Preclinical data suggest that cetuximab should be continued after end of concurrent RT/cetuximab due to its efficacy against residual tumor cells and angiogenesis in the irradiated tumor niche. Based on this concept the phase II add-on cetuximab (AOC) study was designed. Altogether 59 patients with advanced head and neck cancer, stages T3-4 Nx or Tx N2b-c, were treated with radiochemotherapy (70 Gy, cisplatin 40mg/m2 weekly) in combination with concurrent cetuximab (loading dose 400mg/m2, then 250mg/m2 weekly). Thereafter patients were randomized to cetuximab consolidation (500 mg/m2 biweekly x 6) or no further treatment. The primary endpoint was the 2-year locoregional control (LRC) rate. As translational research endpoints serum markers were analyzed before and during treatment and CT-based quantitative image analysis (radiomics) of the 3D primary tumor volume was performed. The 2-year LRC rates were 67.9% and 67.7% in the treatment arms with and without consolidation cetuximab. Higher than median levels prior to treatment of three serum markers were negatively associated with the 2-year LRC rate in the overall patient cohort: Osteopontin, IL8 and FasL2 (p<0.05). A radiomics model consisting of 2 radiomic features could be built showing that higher entropy and higher complexity of tumor Hounsfield unit distribution indicates worse LRC (CI 0.66). No correlation was found between biological and imaging markers. Consolidation cetuximab does not improve LRC when continued after end of RCT/cetuximab. Independent prognostic biological and imaging markers could be identified for the overall patient cohort. Studies with larger patient numbers are needed to identify the biological basis of imaging features.

Background: Acute respiratory decompensation can occur on a background of slowly progressive airway compromise, for example in laryngeal squamous cell cancer. Surgeons in ENT, together with anaesthetists, are often asked to evaluate airway risk and as yet there is no widely adopted standardised approach. Case report: This paper reports the case of an 82-year-old male, who presented with acute airway compromise due to both endolaryngeal obstruction from a squamous cell cancer and extralaryngeal compression from massive subcutaneous emphysema. Results: Primary total laryngectomy was performed, but the patient declined adjuvant radiotherapy. He died a year later from a heart attack without evidence of recurrence. Conclusion: To the best of our knowledge, this is the first case report of acute airway compromise from extralaryngeal subcutaneous emphysema secondary to laryngeal cancer. Options for acute airway management are discussed.

Taenia crassiceps is a tapeworm whose adult form infests the intestine of carnivores of the Northern Hemisphere. Rodents and moles are natural intermediate hosts and larval stages of the tapeworm develop as cyst-like structures (cysticerci) subcutaneously or in their body cavities. Infection in humans is rare, with only eight cases having been reported to date. Most infections have occurred in immunosuppressed patients, skeletal muscles and subcutaneous tissue being the sites for parasite accumulation. In addition, a few immunocompetent patients with intraocular infection have been described. In this article, we report a soft tissue infection with this tapeworm in an immunocompetent patient. It is the first case diagnosed on a fine needle aspiration (FNA) cytology sample, confirmed by PCR.

Objective: Lymphoepithelial carcinoma (LEC) of the salivary glands is a very rare condition especially in the Caucasian population. We herewith contribute a case of a Caucasian woman with LEC of the parotid gland. Furthermore we present a literature review on LEC of the major salivary glands (MSG) of the past 32 years. Materials and Methods: We reported an own case and performed a review of the medical literature (1980-2012). Results: Case report: After a Follow-up (FU) of 5 years after treatment with postoperative IMRT and concomitant carboplatin, our patient has no signs of relapse. Review: We found 404 cases with LEC of the MSG published in 33 articles over the last 32 years. The mean age of the patients was 49 years (16-86); 56% were female. 95% of the cases have been treated and published in Asia (China, Taiwan). Only 17 of the 404 cases (4%) reported in 15 publications, Caucasian patients were affected. Information on treatment and outcome was not available in 75% and 72%, respectively. After a mean FU of 5.5 years (range 0.5-14) 66 of 84 (79%) patients with reported FU showed no evidence of disease. Conclusion: LEC of the salivary glands is a rare disease especially in Caucasian populations with only 4% of published LEC cases in the reviewed literature. In only 21% of the reports, clinical information on treatment and outcome was provided. Based on the limited information available from published reports, the value of radiotherapy remains unclear.

