Epstein-Barr virus-associated diffuse large B-cell lymphoma of the hypopharynx.

PROBLEMS/OBJECTIVES: Epstein-Barr virus (EBV) is commonly associated with nasopharyngeal carcinoma and Burkitt's lymphoma, but association with hypopharyngeal and laryngeal tumours is rare. To the best of our knowledge, this is the first case report of an EBV-associated diffuse large B-cell lymphoma (DLBCL) of the hypopharynx. METHODOLOGY: A 63-year-old male patient suffering from chronic lymphocytic leukemia presented with swallowing disorders and a sore throat. Panendoscopy with laser surgical resection of tissue specimens was performed. RESULTS: Immunohistochemical and molecular genetic diagnostics, including EBV-encoded small RNA in situ hybridization, confirmed the diagnosis of an EBV-associated DLBCL of the hypopharynx. Ten weeks after the diagnosis, the patient died of disease related to multiple complications. CONCLUSIONS: We hypothesize that the EBV infection was triggered by long-term immunosuppressive therapy that led secondarily to the development of a DLBCL. Otorhinolaryngologists should keep in mind that lymphomas might develop in the entire pharynx.

Molecular and immunological aspects of p53 and p53-autoantibodies in head and neck squamous cell carcinoma.

The tumour suppressor protein p53 (wild-type = wt-p53) is of major importance in the genetic integrity of the cell. Mutations of the p53-gene (mt-p53) are the most frequent genetic aberrations identified in different tumour entities. As analyzed in a wide variety of human malignomas, mt-p53 evokes a specific immune response. Yet, the possible occurrence of p53-autoantibodies in patients with head and neck squamous cell carcinomas (HNSCC) correlated to p53-mutations, p53 in sera and p53-overexpression in tissue has not been previously investigated. For the first time, the p53 status in 24 HNSCC patients was analyzed in the present study. The following parameters were investigated: analysis of mutation frequency of the p53-gene by direct sequencing of the exons 5-9, immunohistochemical detection of p53, measurement of the wt- and mt-p53-protein in sera by ELISA and p53-autoantibodies in sera by ELISA. Mutations of the p53-gene were detected in four (17%) patients. Overexpression of wt-p53 was detected by immunohistochemistry in 18 out of 24 (75%) tumours. In 8 (33%) patients the p53-protein was also detectable in sera, whereas in just one of these eight patients p53-autoantibodies were detectable simultaneously. Overall 6 out of 24 (25%) patients were found to be positive for serum p53-autoantibodies. Of these 6 cases, 5 could be assigned to tumours with immunohistochemically measurable wt-p53-overexpression. There was no correlation between p53-overexpression in tissue and p53-protein levels in sera or between p53-autoantibody levels in sera, nor in mutation frequency of the p53-gene and p53-overexpression in tissue. The results presented herein support the hypothesis that strong accumulation of p53 in the tissue is an important prerequisite for development of p53-autoantibodies. However, there must be further, yet unknown factors that influence the p53-autoantibody production because p53-autoantibodies were not identified in sera in each case of p53-accumulation in the tissue.

Selective upregulation and amplification of the lysyl oxidase like-4 (LOXL4) gene in head and neck squamous cell carcinoma.

Members of the lysyl oxidase family (LOX) are copper and lysyl-tyrosine quinone cofactor-containing amine oxidases that are important for the assembly and maintenance of components of the extracellular matrix. Our previous results demonstrated that a novel member, LOXL4, is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. Results of the current study showed overexpression of the LOXL4 transcript in 74% (46 of 62) of invasive HNSCC tumours and 90% of both primary and metastatic HNSCC cell lines. Significant correlation was found between LOXL4 expression and local lymph node metastases versus primary tumour types (p<0.01) and higher tumour stages (p<0.01). Immunocytochemistry demonstrated cellular overexpression of the LOXL4 protein that correlated with the increased mRNA transcription in HNSCC cells. HNSCC cell lines displayed in significant subset of nuclei increased copies of the LOX4 gene locus on chromosome 10q24, demonstrated by fluorescence in situ hybridization (FISH). Extensive metaphase cytogenetic analysis was performed on UTSCC19A cells, identifying an isochromosome i(10)(q10). Taken together, these results highlight LOXL4 expression as a distinctive trait and suggest a functional role for LOXL4 in the molecular pathogenesis of invasive head and neck carcinomas.

