A 73-year-old male patient was referred from his general dentist for further evaluation of whitish lesion on the attached gingiva and associated peri-implantitis. A panoramic view shows generalized alveolar bone loss and calculi deposition in the peri-implant region (#42, #43, #44), and the right mandibular anterior and premolar area showed peri-implant crestal bone loss. Laboratory findings were within normal limits. Other several oncogenic protein elevation situations, including tobacco and/or alcohol use, no nutritional deficiencies, no findings of ionizing radiation exposure, no immunodeficiency or immunosuppressant, and other removal prosthesis irritations, were all excluded. A whitish lesion was excised, and the specimen was sent to an oral pathologist. The contaminated implant surface was treated with a laser. The pathologic diagnosis was confirmed as oral candidiasis. The patient underwent laser treatment three times to treat the peri-implantitis lesion. One year later, his implant of #42, #43, and #44 area were removed due to peri-implantitis at the local clinic. From the referral letter of local clinic, the implant system was internal frictional connection type having a SLA surface, which was installed for more than 10 years.
About 3 years later after the laser operation, a bulging mass was identified on the lingual side of #43 and #44 area. An incisional biopsy was performed and was diagnosed as a SCC (Fig. 1). Further work-up was performed including lab, chest X-ray, ECG, MRI, contrast CT, PET-CT, and neck sonography. The patient was diagnosed with cT4aN2cM0 stage IVA according to the TNM staging system proposed by the American Joint Committee on Cancer (AJCC, 2018). He was immediately scheduled for an operation that included selective neck dissection, mass resection with marginal mandibulectomy, and reconstruction with a radial forearm free flap. The final pathologic report was well-differentiated squamous cell carcinoma, with a 1.5 × 2.0-cm size of tumor, no metastasis to any of the 27 regional lymph nodes, and clear surgical resection margin. Vascular and perineural invasions were not observed; thus, he was diagnosed as pT1N0M0, stage I. Especially, rather than on the interface between the implant and the bone, tumor cells occurred on the surface of the mucosal soft tissue first and penetrated deeply along the implant threads. Neither adjuvant radiotherapy nor chemotherapy was not administered to the patient.
A metastatic lymph node was found at the right ipsilateral level IV on enhanced CT taken 4 months postoperatively. Selective neck dissection, including right level IV, was performed, and a newly developed suspicious for malignant lesion was found on the right maxilla. The patient’s maxilla lesion was confirmed for SCC by incisional biopsy; therefore, he underwent an additional surgery 13 days after second selective neck dissection (Fig. 2). The final pathologic report on the maxillary lesion was well-differentiated SCC with a 2.0 × 1.4-cm size of tumor; depth of invasion was 0.7 cm. Involvement of underlying bone was present. Surgical resection margins were clear.
The surgically removed specimens were fixed in 10% neutral buffered formalin, processed following a routine protocol, and serial micro-sections with different antisera were also prepared for immunohistochemical staining. All data files of the patient were selected from the files of the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital under the approval of the institutional review board of Seoul National University (S-D20170026).
IP-HPLC analysis for the protein extract obtained from the serum of patients
The patient’s blood was collected preoperatively, 10 days postoperatively, and 3 months postoperatively. After precipitation at room temperature, the samples were centrifuged at 4000 rpm for 20 min. Only the supernants were collected and mixed with lysis buffer and used for IP-HPLC. We applied 100 μg of each protein extract to the immunoprecipitation procedure using a protein A/G agarose column (Amicogen Co., Korea). The protein A/G agarose columns were separately pre-incubated with 1 μg of each of the 20 different antisera, including p53, muc1, muc4, TGF-β1, survivin, Wnt1, E-cadherin, β-catenin, matrix metalloproteinase (MMP)-3, MMP-10, TNFα, HER1, HER2, PAI-1, NRAS, KRAS, CEA, Met, FASL, and ERb. Briefly, the protein samples were mixed with 5 ml of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, and 0.5% NP-40) and incubated in the protein A/G agarose columns at 10 °C for 1 h. The columns were placed on a rotating stirrer during the incubation. After washing each column with a sufficient amount of PBS solution (pH 7.3, 137 mM NaCl, 2.7 mM KCl, 43 mM Na2HPO4-7H2O, and 1.4 mM KH2PO4), the target protein was eluted with 150 μl of IgG elution buffer (Pierce Co., USA). The immunoprecipitated proteins were analyzed by HPLC (1100 series®, Agilent, USA) using a reverse-phase column and micro-analytical detector system (SG Hightech Co., Korea), operated with a 0.15-M NaCl and 20% acetonitrile solution at 0.4 mL/min for 30 min, and analyzed via UV spectroscopy at 280 nm. In the IP-HPLC results, the sample protein peak areas obtained from the HPLC analysis in the negative control were used to eliminate the antibody peak area (mAU*s) [7,8,9]. To compare preoperative and postoperative serum protein, the protein peak area values were proportionally normalized by the α-tubulin value and plotted as a bar.
