5-Methylcytosine immunohistochemistry for predicting cutaneous melanoma prognosis.

There is a correlation between DNA methylation and the diseased stage and poor survival. 5-methylcytosine (5-mC) is one of the epigenetic modifications of bases that researchers focus on. Staining with 5-mC immunohistochemistry was used to examine pathological samples taken from individuals diagnosed with cutaneous melanoma. Between Breslow levels 2 and 4, there was a significant difference in the H-score of 5-mC expression (p = 0.046). A significant reduction in 5-mC expression H-scores was seen in patients who were diagnosed with ulcers (p = 0.039). It was shown that patients with low 5-mC had a significantly worse overall survival rate (p = 0.027).

Evaluation of P53 immunostaining in patients with cutaneous melanoma.

P53 is a tumor suppressor gene that is mutated in numerous types of cancer. The aim of the present study was to determine the frequency of this mutation in cutaneous melanomas and to conduct clinicopathological characteristics and clinical outcome association analyses with the P53 mutation. P53 immunohistochemical staining was used as a surrogate marker for P53 mutation analysis to assess P53 status. In the present study, 50 pathological samples of cutaneous melanoma from 2012 to 2018 at Chulalongkorn University (Bangkok, Thailand), were subjected to anti-P53 immunohistochemistry, followed by an examination of the association between P53 statuses and clinical and pathological characteristics, along with clinical outcomes. A positive staining for anti-P53 antibody was detected in 30% of patients (15/50) with cutaneous melanomas. Positivity was significantly associated with female sex, nodular histological subtype and Breslow level 4. Cox regression analysis revealed that an age >65.5 years and Breslow grade 4 disease were associated with mortality. The Kaplan-Meier curve revealed a shorter duration of recurrence time in the P53 mutation than P53 wild type. In the present study, P53 mutations in specific cases of cutaneous melanoma were identified. Notably, patients who were older and/or had a Breslow score of 4 exhibited an increased risk of mortality. These findings suggested the potential involvement of P53 mutations in cutaneous melanoma, highlighting the necessity for further investigations to improve understanding of their roles.

Protein expression analysis for predicting recurrent laryngeal squamous cell carcinoma.

The expression of a number of proteins plays a major role in predicting recurrent laryngeal squamous cell carcinoma (LSCC). Thus, the aim of the present study was to investigate the expression of 16 selected proteins as prognostic indicators for recurrent and non-recurrent LSCC. Samples from a total of 41 patients with LSCC were investigated by immunohistochemistry. Digital image analysis was performed, and various associated factors were calculated. Histoscore (H-score) and receiver operating characteristic curves were used to divide protein expression in high and low for predicting disease recurrence. Disease-free survival (DFS) curves, crude hazard ratios (HRs) and adjusted HRs were analyzed and compared. Significantly different H-scores were found between the recurrent and non-recurrent groups in terms of pRb and c-Met expression. pRb was expressed at high levels in recurrent LSCC, while c-Met was expressed at low levels. Patients with low pRb expression had a longer DFS than those with high pRb expression (log-rank χ2, 5.161; P=0.023). Patients with high c-Met expression had a longer DFS than those with low c-Met expression (log-rank χ2, 6.441; P=0.011). Moreover, patients with high pRb expression and low c-Met expression had the shortest DFS (log-rank χ2, 11.827; P=0.008). Differentiated histological factors had an impact on the risk of recurrence (Cox regression test; crude HR, 9.53; 95% confidence interval, 1.214-74.819; P=0.032). The present study demonstrated that the grading of differentiated squamous cell carcinoma, pRb and c-Met expression are the most useful prognostic factors for the prediction of recurrent LSCC. These might be further applied as potential markers for clinical use.

Inhibition of non-small cell lung cancer (NSCLC) proliferation through targeting G6PD.