Head and neck manifestations of Wegener's granulomatosis.

Wegener's Granulomatosis (WG) is a necrotizing granulomatous angiitis that presents the classic ELK triad of ear, nose, throat (E), lung (L), and kidney (K) involvement. Its potential rapid and fatal outcome makes the early recognition--before irreversible organ involvement occurs--mandatory. The aetiology is still unknown. Today, immunosuppressive therapy makes WG a treatable disease with a chronically relapsing course. The otorhinolaryngologist plays an important role in early diagnosis of WG, because in up to 95% of the patients initial WG symptoms are observed in the head and neck region. The majority of these patients show nasal or sinunasal involvement. Common manifestations are sinusitis, crusting of the nose, and development of saddle nose deformity. Other head and neck problems are middle and inner ear symptoms and subglottic stenosis. Follow up and activity assessment of the disease are also important roles to play for the otorhinolarygologist.

[Wegener's granulomatosis in the head and neck region]. / Morbus Wegener im Kopf-Hals-Bereich.

Wegener's granulomatosis (WG) is defined as a granulomatous inflammation of the upper and lower respiratory tract and systemic vasculitis of small and medium sized vessels which is often accompanied by a necrotizing glomerulonephritis. The etiology of the disease is still unknown. In former times untreated WG usually ended deadly. Immunosuppressive therapy made WG a treatable disease with a chronically relapsing course. Therefore an early diagnosis of WG is of utmost importance. WG usually starts as a limited and localized organ manifestation in the upper respiratory tract and it generalizes, if untreated, with pulmonary and renal involvement. Symptoms in the head and neck region are observed in up to 95% of the patients with WG. Sinusitis, crusting of the nose, development of a saddle nose, middle and inner ear symptoms and subglottic stenosis are common manifestations. Due to the early and common manifestation of WG in the head and neck region the otorhinolaryngologist plays an important role for the early diagnosis and the fast initiation of immunosuppressive therapy but also during follow-up for activity assessment.

Sentinel lymph-node biopsy in head and neck cancer.

The aim of the study was to assess the diagnostic value of the sentinel node method in patients suffering from squamous cell carcinoma of the upper aerodigestive tract. In 50 patients with oral, pharyngeal or laryngeal carcinomas staged N0 up to 50 MBq technetium-99m colloid were injected peritumorally. Sentinel nodes were localised using a gamma-probe in the setting of an elective neck dissection. Pathological findings of sentinel nodes and corresponding neck specimens were compared. In 46 patients sentinel nodes were detected. Of these 34 patients were free of metastatic disease in the sentinel nodes and in the neck specimens. In 12 patients clinically occult metastases were found in the sentinel nodes. Three metastases were detected only after additional sectioning of the sentinel nodes. In four patients, a sentinel lymph node could not be localised. Our results support the sentinel node concept in head and neck cancer and a definition of the sentinel nodes as the three nodes with the highest activity. Careful clinical staging of the neck and thorough pathological evaluation of the sentinel nodes are necessary to avoid false-negative results.

[Collagenase 3 mRNA expression in squamous epithelial carcinomas of the oropharynx]. / Kollagenase-3-mRNA-Expression in Plattenepithelkarzinomen des Oropharynx.

