CD8+ T Cells and PD-L1 Expression as Prognostic Indicators in a Low Prevalence of HPV-Associated Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV) infection detected in oropharyngeal squamous cell carcinoma (OPSCC) is associated with a better survival outcome from previous literature. However, Thailand and several Asian countries have a low prevalence of HPV-associated OPSCC and, therefore, have a low positive rate of immunostaining with p16. Tumor microenvironments (TME), including tumor-infiltrating CD8+ lymphocytes (CD8+ TIL) and programmed death ligand 1 (PD-L1), are proposed as significant prognostic indicators in addition to p16. OBJECTIVES: Explore the expression p16, CD8+ TIL, and PD-L1 and its value as prognostic indicators for overall survival (OS) in patients with OPSCC. MATERIALS AND METHODS: Data from patients with OPSCC diagnosed from 2012 to 2018 were recovered from medical records and national registry. All available glass slides and slides of immunohistochemistry (IHC) of p16, CD8, and PD-L1 were reviewed. The TME was classified into four types according to the expression pattern of PD-L1 and CD8+TIL. Overall survival (OS) was assessed using the Kaplan-Meier method and Cox regression model analysis. RESULTS: In 160 OPSCC patients, p16 was positive in 27 (16.88%). The density of CD8+ TIL was higher in the p16+ and PD-L1+ groups (p = 0.005, 0.039); however, there was no association between p16 and the status of PD-L1. P16 and CD8+ TIL were significant prognostic factors for better OS (p = 0.007, 0.001), but not PD-L1 status (p = 0.317). Among the four types of TME, carcinoma showed mainly type IV TME (PD-L1-/TIL+), while OPSCCs with type I TME (PD-L1+/TIL+) had the best survival outcome. CONCLUSIONS: The positivity of p16 and the density of CD8+ TIL were associated with better OS in OPSCC, while the status of PD-L1 was not significantly related to OS. OPSCC with type I TME (PD-L1+/TIL+) showed the best prognosis of all types of TME.

Prognostic value and clinicopathological status of PD-L1 expression and CD8+ TILs in oral squamous cell cancer patients with or without traditional risk factors.

BACKGROUND: Non-smokers, non-drinkers, and non-betel quid chewers (NSNDNBs) with oral squamous cell carcinoma (OSCC) have poor survival outcomes. Tumor microenvironment based on PD-L1/CD8+ T cell infiltrated lymphocyte (TIL) proportion is proposed as a prognostic indicator. METHODS: Immunohistochemistry staining was performed on OSCC samples from 64 patients. The PD-L1/CD8+ TILs were scored and stratified into four groups. Disease-free survival (DFS) was analyzed using a Cox regression model. RESULTS: OSCC in NSNDNB patients was associated with female sex, T1-2 classification, and PD-L1 positivity. Low CD8+ TILs correlated with perineural invasion. High CD8+ TILs correlated with improved DFS. PD-L1 positivity was not correlated with DFS. Type IV tumor microenvironment yielded the highest DFS (85%). CONCLUSION: NSNDNB status relates to PD-L1 expression regardless of CD8+ TILs infiltration. Type IV tumor microenvironment was associated with the best DFS. High CD8+ TILs resulted in better survival, while PD-L1 positivity alone was not correlated to DFS.

The Association Between Radioiodine Refractory in Papillary Thyroid Carcinoma, Sodium/Iodide Symporter Expression, and BRAF V600E Mutation.

OBJECTIVE: To study the association between radioiodine refractory papillary thyroid carcinoma, sodium/iodide symporter (NIS) expression, and the BRAF V600E mutation. METHODS: A study was conducted on 30 radioiodine refractory papillary thyroid carcinoma patients and 30 radioiodine-avid papillary thyroid carcinoma patients. The expressions of sodium/iodide symporter and BRAF V600E mutated protein were determined by immunohistochemistry using formalin-fixed, paraffin-embedded tissue. RESULTS: The mutated BRAF V600E protein was identified in 26 radioiodine refractory papillary thyroid carcinoma subjects (86.7%) and 22 radioiodine-avid papillary thyroid carcinoma subjects (73.3%), with no significant difference between the 2 groups (P = 0.3). Sodium/iodide symporter expression was detected in 4 of 30 cases (13.3%) from the radioiodine-avid papillary thyroid carcinoma group but was negative for all radioiodine refractory cases. There was no association between sodium/iodide symporter expression and radioiodine refractory papillary thyroid carcinoma (P = 0.11). Cases with positive NIS expression were likely negative for BRAF V600E mutation (3/4; P = 0.02). CONCLUSION: Papillary thyroid carcinomas with BRAF V600E mutation were more likely to be negative for NIS expression. BRAF V600E mutation and NIS expressions cannot be used to predict radioiodine sensitivity.

