CXC ligand 13 orchestrates an immunoactive microenvironment and enhances immunotherapy response in head and neck squamous cell carcinoma.

Objectives: This study aims to systematically explore the role of chemokine CXC ligand 13 (CXCL13) in head and neck squamous cell carcinoma (HNSCC). Methods: The Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases provided the RNA-seq data for cancer and normal tissues, respectively. Gene set enrichment analysis was applied to search the cancer hallmarks associated with CXCL13 expression. TIMER2.0 was the main platform used to investigate the immune cell infiltration related to CXCL13. Immunohistochemistry was applied to explore the relationship between CXCL13 and patients' prognosis and the relationship between CXCL13 and tertiary lymphoid structures (TLSs). Results: The expression of CXCL13 was upregulated in most tumors, including HNSCC. The higher expression of CXCL13 was closely related to the positive prognosis of HNSCC. CXCL13 was mainly expressed in B cells and CD8 + T cells, revealing the relationship between its expression and immune activation in the tumor microenvironment. Furthermore, immunohistochemistry and multiple fluorescence staining analysis of HNSCC samples showed a powerful correlation between CXCL13 expression, TLSs formation, and positive prognosis. Finally, CXCL13 significantly increased the response to cancer immunotherapy. Conclusions: CXCL13 may function as a potential biomarker for predicting prognosis and immunotherapy response and associate with TLSs in HNSCC.

Phase II Randomized Trial of Transoral Surgery and Low-Dose Intensity Modulated Radiation Therapy in Resectable p16+ Locally Advanced Oropharynx Cancer: An ECOG-ACRIN Cancer Research Group Trial (E3311).

PURPOSE: Definitive or postoperative chemoradiation (CRT) is curative for human papillomavirus-associated (HPV+) oropharynx cancer (OPC) but induces significant toxicity. As a deintensification strategy, we studied primary transoral surgery (TOS) and reduced postoperative radiation therapy (RT) in intermediate-risk HPV+ OPC. METHODS: E3311 is a phase II randomized trial of reduced- or standard-dose postoperative RT for resected stage III-IVa (American Joint Committee on Cancer-seventh edition) HPV+ OPC, determined by pathologic parameters. Primary goals were feasibility of prospective multi-institutional study of TOS for HPV+ OPC, and oncologic efficacy (2-year progression-free survival) of TOS and adjuvant therapy in intermediate-risk patients after resection. TOS plus 50 Gy was considered promising if the lower limit of the exact 90% binomial confidence intervals exceeded 85%. Quality of life and swallowing were measured by functional assessment of cancer therapy-head and neck and MD Anderson Dysphagia Index. RESULTS: Credentialed surgeons performed TOS for 495 patients. Eligible and treated patients were assigned as follows: arm A (low risk, n = 38) enrolled 11%, intermediate risk arms B (50 Gy, n = 100) or C (60 Gy, n = 108) randomly allocated 58%, and arm D (high risk, n = 113) enrolled 31%. With a median 35.2-month follow-up for 359 evaluable (eligible and treated) patients, 2-year progression-free survival Kaplan-Meier estimate is 96.9% (90% CI, 91.9 to 100) for arm A (observation), 94.9% (90% CI, 91.3 to 98.6]) for arm B (50 Gy), 96.0% (90% CI, 92.8 to 99.3) for arm C (60 Gy), and 90.7% (90% CI, 86.2 to 95.4) for arm D (66 Gy plus weekly cisplatin). Treatment arm distribution and oncologic outcome for ineligible or step 2 untreated patients (n = 136) mirrored the 359 evaluable patients. Exploratory comparison of functional assessment of cancer therapy-head and neck total scores between arms B and C is presented. CONCLUSION: Primary TOS and reduced postoperative RT result in outstanding oncologic outcome and favorable functional outcomes in intermediate-risk HPV+ OPC.

SHH Expression Is Significantly Associated With Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a highly invasive malignancy with poor prognosis. Recent reports suggest that Sonic Hedgehog (SHH) plays a key role in tumor progression and worsens the response to therapy, possibly through an association with a cancer stem cell (CSC) phenotype. The objective of our study was to investigate the relationship between SHH expression and CSC markers in OSCC. MATERIALS AND METHODS: A total of 67 OSCC specimens were immunostained for SHH and CSC markers using specific antibodies and expression was correlated with clinicopathological parameters. RESULTS: SHH expression was significantly correlated with CD133 (p=0.026, r=0.272) and SRY-box transcription factor 2 (SOX2; p<0.001, r=0.793). SHH and SOX2 expression were associated with worse survival in OSCC (p=0.003 and p=0.003, respectively). In multivariate analysis SHH and CD44 were independent prognostic biomarkers in patients with OSCC (p=0.001 and p=0.008, respectively). CONCLUSION: Our study revealed that SHH overexpression is closely associated with CSC markers, contributing to tumor progression and worse outcomes of patients with OSCC.