BACKGROUND: Metalloproteinases (MMP) are endopeptidases, which are able to degrade extracellular matrix. It is assumed that MMP play an important role in invasion and metastasis of malignomas. The expression of collagenase-3, also named MMP-13, was already detected in squamous cell carcinoma of the head and neck, but the significance in the process of metastasis is still unclear. PATIENTS AND METHODS: 36 tumor biopsies of oropharyngeal squamous cell carcinoma (10 cases N0, 26 patients N+) and 12 biopsies of normal oropharyngeal mucosa were analysed with reverse transcriptase-PCR and Northern Blot for their mRNA-expression of MMP-13 and TIMP-1, the physiological inhibitor of MMP-13. RESULTS: In 30 of 36 (83.3%) tumor biopsies a MMP-13-mRNA-expression was detected. In 9 of 10 (90%) cases with N0-status and 21 of 26 (80.7%) cases with N(+)-neck the mRNA-expression could be shown. There was no correlation between MMP-13-mRNA-expression and N-status. In 34 tumor biopsies (94.4%) a TIMP-1-expression was detected. MMP-13-mRNA was not detected in normal oropharyngeal mucosa. CONCLUSIONS: It seems that MMP-13-mRNA-expression is not a prognostic factor for metastatic behavior of oropharyngeal cancer and therefore not helpful for further decisions on the therapy.

Experimental and clinical results of Er:YAG laser stapedotomy.

BACKGROUND AND OBJECTIVE: At the beginning of the 1980s, different laser types were used for stapes surgery to reduce potential harm to inner ear structures through manipulation with conventional instruments during stapedotomy. Most clinical studies were carried out with the CO2 or the argon laser. The Er:YAG laser has been used rarely in patients with otosclerosis. STUDY DESIGN/MATERIALS AND METHODS: In an experimental study on 54 human petrous bones, the optimal laser energy parameter for dissection of the posterior stapes crus and the footplate perforation were determined. With these parameters, stapedotomy was carried out with the Er:YAG laser in 29 patients with otosclerosis with a conventional dissection of the incudostapedial joint and the stapedius muscle tendon. The Er:YAG laser was used (60 or 100 mJ, 3-6 pulses) for dissection of the posterior stapes crus and footplate perforation. RESULTS: No intra- or postoperative complications were observed in all 29 patients. Vertigo and hearing loss were not observed intra- or postoperatively. The postoperative hearing results (improvement of the air-bone gap) was in all cases satisfactory (median remaining air bone gap, 8.1 dB). The median operation time was 29 minutes (15-42 minutes) and did not show a significant prolongation in comparison to the conventional technique. In 1 of the 29 patients, the footplate perforation needed to be carried out conventionally. CONCLUSION: For the first time, Er:YAG laser parameters have been optimized and refined in a human petrous bone model and were then used in a clinical setting. According to the presented results, the Er:YAG laser seemed to be a very suitable instrument for stapedotomy.

Transcriptional repression of the human fibronectin gene in laryngeal squamous cell carcinoma cells.

PURPOSE: The aim of the experiments was to analyze the mRNA expression pattern and verify the repression of FN gene expression in laryngeal squamous cell carcinoma (SCC) cells in comparison with benign mucosal keratinocytes. METHODS: Messenger RNA from SCC cells and benign keratinocytes was reverse transcribed and subjected to PCR following differential display (DD) analysis of the amplicons. Northern hybridization was carried out to confirm the reduction of the FN-mRNA expression in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. Quantitation of protein synthesis was performed with homogenates of fresh tumor biopsies and their normal phenotypes, as well as of benign keratinocytes and laryngeal SCC cell lines, respectively, using ELISA. In the liposome-mediated transient transfection assay, FN promoter activity was analyzed by linking the FN promoter sequence to the chloramphenicol acetyltransferase (CAT) reporter gene. Transfection efficacy was monitored by co-transfection with pGL3 control vector. RESULTS: A 191 bp mRNA fragment revealing a 99% homology with the human FN-mRNA was detected, the expression of which was repressed 20 times as much in SCC cells as compared to benign phenotypes. Northern hybridization confirmed the distinctly reduced expression of FN-mRNA in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. The quantitation experiments showed a correlation between the range of FN synthesis and the expression of FN-mRNA in cell lines and the biopsies which were used. The 1.28 kb FN gene promoter drove expression of the CAT reporter gene, which was similar to the FN-mRNA expression showed by DD and Northern hybridization. CONCLUSIONS: The mechanisms leading to the low level of FN in many tumors have not yet been sufficiently investigated. Our findings suggest that the decrease of FN in laryngeal SCC cells is transcriptionally regulated.