Identification of novel mutation in RANKL by whole-exome sequencing in a Thai family with osteopetrosis; a case report and review of RANKL osteopetrosis.

BACKGROUND: Osteopetrosis is a rare form of skeletal dysplasia characterized by increased bone density that leads to bone marrow failure, compressive neuropathy, and skeletal dysmorphism. Molecular diagnosis is essential as it guides treatment and prognosis. We report Thai siblings with an ultra-rare form of osteopetrosis. METHODS: The older brother and the younger sister presented with chronic mandibular osteomyelitis in their 20s. Since childhood, they had visual impairment, pathological fracture, and skeletal dysmorphism. Quadruplet whole-exome sequencing was performed and confirmed with Sanger sequencing. Novel mutation in TNFSF11 (RANKL) c.842T>G, p.Phe281Cys was identified in a homozygous state in both siblings. RESULTS: Surgical debridement, antibiotic, and hyperbaric oxygen therapy were used and discontinued over a 6-month period with normalization of C-reactive protein. Hematopoietic stem cell transplantation candidacy was excluded by molecular diagnosis. CONCLUSION: We report a novel mutation in an ultra-rare form of osteopetrosis. Our siblings manifested with a milder phenotype in comparison with nine cases previously published.

Intraoperative nerve monitoring in thyroid surgery: Analysis of recurrent laryngeal nerve identification and operative time.

OBJECTIVE: To evaluate the clinical value of intraoperative nerve monitoring (IONM) by comparing the procedure times for thyroidectomies performed with and without IONM. METHODS: A prospective, randomized, controlled study was conducted on 32 patients (representing 41 nerves at risk) undergoing thyroidectomies carried out by two experienced head and neck surgeons (CC & WP). Sixteen thyroidectomies were performed without IONM (the "non-IONM group"), while 16 thyroidectomies were performed with IONM (the "IONM group"). The measured datapoints were setup time, time to visual identification of the recurrent laryngeal nerve (RLN), time to confirm the RLN electrophysiologically, dissection time, and total operative time. RESULTS: With both surgeons, the IONM group had shorter visual times to RLN identification than the non-IONM group (CC: 3.7 minutes vs 5.3 minutes; WP: 3.4 minutes vs 9.7 minutes). Additionally, the electrophysiological identification time for the IONM group was shorter than the visual identification time for the non-IONM group. The setup times, dissection times, and total operative times of the 2 groups did not significantly differ (P > .05). No RLN injuries were observed. CONCLUSIONS: IONM reduces the time needed for RLN identification in thyroidectomies. Functional RLN confirmation can reassure surgeons of the operative results. Moreover, use of IONM does not significantly impact setup and total operative times. LEVEL OF EVIDENCE: 2.

The implementation of TORS for head and neck surgery in Thailand.

Transoral robotic surgery (TORS) is a novel surgical treatment of head and neck cancers, mainly for limited tumor in oropharynx and supraglottis. Despite the major advantage of favorable postoperative functional outcomes, many obstacles exist during the implementation of TORS, especially in a country where financial resources are modest. We demonstrated our experience of initiating this sophisticated technology at the largest tertiary hospital in Thailand. A retrospective review study was conducted in patients with benign or malignant lesions during 2014-2020 at Siriraj Hospital. Different periods of operation time between initial and subsequent cases were compared to evaluate learning-curve improvement. A total of 36 patients underwent TORS, with median follow-up time of 18 months. The average time of room set-up, anesthesia, and positioning was 37 ± 14, 13 ± 7, and 15 ± 7 min, respectively. Whilst, the average robotic procedure time and total time in room were 44 ± 19 and 118 ± 31 min, consecutively. There was no significant difference in any time interval, except the set-up time between initial and subsequent cases. The worthwhile utilization of TORS could be administered cost-effectively despite the complicated and daunting implementation of TORS. Whilst, meticulous planning and sufficient training prior to the initiation of TORS can favorably shorten the learning curve of operative staffs in the TORS team.