Double Positivity for HPV DNA/P16INK4a Does Not Influence Survival of Patients With Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: We investigated the prevalence of human papillomavirus (HPV) in a prospective cohort of patients with squamous cell carcinoma of the oral cavity (OSCC) using both p16INK4a and HPV DNA, i.e., double positivity, as a definition criterion. Additionally, we examined the association of HPV with survival. PATIENTS AND METHODS: Samples from 280 OSCC patients were analyzed for HPV-positivity using p16INK4a immunohistochemistry (IHC) and in situ hybridization (ISH)/LCD arrays, for HPV low and high-risk types. Only patients positive for both p16INK4a and HPV DNA were considered as HPV-positive. Survival probabilities and 95% confidence intervals were estimated using the Kaplan-Meier method. Cox proportional hazards models were used to assess HPV association with disease-free survival (DFS), cause-specific survival (CSS) and overall survival (OS) in a competing risks scenario. RESULTS: Specimen from 30 (10.7%) patients were p16+ and HPV DNA+, while 31 (11.0%) were either p16+ or HPV DNA+ only. OS probabilities at five years for HPV-positive and -negative groups were 50.9% (35.4%-73.1%) and 52.9% (47.0%-59.5%), respectively. HPV double positivity influenced neither OS, CSS nor DFS: HR=0.84 (0.43-1.63), 1.64 (0.76-3.54) and 1.13 (0.55-2.35), respectively. CONCLUSION: In contrast to oropharyngeal cancer, the prevalence of HPV in OSCC is low and the presence of HPV does not influence survival outcomes. Hence, there is no evidence to support a parallel transfer of therapy regimen for HPV-positive OPC to OSCC, in terms of therapy de-escalation and/or vaccination.

The neuropeptide calcitonin gene-related peptide links perineural invasion with lymph node metastasis in oral squamous cell carcinoma.

OBJECTIVE: Although perineural invasion (PNI) is well-known to be correlated with and able to predict lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC), the clinical and molecular correlation between PNI and LNM has not been elucidated, and preoperative biomarkers for LNM prediction in OSCC are urgently needed. MATERIALS AND METHODS: The correlation between PNI and LNM was retrospectively evaluated using a cohort of 218 patients diagnosed with OSCC. Candidate neuropeptides were screened based on TCGA database and verified via immunohistochemistry and Western blot analyses. ELISA was used to detect calcitonin gene-related peptide (CGRP) in patient plasma. In vitro assays were used to explore the effects of CGRP on OSCC cells. RESULTS: OSCC patients with PNI had a higher incidence of LNM (69.86% vs. 26.2%, P < 0.0001, n = 218). CGRP expression was upregulated in the PNI niche and in metastatic lymph nodes, and was correlated with poor overall survival of OSCC patients. Preoperative plasma CGRP levels were higher in OSCC patients (n = 70) compared to healthy donors (n = 60) (48.59 vs. 14.58 pg/ml, P < 0.0001), and were correlated with LNM (P < 0.0001) and PNI (P = 0.0002). Preoperative plasma CGRP levels alone yielded an AUC value of 0.8088 to predict LNM, and CGRP levels combined with preoperative T stage reached an AUC value of 0.8590. CGRP promoted proliferation and migration abilities of OSCC cells, which could be antagonized by either pharmacological or genetic blockade of the CGRP receptor. CONCLUSIONS: The neuropeptide CGRP links PNI and LNM in OSCC, and preoperative plasma CGRP levels can be used to predict LNM in OSCC.

The expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma.

