Targeted next-generation sequencing identifies molecular subgroups in squamous cell carcinoma of the head and neck with distinct outcome after concurrent chemoradiation.

BACKGROUND: Based on epidemiological (HPV status, smoking habits) and clinical risk factors (T/N stage), three subgroups of patients suffering from locally advanced oropharyngeal carcinoma with significantly different outcome after concurrent chemoradiation (cCRTX) can be distinguished. Mutational profiling by targeted next-generation sequencing (NGS) might further improve risk stratification. PATIENTS AND METHODS: Patients with stage IV squamous cell carcinoma of the oropharynx and hypopharynx who had been enrolled in a randomized phase III trial (ARO-0401) comparing two regimens of cCRTX and from whom archival tumor specimens were available were included. The HPV status was determined by p16 immunostaining and detection of HPV DNA. Targeted NGS covering 45 genes frequently altered in squamous cell carcinoma of the head and neck (SCCHN) was applied for detection of non-synonymous somatic and germline mutations. Interference of mutational profiles with cCRTX efficacy was determined. RESULTS: The prognostic value of the 'Ang' risk model could be confirmed in the total biomarker study cohort (N = 175) as well as the patient subgroup for which mutational profiles could be established (N = 97). Mutations in genes involved in phosphoinositide 3-kinase (PI3K), receptor tyrosine kinase (RTK), and p53 signaling pathways were significantly enriched in the low- (N = 7), intermediate- (N = 20), and high-risk group (N = 70), respectively. Mutations in TP53 identified a subgroup of high-risk patients with dismal outcome after cCRTX. No prognostic relevance was observed for mutations in PI3K and RTK signaling pathways in the low- and intermediate-risk groups, respectively. Mutated NOTCH1 and two functional KDR germline variants (rs2305948, rs1870377) were associated with improved outcome in all risk groups. All genetic markers (TP53, NOTCH1, KDR) remained independent prognosticators of OS in the multivariate model. CONCLUSION: A potential of targeted NGS for risk classification of SCCHN cases beyond HPV status and clinical factors was demonstrated.

[Cells of the tumor front : a potential therapeutic target in head and neck cancer therapy?]. / Zellen der Tumorfront : Ein mögliches therapeutisches Ziel bei der Behandlung von Kopf-Hals-Karzinomen?

BACKGROUND: With a frequency of about 90 %, head and neck squamous cell carcinomas (HNSCCs) are the most common malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis postulates that CSCs are the dangerous part of the tumor and are relevant to metastasis, invasiveness and resistance to chemotherapy. METHODS: Tissue samples taken from HNSCCs and normal mucosa were tested for the expression of several established CSC markers. The expression and activity of the matrix metalloproteinase MMP-9 was also investigated. RESULTS: Cells of the invasive tumor front expressed the basal stem cell markers CD44, ALDH1 and CK14. However, in contrast to the noninvasive basal cell layer of normal mucosa, HNSCC samples were also positive for active MMP-9, which lends the tumor its gelatinolytic activity. CONCLUSION: These observations suggest a model in which cells of the invasive front are derived from the basal cell layer of normal mucosa and harbour the CSCs. Future studies should thus focus on the cells of the invasive front in particular, since the activity of these cells may form the basis for tumor recurrence and therapy resistance.

Effect of thirst challenge on ADH levels in patients with bilateral Menière's disease.

The aim of the study was to investigate plasma ADH levels and plasma/urine osmolality in patients suffering from bilateral Menière's disease since a disturbance in the water household after thirst challenge is a suspected pathogenic factor in the development of this disease. In this study the plasma ADH levels and plasma/urine osmolality of bilateral Menière's disease patients under thirst challenge were investigated to show whether the water balance is affected. 9 patients with bilateral Menière's disease and 9 healthy controls skipped water intake for 12 h. Plasma ADH, plasma/urine osmolality, and electrolytes were measured after this thirst period as well as 8 h later after food and fluid intake. During food and fluid intake the patients demonstrated a slightly higher plasma ADH level and plasma osmolality than controls, whereas at the end of the thirst period patients and the controls showed no significant change. Instead the urine osmolality differed significantly (p<0.001): showing a high urine osmolality in controls and an almost stable urine osmolality in patients after thirst challenge. This indicates that the water balance in patients is likely different from that of controls. These observations point to ADH and its target aquaporine 2 as keyplayers in the pathophysiological events leading to the development of Menière's disease.

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells.

BACKGROUND AND PURPOSE: Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands. EXPERIMENTAL APPROACH: Tissue specimens of the right submandibular gland taken from 18 Wistar rats after intraglandular injection of BoNT A, BoNT B, or a combination of both were examined. As a sham control, an equivalent volume of saline was injected into the left submandibular gland. Morphometric measurements, immunohistochemistry, electron microscopy and western blot analysis were used to analyse the morphological and functional changes of the denervated glands. KEY RESULTS: Morphological and ultrastructural analyses of the cell organelles and secretory granula showed a clear atrophy of the acini, which was more prominent in glands injected with the combination of BoNT/A and B. Morphometric measurements of the glandular acini revealed a significant reduction of the area of the acinar cells after injection of BoNT (P=0.031). The expression of amylase was significantly reduced in BoNT treated glands. CONCLUSIONS AND IMPLICATIONS: Intraglandular application of BoNT induces structural and functional changes of the salivary glands indicated by glandular atrophy. These effects may be due to glandular denervation induced by the inhibition of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) involved in acetylcholine release at the neuroglandular junction and also specially inhibition of those involved in exocytosis of the granula of the acinar cells.

RT-PCR expression profiling of matrix metalloproteinases and their specific inhibitors in cell lines and fresh biopsies of squamous cell carcinomas of the head and neck.

The expressions of MMP2, -7, -9, -13 and TIMP1, -2, -3 were examined in biopsies and cell lines of head and neck squamous cell carcinomas (HNSCC) to determine the association between the expression profile and TNM-staging of the primary. The expressions of MMP2, -7, -9, -13 and TIMP1, -2, -3 were analyzed in 30 HNSCC biopsies, 7 HNSCC cell lines and 1 keratinocyte cell line using RT-PCR. Negative correlation was determined between N-status and MMP13-RNA expression [Kendall-tau-b -0.404 (p = 0.016), Spearman-rho -0.448 (p = 0.014)], histological grading [Kendall-tau-b -0.291 (p = 0.049), Spearman-rho -0,333 (p = 0.048)], and MMP7 and TIMP2 expression [Kendall-tau-b -0.318 (p = 0.045); Spearman-rho -0.353 (p = 0.045)]. Positive correlation was determined between M-status and MMP9-RNA expression [Kendall-tau-b 0.341 (p = 0.025), Spearman-rho 0.377 (p = 0.024)] and MMP13 and TIMP2 expression [Kendall-tau-b 0.727 (p = 0.037), Spearman-rho 0.850 (p = 0.016)]. The results point to a role of the tested MMPs and TIMPs in the metastatic spread of HNSCC.

Sialadenosis of the major salivary glands in a patient with central diabetes insipidus--implications of aquaporin water channels in the pathomechanism of sialadenosis.

Sialadenosis, also referred to as sialosis, is a disease of unknown aetiology. It regularly manifests itself as a massive swelling in both parotid regions involving the major salivary glands, preferably the parotid glands and is characterized by lack of any detectable, underlying pathologies. In this case report we describe a 24-year-old white female patient with diabetes insipidus who developed sialadenosis of the major salivary glands during a period of enhanced water requirement, which the patient tried to compensate for by more frequent nasal ADH application. Since ADH acts on aquaporins (AQPs) in the kidney, we were interested if AQP expression in the patients salivary glands was affected. Surprisingly, compared to normal control tissues we observed an extensively high signal for AQP5, which is the dominant AQP found in salivary acinar cells. Interestingly, previous studies on AQP5 knock out mice found AQP5 to be required for cell volume regulation. We therefore suggest that aquaporin water channels and antidiuretic hormone together with a disturbance in the body's water household are potential key-factors in the pathophysiological events leading to the development of the disease entity called sialadenosis.

The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck.

The ability of tumors to infiltrate the surrounding tissue is one of the major characteristics of a malignancy. This process is based on the tumors ability to destroy the extracellular matrix (ECM) including the basement membrane (BM). Several previous studies identified matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases to be key players in this process. Since then multiple investigations focused on the expression and activation levels of their extracellular regulators and to a lesser extent of their transcriptional modulators. However, the exact diagnostic and prognostic values of these regulators still remain unclear. Squamous cell carcinomas of the head and neck (HNSCC) are known for their infiltrative growth and there is strong evidence that at least some members of the MMP-family play a crucial role in this process. It turned out that MMP-2, -9, -13 and to a lesser extent MMP-7 are related to the metastatic potential of HNSCC but further studies will be required to establish the exact role of MMPs in HNSCC. This Review will discuss the current literature concerning the role of MMPs in HNSCC.

Lymphogenic metastatic spread of auricular VX2 carcinoma in New Zealand white rabbits.

INTRODUCTION: The auricular VX2 carcinoma of the New Zealand white rabbit serves as an animal model for human squamous cell carcinomas of the head and neck region (HNSCC), since both tumors tend to metastasize lymphatically, leading to early lymph node and subsequent distant metastasis. The aim of this study was to examine the pattern of lymphogenic metastatic spread in untreated auricular VX2 carcinomas, since the resulting knowledge potentially could help in the development of new treatment strategies for human HNSCC. MATERIALS AND METHODS: VX2 carcinomas were implanted into both ears of 22 New Zealand white rabbits. The animals were sacrificed at days 7, 14, 21, 28 or 32 after tumor implantation, followed by a detailed histopathological examination of their head and neck lymph nodes. RESULTS: On day 7 after tumor implantation 25% of the animals had metastases in the parotid lymph node, which is the first draining lymph node of the tumor region. This number rose to 87.5% by day 28. At this time 12.5% of all animals also had an additional metastasis in the second echelon node. CONCLUSION: A reproducible metastatic spread into the first draining lymph node could be demonstrated for the auricular VX2 carcinoma of the New Zealand white rabbit. The VX2 carcinoma therefore appears to be a highly suitable animal model for studying the sentinel node concept in the context of human HNSCC.

