate primers and probes can be chosen for quantification. This is necessary because of length restrictions for reliable amplification by real-time PCR systems. For example, primers and probes for the detection and quantification of the Type A breakpoint in MYH11 would be inappropriate for the detection of the Type E or D breakpoints due to length restrictions. Similarly primers and probes optimized for Type E or D breakpoints based on short amplicon sizes (less than 250 bp) would be inappropriate to detect the Type A breakpoint. Hence, we believe that qualitative analysis with a comprehensive assay as the one reported herein should be the first step in the analysis of any patient suspected of having CBFß-MYH11 fusion bearing leukemia. This information will assist in the rational choice of the appropriate primer-probe combinations for quantitative analysis by real time PCR and also help to determine the prognostic significance of various fusion transcripts in different patients.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 2. Algorithm for the comprehensive analysis of CBFß-MYH11 fusion transcripts.

 

   Acknowledgments
 
We thank Dr. Guiseppina Nucifora for her comments and suggestions.


   Footnotes
 
Address reprint requests to ShriHari S. Kadkol, M.D., Ph.D., Assistant Professor of Pathology, Department of Pathology (MC 847), University of Illinois at Chicago, Room 446 CMW, 1819 W. Polk Street, Chicago, IL 60612. E-mail: skadkol{at}uic.edu