Background: Mounting evidence has linked cancer metabolic reprogramming with altered redox homeostasis. The pentose phosphate pathway (PPP) is one of the key metabolism-related pathways that has been enhanced to promote cancer growth. The glucose 6-phosphate dehydrogenase (G6PD) of this pathway generates reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for controlling cellular redox homeostasis. Objective: This research aimed to investigate the growth-promoting effects of G6PD in non-small cell lung cancer (NSCLC). Methods: Clinical characteristics and G6PD expression levels in lung tissues of 64 patients diagnosed with lung cancer at the King Chulalongkorn Memorial Hospital (Bangkok, Thailand) during 2009-2014 were analyzed. G6PD activity in NSCLC cell lines, including NCI-H1975 and NCI-H292, was experimentally inhibited using DHEA and siG6PD to study cancer cell proliferation and migration. Results: The positive expression of G6PD in NSCLC tissues was detected by immunohistochemical staining and was found to be associated with squamous cells. G6PD expression levels and activity also coincided with the proliferation rate of NSCLC cell lines. Suppression of G6PD-induced apoptosis in NSCLC cell lines by increasing Bax/Bcl-2 ratio expression. The addition of D-(-)-ribose, which is an end-product of the PPP, increased the survival of G6PD-deficient NSCLC cell lines. Conclusion: Collectively, these findings demonstrated that G6PD might play an important role in the carcinogenesis of NSCLC. Inhibition of G6PD might provide a therapeutic strategy for the treatment of NSCLC.

Identifying potential immuno-oncology targets in salivary gland mucoepidermoid carcinoma based on inflammatory status and treatment response.

BACKGROUND: Mucoepidermoid carcinoma is a rare salivary gland malignant tumour. This study aimed to investigate inflammatory and immune signatures of mucoepidermoid carcinoma by identifying potential proteo-transcriptomic biomarkers towards the development of precision immuno-oncology treatment strategies. METHODS: A total of 30 biopsies obtained from patients diagnosed with mucoepidermoid carcinoma between 2013 and 2022 were analysed after H&E staining for scoring of histological inflammatory stroma subtypes and inflammatory hotspots with QuPath. Multiplex immunofluorescence staining and NanoString nCounter PanCancer IO 360™ panel were used to assess stroma and tumour inflammation signatures in high grade mucoepidermoid carcinoma cases in the tumour microenvironment via proteomics and transcriptomics, respectively. RESULTS: Inflammatory cells within the histological inflammatory stroma inflammatory (HIS-INF/hot) tumour neighbourhoods were greater compared to the histological inflammatory stroma-immune desert (HIS-ID/cold) (p = 0.001). A similar trend was observed between treatment non-responders and responders in stroma neighbourhoods (p = 0.0625) and in stroma-to-interface inflammatory hotspots (p = 0.0081), indicating an augmented inflammatory response in hot tumours and non-responders. Furthermore, there were striking differences in the expression of pan-immune leukocyte marker CD45 between responders and non responders particularly in the tumour neighbourhoods (p = 0.0341), but such were not robust for PD-1 and macrophage fractions. Additionally, transcriptomic analysis revealed key differences in leukocyte activation profiles between responders and non-responders. CONCLUSION: This preliminary report unveils the importance of assessing immune leukocyte cellular fractions and pathways for future prognostic biomarker discoveries in mucoepidermoid carcinoma as per the involvement of CD45-driven inflammatory and immune mediators in high grade mucoepidermoid carcinoma in non-responders to treatment. These findings will potentially contribute to the development of novel personalised immunotherapies.

High 4-1BB Expression in PBMCs and Tumor Infiltrating Lymphocytes (TILs) in Patients with Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: 4-1BB is a costimulatory immune-activating molecule. Increased amounts of this protein have previously been found in the plasma of patients with oropharyngeal and oral cancer. Here, we focused on this molecule that functions as part of the immune system. We investigated 4-1BB in the peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) of patients with head and neck squamous cell cancer (HNSCC). MATERIALS AND METHODS: The expression level of 4-1BB in the PBMCs was determined using real-time polymerase chain reaction (PCR). The TIMER (Tumor Immune Estimation Resource) web server was utilized to approximate the 4-1BB level in HNSCC TILs. Moreover, 4-1BB immunohistochemistry (IHC) was used to validate TILs in four organs of HNSCC, including oral cancer (OC), oropharyngeal cancer (OPC), sinonasal cancer (SNC), and laryngeal cancer (LC), in both the tumor area and adjacent normal epithelium. The difference in 4-1BB expression levels in various groups was assessed using a Kruskal-Wallis test and an independent sample t-test. RESULTS: The level of 4-1BB expression in PBMCs was highest in OPC, followed by OC and healthy controls (HC). Significant differences were discovered between HC and OPC and between OC and OPC. Bioinformatics revealed a substantial correlation between 4-1BB expression level and lymphocyte infiltration in HNSCC, including B cells, CD8+ T cells, and CD4+ T cells. IHC validation in HNSCC tissue revealed that the average number of 4-1BB positive TILs in all four HNSCC subtypes was considerably greater than the number of lymphocytes seen in adjacent normal tissue. Interestingly, the number of lymphocytes that were 4-1BB positive increased in relation to the TIL level. CONCLUSION: A higher number of 4-1BB expression levels were found in the PBMCs and TILs of HNSCC patients, implying that 4-1BB may be a promising approach for HNSCC patients to improve their immune function. It is important to study and create a treatment that uses 4-1BB medicine as well as existing drugs.

