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© 2001 American Society for Clinical Oncology Comparison of Cytogenetic and Molecular Genetic Detection of t(8;21) and inv(16) in a Prospective Series of Adults With De Novo Acute Myeloid Leukemia: A Cancer and Leukemia Group B StudyFrom the Division of Hematology and Oncology, Comprehensive Cancer Center, and Department of Pathology, Ohio State University, Columbus, OH; University of Alabama at Birmingham, Birmingham, AL; North Shore University Hospital, Manhasset, NY; Wake Forest University Medical Center, Winston-Salem, NC; and Department of Pathology, University of Chicago, Chicago, IL. Address reprint requests to Krzysztof Mrózek, MD, PhD, Division of Hematology and Oncology and the Comprehensive Cancer Center, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Room 1248B, Ohio State University, 300 West 10th Ave, Columbus, OH 43210-1228; email: mrozek-1{at}medctr.osu.edu
PURPOSE: To prospectively compare cytogenetics and reverse transcriptase–polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFß/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFß/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P = .83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFß/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR–positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFß/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFß/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.
ACUTE MYELOID leukemia (AML) is a heterogeneous disease with regard to the morphology, immunophenotype, and genetic rearrangements acquired by leukemic blasts. Multiple recurrent chromosome and gene rearrangements have been identified in AML, and these alterations have been correlated with biologic and clinical features of the disease resulting in delineation of prognostically distinct categories of AML.1-6 One such category is core-binding factor (CBF) AML. Leukemic cells of patients with CBF AML most commonly contain either t(8;21)(q22;q22) or inv(16)(p13q22), chromosome aberrations that result in disruption of genes encoding CBF subunits, CBF  (also known as AML1) or CBFß, respectively.1,6 Translocation (8;21) leads to the fusion of the AML1 gene, located at 21q22, with the ETO gene at 8q22 and creation of a chimeric gene AML1/ETO. Similarly, a fusion gene CBFß/MYH11 is produced by juxtaposition of bands 16q22 (containing CBFß) and 16p13 (containing MYH11) as a result of inv(16) or, less frequently, t(16;16)(p13;q22).1,6 Patients with CBF AML constitute approximately 15% to 20% of adults younger than 60 years with de novo AML. It has been demonstrated that the prognosis of these patients is significantly better than that of patients with AML with other chromosome aberrations or a normal karyotype. This is especially true for patients with CBF AML who receive intensive postremission treatment with high-dose cytarabine.7 Consequently, in the Cancer and Leukemia Group B (CALGB) 9621 treatment protocol, the consolidation therapy of adults with AML has been administered in a risk-adapted fashion. Patients with CBF AML receive three courses of high-dose cytarabine, whereas patients with AML negative for the presence of t(8;21), inv(16) or their molecular equivalents receive autologous peripheral stem cell transplantation. Therefore, for both CALGB 9621 and similar ongoing clinical studies, the accurate identification of all patients with CBF AML is of utmost importance. Patients with CBF AML can be identified by means of standard cytogenetic analysis that can reveal t(8;21) or inv(16) in metaphase cells. The t(8;21) is relatively easy to detect, even if the quality of chromosome preparations is suboptimal. In contrast, inv(16) is a subtle rearrangement that can sometimes be overlooked by less experienced cytogeneticists, especially in preparations of suboptimal quality.8 Occasionally, an inv(16) may be misinterpreted as del(16)(q22).9 Another method of identifying CBF AML is a reverse transcriptase–polymerase chain reaction (RT-PCR). The fusion gene AML1/ETO produces a transcript that is consistently detected by means of RT-PCR.10,11 Although the breakpoints within both the MYH11 and CBFß genes are more variable, resulting in the creation of several molecular variants of the CBFß/MYH11 chimeric gene, RT-PCR detects most patients with the CBFß/MYH11 fusion.12-14 In rare patients, however, RT-PCR results are negative despite the presence of microscopically detectable inv(16) and CBFß rearrangement, as shown by Southern blot analysis.12,13 It has nevertheless been suggested that RT-PCR is more sensitive than cytogenetic analysis in detecting CBF AML because the respective fusion genes have been discovered in patients with AML without visible t(8;21)15-19 or inv(16)/t(16;16).18,20-23 Two recent studies from the Medical Research Council (MRC) Adult Leukaemia Working Party reported a high incidence, 37% and 36%, of patients with AML who were positive for AML1/ETO or CBFß/MYH11, respectively, but in whom t(8;21) or inv(16)/t(16;16) was not found by cytogenetic analysis.16,21 In other studies, however, the frequency of such patients was markedly lower.17,18,20,23 To date, most of the studies correlating cytogenetic and molecular genetic results have been performed retrospectively.15-17,20,21,23 To our knowledge, only two prospective series, comprising 141 and 121 patients with de novo AML, respectively, have been published.18,22 In the present study, we compared the results of cytogenetic analysis with the results of an RT-PCR assay for detection of t(8;21), inv(16) and their variants in what is to our knowledge the largest prospective series of 284 adults diagnosed with de novo AML to date.
