PTPRG is emerging as a key tumor suppressor gene in many types of neoplasia [
5,
7,
23‐
25]. More recently, an oncosuppressive role has been described in selected hematologic malignancies [
8,
26], suggesting that the availability of an antibody suitable for flow cytometric applications would represent a valuable tool with potential clinical applications. In this study, we present the characterization of the first monoclonal antibody raised against the ECD of human PTPRG, expressed and purified from the HEK293F human cell line, which was found suitable for different immunoassay applications, including flow cytometry. This monoclonal antibody (named TPγ B9-2) appears capable of recognizing murine and human PTPRG by immunofluorescence, immunohistochemistry, and flow cytometry and its reactivity overlaps almost entirely with the tissue structures recognized by another validated polyclonal antibody, chPTPRG, raised against a peptide present in the ECD of PTPRG [
10].
Of interest is the observation that TPγ B9-2 displays a slightly different subcellular reactivity, although recognizing overlapping structures in human and murine tissues, suggesting its capability to recognize a distinct epitope associated with a specific isoform. The presence of PTPRG isoforms was described in previous work carried out in rats [
22], humans, and mice [
19,
21]. This observation might also help to explain slight differences in reactivity found in normal and neoplastic samples (in both hematological and solid tissue samples) that will need to be further evaluated in future studies. Importantly, both antibodies confirmed that PTPRG is downregulated in the leukocytes of patients affected by CML, including CD34
+CD38
bright and CD34
+CD38
dim hematopoietic precursors. This observation implies that its loss occurs at the stage of early hematopoietic precursors. PTPRG is a tumor suppressor gene capable of interfering with BCR-ABL1 signaling by decreasing BCR-ABL1-dependent tyrosine phosphorylation in CML cells [
8]. Its downregulation in all CD34
+ fractions of CML patients along with the demonstration that its forced up- and downregulation in CML cells reduces and increases, respectively, clonogenic capacity [
8], suggesting a key role in the maintenance of the disease. The role of PTPRG in controlling hematopoietic cell survival/differentiation is further supported by an early study [
3], while the most recent evidence linking PTPRG to the pathogenesis of leukemia would be in keeping with inhibition of PTPRG expression by epigenetic regulation consisting in methylation of selected promoter regions and the fact that its downregulation cooperates with RAS gene activation in childhood acute lymphoblastic leukemia (ALL) [
26]. This last study adds another hematological malignancy to the number of diseases where deregulated PTPRG is disease associated. Of additional interest is the new observation that TPγ B9-2 antibody is capable of detecting PTPRG expression in non-CML hematopoietic cells re-emerging in the same patient after effective therapy, thus confirming that normal hematopoiesis does require an appropriate level of PTPRG expression, and the latter is specifically downregulated in the diseased cells. Under these carefully controlled conditions (same individuals-same antigen) an antibody found specific for PTPRG binds to the cell surface after TKI therapy. This suggests that the antigen is now expressed as happens for normal individuals. Very few examples of cell surface biomarkers specifically expressed in CML are available in the literature; however, the ones described are expressed on the cell surface and not downregulated or lost, like PTPRG, in CML cells. Among these, RNA expression analysis identified an association between low CD7 expression along with high expression of proteinase 3 or elastase with longer survival in CML [
27]. An association was described between hypomethylation of CD7 promoter region and its increased expression [
11] The orphan receptor tyrosine kinase ROR1 was shown to be expressed in follicular lymphoma. Also, chronic lymphocytic leukemia, hairy cell leukemia, and CML had significantly higher numbers of ROR1
+ cells [
13]. Sperm-associated antigen 9 (SPAG9) mRNA and protein were found to be expressed in CML patients (88%) and have been associated with humoral response against this antigen in 90% of the cases, suggesting it as a potential biomarker and/or immunotherapeutic target [
12]. No specific functional roles in CML have been associated with this gene product. A proteomic survey of cell surface proteins in leukemia cell lines identified 25 proteins, including CD43 and CD98, which appeared to be differentially expressed in CML cell lines, with CD43 expressed uniquely in K562 cells [
28]. More recently, a series of genes were identified by transcriptome analysis [
14]. Both IL1RAP and CD26 can be regarded as promising candidates as CML-specific antigens in the CD34
+CD38
− CML LSC population [
15‐
17]. Within this context, PTPRG has unique features as it is specifically downregulated in CML and has been demonstrated to have a functional role being capable of binding and dephosphorylating the driving oncoprotein BCR-ABL1 and consequently reducing total and specific phosphotyrosine levels as well as clonogenic capacity in various CML cells [
8]. Of interest is the recent observation that JAK2 kinase, a key signaling molecule involved in BCR-ABL1-driven pathways [
29], represents a bona fide PTPRG substrate [
30].