BRAF mutation in cytologically indeterminate thyroid nodules: after reclassification of a variant thyroid carcinoma.

PURPOSE: Fine-needle aspiration biopsy (FNAB) is regarded by the Bethesda system as the gold-standard investigation for stratifying the risk of malignancy of a thyroid nodule. However, some limitations affect the adequacy of the obtained materials, resulting in 30% of the cytological results remaining in the indeterminate category. We aimed to investigate the diagnostic value of the BRAF mutation in cytologically indeterminate thyroid nodules after the reclassification of a variant thyroid carcinoma. PATIENTS AND METHODS: In this prospective diagnostic study, 76 patients with FNAB findings of atypia of undetermined significance (AUS) and suspicious for malignancy (SUS) were included. The BRAF V600 mutation from FNAB was confirmed by a PCR-based method (Sanger sequencing combined with allele-specific real-time PCR techniques) and immunohistochemistry (IHC). Pathological specimens and features, including noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), were reviewed and compared to the FNAB results. RESULTS: Using the PCR-based method, the BRAF mutation was positive in 13/76 cases (17.1%), with the diagnostic values of 16.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 82.8% negative predictive value (NPV) in the AUS compared to 73.3% sensitivity, 100% specificity, 100% PPV, and 20% NPV in the SUS. For the IHC technique, only 20 of the 76 cytological specimens were qualified for testing. The BRAF mutation was positive in 13/20 cases, with the diagnostic values of 100% sensitivity, 63.6% specificity, 42.9% PPV, and 100% NPV in the AUS compared to 100% sensitivity and PPV in the SUS. The BRAF mutation was not found in the pathological reports for NIFTP. CONCLUSION: The malignancy rate is high in our data, with specific and acceptable accuracy rates for the BRAF mutation from FNAB found by using the PCR-based method. NIFTP has been introduced after the pathological reclassification. Molecular diagnosis might be useful to establish the nature of the disease.

Oncoproteomic and gene expression analyses identify prognostic biomarkers for second primary malignancy in patients with head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinoma are at increased risk of developing a second primary malignancy, which is associated with poor prognosis and early death. To help improve clinical outcome, we aimed to identify biomarkers for second primary malignancy risk prediction using the routinely obtained formalin-fixed paraffin-embedded tissues of the index head and neck cancer. Liquid chromatography-tandem mass spectrometry was initially performed for candidate biomarker discovery in 16 pairs of primary cancer tissues and their matched normal mucosal epithelia from head and neck squamous cell carcinoma patients with or without second primary malignancy. The 32 candidate proteins differentially expressed between head and neck cancers with and without second primary malignancy were identified. Among these, 30 selected candidates and seven more from literature review were further studied using NanoString nCounter gene expression assay in an independent cohort of 49 head and neck cancer patients. Focusing on the p16-negative cases, we showed that a multivariate logistic regression model comprising the expression levels of ITPR3, KMT2D, EMILIN1, and the patient's age can accurately predict second primary malignancy occurrence with 88% sensitivity and 75% specificity. Furthermore, using Cox proportional hazards regression analysis and survival analysis, high expression levels of ITPR3 and DSG3 were found to be significantly associated with shorter time to second primary malignancy development (log-rank test P = 0.017). In summary, we identified a set of genes whose expressions may serve as the prognostic biomarkers for second primary malignancy occurrence in head and neck squamous cell carcinomas. In combination with the histopathologic examination of index tumor, these biomarkers can be used to guide the optimum frequency of second primary malignancy surveillance, which may lead to early diagnosis and better survival outcome.

Notch Intracellular Domain (NICD) Expression and Clinical Manifestations of Second Primary Tumor at Esophagus in Patients with Head and Neck Squamous Cell Carcinoma.

