Panhandle and reverse-panhandle PCR enable cloning of der(11) and der(other) genomic breakpoint junctions ofMLLtranslocations and identify complex translocation ofMLL,AF-4, andCDK6

We used panhandle PCR to clone the der(11) genomic breakpoint junction in three leukemias with t(4;11) and devised reverse-panhandle PCR to clone the breakpoint junction of the other derivative chromosome. This work contributes two elements to knowledge onMLLtranslocations. First is reverse-panhandle PCR for cloning breakpoint junctions of the other derivative chromosomes, sequences of which are germane to understanding theMLLtranslocation process. The technique revealed duplicated sequences in one case of infant acute lymphoblastic leukemia (ALL) and small deletions in a case of treatment-related ALL. The second element is discovery of a three-way rearrangement ofMLL,AF-4, andCDK6in another case of infant ALL. Cytogenetic analysis was unsuccessful at diagnosis, but suggested t(4;11) and del(7)(q21q31) at relapse. Panhandle PCR analysis of the diagnostic marrow identified a breakpoint junction ofMLLintron 8 andAF-4intron 3. Reverse-panhandle PCR identified a breakpoint junction ofCDK6from band 7q21-q22 andMLLintron 9.CDK6encodes a critical cell cycle regulator and is the first gene of this type disrupted byMLLtranslocation. Cdk6 is overexpressed or disrupted by translocation in many cancers. The in-frameCDK6-MLLtranscript is provocative with respect to a potential contribution of the predicted Cdk6-MLL fusion protein in the genesis of the ALL, which also contains an in-frameMLL-AF4transcript. The sequences in these three cases show additionalMLLgenomic breakpoint heterogeneity. Each breakpoint junction suggests nonhomologous end joining and is consistent with DNA damage and repair.CDK6-MLLis a new fusion of both genes.