Patients Two hundred eighty-nine consecutive patients diagnosed with de novo AML were enrolled on the CALGB 9621 treatment trial between April 1, 1997, and July 12, 1999. The CALGB Statistical Center managed patient registration. Eligibility criteria for entry on CALGB 9621 included the following: diagnosis of de novo AML of the French-American-British subtype M0-M7, excluding AML M3; no history of myelodysplasia or other antecedent hematologic malignancy; aged 15 years or more, but younger than 60 years; and no previous treatment for leukemia or another malignancy. Leukemias were classified morphologically according to the French-American-British Cooperative Group criteria after assessment of Wright’s stained bone marrow, blood smears, or both.24 Initial central review of slides was performed in the Department of Pathology of the University of Chicago Medical Center, Chicago, IL. There were 17 patients with AML M0, 42 with AML M1, 83 with AML M2, three with AML M2Eo, 56 with AML M4, 28 with AML M4Eo, five with AML M5a, 20 with AML M5b, five with AML M6, and 14 with AML not otherwise specified. Central review data were not available for 11 patients; their institutional morphologic diagnoses were as follows: AML M0 in one patient, AML M2 in four, AML M4 in three, AML M6 in one, and unclassifiable AML in two. In five patients, the initial diagnosis of AML was revised on central review to acute lymphoblastic leukemia L3 in one patient; to myelodysplastic syndrome in one patient; to AML M3 in two patients; and at the local institution to myelodysplastic syndrome in one patient. These patients were excluded from the study. Thus, our analysis has been performed on 284 patients with AML, 135 men and 149 women, with a median age of 43 years (range, 17 to 59 years). Informed consent was obtained from all patients according to the guidelines set forth by institutions participating in the study.
Cytogenetic Studies
Molecular Analyses The PCR reactions were performed by use of 75 ng each of the appropriate sense and antisense primers in a 50-µL reaction mixture containing 1 U Taq polymerase (Applied Biosystems Inc., Foster City, CA), 0.5 mmol/L of each deoxynucleotide triphosphate 3 mmol/L MgCl2, and 5 µL 10x PCR buffer (670 mmol/L Tris, 100 mmol/L ß-mercaptoethanol, 166 mmol/L ammonium sulfate, 67 mmol/L EDTA, and 0.5 mg/mL bovine serum albumin). PCR cycling consisted of an initial denaturation step at 94°C for 3 minutes, then 44 cycles at 95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. Two microliters of cDNA (equivalent to 0.2 µg of total RNA) were amplified in a 50-µL reaction mixture. Twenty microliters of PCR product were mixed with 4.0 µL of 5x gel loading buffer and electrophoresed on an agarose gel for 1.7 hours at 100 V. The gel was stained in a solution of 1x Tris-acetate/EDTA electrophoresis buffer containing 1 µg/mL ethidium bromide and photographed under ultraviolet light. The 166–base pair (bp) t(8;21) fusion product and the 416-bp inv(16) fusion product were confirmed by an ApaI restriction digest yielding 85 + 81 bp and 251 + 165–bp fragments, respectively. Eight microliters of product were digested in 1 µL of 10x digestion buffer and 1 µL ApaI (10 U/µL; New England Biolabs, Beverly, MA). Two microliters of 5x loading buffer were added to digestion and loaded on a 12% polyacrylamide gel (10 cm). An uncut control (8 µL + 2 µL 5x loading buffer) was loaded for each lane. The gel was electrophoresed for 1 hour at 175 V and stained and photographed as described above. The transcript of the hypoxanthine phosphoribosyl transferase (HPRT) gene was used as an RNA control. Primers for amplification of HPRT (HPRT F 5'-TGTAATGACCAGTCAACAGG-3'; HPRT R1 5'-ATTGACTGCTTCTTACTTTTCT-3') yielded a 450-bp product. Standard precautions were undertaken to eliminate the risk of any sample or product