Expression of MMP-3, MMP-13, TIMP-2 and TIMP-3 in the VX2 carcinoma of the New Zealand white rabbit.

BACKGROUND: Squamous cell carcinomas of the head and neck region (HNSCC) are among the most common malignancies in this area. The VX2 carcinoma of the New Zealand white rabbit metastasizes lymphatically as is the case in HNSCC and therefore, potentially, could be used as a model for HNSCC. Since the family of matrix-metalloproteinases (MMPs) is involved in the process of HNSCC invasion, the aim of this study was to investigate the expression level of MMPs and their specific inhibitors (TIMPs) in the VX2 carcinoma to evaluate if they also play a role in VX2 tumor invasion as observed in human HNSCC. MATERIALS AND METHODS: The VX2 carcinoma was generated by tumor implantation in the rabbit's ear as previously described. Western blots were performed under standard conditions, utilizing antibodies against MMP-3, MMP-13, TIMP-2 and TIMP-3. Immunohistochemical staining was performed with the ABC-complex method. RESULTS: A positive immunohistochemical signal could be detected for MMP-3, TIMP-2 and TIMP-3 with no significant signal for MMP-13. In the Western blots immunoreactive bands could be observed for MMP-3, MMP-13, TIMP-2 and TIMP-3. CONCLUSION: MMP-3, MMP-13, TIMP-2 and TIMP-3 were found to be expressed in VX2 carcinomas of the New Zealand white rabbits. The VX2 carcinoma therefore resembles HNSCC tumors not only in its metastatic behavior, but also regarding the expression of MMPs and TIMPs, which are the probable keyplayers during the event of invasion. These observations further underline the significance of the VX2 carcinoma as a model tumor of human HNSCC.

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity.

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

Variations of EGF-R surface expression in squamous cell carcinomas of the head and neck region.

BACKGROUND: It has been previously found that the epidermal growth factor receptor (EGF-R) is highly expressed in squamous cell carcinomas of the head and neck region (SCCHN) and that this expression correlates with malignant transformation and tumor development. MATERIALS AND METHODS: We examined 9 SCCHN cell lines and a control keratinocyte cell line for EGF-R expression, utilizing fluorescence-activated cell sorting (FACS) and Western blot analysis. Immuno-cytochemistry was performed to evaluate the receptor's cellular distribution. RESULTS: We found the EGF-R to be up-regulated in SCCHN. Surprisingly we did not see a clear correlation between tumor staging, grading or Stat3 signaling and EGF-R level but we observed up to three distinct EGF-R specific bands in our Western blots, implicating the presence of different protein isoforms. CONCLUSION: Our data suggest that not only the amount but also the receptor function, including the presence of mutations, need to be considered in SCCHN malignancies.

Characterization of an alternatively spliced isoform of rat vesicle associated membrane protein-2 (VAMP-2).

VAMPs are vesicle associated membrane proteins that are essential for secretion. A spliced isoform of rat VAMP-2, called VAMP-2B, is characterized in this study. The VAMP-2B transcript is the result of alternative RNA splicing in which an intron is retained. The predicted amino acid sequence of VAMP-2B differs from VAMP-2 at its carboxy-terminal end. Because recent studies have shown that VAMP's carboxy-terminal end influences the protein's sorting, the location of myc-epitope tagged VAMP-2B in PC12 cells was determined. Subcellular fractionation showed colocalization of myc-VAMP-2B and endogenous VAMP-2. Thus alternative RNA splicing does not affect VAMP-2 sorting in PC12 cells.

Tissue-specific alternative RNA splicing of rat vesicle-associated membrane protein-1 (VAMP-1).

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.

Anemia with LDH-elevation in a patient with aortic valve replacement.

In a 63-year old patient with a history of aortic valve replacement in 1986, a reduced hemoglobin of 91 g/l was found by a family physician. Since serum LDH was also increased, the patient was diagnosed to suffer from mechanically induced, hemolytic anemia and presented at our hospital for further diagnosis and evaluation of the aortic valve prosthesis.

[Juxta-vesical ureterolithotomy with intubation of the distal part of the ureter without opening the urinary bladder]. / Jukstavezikalna ureterolitotomija sa intubacijom distalnog dela uretera bez otvaranja mokracne besike.

A new surgical method in the treatment of juxtavesical ureterolithiasis is described; its advantage in relation to the classical juxtavesical ureterolithotomy is in the intubation of distal part of the ureter without incision of the bladder. In this way a stenosis at the place of ureterolithotomy is prevented, and on the other side the existent stenosis is dilated. 23 patients have been operated upon, in 9 of whom there was a blockage of the kidney caused by a stone in juxtavesical part of the ureter, with uretero-hydronephrosis in 14 patients. In 14 patients the urine was infected, and in 9 ones sterile; the stone has been in the ureter about 2-3 months in 15 cases. In all patients an extraction of the stone by a Dormy catheter has been tried, but without success. The follow-up of the patients, 8-12 months after surgery showed that the kidney does secrete well, ureter was with a fair passage at the x-ray, and without stenosis of the ureter.