Role of immunocytochemistry in cervical cancer screening

Abstract

The cervical cancer screening has been based conventionally on cytologic analysis. With advances in understanding the role of human papillomavirus, cotesting has been applied. But most of the patients subjected to colposcopy did not benefit, except in cases with HSIL [high-grade squamous intraepithelial lesion] cytology. Because of this, a step to increase the sensitivity to detect cancers and pre-cancers but with higher specificity with minimal overdiagnosis leading to prevention of unindicated cervical biopsies is highly desired. Such triaging step in cases with abnormal screening results is expected to minimize invasive interventions because of low false positivity. With availability of methodologies leading to quantitatively and qualitatively enhanced cell-blocks from residual liquid based cytology specimens, immunostaining can be performed for multiple immunomarkers with potential objectivity to triage initial screening test results. This is enhanced further with inclusion of AV marker in the cell-blocks and application of SCIP (subtractive coordinate immunoreactivity pattern) approach. The cell-blocks are also resource for performing other ancillary studies including molecular pathology and proteomics/metabolomics as potential tests in future. This review explores application of residual liquid based cytology specimen for cell-blocking with application of ancillary studies in algorithmic manner as adjunct to ASCCP management guidelines for improved patient care.

Keywords: Cell-block; Cervical cancer; Cervical cytology; HPV; Immunocytochemistry; Immunohistochemistry; Ki-67; LBC; Liquid based cytology; PAP test; Papanicolaou smear; p16.

Figure 1:

Comparison of cellularity of sections of cell-block with and without AV marker. (Reproduced under Open-Access charter from: Varsegi GM, Shidham V. J Vis Exp 2009;29:1316. doi: 10.3791/1316).^[1]

Figure 2:

Algorithm showing cases which may be triaged to make cell-block from residual liquid-based cytology specimens with enhanced methods such as with Nano NextGen CelBloking™ kits.^[5] LG: Low grade, HG: High grade, HPV: Human papillomavirus.

Figure 3:

Algorithm showing application of immunostaining cell-block sections with subtractive coordinate immunoreactivity pattern. The evaluation may be refined further using digital imaging methodologies and application of high-dimensional imaging of multiplexed ion beam imaging by time-of-flight.^[31] AGC: Atypical glandular cells, AIS: Adenocarcinoma in situ, AdCa: Adenocarcinoma. *May also be used for Molecular pathology studies including miRNA testing and proteomics **Depending on clinical scenario including known history of cancer, imaging findings etc

Figure 4:

Interpretation of immunostaining with subtractive coordinate immunoreactivity pattern approach for definitive management. The evaluation may be refined further using digital imaging methodologies and application of high-dimensional imaging of multiplexed ion beam imaging by time-of-flight.^[31]

Figure 5:

p16/Ki-67 dual-staining positive cells with morphological features of HSIL. (a) Liquid-based cytology (SurePath). (b) Slide A was dual stained with p16/Ki-67. Cell with p16 staining alone (blue arrow) is characterized by a brown cytoplasmic/nuclear signal and cell with Ki-67 staining alone (red arrow) is presented in red nuclear signal. The positive p16/Ki-67 dual-staining cells (dark arrow) are characterized by a brown cytoplasmic signal for p16 overexpression and a dark red nuclear signal for p16/Ki-67 coexpression in the same cell (a 45-year-old woman, CIN3, HPV16+, p16/Ki-67+). (Reproduced from open-access publication: Yu L et al. J Cancer 2019;10:2654-60. doi:10.7150/jca.32743).^[23]

Figure 6:

Left: A case interpreted as LSIL (Pap, ×200); Right: Nuclear ProExC immunostaining in the same case (ProExC, ×100). (Reproduced from open-access publication: Tosuner Z et al. J Cytol 2017;34:34-8. doi:10.4103/0970-9371.197605).^[29]

Figure 7:

(a) Pap smear interpreted as LSIL, (b) H and E cell-block sections, (c) p16-stained cell-block sections, and (d) biopsy showing CIN II-III. (Reproduced under open-access charter from: Shidham VB et al. CytoJournal 2011;8:1).^[4]

Figure 8:

(a) ASC-H (rare single cells with hyperchromatic nuclei and high N: C ratios), (b) H and E stained cell-block sections, (c) p16-stained sections highlighting scattered high-grade cells, and (d) biopsy showing CIN III with extensive endocervical glandular involvement. (Reproduced under open-access charter from: Shidham et al. CytoJournal 2011;8:1).^[4]

Figure 9:

(a) Pap smear interpreted HSIL, (b) H and E cell-block section containing “microbiopsies,” (c) p16-stained cell-block section showing true nuclear immunoreactivity, and (d) biopsy showing invasive squamous cell carcinoma. (Reproduced under open-access charter from: Shidham VB et al. CytoJournal 2011;8:1).^[4]

Figure 10:

miRNAs and cervical neoplasia progression. Reproduced from open-access publication: Pardini B et al. BMC Cancer 2018;18:696. 10.1186/s12885-018-4590-4 .^[38]

Figure 11:

Differential expression of miRNA in exfoliative cytology of cervical lesions with diagnostic accuracy (modified from^[32]). *Six miRNAs (miR-424, miR-375, miR-34a, miR-218, miR-92a, and miR-93). Bold-Trials with the best diagnostic performance. LSIL: Low-grade squamous intraepithelial lesion (milder than CIN2), HSIL: High-grade squamous intraepithelial lesion (CIN2, CIN3, and CIS), AUC: Area under the ROC (receiver operating characteristic) curve, Sn: Sensitivity, Sp: Specificity.

Figure 12:

The workflow in proteomic studies in cervical cancer. This facilitates search for biomarkers that could be applied in the management of cervical cancers from diagnosis to application of new therapeutic targets. The steps include: (1) Biological samples from patients in various forms including FFPE of cell-blocks of cervical specimens are processed to lyse the cells mechanically. (2) Purification of total proteins. (3) The storage at −20°C or −70°C (with or without protease inhibitor) depending on the method selected for proteomic analysis (to avoid any interference). (4) Selection of advanced techniques such as protein microarray, mass spectrometry, 2D gel, 2D-DIGE, and Edman sequencing; quantitative techniques such as ICAT, SILAC, and iTRAQ; high-throughput techniques such as X-ray crystallography and NMR spectroscopy. (5) Analysis with databases and validation of candidate biomarkers by ELISA, western blot, or immunohistochemistry. (Reproduced after minor modification from open-access publication: Martínez-Rodríguez F et al. Cells 2021;10:1854. 10.3390/cells10081854 .^[10]