The aim was to quantify the rate of severe late term effects in our single institution head neck cancer IMRT cohort. Between 01/2002 and 12/2011, 1077 IMRT patients have consecutively been treated in curative intention at our institution. 62% of all patients presented with locoregionally advanced disease (T3/4 and/or N2c/N3). 65% of all patients were referred for curative definitive IMRT (66Gy-72Gy total dose in 30-35 daily fractions); 25% had very large gross tumor volumes >70cc. The remaining patients underwent postoperative IMRT (60-66Gy in 30-33 daily fractions). Systemic concomitant therapy was given in 85%. Highly consistent treatment procedures were performed with respect to contouring, dose constraints, radiation schedules, and the use of systemic therapy. Grade 3/4 late term effects were prospectively assessed. Included in the following late tolerance analysis was the subgroup of patients with no signs of loco-regional disease for at least 12 months of follow up. Seven hundred twenty out of 1077 treated patients (67%) met our inclusion criterion at the time of this analysis. After a mean/median follow up of 39/34 months (range 12-118), only ∼5% persisting grade 3/4 late term effects have been observed: grade 3 radioosteonecrosis of the mandible in 3 patients (.5%), 6 patients with grade 3 and one with grade 4 dysphagia (1%) and/or persisting dependence of a feeding tube (2%), 7 patients with grade 3 xerostomia (1%), 2 patients with need of a laryngo-pharyngectomy. Arteriovascular bleeding (non-fatal), and need of a tracheostoma occurred in 3 patients each (1%). One patient suffers from persisting taste loss. An analysis of the here summarized side effects related to the specific sites at risk and the doses will be provided. IMRT lead to a high late term tolerance in head neck cancer patients. The question of the cervical vessel tolerance in disease free long time survivors is still open, and is currently under evaluation in our institution; first results may be presented at the upcoming ASTRO meeting.

OBJECTIVE:Diagnosis of low grade non-Hodgkin B-cell lymphomas on cytological material may be problematic and in the past frequently required lymph node excision. We analysed our experience of the value of flow cytometry (FC) as an additional tool for the diagnosis of lymphoproliferative processes in the setting of a university cytology division with a busy fine needle cytology service.METHODS:Consecutive cytological specimens with FC over a period of 3 years were retrospectively analysed and correlated with histology and follow-up if available. FC was performed with the following antibodies: CD3, CD4, CD8, CD2, CD7, CD19, CD5, CD10, CD23, lambda and kappa chains.RESULTS:Of 299 probes (273 fine needle aspirations and 26 fluids from 285 patients), 179 cases (60%) were diagnosed as reactive, 91 cases (30%) as malignant or suspicious and 29 cases (10%) as inconclusive. The results of histological examination of the lymph nodes were available in 41 of 91 (45%) malignant or suspicious cases and in 13 of 179 (7%) reactive cytological diagnoses. Cytologically diagnosed malignancy was confirmed in all histologically examined cases. In 12 of 13 reactive cytological cases (92%), a benign process was diagnosed histologically. In 34 of 299 cases (11%) additional molecular investigations of B-cell clonality or specific translocations were performed. The lymphomas most frequently diagnosed were follicular lymphoma and lymphocytic lymphoma, followed by mantle cell and marginal zone lymphomas. Correlation with histology showed a sensitivity of 98% and a specificity of 100% for cytology in our series.CONCLUSIONS:FC is an important additional tool in the cytological diagnosis of lymphoproliferative disorders. The combined approach has a high diagnostic value that allows a reliable subclassification of low grade B-cell non-Hodgkin lymphomas.

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

Background Tracing the origin of a metastasis of a neuroendocrine carcinoma is a challenge. The transcription factors Cdx2 and TTF1 have been found to be helpful in identifying well-differentiated neuroendocrine tumors of gastrointestinal and pulmonary origin, respectively. So far, such a marker is lacking for pancreatic neuroendocrine tumors (PETs) and metastases thereof. Islet1 (Isl1) is a transcription factor expressed in pancreatic islet cells. The aim of this study was (1) to test the specificity and sensitivity of Isl1 as a marker of PETs, and (2) to test the specificity and sensitivity of a panel of markers, including Isl1, Cdx2, and TTF1, for the localization of the primary. Design One hundred eighty-eight primary gastroenteropancreatic and pulmonary endocrine tumors and 49 metastases thereof were examined. Immunohistochemistry using antibodies directed against Isl1, Cdx2, and TTF1 was performed and the staining results were scored semiquantitatively. Results Isl1 proved to be a highly specific marker for pancreatic endocrine tumors. In 84 primary PET its specificity was 78.4% (sensitivity 74.3%) and in 18 metastases of PET the specificity reached 100% (sensitivity 77.8%). Strong Cdx2 staining showed a specificity for gastrointestinal origin of 83.9% (sensitivity 82%) in primary tumors and of 100% (sensitivity 40%) in metastases. Including weakly positive tumors lead to a decreased specificity but an increased sensitivity. TTF1 expression was detected in 2 PET and 1 ileal primary tumor only and was absent in all metastases of gastroenteropancreatic endocrine tumors. Conclusions Isl1 is a reliable marker of pancreatic endocrine tumors and metastases thereof. It shows a comparable sensitivity and specificity as Cdx2 as a marker of ileal and appendiceal neuroendocrine tumors and their metastases. TTF1 is very rarely expressed in well-differentiated gastroentero-PETs. Therefore, the panel of Isl1, Cdx2, and TTF1 seems useful for examining metastases of well-differentiated endocrine carcinomas of unknown origin.