INTRODUCTION: Second primary tumor (SPT) is a major factor that affects the survival of head and neck squamous cell carcinoma (HNSCC) patients, and the esophagus is a common site. Detection of SPT is essential for optimal HNSCC treatment planning and follow-up. Mutation of the NOTCH1 gene is common in head and neck cancer. However, details relating to Notch signaling and clinical outcomes among different primary tumors are still inconclusive. This study aimed to identify the role of the Notch signaling pathway in HNSCC, and to compare NOTCH1 expression in HNSCC compared between those with and without SPT at esophagus while focusing on the Notch intracellular domain (NICD). METHODS: Twenty-three cases of esophageal SPT and 47 non-SPT controls that were treated at Siriraj Hospital during 2006-2017 were included. Patient information and clinical outcomes were analyzed. NICD expression demonstrated by immunohistochemistry technique in formalin-fixed paraffin-embedded specimens was studied. RESULTS: Mean age of SPT and non-SPT was 55.13 and 62.09 years, respectively, and 94.3% of patients were male. Regarding SPT detection, 82.6% were synchronous and 17.4% were metachronous. There was significantly more active smoking among SPT than among non-SPT (87.0% vs 51.1%, p=0.01). Active alcohol use was also significantly greater among SPT than among non-SPT (87.0% vs 61.7%; p=0.04). Hypopharynx was the most common primary tumor site among SPT. Three-year and 5-year survival among SPT patients was 38.0% and 25.3%, respectively. NICD expression was absent in 52.2% of SPT, and in 53.3% of non-SPT. NICD expression intensity was mostly weak or moderate. CONCLUSION: Active smoking and alcohol use were found to be significantly associated with SPT development. A high percentage of NICD inactivation was noted in HNSCC with no significant difference between groups. The Notch signaling pathway is involved in HNSCC tumorigenesis, but may not be a suitable molecular marker for SPT development.

Prevalence of HPV infection in hypopharyngeal and laryngeal squamous cell carcinoma at Thailand's largest tertiary referral center.

BACKGROUND: Following the well-established relationship between human papillomavirus (HPV) and cervical carcinoma, the carcinogenicity of this virus has also been confirmed in subsets of head and neck carcinoma (HNCA), but mainly in the oropharynx. Other subsites of HNCA with less known association to HPV have never been studied in Thailand. Accordingly, the aim of this study was to investigate the prevalence of HPV DNA in hypopharyngeal and laryngeal squamous cell carcinoma in Thai population. METHODS: This cross-sectional study included hypopharyngeal and laryngeal squamous cell carcinoma patients diagnosed and treated at the Department of Otorhinolaryngology, Siriraj Hospital during the September 2011-December 2013 study period. Presence of HPV genome was confirmed by polymerase chain reaction from pathologically-confirmed fresh specimens. Demographic data and risk factors of HPV infection were evaluated. RESULTS: Eighty patients were included, and 95% of those were male. Only one patient was noted with positive HPV-62 serotype. Most patients consumed tobacco and/or alcohol. Five patients had no risk factors for cancer development. Risk of HPV infection was evaluated by self-reporting questionnaire. The mean age of sexual debut was 20.17 years. Forty-eight patients had multiple sexual partners. Sixteen and seven patients had history of sexually transmitted disease infection and habitual oral sex contact, respectively. CONCLUSION: There was no oncogenic HPV DNA detected within pathologic specimens of laryngeal and hypopharyngeal cancers in this study. Compared to rates reported from developed countries, the prevalence of HPV-related HNCA in Thailand is very low.

Human Papillomavirus Infection in Oral Cavity and Oropharyngeal Cancers: Are They the Same Story?

Background: Nowadays, human papillomavirus (HPV) infection becomes the main risk factor for head and neck cancer development. In Thailand, the related role of this viral infection to head and neck cancer is still unknown and not well established. Objective: To identify different characteristics of oral cavity and oropharyngeal cancer, and to determine the HPV-associated prevalence of these two tumor types in Thailand, which is unlike the Western countries. Material and Method: Between 2010 and 2012, a cross-sectional study was performed in 23 oral cavity and 23 oropharyngeal cancer patients. HPV genome was studied in all of them from pathological confirmed fresh specimens. Risks of HPV infection were collected using self-reported questionnaire. Results: The prevalence of HPV-related oropharyngeal cancer was significantly noted in 26.09% (p = 0.009), while no demonstrable HPV-associated prevalence in oral cavity cancer. In addition, the routes of HPV infection were not identifiable. Conclusion: Oral cavity and oropharygeal cancers are not only anatomically distinct, but also greatly differed in their characteristics and pathophysiology. The percentage of HPV-related tumors in Thailand is considerably low when compared to the Western countries. However, the impact on treatment modification cannot yet be universally applied.

Clinical Effectiveness of Hyperbaric Oxygen Therapy in Complex Wounds.

