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, 10 (1), 129

A New Monoclonal Antibody Detects Downregulation of Protein Tyrosine Phosphatase Receptor Type γ in Chronic Myeloid Leukemia Patients

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A New Monoclonal Antibody Detects Downregulation of Protein Tyrosine Phosphatase Receptor Type γ in Chronic Myeloid Leukemia Patients

Marzia Vezzalini et al. J Hematol Oncol.

Abstract

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients.

Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry.

Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells.

Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions.

Keywords: BCR-ABL1; Chronic myeloid leukemia; Monoclonal antibody; Protein tyrosine phosphatase; Tumor suppressor gene.

Figures

Fig. 1
Fig. 1
TPγ B9-2 specifically recognizes PTPRG. Downregulation of PTPRG by siRNA demonstrates the specificity of TPγ B9-2 monoclonal antibody for the antigen. Immunoblotting was performed with the indicated antibodies after transfection with a specific PTPRG siRNA (siRNA) and with a negative control siRNA (scrambled: SCR). Cell lines were DBTRG and K562, and antibodies were Mab TPγ B9-2 or the reference rabbit polyclonal antibody RbtP4 and chicken (ch) anti PTPRG [9, 10]. a Immunoprecipitation of PTPRG by TPγ B9-2 monoclonal antibody. K562 and DBTRG cell lines, respectively negative and positive for PTPRG mRNA expression, were subjected to immunoblotting analysis with chPTPRG antibody after immunoprecipitation with TPγ B9-2 antibody. Left side: Black arrow, full-length PTPRG; gray arrows indicate putative processed forms. No signal was detectable using an irrelevant antibody for IP (data not shown). b Western blotting with mab TPγ B9-2 or the reference rabbit polyclonal antibody RbtP4 in PTPRG expressing DBTRG cell line treated with scrambled (SCR) or PTPRG-specific siRNA. Both antibodies detect the downregulation of PTPRG. Anti-β-actin was used as a loading control. c Western blotting with mab TPγ B9-2 or the reference rabbit polyclonal antibody RbtP4 in PTPRG silenced K562 cell lines overexpressing PTPRG (K562 PTPRG+). Downregulation of the 180 kDa band is apparent in silenced cells using both antibodies. Differences in signal intensities are due to the combined effect of individual affinities of primary antibodies toward the native or cDNA-transfected antigens and secondary antibodies toward the murine or rabbit Igs
Fig. 2
Fig. 2
In situ analysis of PTPRG expression. a Immunohistochemistry using monoclonal TPγ B9-2 and chPTPRG antibodies on cryostatic sections of mouse testis. In the heterozygous PTPRG (+/−) mice, shown in the insets, the two antibodies recognize the same structures while in Ptprg-null (KO) (−/−) mice, shown as the main figure, no staining was detectable. b Comparison between TPγ B9-2, chPTPRG, and RbtP4 in normal human lung (ac) and pancreas (df). Formalin-fixed, paraffin-embedded sections were stained with anti-PTPRG antibodies indicated. All the antibodies recognize lung alveolar macrophages (ac, arrows). In pancreas (df) TPγ B9-2 preferentially stains Islets of Langerhans (white arrows) and centroacinar regions (black arrows), while chPTPRG stains the tissue more diffusely, including the exocrine glands. Matched irrelevant antibodies controls (murine IgG1, IgY, and rabbit IgG) are shown in the insets
Fig. 3
Fig. 3
Co-localization of different anti-PTPRG antibodies on the same histo-cytologic structures: murine cerebral cortex (ad) and vessel sections (eh) were analyzed using TPγ B9-2 and chPTPRG antibodies. White arrow, a dendrite staining positive for chPTPRG and negative with TPγ B9-2 (b). A slightly different subcellular distribution is also observed in small vessels as shown by overlapping of both stains (in colocalization and merge mode, c, d, g, h). In colocalization mode, yellow regions indicate only the overlapping epitopes. Merge mode shows signals from the individual antibodies highlighting the capability of chPTPRG (in red) to identify dendrites that are not recognized by TPγ B9-2 (in green). Slight differences in staining are present also in vessel sections. These results confirm the presence of different Ptprg isoforms recognized by the antibodies and localized in different cellular structures. DAPI (blue) stains nuclei. Scale bars, 30 μm in ad, 10 μm in eh
Fig. 4
Fig. 4
Flow cytometric analysis of healthy human donor peripheral blood samples using two anti-PTPRG antibodies. Dot plots show gating strategy for each cell subset analyzed in histograms. N = 34 monocytes, N = 20 T cells, N = 26 B cells, N = 16 neutrophils, N = 10 eosinophils, and N = 18 CD34+ cells. PTPRG expression in peripheral blood circulating monocytes (CD45+; CD14+), T cells (CD45+; CD3+), B cells (CD45+; CD19+), PMN neutrophils (CD45+; CD16+), PMN eosinophils (CD45+; CD16), CD34+ progenitor cells (CD45low; CD34+), using the reference antibody chPTPRG and TPγ B9-2. Data are expressed as mean fluorescence intensity (MFI) with appropriate isotype controls (preimmune chicken IgY and irrelevant murine IgG1) as references
Fig. 5
Fig. 5
Flow cytometric analysis using TPγ B9-2 antibody of normal individuals and CML patients. Representative flow cytometric analysis of a normal individual (a) and a CML patient (b) performed with both chicken and Mab for comparison. c Comparison made using mean fluorescence intensities (MFI) values obtained with TPγ B9-2. d PTPRG expression in CD34+, CD34+CD38bright, CD34+CD38dim subpopulations from 13 G-CSF mobilized normal individuals and 24 CML patients. MFI values obtained calculating the ratio between the signal derived from TPγ B9-2 and irrelevant murine IgG1, respectively (p values for statistic derive from Mann-Whitney test)
Fig. 6
Fig. 6
Evaluation of sensitivity and specificity of the test. A receiver operating characteristic curve (ROC curve) was built for normal individuals vs CML patients. Data obtained with TPγ B9-2 derived from patients shown in Fig. 5. A statistically significant capability to discriminate between CML and healthy subject is achieved when monocytes and PMN are evaluated. No/negligible expression of PTPRG in lymphocytes renders these cells unsuitable for the purpose
Fig. 7
Fig. 7
PTPRG mRNA expression in paired samples of patients treated with TKI. RQ-PCR analysis of PTPRG expression in peripheral blood (gray symbols) and bone marrow (black symbols) calculated as % vs ABL1 mRNA. Major molecular response (MMR) is associated with recovery of PTPRG expression. Only the few non-responder patients failed to recover PTPRG expression
Fig. 8
Fig. 8
PTPRG expression in various leukocyte populations of CML patients at diagnosis and after treatment. a Representative flow cytometric analysis of PTPRG protein at diagnosis and at follow-up following successful TKI-based treatment. At diagnosis, monocytes and PMN downregulate PTPRG expression. After an average of 6 months of treatment, the levels of phosphatase expression recover and become similar to healthy donors. b Box-Whisker diagram displays significant differences between PTPRG expression in monocytes and PMN populations in three different CML patients at diagnosis and after treatment (statistical analyses are indicated on graphs). c The table summarizes the mean fluorescence intensity (MFI) values related to PTPRG expression shown in panel b

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