Chromosome 17 In Situ Hybridization Grid-based Analysis in Oral Squamous Cell Carcinoma

Materials and Methods

Study group. For the purposes of our study, fifty (n=50) archived, formalin-fixed and paraffin-embedded tissue specimens of histologically confirmed primary OSCC were used. The hospital ethics committee consented to the use of these tissues in the Department of Pathology, Hippocration Hospital, University of Athens, Greece for research purposes, according to World Medical Association Declaration of Helsinki guidelines (2008, revised in 2014). The tissue samples were fixed in 10% neutral-buffered formalin. Hematoxylin and eosin (H&E)-stained slides of the cor¬responding samples were reviewed for confirmation of histopathological diagnoses. All lesions were classified according to the histological typing criteria of the World Health Organization (WHO) Pathology Series (9). The information regarding HPV DNA status (positivity or not) was derived from the patients' medical file records. Among them, eighteen (n=18) HPV DNA positive cases were recorded. HPV 16/31/53 High Risk (HR) subtypes were detected mainly by analyzing the corresponding cases. Clinicopathological data of the examined cases are demonstrated in Table I.

Chromogenic in situ hybridization (CISH) assay. For the purposes of our study, we selected and applied the SPOT LIGHT CISH assay (Zymed/InVitrogen, San Fransisco, CA, USA) based on Centromere Enumeration Probe 17 (CEP17). In brief, the sections were deparaffinized and incubated in pre-treatment buffer in a temperature-controlled microwave oven at 92°C for 10 min using a Spot-Light formalin-fixed, paraffin-embedded reagent kit (Zymed Inc. San Francisco, CA, USA). The sections were allowed to cool at room temperature for 20 min and then washed with phosphate-buffered saline. Enzymatic digestion was carried out by applying 100 ml of formalin-fixed, paraffin-embedded digestion enzyme onto the slides for 10 to 15 min at room temperature. The slides were washed with phosphate-buffered saline and dehydrated with graded ethanol. The ready-to-use biotin-labelled probe was applied onto the slides, which were covered with a coverslip. The slides were denatured on a hot plate at 94°C for 3 min, and hybridization was performed overnight at 37°C. After hybridization, the slides were washed with 0.5 ml standard saline citrate for 5 min at 75°C, followed by 3 washes in phosphate-buffered saline 0.2% concentration at room temperature. The probe was detected with sequential incubations with avidin-peroxidase and 3,3 - diaminobenzidine (3,3-DAB) according to the manufacturer's instructions (Zymed Inc., San Francisco, CA, USA). Tissue sections were lightly counterstained with hematoxylin, dehydrated in graded ethanol and embedded. At the end of the process, CISH CEP17 signals were easily visualized as dark brown scattered dots, using a conventional, bright-field microscope (Figure 1). Interpretation of Chr 17 signals was based on Zymed's Evaluation Chart for CISH. According to these guidelines, two centromere copies per nucleus demonstrate normal, diploid pattern, whereas three and more centromere copies per nucleus show chromosome polysomy in ≥50% of isolated nuclei or nuclei in small clusters. These measurements refer to intact, non-overlapped cancerous nuclei on every stained slide (Figure 1c). Additionally, one centromeric signal per nucleus represents chromosome monosomy (loss of one chromosome).

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Figure 1.

Chromosome 17 CISH analysis in OSCC a. Schematic presentation of the grid-based cover slip that was implemented in CISH slide screening-mapping. Note this prototype grid consisting of seventy-two (n=72) rectangular square areas arranged in 12 columns and 6 rows. The dark stained rectangular square represents a potential field of interest under bright-field microscope b. A snapshot of the grid-based coverslip demonstrating a fine cross made by laser inscription that splits its surface in four square segments c. Chromosome 17 polysomy in a malignant tissue section. Note 3 to 5 isolated dark centromeric signals per nucleus in a sub-group of cancerous stained nuclei (black arrows; DAB chromogen; original magnification 400×).

Slide screening process. The screening procedure regarding the corresponding archived CISH stained slides was performed using bright field microscopy (microscope Olympus CX-31, Menvile, NY, USA, with ToupView image analysis software, (ProWay/ToupTek Protonics, Hangzhou, China) with combined 100X/400X magnification. Concerning the CISH slides evaluation, screening was based on a set of novel designed cover slips with integrated spatial rectangular grid (Grid Cover Slip, now GCS) in order to provide an efficient way of slide eye-scanning ensuring the systematic inspection with full visual coverage of the slide. The grid patterns on GCSs were produced by applying a prototype home-built high precision and efficient Femtosecond Laser based Micromachining system (Femtosecond Laser Micromachining-FLM) based on a high power femtosecond fiber laser (Model: HE-1060-1μJ-fs, Fianium, Southampton, UK) Laser inscription techniques can allow direct writing and transfer of predetermined patterns by means of surface or sub-surface micromachining in a variety of materials ranging from glasses to soft polymeric materials. Adjusting the laser writing characteristics, the inscribed grid lines' width could be flexibly defined to a range from 1 μm to 500 μm. The FLM inscription technique has been applied here for the first time towards the fabrication of a visible rectangular grid in a microscope CISH slide's cover slip, for research medical purposes. Commercially available cover slips, by typical borosilicate-based glass, 50×24×0.5 mm (length × width × thick) (Menarini, Florence, Italy) were used in the study. The grid's size and observation windows' density could be modified according to researcher's needs. According to our previous experience, we selected a prototype grid consisting of seventy-two (n=72) rectangular squares, of typical surface area of 4 mm × 4 mm equal to 16 mm², arranged in 12 columns and 6 rows. Each square segment can have also appropriate spatial indexing marks (printed with various techniques like FLM) in a suitable way to assure minimum visual interference under microscope inspection (Figure 1b). The indexing can be provided by sequential numbering of the square cells as shown schematically in Figure 1a. In the specific example, due to the selection of grid's windows size, six residual non-rectangular cells, numbered at the figure as 73-78, appear also at the right part of the grid that do not affect the generalization of the proposed grid's architecture. The developed GCSs were used for the visual detection of Chr 17 centromeric signals on the corresponding CISH slides by perfectly covering the entire conventional coverslips.

Statistical analysis. Statistics software package IBM SPSS v25 (SPSS Inc, Chicago, IL, USA) was used for statistical analysis. Associations between variables were assessed with of Pearson Chi-Square (χ²) test and Fisher's exact. Correlation analysis with Spearman Rank test was performed for variables with significant chi² associations. Two-tailed p-values ≤0.05 were considered statistically significant. Results and correlations (p-values) are described in Table I.