IP-HPLC analysis of extracted tumor protein
Protein extracts were prepared from tumor tissue, 100 μg each protein extract to the immunoprecipitation procedure using a protein A/G agarose column. The protein A/G agarose columns were individually pre-incubated with 1 μg of each of the 9 different antisera, including TNFα, NRAS, HER2, Met, E-cadherin, p53, survivin, TGF-β1, and NFκB. Briefly, the protein samples were mixed with 5 ml of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, and 0.5% NP-40) and incubated in the protein A/G agarose columns at 10 °C for 1 h. To compare the normal gingiva and SCC tissue protein, the protein peak area values were proportionally normalized by the α-tubulin value and plotted as a bar.
Laboratory analysis
Routine laboratory results were collected, including complete blood cell counts (CBC) with differential count, C-reactive protein (CRP) levels, and erythrocyte sedimentation rate (ESR). A modest elevation in the plasma CRP in the range observed in apparently healthy individuals is a strong predictor of future vascular events [10]. Chen et al. [11] reported the presence of elevated preoperative serum CRP level (> 5.0 mg/L) is an independent prognostic indicator of oral cancer. The results from the blood sample tests were compared from the first visit, preoperatively, postoperatively, and at the time of recurrence.
IP-HPLC analysis from the serum of patients
The IP-HPLC analyses revealed that p53, E-cadherin, β-catenin, MMP-10, HER2, NRAS, Met, and ERb had decreased at postoperative day 10. Other protein markers related to oncogenic signaling were increased at postoperative day 10. This suggests that oncogenic proteins, such as p53, E-cadherin, β-catenin, MMP-10, HER2, NRAS, Met, and ERb were released from the primary tumor; therefore, serum levels of these oncogenic proteins decreased after tumor-ablative surgery (Fig. 3). In 3 months postoperatively using the patient’s serum, all oncogenic protein markers were elevated and tumor recurrence or metastasis was suspected (Fig. 4).
IP-HPLC analysis from extracted tumor protein
IP-HPLC analyses revealed that NRAS, Met, p53, and NFκB were overexpressed in the SCC tissue in the comparison of oncogenic protein levels between normal gingiva and SCC tissue (Fig. 5).
Laboratory findings
Inflammatory markers such as white blood cell count (WBC), absolute neutrophil count (ANC), ESR, and CRP were elevated immediately postoperatively. CRP was not conducted preoperatively, and so, that data cannot be compared to other time points; however, CRP levels had not changed significantly before and after the second operation (right level IV selective neck dissection, Additional file 1: Figure S1). Notably, ESR counts were elevated in the preoperative period, as determined when a malignant lesion was confirmed by incisional biopsy and compared with a sample taking at the first visit. That indicates that some inflammatory reactions can affect the potential of malignant transformation.
Perioperative changes in the RBCs, hemoglobin, hematocrit showed that the levels were decreased during the post-surgery, but tended to recover over time (Additional file 2: Figure S2). The proportion of segmented neutrophils increased immediately after surgery, which are characteristic cells of acute inflammatory reactions, because they moved to surgical wound immediately after trauma in several minutes. The graphed curves of lymphocyte, monocyte, eosinophil, and basophil levels showed opposite trends from the segmented neutrophil levels (Additional file 3: Figure S3).