[Analysis of the p53 gene status of lymph node metastasis in the head and neck region in occult primary cancer]. / Analyse des p53-Gen-Status von Lymphknotenmetastasen im Kopf-Hals-Bereich bei okkultem Primärtumor.

BACKGROUND: Lymph node metastasis in the head and neck region with occult primary cancer are a fairly rare tumor entity in head and neck cancer. Tumor biological parameters as well as mechanism and cause for the development of this so called "cancer of unknown primary" (CUP) are not well investigated in head and neck cancer. Mutations of the p53 tumor suppressor gene are the most prevalent genetic alteration of human malignancies and show an incidence of up to 50% in head and neck cancer. For a further tumor biological definition of CUP the p53 status was determined. METHODS: Twenty-three archival formalin fixed paraffined CUP of an occult squamous cell carcinoma of the head and neck region were examined. In all cases a primary cancer was never diagnosed. The DNA extracted from the tumor material was amplified with specific primers for exon 4-9 of the p53 gene and subsequently sequenced. RESULTS: None of the 23 CUP cases showed a mutation or polymorphism in exon 4-9 of the p53 gene. CONCLUSIONS: The total absence of p53 mutations in the so called mutational "hot spots" shows a significant difference to the frequency of primary tumors of the head and neck region. May be this tumor biological characterisation helps to further elucidate the growth behaviour of CUP in the head and neck region and the reason for their development.

p53 analysis of laryngeal cancer in exon 4 to 9.

p53 gene mutations are a common genetic alteration in human cancer and codon 72/exon 4 polymorphism of the p53 gene has been implicated in cancer risk. Therefore in this study the p53 gene status of 32 shock-frozen tumor specimens from larynx carcinomas was analyzed by PCR and sequencing of exon 4 through 9. Four mutations (12.5%) in exon 5, 7, 8 and 9 were detected in the carcinoma specimen. Analysis of codon 72 revealed in eight cases a homozygosity for proline (CCC) and in 24 cases heterozygosity or homozygosity for arginine (CGC). The group with the proline/proline genotype had a median age 10.3 years lower than the remaining patients and included the only two non-smokers. Firstly, these results confirm the p53 mutational status of laryngeal cancer without any clinical correlation and secondly may suggest an oncogenic potential for the proline/proline genotype of codon 72 for laryngeal cancer as has already been assumed for lung cancer.

Cyfra 21-1: a serological help for detection of distant metastases in head and neck cancer.

Local and neck recurrences of squamous cell carcinoma of the head and neck (SCCHN) can mostly be detected early when the patient has regular follow-ups. Distant metastases though usually remain undiscovered until they produce clinical symptoms. Since Cyfra 21-1 correlates with the tumor size and stage in SCCHN, we looked for possible connections between Cyfra 21-1 increases and the development of distant metastases. The sera of 830 patients with SCCHN were tested for Cyfra 21-1. The levels were compared with the clinical run of the patients. When Cyfra 21-1 levels rose above the threshold of 3.3 ng/ml (71 out of 830) staging procedures were performed. Tumor growth was found in 50 out of 71 patients with elevated Cyfra levels (70.4%). Cyfra serum levels in those cases either represented development of distant metastases (27 out of 71), or local and neck recurrences. The results of this study show that Cyfra 21-1 is a suitable and helpful serological parameter for the follow-up of patients with SCCHN. In the event of an elevation of Cyfra 21-1 above the threshold during follow-up, we would recommend the performance of a thoracal CT-scan and abdominal omi ultrasound as staging procedures.