P-cadherin (CDH3) is a cell-to-cell adhesion molecule that regulates several cellular homeostatic processes in normal tissues. Lack of CDH3 expression is associated with aggressive behavior in oral squamous cell carcinoma (OSCC). Previous studies have shown that CDH3 is downregulated in high-grade OSCC and its reduced expression is predictive for poorer survival. The aim of this study was to evaluate the expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma (TSCC). A retrospective series of 211 TSCC and 50 lymph node samples were stained immunohistochemically with polyclonal antibody (anti-CDH3). CDH3 expression was assessed semi-quantitatively with light microscopy. Fisher's exact test was used to compare patient and tumor characteristics, and the correlations were tested by Spearman correlation. Survival curves were drawn by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate Cox regression was used to estimate the association between CDH3 expression and survival. CDH3 expression did not affect TSCC patient's disease-specific survival or overall survival. Strong CDH3 expression in the primary tumor predicted poor disease-specific and overall survival in patients with recurrent disease. CDH3 expression in lymph nodes without metastasis was negative in all cases. CDH3 expression was positive in all lymph node metastases with extranodal extension. In contrast to previous report about the prognostic value of CDH3 in OSCC, we were not able to validate the result in TSCC.

Discrepancy in p16 expression in patients with HPV-associated head and neck squamous cell carcinoma in Thailand: clinical characteristics and survival outcomes.

BACKGROUND: Lower prevalence HPV infection has been previously reported in Thai population when compared with Western countries. p16 expression indicates HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but not non-OPSCC. We therefore evaluated the characteristic and association of p16 and HPV in Thai patients with HNSCC. METHODS: We used immunohistochemistry and qPCR, respectively, to detect p16 and HPV DNA in archrival formalin-fixed paraffin-embedded HNSCC tissues. Patient characteristics and survival were analyzed. RESULTS: p16 expression was detected in tumors of 72 of 662 (10.9%) patients with HNSCC and was significantly associated with higher-grade histology, advanced nodal stage, and oropharynx. p16 was expressed in 28 and 6.5% of patients with OPSCC or non-OPSCC, respectively, and HPV DNA was detected in 15.6 and 1% of patients, respectively. Using p16 as a surrogate for HPV status, sensitivities were 80 and 25% in OPSCC and non-OPSCC, respectively. Positive and negative predictive rates of OPSCC were 38 and 95%. Discordance rates between HPV and p16 were 23 and 7% in OPSCC and non-OPSCC, respectively. Overall survival (OS) were significantly longer in both p16-positive OPSCC (p = 0.049), and non-OPSCC (p = 0.003). CONCLUSIONS: Low prevalence of p16 and HPV associated OPSCC and non-OPSCC were confirmed in Thai patients. High discordance and low positive predictive rates of p16 were observed in HPV-associated OPSCC. p16 was a significant prognostic factor for OS for patients with OPSCC or non-OPSCC. Therefore, HPV testing should be performed to assess the association of HPV with HNSCC regardless of p16 expression.

DEK-AFF2 Carcinoma of the Sinonasal Region and Skull Base: Detailed Clinicopathologic Characterization of a Distinctive Entity.

A novel DEK-AFF2 fusion was recently reported in 4 nonkeratinizing squamous cell carcinomas of the sinonasal region and skull base, including 1 with exceptional response to immunotherapy, but it is not yet clear if this rearrangement defines a unique clinicopathologic category or represents a rare event. This study aims to characterize a larger cohort of carcinomas with DEK-AFF2 fusions to assess whether they truly constitute a distinctive entity. Among 27 sinonasal and skull base nonkeratinizing squamous cell carcinoma that were negative for human papillomavirus and Epstein-Barr virus, RNA sequencing identified DEK-AFF2 fusions in 13 cases (48%). Nine were centered in the nasal cavity, 2 in the middle ear/temporal bone, 1 in the nasopharynx, and 1 in the orbit. These tumors displayed recurrent histologic features including (1) complex endophytic and exophytic, frequently papilloma-like growth, (2) transitional epithelium with eosinophilic to amphophilic cytoplasm, (3) absent or minimal keratinization with occasional compact keratin pearls, (4) monotonous nuclei, and (5) prominent tumor-infiltrating neutrophils or stromal lymphocytes. This appearance not only overlaps with high-grade basaloid sinonasal carcinomas but also with benign papillomas and tumors reported as low-grade papillary Schneiderian carcinoma. However, DEK-AFF2 carcinomas showed frequent local recurrence, cervical lymph node metastases, and distant metastasis with 2 deaths from disease, confirming they are aggressive malignancies despite relatively bland histology. Overall, the distinctive molecular, histologic, and clinical features of DEK-AFF2 carcinomas suggest they represent a unique entity in the sinonasal region. This tumor merits increased pathologic recognition to better understand its prognostic and therapeutic implications.

