AKR1C3 expression in T acute lymphoblastic leukemia/lymphoma for clinical use as a biomarker

Abstract

To investigate aldo-keto reductase 1C3 (AKR1C3) expression in T and B acute lymphoblastic leukemia/lymphoma (ALL) patients. Three commercial antibodies were evaluated for AKR1C3 immunohistochemistry (IHC) staining performance: Polyclonal Thermofisher scientific (Clone#PA523667), rabbit monoclonal Abcam [EPR16726] (ab209899) and Sigma/Millipore anti-AKR1C3 antibody, mouse monoclonal, clone NP6.G6.A6, purified from hybridoma cell culture. Initial optimization was performed on cell line controls: HCT116 (negative control); genetically modified cell line HCT116 with AKR1C3 overexpression; Nalm and TF1 cell lines. Twenty normal bone marrows from archival B and T-ALL patient samples were subsequently examined. AKR1C3 expression levels in these samples were evaluated by immunohistochemistry, Protein Wes and quantitative RT-PCR. Sigma/Millipore Anti-AKR1C3 antibody (mouse monoclonal, clone NP6.G6.A6) showed higher specificity compared to rabbit polyclonal antibody by immunohistochemistry. H-score was used to quantify percent of nuclear immunoreactivity for AKR1C3 with varying disease involvement. T-ALL samples had a higher H-score (172-190) compared to B-ALL cases (H-score, 30-160). The AKR1C3 expression in peripheral blood by Protein Wes and RT-qPCR showed concordance in relapsed/refractory and/or minimal residual T-ALL cases. Sigma/Millipore Anti-AKR1C3 antibody and mouse monoclonal, clone NP6.G6.A6 can be used to aid in AKR1C expression of T-ALL and in cases of relapsed/refractory and/or minimal residual disease.

Figure 1

AKR1C3 immunoreactivity of four antibodies on control cell lines. Immunoreactivity of Thermo Fisher Scientific (Clone#PA5-23667), rabbit monoclonal Abcam [EPR16726] (ab209899) and Sigma/Millipore Anti-AKR1C3 antibody, mouse monoclonal, clone NP6.G6.A6, on different control cell lines. HCT116 is a human colorectal cancer cell line. HCT116-AKR1C3 is HCT116 engineered overexpress AKR1C3. Nalm6 is a Pre-B cell ALL cell line with very low endogenous AKR1C3 expression. TF1 is an erythroleukemia cell line with high endogenous AKR1C3 expression.

Figure 2

AKR1C3 IHC H-score correlation with ALL blast percentage. Quantification of singleplex nuclear immunoreactivity of AKR1C3 by the H-score in B and T lymphoblastic leukemia/lymphoma among cases with IHC expression of < or ≥ 20% Grade 2 +. Correlation with disease involvement is significant in T-ALL, but not B-ALL.

Figure 3

AKR1C3 RT-qPCR in blood and bone marrow. RT-qPCR values of diagnostic peripheral blood (A, green), R&R/MRD peripheral blood (A, red), and marrow aspirates (B, red). Diagnostic peripheral blood samples surpassed the calculated cutoff value (Table 3).

Figure 4

AKR1C3 expression by Protein Wes Simple in blood and bone marrow. (A) Increased expression of AKR1C3 by Protein Wes in peripheral blood specimens (indicated by red *) compared to the marrow aspirate specimens and control GAPDH expression. (B) In T-ALL cases with AKR1C3 expression in peripheral blood (PB) versus bone marrow aspirate (BMA), in which elevated expression is observed in PB samples regardless of their timepoint (Protein Wes Area t-test: P = 0.0277; Protein Wes height t-test: P = 0.0166). (C) When PB samples were segregated into diagnostic (PBdx) and samples of relapsed/refractory and/or minimal residual disease (PBR&R/MRD), PBR&R/MRD samples demonstrated elevated expression compared to BMA (Protein Wes Area ordinary one-way ANOVA: P = 0.0003).