Serum p53 autoantibodies in the follow-up of head and neck cancer patients.

p53 autoantibodies (AAB) are a fairly new serological parameter in patients with malignancies. Although the actual mechanism of how they develop is still unclear, it seems that these AAB could be of prognostic relevance. Very few studies demonstrated the usefulness of p53 AAB in the follow-up of cancer patients. In this study, 109 patients with head and neck cancer were investigated using an ELISA for the presence of p53 AAB in their serum and were followed-up for at least 36 months. In 21 of the cancer patients, p53 serum AAB were detected. In 5/21 p53-seropositive AAB patients, a correlation with the clinical course was observed. Sixteen of the p53-positive patients did not show any significant AAB titer changes during the follow-up, and no significant correlation with the clinical course was seen. According to these results, the clinical value of p53 AAB in the follow-up of patients with head and neck cancer seems to be limited.

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in patients with head-and-neck squamous-cell carcinoma.

Human papillomaviruses (HPVs) have been recognized as an essential pathogenic factor in anogenital cancer. HPV DNA has also been found in a subgroup of head-and-neck squamous-cell carcinomas (HNSCCs), and a causative role of the virus in the development of these tumors has been suggested by the concomitant inactivation of the tumor-suppressor protein pRb. Using 4 second-generation ELISAs, we found antibodies against at least 1 of the oncoproteins E6 and E7 of the high-risk HPV types 16 and 18 in 11 of 92 sera (12%) taken from HNSCC patients at or near diagnosis, in 1 of 52 sera (2%) taken from HNSCC patients >6 months after diagnosis and in 10 of 288 sera (3. 5%) taken from sex- and age-matched healthy control individuals of the normal population. In 11 of the 12 seropositive HNSCC cases, antibodies were directed against HPV16 proteins. In patients, the HPV16 antibodies were mostly of high titer, and in 6 cases, antibodies against both HPV16 oncoproteins were present. Seven of the 8 HPV16 antibody-positive sera from the control group were of low titer, and none of the 10 antibody-positive sera reacted with both oncoproteins of the same HPV type. The HPV type of the antigens detected by the antibodies in HNSCC patients correlated well with that of the HPV DNA found in the tumor. Of 19 patients known to have HPV16 DNA-positive tumors, 7 (37%) also had HPV16 E6 and/or E7 antibodies. Our finding suggests that the antibodies were formed in an immune response against HPV E6 and E7 proteins expressed in the HNSCC and thus strongly supports the concept of a biologically active role of HPV in the development of a subgroup of HNSCC.

Tracing human papillomavirus DNA in nasal polyps by polymerase chain reaction.

Human papillomavirus (HPV) infections are related to the genesis of various benign and malignant human neoplasias. The HPV types 16 and 18 seem to be causally related to the development of most squamous cell carcinoma of the anogenital tract and a proportion of carcinomas of the upper aerodigestive tract. The near 100% positivity of the HPV types 6 and 11 in laryngeal papillomatosis is well established. We investigated whether HPV also plays a role in non-neoplastic mucosal entities such as sinunasal polyposis, the genesis of which has been discussed as being triggered by viral infections. On DNA from 39 sinunasal polyps (33 patients), polymerase chain reaction (PCR) was performed using beta-globin primers for demonstration of amplifiable DNA in the tissue extracts. Consensus primers for the detection of several different HPV types were applied to the beta-globin-positive samples. The results were confirmed by Southern blot hybridization using consensus probes. Cycle sequencing was performed on the positive cases. All 39 samples showed positive signals for beta-globin. HPV-DNA investigations showed a slight positive signal in only 1 of the 39 investigated cases (2.6%). Further molecular investigations of this sample, including cycle sequencing, could not confirm this result. All the other tissue samples remained HPV-DNA-negative. Therefore, those HPV types readily detectable with the PCR primers and probes used are not frequently associated with sinunasal polyposis. The data confirm the hypothesis that HPV is correlated to a lesser extent to infectious mucosal lesions than to proliferative lesions. Furthermore, the results emphasize that the presence of HPV in specific lesions does not occur by chance, but represents a specific infection of the mucosa leading to proliferation and even to malignancy.

