Summary of clinical course and methylation studies. The top panel shows clinical response parameters as liver and spleen size (in centimeters below costal margin), blast percentage, platelet count, monocyte count, and cytogenetic studies of bone marrow cells. Diagrams in the bottom panel depict the CALCA gene methylation status in bone marrow cells at diagnosis, before the third cycle and eighth cycle of azacitidine. The methylation of 11 CpG dinucleotides located between +172 bp and +325 bp relative to the CALCA transcription start site was mapped by sequencing multiple cloned alleles obtained from PCR on bisulfite-treated genomic DNA. Each horizontal line represents an individual allele (20-24 per sample). ● represents methylated CpG sites; ○, unmethylated CpG sites. FISH indicates fluorescence in situ hybridization.