Cross-reactivity ofTrichomonas in cervical p16INK4a immunocytochemistry is dependent on antibody, not antigen

Abstract

Immunocytochemical staining for detection of overexpressed cyclin-dependent kinase inhibitor protein p16 INK4a has been suggested to represent a promising approach for the evaluation of cervical histology and cytology. 1 Numerous scientific reports have supported the potential value of p16 INK4a immunochemistry as an adjunct to the evaluation of conventional H&E-and Pap-stained cervical histology and cytology preparations, respectively. [2][3][4][5][6] In a recent manuscript, Pantanowitz and colleagues reported that Trichomonas parasites consistently reveal strong immunoreactivity in cervical liquid-based cytology specimens analyzed for p16 INK4a expression. 7 The authors indicated a potential risk of diagnostic misinterpretations due to the presence of immunoreactive Trichomonads in the liquid-based cytology specimens, as they may be mistaken for small dysplastic or malignant epithelial cells.As we never observed occasional positive immunoreactivity of Trichomonas parasites within a four-digit number of cervical cytology specimens analyzed for p16 INK4a expression over the past, we set out to investigate whether the reported immunoreactivity of Trichomonads may be due to a distinct characteristic feature of the individual p16 INK4a monoclonal antibody clone used by Pantanowitz and colleagues, versus a general phenomenon due to the specific labeling of, for example, conserved cellcycle regulatory protein structures in this parasite. Our analyses revealed that the reported cross-reactivity to Trichomonads is antibody-dependent, and not related to the antigen itself.In fact, when using the p16 INK4a antibody clone G175-405 (Pharmingen), we also observed a strong immunoreactivity in 4 out of 4 archived cervical samples showing highly abundant (2 cases) or moderate infection with Trichomonas (Fig. 1A). However, there was no detectable immunoreactivity for any of these cases on liquid-based cytology specimens that have been prepared in parallel from the identical ThinPrep 1 vials and stained with a different p16 INK4a specific monoclonal antibody, clone mtm-E6H4 (included in CINtec 1 p16 INK4a Cytology Kit, Dako) (Fig. 1B). As a control, thin-layer preparations from known HSIL as well as NILM cases without Trichomonas infections have been immunostained using the same polymer-based protocol, but the different p16 INK4a antibody clones described above. Strong immunoreactivity of dysplastic cervical epithelial cells was observed using both antibodies (Figs. 1C and 1D). Trichomonas negative NILM cases were negative for p16 INK4a immunoreactivity for either antibody (data not shown).The reported finding of a putative p16 INK4a immunoreactivity of Trichomonas parasites in cervical cytology specimens underlines the importance of a careful validation of reagents used for immunocytochemistry. Pantanowitz and colleagues have described various control experiments that have been performed to demonstrate the specificity of the staining results, including the use of various secondary reagent systems 7 or primary antibodies against ...

Functional biomarkers in cervical precancer: An overview

“…48,49 p16 has been shown to be expressed in tubal metaplasia and also in trichomonas, which can be a potential pitfall while evaluating the cytologic samples. [50][51][52] Also they detected specific viral mRNA transcripts derived from integrated HPV genomes to identify preneoplastic lesions with a particularly high risk for progression to invasive cancers by APOT-assay. 46 It is now widely accepted that p16 INK4a is a sensitive and specific marker of squamous and glandular dysplastic cells of the cervix and also a surrogate marker of HR-HPV, suggesting a valuable adjunctive test in cervical cancer screening.…”

RET gene rearrangements (RET/PTC1 and RET/PTC3) in papillary thyroid carcinomas from an iodine‐rich country (Japan)

“…In cytology, staining with p16 can improve the identification of suspicious cells on cytological slides 47. p16 cytology has been successfully used as a triage marker for ASCUS and LSIL cytology results 48, 49. p16 has been shown to be expressed in tubal metaplasia and also in trichomonas, which can be a potential pitfall while evaluating the cytologic samples 50–52…”