Hyperbaric Oxygen (HBO, HBO2) Therapy is a non-invasive therapy. It has been applied as adjuvant treatment in many medical conditions over the past 50 years. Different treatment protocols have been proven effective for specifically indicated conditions. To evaluate the clinical effectiveness of Hyperbaric Oxygen (HBO) Therapy as an adjunctive treatment for patients with complex wounds. In this prospective cohort study, 40 patients with complex wounds were included. All patients received HBO. HBO was delivered with 100% oxygen for 90 min at 2.0-2.4 ATA. Wound sizes were assessed by one wound surgeon before, during, and every 2 weeks for a total of 12 months after HBO. An analysis of demographic data, wound size and wound photography was performed. Over the 22-month period ending October 31, 2013, 40 patients (21 men and 19 women) with a mean age of 59.73 (range, 29-88) with complex wounds were included. All complex wounds studied were at least 6 months old. The mean wound size was 16.72 cm(2) in diameter. Thirty-one patients with complex wounds healed after the completion of a series of HBO treatments (77.5%). Two orocutaneous fistulas were completely closed without further surgery. After 5 HBO treatments, the wound size reduced by 29.7% on average (p = 1.24 × 10(-6)). After 10 HBO treatments, the wound size statistically significantly reduced by an additional 16.9% (p = 0.0002). There were no complications in this study. Wound healing process was accelerated by HBO. Significant wound size reduction was noted after 5 HBO treatments. Because the biggest reduction in wound size occurred within the first 10 HBO treatments, it is important to conduct these first treatments without interruption. HBO is an effective and safe treatment modality for complex wounds.

Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells.

Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.

Pharmacological genome demethylation increases radiosensitivity of head and neck squamous carcinoma cells.

Aberrant inactivation of tumor suppressor genes by promoter hypermethylation has been recognized as a crucial step of tumor development and is related to aggressiveness and therapy resistance. To identify potential novel treatment strategies, we evaluated pharmacological genome demethylation for the increase of irradiation treatment effectiveness in head and neck squamous cell carcinoma (HNSCC) in this in vitro study. HNSCC cells were cultured with 2 different concentrations of 5-azacytidine (5-Aza) for 72 h, followed by a single fraction irradiation with 4 or 50 Gy, respectively. To show successful genome demethylation, the methylation status of the tumor suppressor gene hic1 (hypermethylated in cancer) promoter was analyzed by methylation specific PCR (MSP) as well as hic1 transcription by quantitative RT-PCR. Survival, apoptosis, viability, and migration of the tumor cells were analyzed as functional parameters of combined treatment response. After 5-Aza treatment the hic1 promoter was demethylated and gene transcription restored demonstrating genome demethylation. 5-Aza treated cells tended to be less viable and showed decreased survival indicated by lower colony numbers. Apoptosis and migration were not affected. The combined application of irradiation and 5-Aza significantly reduced survival compared to the single treatments. Accordingly, apoptosis was strongly increased after combined 4 Gy/5-Aza treatment. Viability was not additionally affected by combined treatment. Migration was affected weakly by combined high dosage irradiation/5­Aza treatment. Our data show that the combined application of 5-Aza and irradiation is effective in vitro. A demethylating concept prior to irradiation should be further evaluated for its potential to reduce irradiation resistance.

Demethylation treatment restores hic1 expression and impairs aggressiveness of head and neck squamous cell carcinoma.

Promoter hypermethylation of tumor suppressor genes is a common feature of primary cancer cells. However, at date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis are not yet been well defined. In the present study we analysed the methylation status of the gene hypermethylated in cancer-1 (hic1), a gene located on chromosome 17p13.3, a region frequently lost in HNSCC. We analysed 22 HNSCC samples and three cell lines using methylation specific PCR (MSP). We found hic1 methylated in 21 out of 22 samples and in all three cell lines. Treatment of the cell lines with the demethylating agent 5-Azacytidin (5-Aza) resulted in the demethylation of the hic1 promoter and reactivation of hic1 expression as determined by MSP, qPCR and Western blot. Functional analyses revealed decreased proliferative activity and colony forming ability of treated cells. In summary, we found in HNSCC hic1 regulated by promoter methylation. 5-Aza application resulted in the reexpression of hic1 and was followed by decreased aggressiveness of the cancer cells. Our data indicate that hic1 might be a player in HNSCC development and suggest further evaluation of 5-Aza for HNSCC treatment.