Oropharyngeal Squamous Cell Carcinoma With Discordant p16 and HPV mRNA Results: Incidence and Characterization in a Large, Contemporary United States Cohort.

Early studies estimate that 5% to 10% of oropharyngeal squamous cell carcinomas overexpress p16 but are unassociated with transcriptionally-active high-risk human papillomavirus (HPV). Patients with discordant HPV testing may experience clinical outcomes that differ from traditional expectations. To document the rate of p16 and HPV mRNA positivity, characterize patients with discordant testing, and identify features that may warrant selective use of HPV-specific testing after p16 IHC, a multi-institutional, retrospective review of oropharyngeal squamous cell carcinoma patients with p16 IHC and HPV mRNA testing by reverse transcriptase polymerase chain reaction was performed. Of the 467 patients, most had T1 or T2 tumors (71%), 82% were p16 positive, and 84% were HPV mRNA positive. Overall, most tumors were nonkeratinizing (378, 81%), which was strongly associated with p16 and HPV positivity (93% and 95%, respectively). Overall, 81% of patients were double positive, 14% double negative, and 4.9% discordant (3.4% p16 negative/HPV mRNA positive and 1.5% p16 positive/HPV mRNA negative). The survival rates of these discordant patient groups fell squarely between the 2 concordant groups, although in multivariate analysis for both disease-free survival and overall survival, discordant patients were not found to have statistically significantly different outcomes. Reclassifying patients by applying HPV mRNA testing when p16 results and morphology do not match, or when p16 results are equivocal, improved prognostication slightly over p16 or HPV mRNA testing alone. Patients with discordant testing demonstrate a borderline significant trend toward survival differences from those with concordant tests. When evaluated independently, patients who were p16 negative but HPV mRNA positive had a prognosis somewhat closer to double-positive patients, while those who were p16 positive, but HPV mRNA negative had a prognosis closer to that of double-negative patients. We suggest an algorithm whereby confirmatory HPV mRNA testing is performed in patients where p16 status is not consistent with tumor morphology. This captures a majority of discordant patients and improves, albeit modestly, the prognostication.

Systematic Review of Second Primary Oropharyngeal Cancers in Patients With p16+ Oropharyngeal Cancer.

OBJECTIVE: To systematically review the literature to determine the prevalence and clinical outcomes of second primary oropharyngeal squamous cell carcinoma (OPSCC). DATA SOURCES: Search strategies created with a medical librarian were implemented using multiple databases in October 2019. REVIEW METHODS: The population of interest included adults age >18 years with a p16+ or human papillomavirus-positive OPSCC. The outcome was a synchronous or metachronous second primary OPSCC. Inclusion and exclusion criteria were designed to capture all study designs. In total, 685 records were identified by the search strategy. Two reviewers independently performed the review, extracted data, and performed a quality assessment. Primary Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. A random-effects model was used for the meta-analysis. RESULTS: A total of 2470 patients with 35 second primary OPSCCs from 15 studies were identified. The pooled prevalence of second primary OPSCC was 1.4% (range, 0%-14.3%). In the random-effects model, the prevalence was estimated at 1.3% (95% CI, 0.7%-2.3%; P = .51, I2 = 52%). Of the 30 patients with treatment information, 26 (86.7%) received surgical treatment, while 4 (13.3%) underwent nonsurgical therapy. Of the 29 patients with available survival information, 22 (75.9%) had no evidence of disease at last follow-up, 5 (17.2%) ultimately died of disease, and 2 (6.9%) were alive with disease. CONCLUSION: Overall, the rate of second primary OPSCC in patients with an index p16+ OPSCC is low, and most patients are successfully treated. Insufficient evidence currently exists to recommend routine elective tonsillectomy during surgical treatment of p16+ OPSCC.

Expression of invadopodia markers can identify oral lesions with a high risk of malignant transformation.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity and is usually preceded by a range of premalignant tissue abnormalities termed oral potentially malignant disorders. Identifying malignant transformation is critical for early treatment and consequently improved survival and decreased morbidity. Invadopodia (INV) are specialized subcellular structures required for cancer cell invasion. We developed a new method to visualize INV in keratinocytes using fluorescent immunohistochemistry (FIHC) and semi-automated images analysis. The presence of INV was used to determine the risk of malignant transformation. We analyzed 145 formalin-fixed, paraffin-embedded (FFPE) oral biopsy samples from 95 patients diagnosed as nondysplastic, dysplastic, and OSCC including 49 patients whose lesions transformed to OSCC (progressing) and 46 cases that did not transform to OSCC (control). All samples were stained for Cortactin, tyrosine kinase substrate with five SH3 domains (Tks5) and matrix metallopeptidase 14 (MMP14) using FIHC, imaged using confocal microscopy and analyzed using a multichannel colocalization analysis. The areas of colocalization were used to generate an INV score. Using the INV score, we were able to identify progressing lesions with a sensitivity of 75-100% and specificity of 72-76%. A positive INV score was associated with increased risk of progression to OSCC. Our results suggest that INV markers can be used in conjunction with the current diagnostic standard for early detection of OSCC.

Primary Cutaneous Myxoid Spindle Cell Squamous Cell Carcinoma of the Scalp: A Case Report.

Cutaneous squamous cell carcinomas are the second most frequent type of non-melanoma skin cancer. A 78-year-old man with a slow-growing but large nodular lesion on his scalp presented to the hospital. The nodular lesion was excised. Histologically, the lesion was diagnosed as a primary cutaneous myxoid spindle cell squamous cell carcinoma, which is the subject of this case report. Extracellular mucin production is an even less common finding in SCC. We also aim to discuss the histological and immunohistochemical findings for distinguishing MSC SCC from other primary cutaneous and metastatic spindle cell neoplasms with myxoid stroma.

5-Nitro-2,4-Dichloropyrimidine as an Universal Model for Low-Energy Electron Processes Relevant for Radiosensitization.

We report experimental results of low-energy electron interactions with.

A novel immunohistochemical scoring system reveals associations of C-terminal MET, ectodomain shedding, and loss of E-cadherin with poor prognosis in oral squamous cell carcinoma.

Using tissue microarrays, it was shown that membranous C-terminal MET immunoreactivity and ectodomain (ECD) shedding are associated with poor prognosis in oral cancer. Seen the potential diagnostic value, extrapolation of these results to whole-tissue sections was investigated. Because MET orchestrates epithelial-to-mesenchymal transition (EMT), the results were benchmarked to loss of E-cadherin, a readout for EMT known to be associated with poor prognosis. C-terminal MET, N-terminal MET, and E-cadherin immunoreactivities were examined on formalin-fixed paraffin-embedded parallel sections of 203 oral cancers using antibody clones D1C2, A2H2-3, and NCH-38. Interantibody and intra-antibody relations were examined using a novel scoring system, nonparametric distribution, and median tests. Survival analyses were used to examine the prognostic value of the observed immunoreactivities. Assessment of the three clones revealed MET protein status (no, decoy, transmembranous C-terminal positive), ECD shedding, and EMT. For C-terminal MET-positive cancers, D1C2 immunoreactivity is independently associated with poor overall survival (hazard ratio [HR] = 2.40; 95% confidence interval [CI] = 1.25 to 4.61; and P = 0.008) and disease-free survival (HR = 1.83; 95% CI = 1.07-3.14; P = 0.027). For both survival measures, this is also the case for ECD shedding (43.4%, with HR = 2.30; 95% CI = 1.38 to 3.83; and P = 0.001 versus HR = 1.87; 95% CI = 1.19-2.92; P = 0.006) and loss of E-cadherin (55.3%, with HR = 2.21; 95% CI = 1.30 to 3.77; and P = 0.004 versus HR = 1.90; 95% CI = 1.20-3.01; P = 0.007). The developed scoring system accounts for MET protein status, ECD shedding, and EMT and is prognostically informative. These findings may contribute to development of companion diagnostics for MET-based targeted therapy.

Evaluation of p16INK4a expression as a single marker to select patients with HPV-driven oropharyngeal cancers for treatment de-escalation.

BACKGROUND: A remarkably better prognosis is associated with oropharyngeal squamous cell carcinomas (OPSCC) driven by human papillomaviruses (HPV) compared with HPV-negative OPSCC. Consequently, de-escalation of standard treatment has been suggested. Due to modest specificity rates, debates are ongoing, whether p16INK4a, a surrogate marker for HPV-driven OPSCC, is sufficient to correctly identify those tumours and avoid substantial HPV misattribution and thus undertreatment of patients by de-escalation. Robust data estimating the proportion of potentially undertreated patients are missing. METHODS: We assessed a large-scale cohort of consecutively included OPSCC diagnosed between 2000 and 2017 for HPV-DNA, HPV genotypes, p16INK4a expression and multiple tumour- and patient-related risk factors, and investigated their impact on patients' survival in comprehensive uni- and multivariate analyses. RESULTS: Aetiological relevance of HPV (p16INK4a- and high-risk HPV-DNA-positivity) was detected in 27.1% (n = 192) of OPSCC, with HPV16 being the most abundant HPV type (94.6%). In 5.5% patients (n = 39), p16INK4a overexpression but no HPV-DNA was detected. Principal component and survival analyses revealed that 60.6% of these p16INK4a-positive OPSCC lacking HPV-DNA did not resemble HPV16-driven but HPV-negative OPSCC regarding risk-factor profile and overall survival. Notably, this group represented 10.6% of all p16INK4a-overexpressing OPSCC. CONCLUSIONS: p16INK4a as a single marker appears insufficient to indicate OPSCC patients suitable for treatment de-escalation.

Volatile organic compounds in head and neck squamous cell carcinoma-An in vitro pilot study.

Owing to the lack of specific symptoms, diagnosis of head and neck squamous cell carcinoma (HNSCC) may be delayed. We evaluated volatile organic compounds in tumor samples from patients suffering from HNSCC and tested the hypothesis that there is a characteristic altered composition in the headspace of HNSCC compared with control samples from the same patient with normal squamous epithelium. These results provide the basis for future noninvasive breath analysis in HNSCC. Headspace air of suspected tumor and contralateral control samples in 20 patients were analyzed using ion-mobility spectrometry. Squamous cell carcinoma was diagnosed in 16 patients. In total, we observed 93 different signals in headspace measurements. Squamous cell carcinomas revealed significantly higher levels of volatile cyclohexanol (0.54 ppbv , 25th to 75th percentiles 0.35-0.86) compared with healthy squamous epithelium (0.24 ppbv , 25th to 75th percentiles 0.12-0.3; p < 0.001). In conclusion, head and neck squamous cell carcinoma emitted significantly higher levels of volatile cyclohexanol in headspace compared with normal squamous epithelium. These findings form the basis for future breath analysis for diagnosis, therapy control and the follow-up of HNSSC to improve therapy and aftercare.

Molecular profiling of circulating tumor cells predicts clinical outcome in head and neck squamous cell carcinoma.

OBJECTIVES: The relationship between the molecular profiling of circulating tumor cells (CTCs) and clinical factors is a challenge. In this study, we performed molecular detection and characterization of CTCs in patients with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: CTCs captured by microfilter were analyzed for the expression of multiple epithelial markers (EPCAM, MET, KRT19, and EGFR) by RT-qPCR. The CTCs-positive samples were further analyzed for the expression of 10 genes (PIK3CA, CCND1, SNAI1, VIM, CD44, NANOG, ALDH1A1, CD47, CD274, and PDCD1LG2). Finally, we analyzed whether the molecular profiling of CTCs was associated with clinical factors. RESULTS: Twenty-eight (63.6%) of the 44 HNSCC patients were positive for at least one epithelial-related gene. CTC-positivity was significantly correlated with treatment resistance (p = 0.0363), locoregional recurrence (p = 0.0151), and a shorter progression-free survival (PFS) (p = 0.0107). Moreover, the expression of MET in CTCs was associated with a shorter PFS (p = 0.0426). Notably, patients with CD274-positive CTC showed prolonged PFS (p = 0.0346) and overall survival (p = 0.0378) compared to those with CD274-negative CTC. CONCLUSION: Our results suggest that molecular profiling characterized by the gene expression of CTCs influences clinical factors in patients with HNSCC.

HPV-positive Squamous Cell Carcinoma of the Larynx, Oral Cavity, and Hypopharynx: Clinicopathologic Characterization With Recognition of a Novel Warty Variant.

Human papillomavirus (HPV) is a principal driver for most oropharyngeal squamous cell carcinomas (OPSCCs), where it is strongly associated with improved survival. HPV is much less frequently detected in squamous cell carcinomas arising in nonoropharyngeal sites (non-OPSCCs), and its pathogenic role and prognostic value in these tumors is unclear. We evaluated the clinicopathologic features of 52 non-OPSCCs considered HPV-positive based upon p16 immunohistochemistry and direct HPV detection using RNA in situ hybridization (ISH), DNA ISH, or real-time DNA polymerase chain reaction. The HPV-positive non-OPSCCs were from the larynx (n=27), oral cavity (n=21), and hypopharynx (n=4). While most cases (n=34, 65%) showed classic histologic features of HPV-positive OPSCC, including endophytic growth, minimal keratinization, and hyperchromatic nuclei without koilocytic changes, a subset (n=13, 25%) were characterized by exophytic growth, exuberant surface hyperkeratosis and parakeratosis, marked nuclear pleomorphism, and prominent koilocytic atypia. These antithetical features were highly reminiscent of the warty variant of HPV-positive squamous cell carcinoma described in anogenital sites. Compared with tumors without warty features, the warty tumors presented at lower stage and were not associated with lymph node metastasis, local recurrence, or distant spread (4 y disease-free survival of 100% vs. 66%, P=0.069). The presence of transcriptionally active HPV as detected by RNA ISH suggests a pathogenic role for HPV in these nonoropharyngeal sites. While most HPV-positive non-OPSCCs are morphologically similar to their tonsillar counterparts, this study highlights a previously unrecognized warty variant that may be associated with a highly favorable clinical outcome.

Chrysotile fibers in tissue adjacent to laryngeal squamous cell carcinoma in cases with a history of occupational asbestos exposure.

Asbestos describes a group of naturally occurring fibrous silicate mineral compounds that have been associated with a number of respiratory maladies, including mesothelioma and lung cancer. In addition, based primarily on epidemiologic studies, asbestos has been implicated as a risk factor for laryngeal and pharyngeal squamous cell carcinoma (SCC). The main objective of this work was to strengthen existing evidence via empirical demonstration of persistent asbestos fibers embedded in the tissue surrounding laryngeal and pharyngeal SCC, thus providing a more definitive biological link between exposure and disease. Six human papillomavirus (HPV)-negative laryngeal (n = 4) and pharyngeal (n = 2) SCC cases with a history working in an asbestos-exposed occupation were selected from a large population-based case-control study of head and neck cancer. A laryngeal SCC case with no history of occupational asbestos exposure was included as a control. Tissue cores were obtained from adjacent nonneoplastic tissue in tumor blocks from the initial primary tumor resection, and mineral fiber analysis was performed using a scanning electron microscope equipped with an energy dispersive X-ray analyzer (EDXA). Chrysotile asbestos fiber bundles were identified in 3/6 of evaluated cases with a history of occupational asbestos exposure. All three cases had tumors originating in the larynx. In addition, a wollastonite fiber of unclear significance was identified one of the HPV-negative pharyngeal SCC cases. No mineral fibers were identified in adjacent tissue of the case without occupational exposure. The presence of asbestos fibers in the epithelial tissue surrounding laryngeal SCC in cases with a history of occupational asbestos exposure adds a key line of physical evidence implicating asbestos as an etiologic factor.

Prognostic impact of p16 and PD-L1 expression in patients with oropharyngeal squamous cell carcinoma receiving a definitive treatment.

AIMS: Limited information is available regarding the precise differences in the tumour immune microenvironment (TIM) of patients with human papilloma virus (HPV)-associated and non-HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Here, we retrospectively reviewed 137 patients with OPSCC treated with a definitive treatment to identify molecular relationships in the TIM. MATERIALS AND METHODS: We used immunohistochemical analysis to assess p16 status, programmed death ligand 1 (PD-L1) level, and/or CD8+ tumour-infiltrating lymphocyte (TIL) density, followed by prognostic evaluation of these immune-related parameters. RESULTS: Multivariate analyses demonstrated that PD-L1 level on immune cells but not on tumour cells or CD8+ TIL density was a significant predictive factor of disease-free survival (DFS) and overall survival (OS). Additionally, subgroup analyses demonstrated that patients positive for p16 and PD-L1 expression on immune cells had favourable DFS and OS, whereas patients negative for p16 and PD-L1 expression on immune cells showed worse DFS and OS. CONCLUSIONS: We demonstrated that PD-L1 expression on immune cells but not tumour cells might represent a useful prognostic biomarker in patients with OPSCC receiving a definitive treatment. We propose that a co-assessment of p16 and PD-L1 expression on immune cells would have greater prognostic potential compared with evaluation of each factor alone in patients with OPSCC.