DEK::AFF2 Fusion Carcinomas of Head and Neck.

A novel DEK::AFF2 fusion carcinoma was recently described in 29 patients who originally presented with non-viral-associated nonkeratinizing squamous cell carcinoma. The tumors occurred at multiple sites in the head and neck including in the sinonasal tract, middle ear, and temporal bone. This tumor behaves aggressively involving adjacent vital structures, frequently recurs, and is inclined to develop lymph node and distant metastasis. This review aims to summarize the demographic, clinical, pathologic, immunophenotypic features, and pattern of molecular alterations as well as to discuss the differential diagnosis of DEK::AFF2 fusion carcinoma.

BRAF V600E Immunohistochemistry Predicts Prognosis of Patients with Cutaneous Melanoma in Thai population.

The BRAF V600E mutation in the Thai population has been identified in a considerable percentage of people with cutaneous melanoma. The objectives of this study were to determine the prevalence of this mutation in cutaneous melanomas, conduct a clinicopathological association analysis with the BRAF V600E mutation, and develop a treatment strategy for patients with this mutation that would take advantage of the medications currently available to treat them. Methods: Anti-BRAF V600E (clone VE1) immunohistochemistry was performed on 50 pathological samples of cutaneous melanoma after excluding the samples with a low amount of pathologic tissue, a lack of clinical data' and poor follow-up. BRAF V600E expression DNA sequencing was performed to confirm the results of several cases. Results: Anti-BRAF V600E antibody positivity was noted in 56% (28/50) of cutaneous melanoma cases. DNA sequencing results were consistent with immunohistochemistry results. In cutaneous melanoma, the BRAF V600E mutation was significantly associated with adverse prognosis of patients, including reduced overall survival and disease-free survival. Conclusions: An increased prevalence of the BRAF V600E mutation was determined in a collection of cutaneous melanomas in the Thai population, implying that BRAF-targeted therapy may be a promising strategy for patients with BRAF-mutated cutaneous melanoma. This study revealed an association between the clinicopathological aspects of cutaneous melanoma and overall survival, disease-free survival, and overall mortality. A treatment with anti-BRAF-targeted therapy, which incorporates the already available medications' is being researched and developed.

MCM-2, Ki-67, and EGFR downregulated expression levels in advanced stage laryngeal squamous cell carcinoma.

We present the conceptual study investigated the capacity of minichromosome maintenance-2 (MCM-2), Ki-67, and epidermal growth factor receptor (EGFR) to assess the severity and progression of laryngeal squamous cell carcinoma (LSCC) disease and to study the correlations among these markers. A total of 30 patients with LSCC with immunohistochemistry (IHC) staining for MCM-2, Ki-67 and EGFR were examined. Mean expression levels of the three markers were evaluated for comparing between early and advanced stages of LSCC. The mean MCM-2, Ki-67, and EGFR expression levels were significantly decreased in advanced-stage compared with early-stage LSCC. Pearson correlation analysis showed a statistically significant correlation between the MCM-2 and Ki-67. Regarding subgroup analyses, MCM-2, Ki-67, and EGFR showed significant differences between early- and advanced-stage LSCC with non-recurrence, while for the recurrent subgroup LSCC, only MCM-2 revealed a significant difference between early- and advanced-stage LSCC. Altogether, these results support the role for downregulation of MCM-2, Ki-67 and EGFR in advanced-stage LSCC and correlation of MCM-2 and Ki-67 expressions that would be a promising strategy to predict prognosis of LSCC including severity and progression. We contextualize our findings and advocate the position of the biological markers, especially MCM-2, as an emerging evaluation tool for LSCC disease.

Vimentin expression status is a potential biomarker for brain metastasis development in EGFR-mutant NSCLC patients.