Transcriptional repression of the human galactocerebrosidase gene in squamous cell carcinomas of the larynx.

Alterations of gene expression in squamous cell carcinoma (SCC) cell lines derived from the larynx and keratinocytes derived from adjacent normal mucosa of the larynx have been studied using the mRNA differential display technique. Lane-to-lane comparison of reverse transcribed mRNA showed a strong repression of a 148 bp fragment in SCC cells. The fragment was reamplified and cloned. Sequencing revealed a 99.3% homology with a region in exon 17 of the human galactocerebrosidase (GALC) gene. Northern blot analysis confirmed the differential expression of this gene in both carcinoma cell lines and laryngeal SCC biopsies in contrast with corresponding normal mucosa. To provide further evidence for the differential expression rate, both types of cells were transiently transfected with a 152 bp (-176 to -24) high regulatory promoter element of the 5' flanking region of the GALC gene. Results of 3 independent transfection experiments indicated a 16-fold repression of the GALC gene expression in SCC cells compared with benign keratinocytes. However, neither mutation nor other alterations of the promoter sequence were detected. Expression of the GALC gene is thus greatly affected in SCCs of the larynx.

A new prognostic indicator for head and neck cancer--p53 serum antibodies?

BACKGROUND: p53 antibodies are a new serological parameter of unknown potential in patients with malignancies. Their occurrence has been described in various types of cancer patients and several studies in head and neck cancer patients and other cancer patient groups have indicated its prognostic value. MATERIAL AND METHODS: We investigated the incidence of p53 serum antibodies in 271 head and neck cancer patients with an ELISA and investigated a possible correlation with clinical parameters like tumor staging and grading. RESULTS: Sixty-seven head and neck cancer patients (24.7%) were seropositive for p53 antibodies. No correlation between p53 antibody status, tumor staging and grading was found. CONCLUSIONS: These results indicate that the occurrence of p53 antibodies does not correlate with the most relevant prognostic factors, rate of regional metastasis and primary tumor size, in patients with head and neck cancer.

CYFRA 8/18 in head and neck cancer.

BACKGROUND: Cytokeratins (Ck) 8, 18 and 19 that are normally expressed in head and neck tissue in only small amounts, become overexpressed in head and neck squamous cell cancerous tissue. It was questioned whether Ck 8 and 18, which occur together, were also detectable and of value as a tumor markers for this cancer entity. MATERIALS AND METHODS: One hundred forty-nine sera from patients with squamous cell carcinoma of the head and neck (SCCHN) were investigated with a novel ELISA kit for the detection of fragments of Ck 8 and 18. Twenty-five sera from healthy volunteers and 39 patients with benign diseases of the head and neck region served as controls. RESULTS: It was found that CYFRA 8/18 had a sensitivity of 7%. CYFRA 8/18 values did not show a correlation with clinical parameters. CONCLUSIONS: Because of a low sensitivity CYFRA 8/18 seems not to be of value as a tumor marker for SCCHN.

The influence of differing dissection techniques on p53 as a possible indicator for local recurrences in carcinomas of the upper aerodigestive tract.

BACKGROUND: A correlation between the occurrence of p53 protein in margins after tumor resection has been proposed as indicator for recurrence. This study should clarify whether differences occur in p53 protein detection after laser surgery as opposed to surgery with the scalpel. MATERIAL AND METHODS: Healthy mucosa of the oropharynx (n = 28) and squamous cell carcinoma of the head and neck (SCCHN) (n = 19) were excised with scalpel, CO2 and Nd:YAG laser and then examined for the presence of p53 protein with immunohistochemistry and ELISA. RESULTS: The occurrence of p53 protein in SCCHN was dependent on the applied resection technique. In lasersurgically excised tissue only a small zone adjacent to the cutting edge was found to be negative for p53. This zone of necrosis was smaller in margins after CO2 than after Nd:YAG laser resection. CONCLUSIONS: Lasersurgical excision does only inhibit the detection of p53 in the zone of necrosis.