Pheochromocytomas (PCCs) are rare tumors that arise from chromaffin tissue in the adrenal medulla, but can also occur in the abdomen outside the adrenals and are then called sympathetic paragangliornas (sPGLs). According to the literature, between 15 and 25% of apparently sporadic adrenal PCC and sPGL are caused by germline mutations in RET, von Hippel-Lindau disease (VHL), succinate dehydrogenase subunit B (SDHB), or subunit D SDHD. However, few studies have addressed the mutation frequency of these candidate genes in selected subgroups of PCCandsPGL, such as bilateral adrenal PCC or extra-adrenal sPGL, and none have looked at somatic mutations by analyzing tumor tissue. Therefore, we have investigated the occurrence of germline and somatic mutations in RET, VHL, SDHB, and SDHD in comparatively large series of bilateral adrenal PCC (n= 33 patients) and sPGL (n= 26 patients), with the aim of determining the mutation frequency of each of these genes and to establish a genetic testing algorithm. Twenty-one RET, two VHL germline, and one SDHD mutations were found in the patients with bilateral adrenal PCC. In sPGL, one novel SDHB germline and one novel SDHB somatic mutation were observed. In addition, two SDHD germline mutations were found. We conclude that germline RET mutations are predominantly found in bilateral PCC, and that somatic and germline SDHB and SDHD mutations usually occur in sPGL, which has practical consequences for genetic testing algorithms. We suggest that sequential mutation analysis should be directed first at RET, followed by VHL and SDHD for patients with bilateral adrenal PCC at diagnosis, and at SDHB and SDHD for patients with sPGL.

Background It is difficult to predict the biologic behavior of pancreatic endocrine tumors in absence of metastases or invasion into adjacent organs. The World Health Organization (WHO) has proposed in 2004 size, angioinvasion, mitotic activity, and MIB1 proliferation index as prognostic criteria. Our aim was to test retrospectively the predictive value of these 2004 WHO criteria and of CK19, CD99, COX2, and p27 immunohistochemistry in a large series of patients with long-term follow-up. Design The histology of 216 pancreatic endocrine tumor specimens was reviewed and the tumors were reclassified according to the 2004 WHO classification. The prognostic value of the WHO classification and the histopathologic criteria necrosis and nodular fibrosis was tested in 113 patients. A tissue microarray was constructed for immunohistochemical staining. The staining results were scored quantitatively for MIB1 and semiquantitatively for CK19, COX2, p27, and CD99. The prognostic value of these markers was tested in 93 patients. Results The stratification of the patients into 4 risk groups according to the 2004 WHO classification was reliable with regard to both time span to relapse and tumor-specific death. In a multivariate analysis, the CK19 status was shown to be independent of the WHO criteria. By contrast, the prognostic significance of COX2, p27, and CD99 could not be confirmed. Conclusions The 2004 WHO classification with 4 risk groups is very reliable for predicting both disease-free survival and the time span until tumor-specific death. CK19 staining is a potential additional prognostic marker independent from the WHO criteria for pancreatic endocrine tumors.

The tumorigenesis of sporadic endocrine tumors is still not fully understood. It is well known that patients with von Recklinghausen syndrome (NF-1) (OMIM 162200) carrying NF1 germline mutations are predisposed to endocrine tumors including pheochromocytomas and duodenal somatostatinomas. It is unclear, however, whether the rarely reported occurrence of pancreatic insulinomas in NF-1 patients represents a coincidental finding or whether insulinomas are a rare manifestation of the NF-1 syndrome. To determine the potential association between the NF-1 syndrome and pancreatic endocrine tumors, we analyzed a NF-1 patient with a well-differentiated pancreatic endocrine carcinoma for NF1 mutation, allelic loss of the NF1 gene and its expression in peripheral blood and tumor cells. The germline mutation c. 499 del TGTT known in the family was confirmed by polymerase chain reaction (PCR) and direct sequencing of exon 4 in DNA extracted from peripheral blood. Loss of heterozygosity (LOH) analysis of the NF1 gene was carried out using 3 intragenic microsatellite markers on 17q11.2. RNA expression was examined by reverse transcription and a consecutive PCR spanning intron 3 of the NF1 gene including the mutated site in exon 4. Immunohistochemistry was used to analyze NF-1 protein expression. Mutation analysis of peripheral blood leukocytes confirmed the 4 base pair deletion in exon 4 starting at codon 167 (499 del TGTT). LOH analysis of tumor tissue revealed retention of both NF1 alleles. While reverse transcriptase-PCR of peripheral blood showed bi-allelic expression of both the wild-type NF1 and the mutated form, reverse transcriptase-PCR of tumor extracts demonstrated expression of the mutated but not the wild-type NF1 allele. Additionally, neurofibromin, the NF1 gene product, was absent in the tumor tissue of the NF-1 patient. These results show that the wild-type NF1 transcrips and protein are reduced, in the reported insulinoma, supposedly by epigenetic mechanisms. This provides strong evidence that there is a relationship between von Recklinghausen disease and the patient's insulinoma. In this line, insulinomas may be viewed as a rare manifestation of the NF-1 syndrome. Furthermore, the NF1 gene must be considered as a candidate tumor suppressor gene for sporadic insulinomas and probably other pancreatic endocrine tumors.