BACKGROUND: Despite advances in systemic therapy and improvements in survival for advanced epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC), brain metastasis (BM) remains a poor outcome. Previous studies on risk factors for BM occurrence included unselected patients and biomarker prediction of BM in these populations were not well studied. We aimed to identify the role of epithelial mesenchymal transition (EMT) marker and clinical factors predicting BM in. EGFR: mutant NSCLC patients. METHODS: Advanced EGFR-mutant NSCLC patients in the King Chulalongkorn Memorial Hospital from January 2013 to December 2017 were included. Vimentin expression was assessed by immunohistochemistry. The correlation between vimentin expression and factors associated with BM occurrence was analyzed by univariate and multivariate analyses. RESULTS: 304 patients were enrolled. Of these, 149 patients (49%) developed BM. In multivariate analysis, the occurrence of BM was associated with age <60 years, metastatic disease at diagnosis, and 3 or more metastatic sites. Moreover, positive vimentin expression was also found more common in patients with BM than those without BM (52.4% vs. 27.6%, respectively) and predicted overall BM development in EGFR-mutant patients (OR 2.53, 95% CI, 1.11-5.77; P=0.027). Overall survival (OS) was shorter in vimentinpositive group than in vimentinnegative group. Median OS was 20.0 months (95% CI, 14.51-25.51) and 30.9 months (95% CI, 20.99-40.84), respectively (HR, 1.57; P=0.04). CONCLUSIONS: Younger patients with EGFR-mutant NSCLC who had high disease burden were more likely to develop BM. Vimentin served as a biomarker for predicting BM and poor prognostic factor in EGFR-mutant patients. EMT pathway may be considered as a therapeutic target in these high-risk populations.

EGFR cut-off point for prognostic impact in laryngeal squamous cell carcinoma.

Background: Various molecular biomarkers, including epidermal growth factor receptor (EGFR), have gained importance as predictors of head and neck cancer disease progression.Aims: This study aimed to investigate the ability of EGFR expression as a prognostic marker for laryngeal squamous cell carcinoma (LSCC).Materials and methods: A total of 31 patients with LSCC with immunohistochemistry (IHC) staining for EGFR were examined. Digital image processing was applied to analyze EGFR staining intensity and percent distribution, which were calculated as the H-score. We used a receiver operating characteristic (ROC) curve to identify the best cut-off point of EGFR expression, with H-score separated into high- and low-grade for cancer recurrence prediction.Results: The cut-off point of EGFR expression for high- and low-grades was an H-score of 170 with a sensitivity of 75% and a specificity of 66.7%. Using this cut-off, 14 (45.16%) and 17 (54.84%) patients were categorized as having high- and low-grades EGFR, respectively. The analysis showed a significant reverse correlation between the EGFR grade and LSCC recurrence (RR, 0.4; 95% CI, 0.17-0.98; p = .02).Conclusions: Our study demonstrated that EGFR grading using H-score with the generated cut-off point by the ROC curve might be further applied as a potential marker for LSCC prognostic prediction.

Clinical evaluation of tumour-infiltrating lymphocytes as a prognostic factor in patients with human papillomavirus-associated oropharyngeal squamous cell carcinoma.

Clinical evaluation of tumour-infiltrating lymphocytes as a prognostic factor in patients with human papillomavirus-associated oropharyngeal squamous cell carcinoma AIMS: The majority of patients with human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OpSCC) have favourable survival outcomes, but a significant minority of individuals will die of their disease. There are currently no definitive criteria with which to identify HPV-associated OpSCC patients with poor outcomes. Recent reports suggest that quantitative evaluation of T-cell subpopulations in OpSCC may be of prognostic value, but the methods used have limited utility in a clinical diagnostic setting. We therefore sought to determine the clinical prognostic utility of tumour-infiltrating lymphocyte (TIL) evaluation in patients with HPV-associated OpSCC within the context of a diagnostic histopathology setting. METHODS AND RESULTS: Representative diagnostic haematoxylin and eosin (H&E)-stained slides from 232 consecutive HPV-associated OpSCC patients were classified as containing a high (TILHi ; diffuse, lymphocytes in >80% of tumour and stroma), moderate (TILMod ; patchy, present in 20-80% of tumour and stroma) or low (TILLo ; sparse or absent, present in <20% of tumour and stroma) TILs. Interobserver reliability was assessed, and TIL category was then correlated with overall survival (OS) and disease-free survival (DFS). Univariate and multivariate analyses showed statistically significant differences in OS and DFS estimates when TILHi and TILMod patients were compared with TILLo patients (P < 0.0001 for TILHi versus TILLo ; P < 0.0001 for TILMod versus TILLo ). Statistical significance was retained when TILHi and TILMod patients were grouped into a single category (TILHi ) and compared with TILLo patients (P < 0.0001). CONCLUSION: We demonstrate the prognostic utility of TILs in patients with HPV-associated OpSCC in clinical practice. A binary system classifying HPV-associated OpSCC into TILHi and TILLo on the basis of routine H&E staining stratifies patients into those with potentially favourable and unfavourable survival outcomes, respectively.