contamination. The CBFß/MYH11 fusion transcript RT-PCR products that by size and restriction analysis were not indicative of type A were purified by the Exonuclease I/shrimp alkaline phosphatase PCR product sequencing kit (USB-Amersham Life Science, Piscataway, NJ) according to the manufacturer’s directions. After purification, 2 µL of the PCR products were sequenced by the BigDye Terminator Amplitaq FS Cycle Sequencing kit (ABI, Foster City, CA). In 10 of 13 patients with apparent discrepancy between results of cytogenetic and molecular studies ( Tables 1 and 2) and in two of three patients with initial AML M4Eo but without inv(16)/t(16;16) or CBFß/MYH11 fusion, additional tests were carried out. These included another "standard" RT-PCR and real-time quantitative RT-PCR29-31 assays performed on RNA isolated from another vial of diagnostic bone marrow sample in patient nos. 5, 6, 9, 10, 11, 12, and 13 ( Tables 1 and 2) and in the two inv(16)/t(16;16)/CBFß/MYH11-negative patients with AML M4Eo; metaphase fluorescence in situ hybridization (FISH) studies detecting inv(16) in patient nos. 8, 9, and 12; and FISH analysis that used painting probes of chromosomes 8 and 21 in patient no. 3. Additionally, multicolor spectral karyotyping, a FISH-based technique that enables simultaneous display of all human chromosomes in different colors,32 was used to confirm a suspected ins(8;21)(q22;q22q22) in patient no. 4.
Statistical Analysis To assess the overall ability of cytogenetic and RT-PCR analyses to accurately classify patients as having CBF AML or not, we calculated the proportions of all patients studied by initial cytogenetic and RT-PCR assays, separately, that were correctly classified. We considered a patient to be incorrectly classified when a given technique produced either a false-negative or false-positive result or if a sample was processed but no result was obtained. A comparison of the overall abilities of the two techniques to accurately classify the patients was performed by a z test to compare two proportions.33 We also assessed the respective abilities of cytogenetic and RT-PCR analyses to detect the presence or absence of inv(16)/t(16;16) and, separately, t(8;21), in patients with AML by calculating their predictive value positive (PVP) and predictive value negative (PVN) and associated 95% confidence intervals. The PVP is the probability that a patient is truly positive given a positive test result; the PVN is the probability that a patient is truly negative given a negative test result.34 The related statistics, sensitivity and specificity, were also examined. Sensitivity is the probability that a test will be positive, given that the abnormality is truly present, whereas specificity is the probability that a test will be negative, given that the abnormality is truly absent.34 The PVP, PVN, sensitivity, and specificity calculations were performed on data from 271 patients with both cytogenetic and molecular results. Additionally, the PVP, PVN, sensitivity, specificity, and associated 95% confidence intervals were calculated to determine how well the initial morphologic diagnosis of AML M4Eo can predict the presence of inv(16)/t(16;16). This analysis was performed on data from 271 patients for whom both central morphologic review results and the inv(16)/CBFß/MYH11 status were available.
Cytogenetic Studies Cytogenetic analyses yielded analyzable metaphase cells in 276 (98%) of 282 patients. Two patients initially considered to have a chromosome abnormality were found on central review to have a normal karyotype, and the karyotype of another patient, submitted as normal, was found to contain a clonal abnormality. None of these three patients was CBFß/MYH11- or AML1/ETO-positive. Consequently, of the 276 patients with a cytogenetic result, 125 (45%) had a normal and 151 (55%) had an abnormal karyotype.