Objective/Hypothesis: Mammalian auditory hair cells that are unable to regenerate and various agents, including gentamicin, can irreversibly damage the hair cells. Erythropoietin, known as the primary regulator of erythropoiesis, exerts also neuroprotective effects by binding to its receptor. We tested whether erythropoietin can protect the hair cells from gentamicin-induced damage. Study Design: This study localized the erythropoietin receptor in the cochlea and analyzed the effect of erythropoietin on gentamicin-damaged hair cells in vitro. Methods: Expression of erythropoietin receptor in the rat cochlea was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Protection of auditory hair cells from gentamicin was tested in vitro by exposing cultured rat organs of Corti with increasing concentrations of erythropoietin (0.1 U/mL, 1 U/mL, and 10 U/mL). Results: We detected erythropoietin and erythropoietin receptor mRNA expression in the organ of Corti, spiral ganglion, and stria vascularis by RT-PCR. Immunohistochemistry revealed that the erythropoietin receptor localizes to the outer and inner hair cells and supporting cells of the organ of Corti, as well as to the spiral ganglion cells and the stria vascularis. Significantly less hair cell loss occurred in the organs of Corti that were pretreated with 0.1 U/mL erythropoietin as compared with samples treated with gentamicin only. Conclusion: Decreased hair cell loss in erythropoietin-treated organs of Corti that had been exposed to gentamicin provides evidence for a protective effect of erythropoietin in aminoglycoside-induced hair cell death.

AIMS:The differentiation of adrenocortical carcinomas from adenomas may be difficult based on morphology alone. Differential expression of insulin-like growth factor (IGF) II and cyclin-dependent kinase (CDK) 4 has recently been described in these tumours. The aim of this study was to investigate the diagnostic usefulness of these markers immunohistochemically.METHODS AND RESULTS:We examined 22 benign and 17 malignant adrenocortical tumours and compared IGFII and CDK4 expression with known immunohistochemical as well as morphological criteria of malignancy. Thirteen of 17 carcinomas showed immunohistochemical reactivity for IGFII, whereas all adenomas but one were negative. Intense CDK4 expression was detected in 11 of 17 carcinomas but was present in only three of 22 adenomas. The MIB1 index was >5% in 14 of 16 carcinomas and was <5% in all adenomas but one. The combination of IGFII immunohistochemistry with MIB1 index led to high sensitivity and specificity in detecting adrenocortical carcinomas.CONCLUSIONS:IGFII and MIB1 are helpful immunohistochemical markers to predict malignancy in adrenocortical neoplasms. These markers can be used in addition to clinical, gross and morphological features to establish a diagnosis in difficult cases.

Objective: To present our experience with successful reversal of laryngotracheal separation in patients with chronic aspiration, to discuss our surgical technique, and to review the literature. Study Design: Retrospective case review. Methods: The medical records of two patients who underwent a successful reversal of laryngotracheal reversal after separation were reviewed. The two patients were evaluated clinically with videostroboscopy and videofluoroscopy. Results: Both patients presented with intractable aspiration despite protective tracheostomy because of impairment of lower cranial nerve function. After laryngotracheal separation, swallowing rehabilitation was safely possible, and neurologic improvement allowed reversal of the separation procedure. For reversal, we present the use of a partial cricotracheal resection with tracheocricothyropexia similar to the technique used for subglottic stenosis. Both patients were successfully reversed. Satisfactory voice and swallowing function 7 and 10 years after reversal, respectively, could be assessed through our evaluation. Review of the literature revealed a total of 13 cases of successful reversal after laryngotracheal separation. Technical details of surgery and functional results are rarely reported.