Precise Immunodetection of PTEN Protein in Human Neoplasia

Abstract

PTEN is a major tumor-suppressor protein whose expression and biological activity are frequently diminished in sporadic or inherited cancers. PTEN gene deletion or loss-of-function mutations favor tumor cell growth and are commonly found in clinical practice. In addition, diminished PTEN protein expression is also frequently observed in tumor samples from cancer patients in the absence of PTEN gene alterations. This makes PTEN protein levels a potential biomarker parameter in clinical oncology, which can guide therapeutic decisions. The specific detection of PTEN protein can be achieved by using highly defined anti-PTEN monoclonal antibodies (mAbs), characterized with precision in terms of sensitivity for the detection technique, specificity for PTEN binding, and constraints of epitope recognition. This is especially relevant taking into consideration that PTEN is highly targeted by mutations and posttranslational modifications, and different PTEN protein isoforms exist. The precise characterization of anti-PTEN mAb reactivity is an important step in the validation of these reagents as diagnostic and prognostic tools in clinical oncology, including their routine use in analytical immunohistochemistry (IHC). Here, we review the current status on the use of well-defined anti-PTEN mAbs for PTEN immunodetection in the clinical context and discuss their potential usefulness and limitations for a more precise cancer diagnosis and patient benefit.

Figure 1.

PTEN gene and protein alterations in human tumors. (A) Frequency of PTEN gene deletion in human tumors and PTEN mutations causing loss of PTEN protein (premature termination codon [PTC] mutations and frameshift small deletions or insertions). (B) Frequency of PTEN messenger RNA (mRNA) down- or up-regulation in human tumors (z-score threshold ±2). In both cases, the alteration frequencies are indicated for 16 different human cancers from data generated by the TCGA Research Network and using the cBioPortal database (cBioPortal for Cancer Genomics; Cerami et al. 2012; Gao et al. 2013). Cancer types are as follows: endometrial, uterine corpus endometrial carcinoma; gliobastoma, glioblastoma multiforme; prostate, prostate adenocarcinoma; lung, lung squamous cell carcinoma; melanoma, skin cutaneous melanoma; gastric, stomach adenocarcinoma; cervical, cervical squamous cell carcinoma; soft tissue, sarcoma; breast, breast invasive carcinoma; ovarian, ovarian serous cystadenocarcinoma; liver, liver hepatocellular carcinoma; colorectal, colorectal adenocarcinoma; bladder, bladder urothelial carcinoma; kidney, kidney renal clear cell carcinoma; thyroid, thyroid carcinoma; pancreas, pancreatic adenocarcinoma. (C) Boxplot of the frequency of PTEN protein loss in human tumors as detected by immunohistochemistry (IHC). Whiskers represent the minimum and maximum of all of the data; boxes represent the values between quartiles 1 and 3, and bands inside the boxes represent the median. Data are compilations from Tables 2–4. Note that the cancer categories in C have a wider coverage of cancer subtypes than the categories in A and B.

Figure 2.

Immunohistochemistry (IHC) staining of formalin-fixed paraffin-embedded (FFPE) tumor tissue sections with different anti-PTEN monoclonal antibodies (mAbs). Bladder urothelial carcinoma samples, immunostained with six different anti-PTEN mAbs, are shown. For each mAb, a positive (+) and a negative (–) case are shown. Magnification, ×200.

Figure 3.

Frequency of PTEN protein loss in human tumors, as detected by IHC using defined anti-PTEN mAbs. Data are compilations from Tables 3 and 4 and are represented as the average of samples with PTEN protein loss clustered by cancer type and the anti-PTEN mAb used. Note that the distinct mAbs have not been used in all cancer types.

Figure 4.

Variability of PTEN amino acid composition and posttranslational modifications in relation with PTEN carboxy-terminal epitopes. (A) Schematic depiction of PTEN isoforms. PTEN long isoforms (PTEN-L, PTEN-M, PTEN-N, and PTEN-O), generated by alternative translation initiation, are shown in the upper part. Amino acid numbering and nomenclature are according to Pulido et al. (2014) and Tzani et al. (2016). PTEN-Δ isoform (PTEN 1-343-Ser), generated by alternative splicing, is shown in the bottom. PTEN (1–403) is shown in the middle with indication of the residues flanking the PTP and C2 domains and the carboxy-terminal intrinsically disordered region (C-tail). (B) Schematic of PTEN posttranslational modifications. The distinct posttranslational modifications undergone in the different PTEN domains are denoted with indications of the identity of the residues modified as reported for PTEN (1–403; see Table 5 for more information). The disordered region in the C2 domain (residues 286–310) is shown as a line loop. Note that the existence of these modifications in the PTEN long isoforms has not been reported. (C) Number of PTEN missense mutations along PTEN protein found in human tumor samples as annotated in the COSMIC database (Catalogue of Somatic Mutations in Cancer, Wellcome Trust Sanger Institute; Forbes et al. 2017). Note that the y-axis is scaled to allow visualization of low-frequency mutations. The total number of missense mutations for residues R130 and R173 is in parentheses. (D) Delimitation of PTEN carboxy-terminal epitopes recognized by the indicated commercial anti-PTEN mAb. PTEN carboxy-terminal amino acid sequence (residues 370–403) is indicated with a one-letter code. Amino acids in red are subjected to phosphorylation or acetylation (K402). Amino acids underlined are targeted by disease-associated mutations (see Table 6 for more information). *, caspase-3 cleavage sites as shown in B. Epitope mapping is from Mingo et al. (2019) and our unpublished observations.