title: Characterization and use of an antibody detecting the CBFbeta-SMMHC fusion protein in inv(16)/t(16;16)-associated acute myeloid leukemias.
abstract: The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFbeta-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFbeta-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR-negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFbeta-SMMHC fusion proteins. Molecular characterization of one RT-PCR-positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, -0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR-positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.
authors.affiliation: Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Viswanatha
authors.forename: I
authors.initials: I
authors.lastname: Chen
authors.forename: P P
authors.initials: PP
authors.lastname: Liu
authors.forename: M L
authors.initials: ML
authors.lastname: Slovak
authors.forename: C
authors.initials: C
authors.lastname: Rankin
authors.forename: D R
authors.initials: DR
authors.lastname: Head
authors.forename: C L
authors.initials: CL
authors.lastname: Willman
mesh_terms: D000208:Acute Disease; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000032:analysis*; D002675:Child, Preschool; D007446:Chromosome Inversion; D002885:Chromosomes, Human, Pair 16 / Q000235:genetics* / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D018365:Neoplasm, Residual; D015514:Oncogene Proteins, Fusion / Q000032:analysis / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C095514:CBFbeta-MYH11 fusion protein; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D012334:RNA, Neoplasm