Identification of acute myeloid leukemia patients with diminished expression of CD13 myeloid transcripts by competitive reverse transcription polymerase chain reaction (RT-PCR)

Abstract

Normal myeloid cells of monocytic and granulocytic origin express the metallopeptidase cluster of differentiation 13 (CD13) on the surface just as leukemic blasts in most acute myeloid leukemias (AML). A minor percentage of AML patients, however, lack the surface expression of CD13 antigen. To study this difference in CD13 surface expression, specific CD13 mRNA from 44 individuals were quantified by competitive reverse transcription polymerase chain reaction (RT-PCR). Absolute values for CD13 transcripts were normalised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels to control for variations in sample preparation and mRNA degradation. By correlating normalised CD13 transcript levels and CD13 surface expression, a subgroup of AML patients was identified, having simultaneous diminished levels of myeloid CD13 transcripts and surface expression of the corresponding antigen. For this subgroup we suggest CD13/aminopeptidase N (APN) gene expression to be restricted primarily by limited amounts of transcripts. For the majority of AML patients determinants in addition to transcript levels must be involved in regulating CD13/APN gene expression.

Single site polymorphisms and alternative splicing of the human CD13 gene – different splicing frequencies among patients with acute myeloid leukaemia and healthy individuals

“…In addition, the frequencies of truncated CD13 mRNA were consistently observed for cells of both BM and PB origin for the five AML patients for whom both cell types were analysed. The total content of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA was determined using a competitive quantitative RT‐PCR (Dybkaer et al, 2000) for all individuals included in this study. The average content of total GAPDH mRNA ranged from 2·2 × 10 9 to 4·8 × 10 9 (transcripts/0·5 µg total RNA) for the groups described in Fig 2, thereby indicating comparable RNA integrity of AML patients with different splicing frequencies of the CD13 transcript.…”

“…Because diagnosing leukaemia and other haematopoietic disorders includes immunophenotyping with monoclonal antibodies directed against the extracellular domain of CD13 (Griffin et al, 1983), altered translation and processing resulting from truncated CD13 mRNA obviously represents a problem. Changed protein structure and epitope accessibility will alter the reactivity of monoclonal antibodies against CD13, and may be involved in the cases of AML where only a diminished CD13 surface antigen expression is observed (Drexler, 1987;Civin, 1990;Bradstock et al, 1994;Dybkaer et al, 2000).…”

“…Amplicons 3 and 5, spanning the alternatively spliced regions, were obtained by PCR performed at 95°C for 10 min to activate the AmpliTAQ Gold (Perkin Elmer‐Roche, CA, USA), then 30 repeating cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 60 s. The last extension step was 72°C for 10 min. The PCR products, stained with ethidium bromide, were separated by agarose gel electrophoresis before intensity of luminescence of full length and truncated forms was measured using a charged‐coupled device (CCD) camera (BioRad, CA, USA) (Dybkaer et al, 2000). Primers SF and SR were only used for sequencing amplicon 5.…”

Applications of enzymatic amplification staining in immunophenotyping hematopoietic cells

“…There are many other examples of antigens expressed at low levels in hematologic malignancies. CD2, CD3, CD4, CD7, CD8 in T cell lymphomas (76,77), CD10 in follicle center lymphomas (78), CD13 and CD33 in acute myeloid leukemia, and CD34 in acute lymphoblastic leukemia (79)(80)(81)(82) would all show increased detection rates with enzymatic amplification staining. CD34 expression is also important for defining hematopoietic stem cells (83).…”

Fusion of Dendritic Cells and CD34+CD38− Acute Myeloid Leukemia (AML) Cells Potentiates Targeting AML-initiating Cells by Specific CTL Induction

“…Many markers associated with L-IC such as CD33, CD13, CD123 also are expressed on normal hematopoietic stem cells or committed myeloid cells. 7,8 Thus, new strategies should be explored to generate L-IC-specific CTLs for leukemia immunotherapy.…”