RT-PCR Analysis AML1/ETO fusion transcripts were detected in all 17 patients with a standard t(8;21), as well as in a patient with a complex variant translocation t(8;20;21)(q22;p11.2;q22) ( Fig 1). AML1/ETO was also found in a patient with t(8;10)(q22;q26) and in a patient with t(1;10;8)(p22;p13;q22) and del(21)(q22), thus indicating that these aberrations constituted variants of t(8;21) (patient nos. 2 and 3 in Table 1, respectively; Fig 1). In addition, a patient with a suspected ins(8;21)(q22;q22q22) (patient no. 4, Fig 1), was AML1/ETO positive. The presence of an insertion of a segment from chromosome 22q22 into band 8q22 was confirmed by multicolor spectral karyotyping (data not shown). Interestingly, loss of a sex chromosome, which is the most common secondary aberration in patients with AML with t(8;21),2 was observed in three of four patients with variants of t(8;21) (patient nos. 1, 2, and 4). Ultimately, there were 21 patients with CBF AML with t(8;21) or its variant who were positive for AML1/ETO. The AML1/ETO fusion transcript was not detected in any of the 124 patients studied successfully by RT-PCR who were reported originally to have a normal karyotype, in the two patients whose karyotypes were revised from abnormal to normal, in any of the four patients with del(9q) but without a microscopically detectable t(8;21), or in any of the 120 remaining patients with other chromosome abnormalities.
In two patients, one with trisomy 8 as the sole chromosome change and another whose bone marrow sample was not submitted for cytogenetic analysis (patient nos. 5 and 6 in Table 1, respectively), the initial RT-PCR assays were positive for AML1/ETO transcript. However, subsequent standard and real-time quantitative RT-PCR of different vials of diagnostic samples were negative in both patients, indicating that the initial molecular results were false. The expression of CBFß/MYH11 chimeric mRNA was found initially in 22 (96%) of 23 patients in whom inv(16) or t(16;16) was originally identified by the submitting cytogenetic laboratory. In addition, CBFß/MYH11 was detected in one patient whose cytogenetic test failed to yield metaphase cells and in two patients initially reported to have an abnormal karyotype but no inv(16)/t(16;16). One of these patients (patient no. 7 in Table 2) was described as having an isolated trisomy 22 that is known to constitute a nonrandom secondary aberration accompanying inv(16).2,8 Indeed, reexamination of his karyotype confirmed the presence of inv(16). In the second patient (patient no. 8), the original karyotype description also included trisomy 22 together with del(16)(q22). Subsequent analysis of microscope slides and metaphase FISH analysis, performed after CBFß/MYH11 had been detected, also resulted in revision of the karyotype, this time to 47,XX,t(16;16)(p13;q22),+22. One patient (patient no. 9 in Table 2) with microscopically typical inv(16) was found to be negative by initial analysis by RT-PCR. Metaphase FISH was performed on samples from this patient, and FISH confirmed the presence of inv(16) (data not shown). The patient had marrow morphology of AML M4Eo. Repeated standard and real-time quantitative RT-PCR analyses of a sample in another vial from the diagnostic sample were both positive for CBFß/MYH11. All but three patients with inv(16)/t(16;16), CBFß/MYH11 transcript, or both were found to have AML of M4Eo type on initial central morphologic review. The three remaining patients were diagnosed with AML M4, M5b, and AML, not otherwise specified, respectively. On rereview of the slides from the patients with M4 and M5b, performed by J.W.V. with the knowledge of cytogenetic and molecular results, abnormal eosinophils were found, and the morphologic diagnosis changed to M4Eo. In addition, M4Eo marrow morphology was also found in three patients who were initially negative for inv(16)/t(16;16)/CBFß/MYH11: one with t(8;21)/AML1/ETO; one with the karyotype 47,XY,+10/47,idem,del(9)(q13q22), and one with a normal karyotype (patient no. 10 in Table 2). Reevaluation of the slides resulted in revision of the morphologic diagnosis to M2 with monocytic component with features of t(8;21) in the t(8;21)/AML1/ETO-positive patient and to atypical M2 with monocytic component in the patient with trisomy 10 and del(9), who was also found to be negative for CBFß/MYH11 on repeated standard and real-time quantitative RT-PCR analyses. In contrast, patient no. 10, whose initial karyotype and standard RT-PCR analysis had been reported originally as normal, was found to be positive for CBFß/MYH11 fusion transcript on repeated standard and real-time quantitative RT-PCR assays. Cytogenetic reexamination of microscope slides confirmed the presence of inv(16) in this patient. Thus, initial negative results of both molecular and cytogenetic analyses were in this instance false. This patient was the only one (0.8%) among 124 patients reported originally to have a normal karyotype that was ultimately found to be positive by means of molecular analysis. Overall, there were 26 patients with CBF AML with inv(16) or t(16;16) and one with cytogenetic failure who were positive for CBFß/MYH11. After reanalysis of chromosome preparations, there was no CBFß/MYH11-positive patient who did not have an inv(16) or t(16;16) on successful cytogenetic analysis. Notably, the initial finding of a CBFß/MYH11 transcript was not substantiated by subsequent analyses in two patients: one with isolated +8 (patient no. 12 in Table 2) and another with a complex karyotype (patient no. 13 in Table 2). In patient no. 12, a subsequent RT-PCR study of another diagnostic sample and a real-time quantitative RT-PCR assay performed on bone marrow obtained 4 weeks after diagnosis were negative, as was the FISH analysis of metaphase cells from the diagnostic sample. Similarly, repeated standard RT-PCR and another analysis by real-time quantitative RT-PCR of a diagnostic sample were both negative in patient no. 13. This suggests that the initial molecular results were false. In addition to patient no. 8, with del(16) revised to t(16;16), described above, two other patients displayed del(16)(q22). Both of them had a complex karyotype that included monosomy 5 and a rearrangement of 17p, and in both of them, the initial RT-PCR analyses were negative for the CBFß/MYH11, as were repeated standard and real-time quantitative RT-PCR studies. Neither patient had AML M4Eo—one was diagnosed with AML M2 and the other with AML M6. Neither patient achieved a complete remission.
Overall Ability of Cytogenetics and RT-PCR to Correctly Classify Patients
Results of Calculations for the Presence of t(8;21) and inv (16)/t(16;16)
In this study, 48 (16.9%) of 284 patients were ultimately identified as having CBF AML. In only one of these patients did both cytogenetic and molecular techniques fail to recognize CBF AML initially, and in no instance did both methods generate a false-positive result simultaneously. Overall, we have demonstrated that detection of CBF AML can be successfully achieved in the setting of a multi-institutional clinical trial, where the cytogenetic analyses were performed at the local institutions (with central review of karyotypes) and RT-PCR assays were performed centrally. Both methods had essentially the same overall ability to correctly classify patients as having or not having CBF AML. The respective PVP and PVN, sensitivity, and specificity also were in agreement for both techniques, with the possible exception of sensitivity of cytogenetic analysis in detection of t(8;21). Sensitivity of cytogenetic analysis was lower than the sensitivity of the respective molecular test because three AML1/ETO-positive patients were considered to be cytogenetically negative for t(8;21), even though all of them had chromosome aberrations involving band 8q22, and in two of the patients, also band 21q22. Although each of these aberrations was suspected of being a variant of t(8;21), and two of them were later revised as three- and four-way variant translocations, none of them could initially be regarded as proof of CBF AML without molecular confirmation. All 17 patients with a standard t(8;21) were found to be positive for AML1/ETO. This result corroborates earlier reports that all patients with cytogenetically detectable t(8;21) express the AML1/ETO chimeric mRNA.11,15-17,22,23,35 Consequently, molecular testing of patients with AML with a straightforward t(8;21) at diagnosis serves only as a confirmatory test, although RT-PCR can be subsequently used to detect minimal residual disease.36 On the other hand, patients suspected of having variant translocations, especially simple variants of t(8;21)—that is, rearrangements involving only 8q22 or 21q22—should always be tested by RT-PCR before they can be diagnosed with CBF AML. Molecular testing is essential because some translocations reported previously as simple variants of t(8;21)—for example, t(16;21)(p11;q22)37—when studied by molecular genetic techniques turned out to be unrelated to the t(8;21) and to confer a poor prognosis.38 In contrast, other rearrangements may indeed represent variants of t(8;21) with the AML1/ETO fusion as shown by us for t(8;10)(q22;q26) and by others for t(8;20)(q22;p13), t(8;21)(p21;q22), and t(8;12)(q22.1;q24.1).39,40 Review of the karyotype in patient no. 2 suggests that t(8;10) likely represents a cryptic, three-way translocation t(8;10;21) (q22;q26;q22) that, to our knowledge, has not been previously reported.41 Demonstration of the AML1/ETO fusion transcript is also essential in patients whose karyotype contains complex rearrangements of 8q22 and 21q22 but no obvious juxtaposition of these bands, akin to the patient we describe who initially was reported to have t(1;10;8) and del(21)(q22) and to two similar patients described in the literature who had rearrangements of 8q22 and del(21)(q22).11,15 AML1/ETO positivity and FISH analysis led to reinterpretation of abnormalities—in our case, to a four-way variant t(1;10;21;8)(p22;p15;q22;q22), hitherto not reported.41 Likewise, FISH analysis in one of the published AML1/ETO-positive patients resulted in the revision of the rearrangements from del(8)(q22),add(19)(?q13),del(21)(q22) to a variant t(8;19;21)(q22;?q13;q22).15,42 RT-PCR should also be performed in patients with AML suspected of insertions involving bands 8q22 and 21q22. Chromosomal insertions in general seem to be less common than translocations in hematologic malignancies.2,39 We are aware of only four previously reported patients with AML M2 with ins(21;8).42-45 The three tested for AML1/ETO were positive.42-44 Thus, patient no. 4 in this study, with ins(8;21)(q22;q22q22), seems to be the fourth case of insertion resulting in AML1/ETO gene fusion and the first in which material from 21q22 was inserted into band 8q22. The proportions of AML1/ETO-positive patients in whom no structural abnormality of chromosome 8 or 21 could be identified by cytogenetic analysis have differed among studies. In our study and one other,22 no such patient was identified. Two other studies found a relatively low percentage (6%) of patients who were found to be positive by molecular analysis but negative by cytogenetic analysis.17,18 In contrast, as many as 37% (19 of 51) of AML1/ETO-positive patients were reported not to have any structural abnormality of either chromosome 8 or 21 in the study of Langabeer et al.16 Such a large difference between the latter study and the results of others17,18,22 and ours is difficult to explain. Possible explanations include the following: the presence of undetected insertions(21;8) or (8;21) (although, as indicated by our findings, the insertions are rare and occur in less than 5% of AML1/ETO-positive patients); false-negative cytogenetic results in some patients because of deterioration during transit of their samples sent by mail to the central MRC AML trial cytogenetics laboratory; differences in technical parameters of the RT-PCR methodology used in different studies, such as primer sequences, amplification conditions, and number of cycles; detection of AML1/ETO fusion transcripts by nested RT-PCR not in leukemic blasts but in a low number of nonmalignant cells, a phenomenon described for other leukemia-associated fusion transcripts46,47; and finally artifactual RT-PCR results. Perhaps RT-PCR reanalysis of CALGB leukemic samples by the MRC molecular laboratory and MRC samples by the CALGB molecular laboratory, along with a joint rereview of karyotypes by CALGB and MRC cytogeneticists, could resolve the discrepancy between results of these research groups in the future. Patients expressing the CBFß/MYH11 chimeric mRNA in the absence of chromosome 16 aberrations have been shown to constitute as many as 33% to 43% of all CBFß/MYH11-positive patients in some18,21,22 but not all (this series; and Poirel et al20 and Mitterbauer et al23) studies. Lack of agreement between studies may in part be attributed to differences in technical parameters of the RT-PCR methodology. It has been shown that the published nested RT-PCR method detecting the CBFß/MYH11 transcript is prone to generate false-positive results.48 Our study demonstrates that despite taking every standard precaution to avoid sample and product contamination, false-positive RT-PCR results may also be occasionally obtained when primers described by Liu et al28 are used. We and others12,13,49 have encountered instances in which initial RT-PCR analysis failed to detect CBFß/MYH11 despite the cytogenetically discernible inv(16). Several potential reasons for the lack of transcript amplification have been offered, such as the existence of rare chimeric products not detectable by the primer sets used, low expression of the CBFß/MYH11 transcript that was below levels detectable by the RT-PCR assay, and partial RNA degradation eliminating the CBFß/MYH11 transcript but not the likely more stable and abundant transcript (eg, ßactin) used as a quality control of RNA extraction.12,13,49 The latter seems to be the most likely explanation for RT-PCR failure in the patients we assessed because the repeated standard and real-time quantitative RT-PCR analyses, performed on RNA isolated from diagnostic marrow samples stored in another vial, were in both of our patients positive for the most common type A fusion transcript. However, it remains to be established whether CBFß/MYH11 is in fact less stable than HPRT used as the control in our study. If so, future false-negative results might be prevented by the use of quality-controlled transcripts with expression levels similar to CBFß/MYH11. In some instances, the discrepancy between cytogenetic and molecular results may be caused by the inherent limitations of cytogenetic methodology. Accurate recognition of inv(16)/t(16;16) is more difficult than that of t(8;21) and is more dependent on the quality of chromosome preparations and the experience of the cytogenetic laboratory performing the analysis.8 This is illustrated by three of our CBFß/MYH11-positive patients in whom the inv(16) was initially missed or misinterpreted. Interestingly, the initial karyotype of two of these patients contained +22, which is the most common secondary abnormality in patients with inv(16)/t(16;16) but is rarely seen with other primary aberrations in AML.2,8,41 Similarly, +22 was found in the CBFß/MYH11-positive patient without any cytogenetic anomaly of chromosome 16 in another study.23 Thus, a finding of +22 should alert the cytogeneticist to the possibility of an overlooked or misinterpreted inv(16)/t(16;16) and warrants RT-PCR testing of a patient’s marrow sample. Although inv(16)/t(16;16) can be occasionally overlooked by cytogenetic investigation, this seems to occur rarely in patients with a normal karyotype. In the two previous studies that provided information on the number of patients with a normal karyotype analyzed molecularly, CBFß/MYH11 was expressed in 1 (1.5%) of 65 and 1 (1.1%) of 87 such patients, respectively.18,23 This percentage was even lower in our study—0.8%. It is therefore debatable whether molecular testing is justified in patients with a successful cytogenetic study and a normal karyotype. This is especially true for cytogenetically normal patients without any other characteristics suggestive of CBF AML, such as M4Eo marrow morphology. In contrast to the findings of some13,23 but in agreement with the results of others,49 we found a strong correlation between the presence of M4Eo and inv(16)/CBFß/MYH11. However, because we found patients with AML M4Eo on initial central review who did not have inv(16)/CBFß/MYH11, cytogenetic and/or RT-PCR analyses should be performed to confirm or refute CBF AML in patients with AML M4Eo. Importantly, two of our patients with del(16)(q22) did not express CBFß/MYH11. In some earlier studies, patients with del(16)(q22) have been grouped together with patients who had inv(16) or t(16;16).2,3 However, in some published reports, especially of cases of AML M4Eo, the rearrangements described as del(16)(q22) may have been in fact misinterpreted inv(16) or t(16;16), as was the case for patient no. 8 in our study and for three patients reported previously in the literature.9 Alternatively, an inverted chromosome 16 with the CBFß/MYH11 fusion may have undergone a subsequent deletion of the region distal to 16q2250 or may have participated in a cryptic translocation with another chromosome that imitated a del(16).51 The RT-PCR assay is critical in detecting such patients and should be performed in all patients with del(16)(q22). Both the clinical findings and negative results of standard and real-time RT-PCR analyses in the two patients we studied with del(16) suggest that cryptic rearrangements involving CBFß and MYH11 genes have not taken place and that the observed chromosome 16 aberrations were true deletions, unrelated to CBF AML. Our findings support an earlier report52 that patients with true del(16)(q22) differ from those with inv(16)/t(16;16) and should not be categorized with them into a prognostically favorable group in risk-adapted therapeutic protocols. In conclusion, we have shown that patients with CBF AML can be successfully identified in the setting of a multi-institutional clinical trial. Both cytogenetic and RT-PCR analyses detect the majority of patients with CBF AML, have essentially the same overall abilities to correctly classify patients, and have similar positive and negative predictive values. However, each method has limitations that may hinder detection of CBF AML in rare instances. In our study, discordant results were considerably less frequent than those in several previously reported studies,16,18,21,22 which may be attributable in part to the high quality of cytogenetic analysis performed in CALGB-designated institutional laboratories participating in this study. Patients with a normal karyotype, especially those with marrow morphology other than M4Eo, are unlikely to be molecularly positive. RT-PCR is required in patients in whom cytogenetic study fails and in determining if patients with del(16)(q22), +22 or suspected variants of t(8;21) represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test because of the possibility of obtaining both false-positive and false-negative results.
APPENDIX
Supported in part by grants from the National Cancer Institute to the Cancer and Leukemia Group B (grant nos. CA31946, CA77658, and CA16058) and the Coleman Leukemia Research Fund. We thank Dr Bert A. van der Reijden for helpful discussions.
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