Addition of tumor microenvironment immune cell composition to improve the performance of a predictive model for oral squamous cell carcinoma.

BACKGROUND: Conventional clinicopathological characteristics insufficiently predict prognosis in oral squamous cell carcinoma (OSCC). We aimed to assess the added predictive value of tumor microenvironment immune cell composition (TMICC) in addition to conventional clinicopathological characteristics. METHODS: Primary tumor samples of 290 OSCC patients were immunohistochemically stained for CD4, CD8, CD20, CD68, CD163, CD57, FoxP3 and Programmed cell Death Ligand 1. Additionally, clinicopathological characteristics were obtained from patients' medical files. Predictive models were trained and validated by conducting Least Absolute Shrinkage and Selection Operator (LASSO) regression analyses with cross-validation. To quantify the added predictive power of TMICC within models, receiver operating characteristic (ROC) analyses were used. RESULTS: Recurrence occurred in 74 patients (25.5%). Conventional clinicopathological characteristics (tumor localization, pathological T-stage, pathological N-stage, extracapsular spread, resection margin, differentiation grade, perineural invasion, lymphovascular invasion) and treatment modality, were used to build a LASSO logistic regression-based predictive model. Addition of TMICC to the model resulted in a comparable AUC of respectively 0.79 (±0.01) and 0.76 (±0.1) in the training and test sets. The model indicated that high numbers of CD4+ T cells protected against recurrence. Lymph node metastasis, extracapsular spread, perineural invasion, positive surgical margins and reception of adjuvant treatment were associated with increased risk for recurrence. CONCLUSIONS: The TMICC, specifically the number of CD4+ T cells, is an independent predictor , however, addition to conventional clinicopathological characteristics does not improve the performance of a predictive model for recurrence in OSCC.

Potential imaging targets in primary head and neck squamous cell carcinoma and lymph node metastases.

PURPOSE: To investigate glycoprotein nonmetastatic melanoma protein B (GPNMB) and vascular endothelial growth factor (VEGF) as potential fluorescent imaging markers by comparing their protein expression to epidermal growth factor receptor (EGFR). MATERIALS AND METHODS: Thirty-eight paired samples of untreated head and neck squamous cell carcinoma (HNSCC) primary tumours (PT) and corresponding synchronous lymph node metastases (LNM) were selected. After immunohistochemical staining, expression was assessed and compared by the percentage of positive tumour cells. Data were analysed using the Mann-Whitney test, effect sizes (ESr) and Spearman's correlation coefficient (r). RESULTS: GPNMB expression was observed in 100 % of PT, and median 80 % (range 5-100 %) of tumour cells, VEGF in 92 % and 60 % (0-100 %), EGFR in 87 % and 60 % (0-100 %) respectively. In corresponding LNM, GPNMB expression was observed in 100 % of LNM and median 90 % (20-100 %) of tumour cells, VEGF in 87 % and 65 % (0-100 %), and EGFR in 84 % and 35 % (0-100 %). A positive correlation was found between expression in PT and LNM for GPNMB (r = 0.548) and EGFR (r = 0.618) (p < 0.001), but not for VEGF (r = -0.020; p = 0.905). GPNMB expression was present in a higher percentage of tumour cells compared to EGFR in PT (p = 0.015, ESr = -0.320) and in LNM (p < 0.001, ESr = -0.478), while VEGF was not (p = 1.00, ESr = -0.109 and - 0.152, respectively). CONCLUSION: GPNMB expression is higher than EGFR in untreated HNSCC PT and corresponding LNM, while VEGF expression is comparable to EGFR. GPNMB is a promising target for fluorescent imaging in HNSCC.

Identification of new head and neck squamous cell carcinoma molecular imaging targets.

