Lymphangiogenesis and Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Patients and Methods

Patients. The present study was a prospective study. This study was conducted in full accordance with the World Medical Association Declaration of Helsinki. All procedures used in this research were approved by the Ethical Committee of Kobe University Hospital (approval number: No. 180171) and informed consent was obtained from each patient. All tumors were resected at the Department of Oral and Maxillofacial Surgery, Kobe University Graduate School of Medicine, Japan, between 2013 and 2017. The inclusion criteria were (i) no preoperative chemotherapy, hormone therapy or radiotherapy, and (ii) sufficient tumor tissues and normal tissues available. Patients that did not want to participate were not included in the study. All patients had surgery as their first line of management. Clinical data including age, gender, smoking, alcohol, primary tumor site, pathological T stage, LN status and histological grade were abstracted from the clinical records and are shown in Table I. The median age of the patients was 69 (range=41-88 years) at the time of diagnosis; the group included 20 (60.6%) male and 13 (39.4%) female. A surgical excision with a clear 10 mm margin from the tumor edge was performed for each patient.

Quantitative real-time RT-PCR. The normal and tumor tissues from 33 previously untreated patients with OSCC were snap-frozen at the time of surgery and stored at −80°C. Briefly, approximately 100 mg was homogenized using 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) at room temperature, and total RNA was extracted. The RNA was further cleaned up with RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The cDNA was synthesized (600 ng of total RNA) using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The mRNA expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), podoplanin and LYVE-1 was analyzed using quantitative real-time polymerase chain reaction (qPCR). GAPDH was used as a reference gene. The quantification of mRNA transcription was performed using a StepOne Real-Time PCR System (Applied Biosystems). Real-time PCR (20 μl) was performed with 0.5 μM forward primer, 0.5 μM reverse primer, and 1 μl of the 10-times diluted cDNA template from the reverse transcription reaction and 10 μl (2×) Power SYBR Green Master Mix (Applied Biosystems). The PCR conditions were as follows: one cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. GAPDH primers were obtained from Invitrogen. QuantiTect® Primer Assays (Qiagen: QT02321263, QT00034566) for Podoplanin and LYVE-1 were obtained from Qiagen (Valencia, CA, USA).

Immunohistochemistry. Specimens of complete resections were selected so that both normal and tumor tissues were present on each slide. The paraffin-embedded tissue blocks were sliced into 4 μm thick sections for subsequent histological examinations. These were deparaffinized with xylene, rehydrated in a graded alcohol series, antigen retrieval was performed by proteinase K, endogenous peroxidase activity was blocked by incubation with 3% H₂O₂, and were incubated overnight at 4°C with the following primary antibodies in Can Get Signal Immuno-stain Solution A (Toyobo, Osaka, Japan); polyclonal sheep anti-human podoplanin (1:200 dilution, AF3670, R&D Systems, Minneapolis, USA), polyclonal goat anti-human LYVE1 (1:200 dilution, AF2089, R&D Systems). Following treatment, sections were incubated with horseradish peroxidase (HRP)-conjugated anti-sheep IgG polyclonal antibody (1:100 dilution in PBS, Abcam, Cambridge, UK) and anti-goat IgG polyclonal antibody (Nichirei, Tokyo, Japan) for 2 h at room temperature. The signal was developed as a brown stain using the peroxidase substrate 3, 3-diaminobenzidine (Nichirei). The sections were counterstained with hematoxylin and were captured under a microscope. The samples were observed with BZ-X700 (Keyence, Osaka, Japan).

Evaluation of stained slides. The slides were analyzed randomly by two authors, blinded to the clinical data. Podoplanin expression was scored as described by Rodrigo et al. (6). The proportion of immunoreactive positive cells was scored as 0 (0 %), 1 (less than 10%), 2 (10-29%), 3 (30-49%), 4 (50-79%), or 5 (more than 80%). The staining intensity was rated on a scale of 0-3 (0=negative, 1=weak, 2=moderate, and 3=strong) (Figure 1). German immunoreactive score (IRS) was calculated by multiplying the podoplamin expression and staining intensity scores. An IRS score above the median (7 or higher) was considered as high reactivity and between 0-6 as low reactivity. The scoring method is described in Table II. LYVE-1 expression was counted by lymphatic vessel density (LVD). Three fields with the highest LVD (hot spots) were identified in each sample at ×40 magnification. The number of lymphatic vessels was counted within 3 microscopic fields at a magnification of ×200 and the median was used for statistical analysis (Figure 2). The section was identified as having LYVE-1 high expression when the number of LYVE-1-positive vessels was higher than the median number. The section was identified as having LYVE-1 low expression when the number of LYVE-1-positive vessels was lower than the median number (7).

Statistical analysis. The expression of each target gene normalized to an endogenous reference gene relative to the calibrator was calculated by using the ΔΔCT method (Applied Biosystems). The calibrator was the 1× sample, and all other quantities were expressed as an n-fold difference. ΔΔCT was calculated (tumor tissues relative to normal tissues) and a two-tailed Mann–Whitney test was used to test statistical significance. The Mann–Whitney test was used for the associations between the mRNA expression levels of each gene and clinicopathological characteristics. The associations between immunohistochemical expression of each gene and primary tumor site were examined using the Mann–Whitney U-test. The associations between immunohistochemical expression of each gene and other clinicopathological variables were examined using the chi-square test. The data collection and statistical analyses were performed using IBM SPSS Statistics for Windows version 22.0 (IBM Corp., Armonk, NY, USA). Data was presented as mean±standard error (SE). A value of p<0.05 was considered statistically significant.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Expression of podoplanin. The staining intensity was rated on a scale of 0-3 (0=negaive, 1=weak, 2=moderate, 3=strong). (A) Weak: faint staining, (B) Moderate: staining between dark brown and weak staining, (C) Strong: dark brown staining of cells. Scale bar=100 μm.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Expression of LYVE-1. (A) LYVE-1-positive lymphatic vessels were observed in normal tissue. (B) LYVE-1-positive lymphatic vessels were observed in tumor tissue. Scale bar=100 μm.