Oncocytic Sialolipoma of Parotid Gland: Case Report and Literature Review.

Oncocytic sialolipoma is a very rare tumor of which only three cases have been reported. This entity is considered to be a variant of sialolipoma which harbours oncocytic nodules within a well-circumscribed lipomatous mass. We report a parotid mass in 73-year-old female that was difficult to diagnose in imaging and on biopsy. Ultrasonography and MRI demonstrated a mass with features thought to be consistent with lipoma. Twice needle core biopsies were performed. Both were indefinite for diagnosis. The first report favoured a lipoma and the second report suggested the lesion represented oncocytic hyperplasia or an oncocytoma. The microscopic examination of the excised surgical specimen demonstrated typical features of oncocytic sialolipoma, characterized by a predominately lipomatous component, sparse normal-appearing salivary gland tissue and multiple oncocytic nodules. This is the second case of oncocytic sialolipoma reported to occur in the parotid gland.

Human papillomavirus in anal biopsy tissues and liquid-based cytology samples of HIV-positive and HIV-negative Thai men who have sex with men.

BACKGROUND: Men who have sex with men (MSM) are at high risk of developing human papillomavirus (HPV)-related anal cancer. We compared HPV genotypes in anal tissues (Bx) and anal liquid-based cytology fluid (LBC) from HIV-positive and HIV-negative MSM. METHODS: Bx (32 normal, 41 low-grade squamous intraepithelial lesions (LSIL) and 22 high-grade squamous intraepithelial lesions (HSIL)), along with LBC from the same visit, were selected from 61 HIV-positive and 34 HIV-negative MSM who enrolled into a prospective cohort in Bangkok, Thailand. HPV genotyping was performed on Bx and LBC. RESULTS: Any HPV and high-risk HPV (HR-HPV) prevalence were 63.2% and 60.0% in Bx and 71.6% and 62.1% in LBC, respectively. HIV-positive MSM had higher rates of HR-HPV genotypes detection (70.5% vs. 47.1%, p=0.03) in LBC than HIV-negative MSM. HPV16 (27%) was the most common HR-HPV found in HSIL tissue. In HIV-positive MSM, the frequency of HR-HPV detection increased with histopathologic grading in both Bx and LBC samples. HSIL was associated with the presence of any HR-HPV(OR 7.6 (95%CI 1.8-31.9); P=0.006) in LBC and in Bx((OR 5.6 (95%CI 1.4-22.7); P=0.02). CONCLUSIONS: Our data strongly support the integration of HR-HPV screening on LBC samples, along with HPV vaccination, into an anal cancer prevention program.

Invasive fungal disease in university hospital: a PCR-based study of autopsy cases.

Invasive fungal disease (IFD) has high mortality rate, especially in the growing population of immunocompromised patients. In spite of introduction of novel diagnostic approaches, the intravital recognition of IFD is challenging. Autopsy studies remain a key tool for assessment of epidemiology of visceral mycoses. We aimed to determine species distribution and trends of IFD over the last 10 years in unselected autopsy series from a large university hospital. Forty-five cases of visceral mycoses, confirmed by histopathology and panfungal PCR, were found in 587 consecutive autopsies. Major underlying diseases were diabetes mellitus (20%), hematologic malignancies (15.6%) and systemic lupus erythematosus (15.6%). There was a high risk for disseminated IFD in immunocompromised patients stayed in the hospital over 1 month with a fever longer than 3 weeks. The most common fungi were Aspergillus spp. (58%), Candida spp. (16%), Mucorales (14%) and Fusarium spp. (10%). We found significant increase in Aspergillus flavus (P = 0.04) and Mucorales (P < 0.01) infections over the last 5 years. Concordance rate between histopathology and panfungal PCR was 89.5% to the genus level. All 6 cases of fusariomycosis were misinterpreted as aspergillosis by histology alone. The precise species identification, necessary for targeted antifungal treatment, was rendered only by the molecular technique. Panfungal PCR showed high performance on formalin-fixed paraffin-embedded specimens, providing important epidemiological data in retrospective autopsy series. Rapid detection of fungi by panfungal PCR assay has high potential for intravital diagnostics of IFD in surgical and biopsy specimens.