Simon F. Preuss

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Genetic Signatures of HPV-related and Unrelated Oropharyngeal Carcinoma and Their Prognostic Implications

Purpose: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. Experimental Design: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16INK4A immunostaining. The results were correlated with HPV status and clinical data from patients. Results: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P = 0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P = 0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P = 0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P = 0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P = 0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P = 0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P = 0.008; disease-free survival, P = 0.01) and none of these patients had a tumor recurrence. Conclusions: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Clinical Predictors for Germline Mutations in Head and Neck Paraganglioma Patients: Cost Reduction Strategy in Genetic Diagnostic Process as Fall-Out

Multiple genes and their variants that lend susceptibility to many diseases will play a major role in clinical routine. Genetics-based cost reduction strategies in diagnostic processes are important in the setting of multiple susceptibility genes for a single disease. Head and neck paraganglioma (HNP) is caused by germline mutations of at least three succinate dehydrogenase subunit genes (SDHx). Mutation analysis for all 3 costs approximately US

Head and neck paragangliomas: clinical and molecular genetic classification

2,700 per patient. Genetic classification is essential for downstream management of the patient and preemptive management of family members. Utilizing HNP as a model, we wanted to determine predictors to prioritize the most heritable clinical presentations and which gene to begin testing in HNP presentations, to reduce costs of genetic screening. Patients were tested for SDHB, SDHC, and SDHD intragenic mutations and large deletions. Clinical parameters were analyzed as potential predictors for finding germline mutations. Cost reduction was calculated between prioritized gene testing compared with that for all genes. Of 598 patients, 30.6% had SDHx germline mutations: 34.4% in SDHB, 14.2% SDHC, and 51.4% SDHD. Predictors for an SDHx mutation are family history [odds ratio (OR), 37.9], previous pheochromocytoma (OR, 10.9), multiple HNP (OR, 10.6), age <or=40 years (OR, 4.0), and male gender (OR, 3.5). By screening only preselected cases and a stepwise approach, 60% cost reduction can be achieved, with 91.8% sensitivity and 94.5% negative predictive value. Our data give evidence that clinical parameters can predict for mutation and help prioritize gene testing to reduce costs in HNP. Such strategy is cost-saving in the practice of genetics-based personalized health care.

High risk for bilateral Warthin tumor in heavy smokers – review of 185 cases

Head and neck paragangliomas are tumors arising from specialized neural crest cells. Prominent locations are the carotid body along with the vagal, jugular, and tympanic glomus. Head and neck paragangliomas are slowly growing tumors, with some carotid body tumors being reported to exist for many years as a painless lateral mass on the neck. Symptoms depend on the specific locations. In contrast to paraganglial tumors of the adrenals, abdomen and thorax, head and neck paragangliomas seldom release catecholamines and are hence rarely vasoactive. Petrous bone, jugular, and tympanic head and neck paragangliomas may cause hearing loss. The internationally accepted clinical classifications for carotid body tumors are based on the Shamblin Class I–III stages, which correspond to postoperative permanent side effects. For petrous-bone paragangliomas in the head and neck, the Fisch classification is used. Regarding the molecular genetics, head and neck paragangliomas have been associated with nine susceptibility genes: NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127. Hereditary HNPs are mostly caused by mutations of the SDHD gene, but SDHB and SDHC mutations are not uncommon in such patients. Head and neck paragangliomas are rarely associated with mutations of VHL, RET, or NF1. The research on SDHA, SDHAF2 and TMEM127 is ongoing. Multiple head and neck paragangliomas are common in patients with SDHD mutations, while malignant head and neck paraganglioma is mostly seen in patients with SDHB mutations. The treatment of choice is surgical resection. Good postoperative results can be expected in carotid body tumors of Shamblin Class I and II, whereas operations on other carotid body tumors and other head and neck paragangliomas frequently result in deficits of the cranial nerves adjacent to the tumors. Slow growth and the tendency of hereditary head and neck paragangliomas to be multifocal may justify less aggressive treatment strategies.

Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

Conclusions. We identified smoking as a significant risk factor for multilocular Warthin tumor development. Therefore, we recommend taking history of smoking into account when making the decisions for surgical strategy. Objectives. Warthin tumor is a common benign neoplasm of the parotid gland. Risk factors for multilocular development have not been defined. Patients and methods. A total of 185 consecutive patients treated for Warthin tumor were included. Charts were reviewed for symptoms, risk factors, and diagnostic and surgical procedures. Patients were followed for facial function and recurrence. Risk factors were evaluated. Results. Overall, in 203 parotid operations, a lateral parotidectomy (77%) was performed in most cases; 94% were primary surgery and 6% were revision surgery. In 89% of patients swelling was the only symptom. Bilateral Warthin tumor was seen in 17% of patients. Of these cases synchronous and metachronous bilateral tumors were observed in 61% and 39%, respectively. The median time period for second contralateral tumor development was 7 years. Postoperative transient facial dysfunction was observed in 31%, which recovered within 3 months in all cases. Evaluation of risk factors revealed that 89% of the subjects were smokers and 66% were heavy smokers. The risk for bilateral Warthin tumors correlated significantly with the amount of nicotine intake (p=0.003).

Nuclear survivin expression is associated with HPV-independent carcinogenesis and is an indicator of poor prognosis in oropharyngeal cancer

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Transoral laser surgery for laryngeal cancer: Outcome, complications and prognostic factors in 275 patients

The relationship between expression of the inhibitor of apoptosis protein survivin and the presence of high-risk human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC) remains unclear. This also accounts for its role as a predictor of survival. Therefore, we conducted a multicentre retrospective study on 106 consecutive oropharyngeal cancer patients. Human papillomavirus sequences were detected by nested PCR protocols. Survivin and p16 expression as a surrogate marker for HPV status were analysed by immunohistochemistry. Sequences of high-risk HPV were detected in 29% of cases. Prominent cytoplasmatic expression of survivin was found in 58% of cases and nuclear expression of survivin was found in 19% of the survivin-positive tumours. Nuclear expression of survivin was significantly correlated with HPV-negative tumours (P=0.023) and with a poor disease-free survival rate with an estimated 3-year disease-free survival probability of 35% for tumours with nuclear expression of survivin vs 78% for tumours with non-nuclear expression of survivin (hazard ratio=8.264; 95% confidence interval (95% CI)=2.510–27.210; P<0.001). In multivariate analysis, p16 expression status as well as nuclear expression of survivin were strong independent and opposing prognostic indicators of disease-free survival (hazard ratio=0.068; 95% CI=0.005–0.892; P=0.041 and hazard ratio=15.975; 95% CI=2.377–107.360; P=0.004, respectively). Our data show that nuclear accumulation of survivin correlates with HPV-independent carcinogenesis and is an independent predictor of poor survival in patients with OSCC.

p16 Expression in carcinoma of unknown primary: Diagnostic indicator and prognostic marker

AIM Curative treatment options for laryngeal carcinoma include primary radiation therapy, open surgical techniques and transoral laser surgery (TLS). In the last decade, TLS has become an important tool in the treatment of laryngeal cancer and has become the standard approach in many institutions. The aim of this study was to review the experience of a single center institution with TLS for early and advanced laryngeal cancer. METHODS We retrospectively analyzed 275 patients who underwent TLS in regard to the survival outcome and surgical complications. RESULTS The 5-year disease-free survival estimate was 90.3% and the 10-year disease-free survival estimate was 88.2%. The 5-year larynx preservation rate estimate was 88.2% and the 10-year larynx preservation rate estimate was 87.3%. The disease-free survival was significantly worsened by the variables T and N (p=0.0003; p<0.001, respectively). Two percent of all patients required intraoperative tracheostomy and the rate of minor postoperative complications was 17%. There were no fatal complications. CONCLUSIONS We conclude that TLS is a valid treatment method for early laryngeal carcinoma. Selected cases of advanced carcinomas may also benefit from TLS.

Long-term results of surgical treatment for recurrent respiratory papillomatosis.

Carcinoma of unknown primary (CUP) of the neck are heterogeneous tumors in their clinical and biological characteristics, and a preoperative prognostic marker is desirable to optimize staging and therapy and to improve outcome and survival. For CUP syndrome, no optimized diagnostic and treatment strategy or biomarker have yet been determined.