OBJECTIVES: Intraoperative fluorescence imaging (FI) of head and neck squamous cell carcinoma (HNSCC) is performed to identify tumour-positive surgical margins, currently using epidermal growth factor receptor (EGFR) as imaging target. EGFR, not exclusively present in HNSCC, may result in non-specific tracer accumulation in normal tissues. We aimed to identify new potential HNSCC FI targets. MATERIALS AND METHODS: Publicly available transcriptomic data were collected, and a biostatistical method (Transcriptional Adaptation to Copy Number Alterations (TACNA)-profiling) was applied. TACNA-profiling captures downstream effects of CNAs on mRNA levels, which may translate to protein-level overexpression. Overexpressed genes were identified by comparing HNSCC versus healthy oral mucosa. Potential targets, selected based on overexpression and plasma membrane expression, were immunohistochemically stained. Expression was compared to EGFR on paired biopsies of HNSCC, adjacent macroscopically suspicious mucosa, and healthy mucosa. RESULTS: TACNA-profiling was applied on 111 healthy oral mucosa and 410 HNSCC samples, comparing expression levels of 19,635 genes. The newly identified targets were glucose transporter-1 (GLUT-1), placental cadherin (P-cadherin), monocarboxylate transporter-1 (MCT-1), and neural/glial antigen-2 (NG2), and were evaluated by IHC on samples of 31 patients. GLUT-1 was expressed in 100 % (median; range: 60-100 %) of tumour cells, P-cadherin in 100 % (50-100 %), EGFR in 70 % (0-100 %), MCT-1 in 30 % (0-100 %), and NG2 in 10 % (0-70 %). GLUT-1 and P-cadherin showed higher expression than EGFR (p < 0.001 and p = 0.015). CONCLUSIONS: The immunohistochemical confirmation of TACNA-profiling results showed significantly higher GLUT-1 and P-cadherin expression than EGFR, warranting further investigation as HNSCC FI targets.

Association of Tumor Microenvironment with Biological and Chronological Age in Head and Neck Cancer.

There is often a mismatch between the chronological and biological age of head and neck squamous cell carcinoma (HNSCC) patients. Treatment is based on chronological age, while biological age seems to be a better prognosticator for treatment toleration. This study investigated whether tumor characteristics are associated with chronological and biological age. The relation with survival was also assessed. Prospectively collected data from 164 newly diagnosed HNSCC patients enrolled in the OncoLifeS database were analyzed. Biological age was assessed by a multidomain geriatric assessment. Several immunological markers were tested by immunohistochemistry on tissue microarray sections from the tumor. Disease-free survival (DFS), adjusted for chronological- and biological age, was assessed by univariable and bivariable analyses. In biologically old patients, a lower infiltration of CD163+ macrophages (p = 0.036) as well as CD4+ (p = 0.019) and CD8+ (p = 0.026) lymphocytes was found in the tumor microenvironment. Chronological older patients showed significantly lower PD-L1 combined positive scores (p = 0.030). Advanced tumor stage and perineural growth were related to a worse DFS. None of the immunological markers showed a significant association with DFS. Biological age might have a stronger influence on tumor microenvironment than chronological age. These findings should initiate clinical studies investigating the response to specific treatment regimens (e.g., immunotherapy) according to the biological age.

The relation between hypoxia and proliferation biomarkers with radiosensitivity in locally advanced laryngeal cancer.

PURPOSE: Treatment decision-making in advanced-stage laryngeal squamous cell carcinoma (LSCC) is difficult due to the high recurrence rates and the desire to preserve laryngeal functions. New predictive markers for radiosensitivity are needed to facilitate treatment choices. In early stage glottic LSCC treated with primary radiotherapy, expression of hypoxia (HIF-1α and CA-IX) and proliferation (Ki-67) tumour markers showed prognostic value for local control. The objective of this study is to examine the prognostic value of tumour markers for hypoxia and proliferation on locoregional recurrent disease and disease-specific mortality in a well-defined cohort of patients with locally advanced LSCC treated with primary, curatively intended radiotherapy. METHODS: In pre-treatment biopsy tissues from a homogeneous cohort of 61 patients with advanced stage (T3-T4, M0) LSCC primarily treated with radiotherapy, expression of HIF-1α, CA-IX and Ki-67 was evaluated with immunohistochemistry. Demographic data (age and sex) and clinical data (T- and N-status) were retrospectively collected from the medical records. Cox regression analysis was performed to assess the relation between marker expression, demographic and clinical data, and locoregional recurrence and disease-specific mortality. RESULTS: Patients with high expression of HIF-1α developed significantly more often a locoregional recurrence (39%) compared to patients with a low expression (21%) (p = 0.002). The expression of CA-IX and Ki-67 showed no association with locoregional recurrent disease. HIF-1α, CA-IX and Ki-67 were not significantly related to disease-specific mortality. Clinical N-status was an independent predictor of recurrent disease (p < 0.001) and disease-specific mortality (p = 0.003). Age, sex and T-status were not related to locoregional recurrent disease or disease-specific mortality. CONCLUSION: HIF-1α overexpression and the presence of regional lymph node metastases at diagnosis were independent predictors of locoregional recurrent disease after primary treatment with curatively intended radiotherapy in patients with locally advanced LSCC.

Inter-assay reliability of programmed cell death-ligand 1 in head and neck squamous cell carcinoma.

OBJECTIVES: The programmed cell death-ligand 1 (PD-L1) 22C3 pharmDx assay is used as a companion diagnostic test to select head and neck squamous cell carcinoma (HNSCC) patients that may benefit from treatment with the checkpoint inhibitor pembrolizumab. Because the Dako platform is not universally available, we studied the performance of a 22C3 laboratory developed test (LDT) performed on a Ventana BenchMark Ultra compared to the 22C3 pharmDx assay. MATERIALS AND METHODS: Serial sections from tissue micro arrays (TMAs) containing tumour tissue from 97 HNSCC patients were stained with the 22C3 pharmDx assay and 22C3 LDT. All TMA cores were scored by three dedicated head and neck pathologists for PD-L1 expression. RESULTS: Substantial interobserver agreement was reported for both the standardized 22C3 pharmDx assay and the 22C3 LDT (respectively Fleiss' κ 0.62, 95% CI 0.57-0.67 and 0.63, 95% CI 0.58-0.68). Concordance between the assays was almost perfect on core and patient level (respectively Weighted κ 0.84, 95% CI 0.79-0.89 and 0.84, 95% CI 0.75-0.92). Intratumor heterogeneity between the cores per patient case was similar in both assays. CONCLUSION: After validation a 22C3 LDT is non-inferior to the standardized 22C3 pharmDx assay and can be safely used to select HNSCC patients for pembrolizumab treatment.

High DNMT1 Is Associated With Worse Local Control in Early-Stage Laryngeal Squamous Cell Carcinoma.

OBJECTIVES/HYPOTHESIS: Early-stage laryngeal squamous cell carcinoma (LSCC) has yielded local control rates of 75% after radiotherapy. DNA methylation, in which DNA methyltransferases play an important role, has influence on tumorigenesis. In this study, we investigated the association between the expression of DNA methyltransferase 1 (DNMT1) and local control in early-stage LSCC treated with radiotherapy. STUDY DESIGN: Retrospective cohort study. METHODS: We analyzed a well-defined homogeneous series of 125 LSCC patients treated with radiotherapy with curative intent. The association of immunohistochemical expression of DNMT1 with local control was evaluated using Cox proportional hazard regression models. RESULTS: With a median follow-up of 58 months, 29 local recurrences (23%) were observed. On univariate analysis, worse local control was associated with high DNMT1 expression (hazard ratio [HR] 2.57, 95% confidence interval [CI] 1.10-6.01). Also, higher T-stage (HR 2.48, 95% CI 1.06-5.80) and positive N-status (HR 2.62, 95% CI 1.06-6.47) were associated with worse local control. Multivariate Cox regression demonstrated that high DNMT1 (HR 2.81; 95% CI 1.20-6.58) was independently associated with worse local control. CONCLUSIONS: We found an association between high DNMT1 expression and worse local control in a homogeneous well-defined cohort of early-stage LSCC patients treated with definitive radiotherapy. The association between DNA methylation status as determined by DNMT1 expression and local control suggests that DNMT1 acts as a potential prognostic tumor marker in treatment decision-making in early-stage laryngeal carcinoma. LEVEL OF EVIDENCE: NA Laryngoscope, 132:801-805, 2022.

Cortactin expression assessment improves patient selection for a watchful waiting strategy in pT1cN0-staged oral squamous cell carcinomas with a tumor infiltration depth below 4 mm.

BACKGROUND: In this feasibility study we aimed to evaluate the value of previously reported molecular tumor biomarkers associated with lymph node metastasis in oral squamous cell carcinoma (OSCC) to optimize neck strategy selection criteria. METHODS: The association between expression of cortactin, cyclin D1, FADD, RAB25, and S100A9 and sentinel lymph node status was evaluated in a series of 87 (cT1-2N0) patients with OSCC treated with primary resection and SLNB procedure. RESULTS: Tumor infiltration depth and tumor pattern of invasion were independent prognostic markers for SLN status, while none of the tumor makers showed a better prognostic value to replace SLNB as neck staging technique in the total cohort. However, in the subgroup of patients with pT1N0 OSCC, cortactin expression (OR 16.0, 95%CI 2.0-127.9) was associated with SLN classification. CONCLUSIONS: Expression of cortactin is a promising immunohistochemical tumor marker to identify patients at low risk that may not benefit from SLNB or END.

High pATM is Associated With Poor Local Control in Supraglottic Cancer Treated With Radiotherapy.

OBJECTIVES: Most early stage laryngeal squamous cell carcinomas (LSCC) are treated with radiotherapy. Discovery of new biomarkers are needed to improve prediction of outcome after radiotherapy and to identify potential targets for systemic targeted therapy. The ataxia telangiectasia mutated (ATM) gene plays a critical role in DNA damage response induced by ionizing radiation. METHODS: The prognostic value of immunohistochemical expression of pATM, pChk2, and p53 were investigated in 141 patients with T1-T2 LSCC curatively treated with external beam radiotherapy. Uni- and multivariable Cox regression analyses were performed to examine the relation between expression levels of markers and local control. RESULTS: Local control was significantly worse in cases with high levels of pATM (HR 2.14; 95% CI, 1.08-4.24; P = .03). No significant associations with local control were found for pChk2 and p53 expression. The association of high pATM expression with poor local control was only found for supraglottic LSCC (HR 10.9; 95% CI, 1.40-84.4; P = .02). CONCLUSION: Our findings suggest a potential role for ATM in response to radiotherapy in early stage supraglottic LSCC and imply ATM inhibition as a possibility to improve response to radiotherapy. LEVEL OF EVIDENCE: NA Laryngoscope, 130: 1954-1960, 2020.

Distinct Biomarker Profiles and Clinical Characteristics in T1-T2 Glottic and Supraglottic Carcinomas.

BACKGROUND: In early stage laryngeal squamous cell carcinoma (LSCC) radiotherapy with curative intent is a major treatment modality. TNM classification is used to define patients eligible for radiotherapy. Studies in early stage glottic LSCC identified several predictive biomarkers associated with local control. However, we recently reported that this predictive value could not be confirmed in supraglottic LSCC. OBJECTIVE: To examine whether clinical behavior and protein expression patterns of these biomarkers differ between glottic and supraglottic LSCC. STUDY DESIGN: Retrospective cohort study. METHODS: Tumor tissue sections of 196 glottic and 80 supraglottic T1-T2 LSCC treated primarily with RT were assessed immunohistochemically for expression of pAKT, Ki-67 and ß-Catenin. Expression data of HIF-1α, CA-IX, OPN, FADD, pFADD, Cyclin D1, Cortactin and EGFR in the same cohort of glottic and supraglottic LSCC, were retrieved from previously reported data. The relationship between glottic and supraglottic sublocalization and clinicopathological, follow-up, and immunohistochemical staining characteristics were evaluated using logistic regression and Cox regression analyses. RESULTS: Glottic LSCC were correlated with male gender (P = .001), hoarseness as a primary symptom (P < .001), T1 tumor stage (P < .001), negative lymph node status (P < .001), and an older age at presentation (P = .004). Supraglottic LSCC patients developed more post-treatment distant metastasis when adjusted for gender, age, and T-status. While supraglottic LSCC was associated with higher expression of HIF-1α (P = .001), Cortactin (P < .001), EGFR (P < .001), and Ki-67 (P = .027), glottic LSCC demonstrated higher expression of CA-IX (P = .005) and Cyclin D1 (P = .001). CONCLUSION: Differences in clinicopathological and immunohistochemical staining characteristics suggest that T1-T2 glottic and supraglottic LSCC should be considered as different entities. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2020.

Standardised Ki-67 proliferation index assessment in early-stage laryngeal squamous cell carcinoma in relation to local control and survival after primary radiotherapy.

OBJECTIVES: Ambiguous results have been reported on the predictive value of the Ki-67 proliferation index (Ki-67 PI) regarding local control (LC) and survival after primary radiotherapy (RT) in early-stage laryngeal squamous cell cancer (LSCC). Small study size, heterogenic inclusion, variations in immunostaining and cut-off values are attributing factors. Our aim was to elucidate the predictive value of the Ki-67 PI for LC and disease-specific survival (DSS) using a well-defined series of T1-T2 LSCC, standardised automatic immunostaining and digital image analysis (DIA). METHODS: A consecutive and well-defined cohort of 208 patients with T1-T2 LSCC treated with primary RT was selected. The Ki-67 PI was determined using DIA. Mann-Whitney U-tests, logistic and Cox regression analyses were performed to assess associations between Ki-67 PI, clinicopathological variables, LC and DSS. RESULTS: In multivariate Cox regression analysis, poor tumour differentiation (HR 2.20; 95% CI 1.06-4.59, P = .04) and alcohol use (HR 2.84, 95% CI 1.20-6.71; P = .02) were independent predictors for LC. Lymph node positivity was an independent predictor for DSS (HR 3.16, 95% CI 1.16-8.64; P = .03). Ki-67 PI was not associated with LC (HR 1.59; 95% CI 0.89-2.81; P = .11) or DSS (HR 0.98; 95% CI 0.57-1.66; P = .97). In addition, continuous Ki-67 PI was not associated with LC (HR 2.03; 95% CI 0.37-11.14, P = .42) or DSS (HR 0.62; 95% CI 0.05-8.28; P = .72). CONCLUSION: The Ki-67 PI was not found to be a predictor for LC or DSS and therefore should not be incorporated in treatment-related decision-making for LSCC.

PTEN Is Associated With Worse Local Control in Early Stage Supraglottic Laryngeal Cancer Treated With Radiotherapy.

OBJECTIVES: The aim of this study was to establish the prognostic value of the epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on local control in patients with early stage supraglottic laryngeal squamous cell carcinoma (LSCC) treated with radiotherapy only. STUDY DESIGN: Retrospective cohort study. METHODS: Immunohistochemical staining for EGFR and PTEN was performed on pretreatment biopsies of a selected well-defined homogeneous group of 52 patients with T1-T2 supraglottic LSCC treated with radiotherapy between 1990 and 2008. Kaplan-Meier analysis and univariate and multivariate Cox Regression analyses were performed to correlate clinical data and expression levels of EGFR and PTEN with local control. RESULTS: Kaplan-Meier survival analysis and Cox Regression analysis showed a significant association between PTEN expression and local control (hazard ratio [HR] = 3.26, 95% confidence interval [CI] = 1.14-9.33, P = .027) and between lymph node status and local control (HR = 3.60, 95% CI = 1.26-10.31, P = .017). Both were independent prognostic factors in a multivariate analysis (HR = 3.28, 95% CI = 1.14-9.39, P = .027 and HR = 3.62, 95% CI = 1.26-10.37, P = .017, respectively). There was no significant association between EGFR expression and local control (HR = 1.32, 95% CI = 1.17-10.14, P = .79). CONCLUSION: This study showed an association between both high PTEN expression and the presence of lymph node metastasis and deteriorated local control in early stage supraglottic LSCC treated with radiotherapy. LEVEL OF EVIDENCE: NA.

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma.

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma.

Phosphorylated FADD is not prognostic for local control in T1-T2 supraglottic laryngeal carcinoma treated with radiotherapy.

OBJECTIVE: The Fas-Associated Death Domain (FADD) gene is located in the chromosome 11q13-region and frequently is amplified in head and neck squamous cell carcinoma. Expression of FADD and its phosphorylated isoform (pFADD) have been associated with aggressive tumor growth, lymph node metastasis, and overall survival. Previously, we demonstrated that pFADD expression was related to a significantly improved local control in early stage (tumor [T]1 to T2) glottic laryngeal squamous cell carcinoma (LSCC). The aim of this study was to examine the prognostic value of pFADD and FADD in T1 to T2 supraglottic LSCC treated with primarily radiotherapy. METHODS: Tumor tissue sections of 60 patients with T1 to T2 supraglottic LSCC treated with primarily radiotherapy were assessed immunohistochemically for expression of pFADD and FADD. Expression percentages and clinical parameters and their associations with clinical outcome were studied using Cox regression and Kaplan-Meier survival analyses. Expression percentages in supraglottic and glottic LSCC were compared using the Mann-Whitney U test. RESULTS: Expression of pFADD and FADD in supraglottic and glottic LSCC did not significantly differ. In supraglottic LSCC, both pFADD and FADD did not show prognostic value for local control (hazard ratio [HR] 1.00, 95% confidence interval [CI] 0.98-1.03; HR 1.03, 95% CI 0.60-1.78, respectively) and overall survival (HR 0.99, 95% CI 0.98-1.01; HR 1.19, 95% CI 0.83-1.71 respectively). In this cohort, lymph node status was the best predictor for local control (HR 3.73, 95% CI 1.30-10.67). CONCLUSION: In this homogeneous cohort of T1 to T2 supraglottic LSCC primarily treated with radiotherapy, lymph node status was associated with local recurrence, whereas the expression of pFADD was not. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E301-E307, 2017.

Identification and validation of WISP1 as an epigenetic regulator of metastasis in oral squamous cell carcinoma.

Lymph node (LN) metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC) patients. However, in approximately one third of OSCC patients nodal metastases remain undetected, and thus are not adequately treated. Therefore, clinical assessment of LN metastasis needs to be improved. The purpose of this study was to identify DNA methylation biomarkers to predict LN metastases in OSCC. Genome wide methylation assessment was performed on six OSCC with (N+) and six without LN metastases (N0). Differentially methylated sequences were selected based on the likelihood of differential methylation and validated using an independent OSCC cohort as well as OSCC from The Cancer Genome Atlas (TCGA). Expression of WISP1 using immunohistochemistry was analyzed on a large OSCC cohort (n = 204). MethylCap-Seq analysis revealed 268 differentially methylated markers. WISP1 was the highest ranking annotated gene that showed hypomethylation in the N+ group. Bisulfite pyrosequencing confirmed significant hypomethylation within the WISP1 promoter region in N+ OSCC (P = 0.03) and showed an association between WISP1 hypomethylation and high WISP1 expression (P = 0.01). Both these results were confirmed using 148 OSCC retrieved from the TCGA database. In a large OSCC cohort, high WISP1 expression was associated with LN metastasis (P = 0.05), disease-specific survival (P = 0.022), and regional disease-free survival (P = 0.027). These data suggest that WISP1 expression is regulated by methylation and WISP1 hypomethylation contributes to LN metastasis in OSCC. WISP1 is a potential biomarker to predict the presence of LN metastases.

Assessment of hypoxic subvolumes in laryngeal cancer with (18)F-fluoroazomycinarabinoside ((18)F-FAZA)-PET/CT scanning and immunohistochemistry.

BACKGROUND AND PURPOSE: (18)F-fluoroazomycinarabinoside ((18)F-FAZA) is a promising hypoxia radiopharmaceutical agent with outstanding biokinetic parameters. We aimed to determine the accuracy of (18)F-FAZA-PET/CT scan in detecting hypoxic regions within the tumor using immunohistochemical markers in a pilot study. PATIENTS AND METHODS: Eleven patients with primary or recurrent laryngeal squamous cell carcinoma were indicated for total laryngectomy (TLE). Patients underwent (18)F-FAZA-PET/CT scan before TLE. Hypoxic regions inside the laryngeal tumor were determined. After TLE, regions with high uptake on (18)F-FAZA-PET scan were selected for immunohistochemical examination for exogenous (pimonidazole) and endogenous (HIF1α, CA-IX and GLUT-1) hypoxia markers. To assess the accuracy of (18)F-FAZA-PET scanning, radiopharmacon accumulation was related with immunohistochemical expression of hypoxia markers. RESULTS: Inter- and intratumoral heterogeneity of tumor hypoxia was observed on (18)F-FAZA-PET scan. Nine of the eleven tumors were hypoxic with (18)F-FAZA-PET. Hypoxia could also be detected with pimonidazole, HIF1α, CA-IX and GLUT-1 expression in some tumors. No clear association was observed between (18)F-FAZA uptake and hypoxia markers. CONCLUSIONS: This pilot study could not prove the accuracy of (18)F-FAZA-PET in determining hypoxic subvolumes in laryngeal cancer. Further study is required to investigate the benefit of (18)F-FAZA-PET imaging in radiotherapy planning.

Clinical course of recurrent respiratory papillomatosis: comparison between aggressiveness of human papillomavirus-6 and human papillomavirus-11.

BACKGROUND: Recurrent respiratory papillomatosis (RRP) is mainly associated with human papillomavirus (HPV)6 or HPV11. The purpose of this study was to compare clinical outcome, aggressiveness, and treatment response between HPV6- and HPV11-associated RRP. METHODS: A retrospective cohort of 55 patients with RRP (1974-2012) was used. Surgical interventions (n = 814) were analyzed, and complications scored. HPV6/11-specific polymerase chain reaction (PCR) was performed on RRP biopsies. RESULTS: Seventy-six percent of patients (42 of 55) were infected with HPV6 and 24% (13 of 55) with HPV11. The HPV11 group had anatomically more widespread disease. The expected number of surgical interventions was higher in the younger age (<22.4 years) HPV11 group, and the older age (<22.4 years) HPV6 group. Regardless of HPV type, earlier age of onset of RRP resulted in a higher number of surgical interventions. CONCLUSION: Anatomically, HPV11-associated RRP behaves more aggressively. Younger patients with HPV11 and older patients with HPV6 experience a worse clinical course of RRP.

Head and neck squamous cell carcinomas do not express EGFRvIII.

PURPOSE: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription-polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. RESULTS: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription-polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. CONCLUSIONS: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.

Clinical validation of the Cervista HPV HR test according to the international guidelines for human papillomavirus test requirements for cervical cancer screening.

This study demonstrates that both the clinical sensitivity and specificity of the Cervista HPV HR test for high-risk human papillomavirus (HPV) detection are not inferior to those of the Hybrid Capture 2 (HC2) test. The intra- and interlaboratory reproducibilities of Cervista were 92.0% (kappa, 0.83) and 90.4% (kappa, 0.80), respectively. The Cervista HPV HR test fulfills all the international HPV test requirements for cervical primary screening purposes.

Comparing the Cervista HPV HR test and Hybrid Capture 2 assay in a Dutch screening population: improved specificity of the Cervista HPV HR test by changing the cut-off.

The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84-91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76-97.37) for the HC2 test with 93% agreement between both tests (κ = 0.5, p<0.001). The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97-96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94-97.43) for the HC2 test with 95% agreement between both tests (κ = 0.7, p<0.001). Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0 (κ = 0.83 and κ = 0.84, p<0.001) and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 (κ = 0.80 and κ = 0.85, p<0.001). In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting.