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Clinical Trial
. 2017 Aug 10;35(23):2674-2682.
doi: 10.1200/JCO.2016.71.2513. Epub 2017 Jun 23.

CD33 Splicing Polymorphism Determines Gemtuzumab Ozogamicin Response in De Novo Acute Myeloid Leukemia: Report From Randomized Phase III Children's Oncology Group Trial AAML0531

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Clinical Trial

CD33 Splicing Polymorphism Determines Gemtuzumab Ozogamicin Response in De Novo Acute Myeloid Leukemia: Report From Randomized Phase III Children's Oncology Group Trial AAML0531

Jatinder K Lamba et al. J Clin Oncol. .
Free PMC article

Abstract

Purpose Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging therapy for acute myeloid leukemia (AML). CD33 single nucleotide polymorphism rs12459419 C>T in the splice enhancer region regulates the expression of an alternatively spliced CD33 isoform lacking exon2 (D2-CD33), thus eliminating the CD33 IgV domain, which is the antibody-binding site for GO, as well as diagnostic immunophenotypic panels. We aimed to determine the impact of the genotype of this splicing polymorphism in patients with AML treated with GO-containing chemotherapy. Patients and Methods CD33 splicing single nucleotide polymorphism was evaluated in newly diagnosed patients with AML randomly assigned to receive standard five-course chemotherapy alone (No-GO arm, n = 408) or chemotherapy with the addition of two doses of GO once during induction and once during intensification (GO arm, n = 408) as per the Children's Oncology Group AAML0531 trial. Results The rs12459419 genotype was CC in 415 patients (51%), CT in 316 patients (39%), and TT in 85 patients (10%), with a minor allele frequency of 30%. The T allele was significantly associated with higher levels of D2-CD33 transcript ( P < 1.0E-6) and with lower diagnostic leukemic cell surface CD33 intensity ( P < 1.0E-6). Patients with the CC genotype had significantly lower relapse risk in the GO arm than in the No-GO arm (26% v 49%; P < .001). However, in patients with the CT or TT genotype, exposure to GO did not influence relapse risk (39% v 40%; P = .85). Disease-free survival was higher in patients with the CC genotype in the GO arm than in the No-GO arm (65% v 46%, respectively; P = .004), but this benefit of GO addition was not seen in patients with the CT or TT genotype. Conclusion Our results suggest that patients with the CC genotype for rs12459419 have a substantial response to GO, making this a potential biomarker for the selection of patients with a likelihood of significant response to GO.

Figures

Fig 1.
Fig 1.
CD33 exon2 single nucleotide polymorphism (SNP) rs12459419 influences the alternative splicing of CD33. (A) CD33 gene and location and potential functional consequences of rs12459419 SNP. The presence of rs12459419 in exon2 affects the exonic enhancer binding site for SRFS2, thereby resulting in the loss of exon2 (shown in red) in the T allele. The loss of exon2 results in a shorter CD33 isoform lacking the IgV domain, which is recognized by gemtuzumab ozogamicin (GO) and currently, used antibodies. (B) Transcriptome-sequencing data from AAML0531 patients (n = 88) shown as percent spliced isoform (PSI) with loss of exon2 for different rs12459419 SNP genotypes. (C) Quantitative real-time polymerase chain reaction (Data Supplement) using isoform-specific primers shown in (A) in an independent set of patient specimens (n = 30) obtained at diagnosis. Log10 relative levels of the D2-CD33 isoform are shown for the rs12459419 genotypes (patient numbers in parentheses). Box plots show medians as a line between the boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside the first and third quartiles by > 1.5 times the interquartile range. Circles outside the whiskers represent outliers. Cell membrane was created using clipart from clker.com. (D) Association of rs12459419 with CD33 expression determined as mean fluorescence intensity (MFI) in diagnostic leukemic blasts from patients treated in AAML0531 (n = 816). CD33 levels were determined in the diagnostic leukemic blasts by multiparameter flow cytometry. Log10 CD33 MFI is shown for different rs12459419 genotypes (patient numbers in parentheses). Correlation plots show the association of CD33 MFI with levels of (E) the CD33 total mRNA levels and (F) D2-CD33 isoform. Plots were created using the GraphPad Prism software. ITIM, immunoreceptor tyrosine inhibitory motif; WT, wild-type.
Fig 2.
Fig 2.
Association of rs12459419 genotypes with clinical response by treatment arm in patients treated in AAML0531. (A) Differences in relapse risk (RR) and (B) disease-free survival (DFS) from the end of course 1 in patients with the CC, CT, or TT genotype in the standard five-course chemotherapy with the addition of two doses of 3 mg/m2 gemtuzumab ozogamicin (GO) versus the standard five-course chemotherapy alone (No-GO) arm.
Fig 3.
Fig 3.
Association of rs12459419 genotypes with relapse risk (RR) for patients treated in AAML0531 in the low-, standard-, and high-risk groups in the standard five-course chemotherapy with the addition of two doses of 3 mg/m2 gemtuzumab ozogamicin (GO) versus the standard five-course chemotherapy alone (No-GO) arm.

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