Accepted for publication August 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Poirel H, Radford-Weiss I, Rack K, Troussard X, Veil A, Valensi F, Picard F, Guesnu M, Leboeuf D, Melle J, Dreyfus F, Flandrin G, Macintyre E: Detection of the chromosome 16 CBF ß-MYH11 fusion transcript in myelomonocytic leukemias. Blood 1995, 85:1313-1322[Abstract/Free Full Text]
  2. Claxton DF, Liu P, Hsu HB, Marlton P, Hester J, Collins F, Deisseroth AB, Rowley JD, Siciliano MJ: Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia. Blood 1994, 83:1750-1756[Abstract/Free Full Text]
  3. Tobal K, Johnson PR, Saunders MJ, Harrison CJ, Liu Yin JA: Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission. Br J Haematol 1995, 91:104-108[Medline]
  4. van der Reijden BA, Lombardo M, Dauwerse HG, Giles RH, Muhlematter D, Bellomo MJ, Wessels HW, Beverstock GC, van Ommen GJ, Hagemeijer A, Breuning MH: RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts. Blood 1995, 86:277-282[Abstract/Free Full Text]
  5. Braess J, Jahns-Streubel G, Schoch C, Haase D, Haferlach T, Fiegl M, Voss S, Kern W, Schleyer E, Hiddemann W: Proliferative activity of leukaemic blasts and cytosine arabinoside pharmacodynamics are associated with cytogenetically defined prognostic subgroups in acute myeloid leukaemia. Br J Haematol 2001, 113:975-982[Medline]
  6. Bloomfield CD, Lawrence D, Byrd JC, Carroll A, Pettenati MJ, Tantravahi R, Patil SR, Davey FR, Berg DT, Schiffer CA, Arthur DC, Mayer RJ: Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype. Cancer Res 1998, 58:4173-4179[Abstract/Free Full Text]
  7. Hart SS, Foroni L: Core binding factor genes and human leukemia. Haematologica 2002, 87:1307-1323[Abstract/Free Full Text]
  8. Shurtleff SA, Meyers S, Hiebert SW, Raimondi SC, Head DR, Willman CL, Wolman S, Slovak ML, Carroll AJ, Behm F, Hulshof MG, Motroni TA, Okuda T, Liu P, Collins FS, Downing JR: Heterogeneity in CBF ß/MYH11 fusion messages encoded by the inv(16)(p13q22) and the t(16;16)(p13;q22) in acute myelogenous leukemia. Blood 1995, 85:3695-3703[Abstract/Free Full Text]
  9. Kundu M, Javed A, Jeon JP, Horner A, Shum L, Eckhaus M, Muenke M, Lian JB, Yang Y, Nuckolls GH, Stein GS, Liu PP: Cbfß interacts with Runx2 and has a critical role in bone development. Nat Genet 2002, 32:639-644[Medline]
  10. Miller JD, Stacy T, Liu PP, Speck NA: Core-binding factor ß (CBFß), but not CBFß-smooth muscle myosin heavy chain, rescues definitive hematopoiesis in CBFß-deficient embryonic stem cells. Blood 2001, 97:2248-2256[Abstract/Free Full Text]
  11. van der Reijden BA, de Wit L, van der Poel S, Luiten EB, Lafage-Pochitaloff M, Dastugue N, Gabert J, Lowenberg B, Jansen JH: Identification of a novel CBFB-MYH11 transcript: implications for RT-PCR diagnosis. Hematol J 2001, 2:206-209[Medline]
  12. Langabeer SE, Walker H, Gale RE, Wheatley K, Burnett AK, Goldstone AH, Linch DC: Frequency of CBF ß/MYH11 fusion transcripts in patients entered into the UK MRC AML trials: The MRC Adult Leukaemia Working Party. Br J Haematol 1997, 96:736-739[Medline]
  13. Ritter M, Thiede C, Schakel U, Schmidt M, Alpen B, Pascheberg U, Mohr B, Ehninger G, Neubauer A: Underestimation of inversion (16) in acute myeloid leukaemia using standard cytogenetics as compared with polymerase chain reaction: results of a prospective investigation. Br J Haematol 1997, 98:969-972[Medline]
  14. Rowe D, Cotterill SJ, Ross FM, Bunyan DJ, Vickers SJ, Bryon J, McMullan DJ, Griffiths MJ, Reilly JT, Vandenberghe EA, Wilson G, Watmore AE, Bown NP: Cytogenetically cryptic AML1-ETO and CBF ß-MYH11 gene rearrangements: incidence in 412 cases of acute myeloid leukaemia. Br J Haematol 2000, 111:1051-1056[Medline]
  15. Harth H, Pees H, Zankl H: Acute myelomonocytic leukemia (M4) with eosinophilia: problems concerning chromosome 16 abnormality. Cancer Genet Cytogenet 1986, 23:127-133[Medline]
  16. Ravandi F, Kadkol SS, Ridgeway J, Bruno A, Dodge C, Lindgren V: Molecular identification of CBFß-MYH11 fusion transcripts in an AML M4Eo patient in the absence of inv16 or other abnormality by cytogenetic and FISH analyses: a rare occurrence. Leukemia 2003, 17:1907-1910[Medline]
  17. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A: The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998, 92:2322-2333[Abstract/Free Full Text]
  18. Mitterbauer M, Laczika K, Noval M, Mitterbauer G, Hilgarth B, Pirc-Danoewinata H, Schwarzinger I, Haas OA, Fonatsch C, Lechner K, Jaeger U: High concordance of karyotype analysis and RT-PCR for CBF ß/MYH11 in unselected patients with acute myeloid leukemia. Am J Pathol 2000, 113:406-410
  19. Grardel N, Roumier C, Soenen V, Lai JL, Plantier I, Gheveart C, Cosson A, Fenaux P, Preudhomme C: Acute myeloblastic leukemia (AML) with inv (16)(p13;q22) and the rare I type CBFß-MYH11 transcript: report of two new cases. Leukemia 2002, 16:150-151[Medline]
  20. Yanagisawa K, Horiuchi T, Fujita S: Establishment and characterization of a new human leukemia cell line derived from M4E0. Blood 1991, 78:451-457[Abstract/Free Full Text]
  21. Marcucci G, Caligiuri MA, Dohner H, Archer KJ, Schlenk RF, Dohner K, Maghraby EA, Bloomfield CD: Quantification of CBFß/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 2001, 15:1072-1080[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kadkol, S. S.
Right arrow Articles by Ravandi, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kadkol, S. S.
Right arrow Articles by Ravandi, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS