The effect of leukocyte interleukin injection (Multikine) treatment on the peritumoral and intratumoral subpopulation of mononuclear cells and on tumor epithelia: a possible new approach to augmenting sensitivity to radiation therapy and chemotherapy in oral cancer--a multicenter phase I/II clinical Trial.

OBJECTIVES/HYPOTHESIS: The main objective of this study was to investigate the effect of the administration of a novel immunoadjuvant, leukocyte interleukin injection, as part of an immuno-augmenting treatment regimen on the peritumoral and intratumoral subpopulations of the tumor infiltrating mononuclear cells and on the epithelial and stromal components, when administered to patients with advanced primary oral squamous cell carcinoma classified as T2-3N0-2M0, as compared with disease-matched control patients (not treated with leukocyte interleukin injection). STUDY DESIGN: Multicenter Phase I/II clinical trial. Fifty-four patients from four clinical centers were included in the dose-escalating study (27 in each group [leukocyte interleukin injection-treated and control groups]). Cumulative leukocyte inter-leukin injection doses were 2400, 4800, and 8000 IU (as interleukin-2 equivalent). METHODS: Paraffin-embedded tumor samples obtained at surgical resection of the residual tumor (between days 21 and 28 after treatment initiation) were used. Histological analysis, necrosis evaluation, and American Joint Committee on Cancer grading were performed from H&E-stained sections. Immunohistochemical analysis was performed on three different tumor regions (surface, zone 1; center, zone 2; and tumor-stroma interface, zone 3). Trichrome staining was used to evaluate connective tissue, and morphometric measurements were made using ImagePro analysis software. Cell cycling was determined by the use of Ki-67 marker. RESULTS: Leukocyte interleukin injection treatment induced a shift from stromal infiltrating T cells toward intraepithelial T cells and posted a significant (P <.05) increase in intraepithelial CD3-positive T cells independent of the leukocyte interleukin injection dose, whereas the increase in CD25 (interleukin-2 receptor alpha [IL-2Ralpha])-positive lymphoid cells was significant only at the lowest leukocyte interleukin injection dose (P <.05). Furthermore, both low- and medium-dose leukocyte interleukin injection treatment induced a significant (P <.05) increase in the number of cycling tumor cells, as compared with control values. CONCLUSION: The results could be highly beneficial for patients with oral squamous cell carcinoma. First, leukocyte interleukin injection treatment induces T-cell migration into cancer nests and, second, noncycling cancer cells may enter cell cycling on administration of leukocyte interleukin injection. This latter effect may modulate the susceptibility of cancer cells to radiation therapy and chemotherapy. The findings may indicate a need to re-evaluate the way in which follow-up treatment (with radiation therapy and chemotherapy) of patients with head and neck cancer is currently approached.

Air caloric computer system application in monitoring vestibular function changes after cochlear implantation.

INTRODUCTION: The computer-based ENG system's analytical routine refinements are available only in research laboratories. The computer-based system contains a stimulator for saccadic eye-movement and an air caloric stimulator interconnected to the registration program and analysis software. Several authors have reported the preoperative and postoperative vestibular functions in cochlear implant patients. The safe examination of the operated ear and comparison of the preoperative and postoperative average slow phase velocities of the caloric nystagmus is possible using the air caloric computer system. METHODS: The authors have used a computer-based ENG system with caloric air stimulation, which is very useful for examination of the operated ear. The vestibular system of patients with total deafness was examined before and after the cochlear implantation. The results of 60 vestibular examinations of 64 patients are reported. RESULTS: The vestibular function in the operated ear was found unchanged in 20 patients. In 14 patients the vestibular function was worse. In 16 patients the postoperative vestibular responsiveness improved. The detailed data analysis of 10 patients in whom preoperative and postoperative vestibular examination was similar was performed by the authors. The preoperative and postoperative average slow phase velocity values and the relative canal paresis was observed. The values demonstrated that the caloric responsiveness of the operated ear improved in few cases. CONCLUSIONS: The computer-based air caloric system is a useful and safe method in evaluation of the vestibular system changes after ear surgery. The reason of the improvement of vestibular responsiveness is not clear. The results need further investigation to solve the problem of vestibular responsiveness improvement after cochlear implantation.

Apoptotic and mitotic activity in squamous cell carcinoma cells after combined modality treatment with gamma-irradiation and dibromodulcitol.

A-431 squamous cell carcinoma cells were treated in vitro with either 4 Gy radiation of 15 (or 45) microg/ml dibromodulcitol (DBD), as well as with combined 4 Gy irradiation and DBD, with the latter as either a pretreatment or post-treatment. DBD alone or in combination with radiation had a greater effect on cell proliferation than the effect of radiation alone. The difference is due to a higher level of apoptosis induced by DBD, especially in conjunction with radiation. Such a combination may therefore be useful in the treatment of squamous cell carcinoma, which in general responds poorly to radiation therapy.

Progression difference between cancers of the larynx and hypopharynx is not due to tumor size and vascularization.

A recent survey of head and neck cancer indicated a sharp difference in survival between cancer of the hypopharynx and cancers formed in other head and neck sites. We have analyzed tumor size relative to clinical stage and vascularization as possible causes for such a difference in a series of 21 patients with cancer of the laryngopharynx (11 glottic and 10 hypopharyngeal). We found that the volume of the smallest cancers of the larynx at stage 2 is significantly larger than the volume of the cancers of the hypopharynx at stage 4 (P < 0.05). Next, we have determined by immunohistochemistry and morphometry the microvessel density, microvessel perimeter, and vascular endothelial growth factor (VEGF) expression of laryngo-hypopharyngeal cancers. Analysis of these data indicates that there is no difference in vascularization and VEGF expression between these two tumor types. These data strongly suggest that the invasive but not the angiogenic phenotype of hypopharyngeal cancer cells could be responsible for the more aggressive biologic behavior of this head and neck cancer subtype.

The diagnostic value of stapedius reflex and stapedius reflex exhaustion in myasthenia gravis.

Authors studies impedance, stapedius reflex thresholds and stapedius muscle exhaustion on 31 ears of 16 MG patients. Investigations were carried out using GSI 33 computer-assisted middle ear analyzator. Stapedius reflex threshold values were increased in 93% of patients. Stapedius exhaustion was observed in 71% of patients. After the administration of the reversible cholinesterase inhibitor Mestinon (60 mg pyridostigmin bromide), reflex threshold decreased and exhaustion occurred in only 50% of cases. Authors review the literature in context with the audiometric diagnostics of MG and also recommend the use of these methods in more complicated ocular and bulbar cases of MG.

Characteristic distribution patterns of tenascin in laryngeal and hypopharyngeal cancers.

OBJECTIVES: Progression of malignant neoplasias is accompanied by alteration of the extracellular matrix (ECM) composition. Tenascin is known as a member of the adhesion-modulating family of ECM macromolecules; thus its expression and distribution may have significant influence on tumor cell proliferation and invasiveness. STUDY DESIGN: The present study was carried out to determine the distribution pattern of tenascin in laryngeal and hypopharyngeal cancer samples. METHODS: In double and triple immunofluorescent staining reactions the detection of tenascin was combined with labelings for cytokeratin (marker protein of epithelial cells), for CD-34 (endothelial cell surface glycoprotein), and for a reaction with Ki-67 monoclonal antibody (nuclear antigen in proliferating cells). RESULTS: In laryngeal cancers, in early stages of tumor growth a markedly enhanced production of tenascin at the tumor host interphase was observed. In the later stages of tumor progression, a high number of blood vessels located in the tumorous tissues were also strongly labeled for tenascin. Around these vessels a significant number of proliferating tumor cells could be detected. In contrast, in hypopharyngeal cancers this vasculature-associated staining pattern could be observed from the very early stage of tumor development. In laryngeal and in hypopharyngeal cancers, tenascin upregulation strongly correlated with metastasis formation, early tumor recurrence, and lethal outcome of the disease. CONCLUSIONS: Clinical and immunohistologic data indicate that the accumulation of tenascin in the tumor blood vessels is an unfavorable prognostic indicator in laryngeal and hypopharyngeal cancers.

Prognostic significance of P53 histochemistry and DNA histogram parameters in head and neck malignancies.

BACKGROUND: The aim of the study was to determine the role of quantitative pathological parameters in prognosis of head and neck malignancies. MATERIALS AND METHODS: 51 head and neck squamous cell carcinoma patients were examined for mutant p53 gene expression (45 out of 51 patients) by immunohistochemistry and for cellular DNA-content (44 out of 51 patients) using digital picture analyzer. Statistical analysis was performed using BMDP package. RESULTS: No correlation with prognosis was found for age, sex, localization, T-classification and therapy. There was significant relationship between N-status and overall survival (p = 0.0008). No correlation was found with overall and disease-free survival for either histologic type or grading. P53: No significant correlation was detected with overall survival. A relationship was found between mutant p53 and metastasis-free time (p = 0.06). Ploidy: There were no significant differences between aneuploid and euploid tumors for either disease-free or overall survival. Synthetic (S)-phase fraction: A correlation was found for both survival rates (p = 0.029) and metastasis-free time (p = 0.05). Polyploid fraction (PF): correlation was shown for both overall survival (p = 0.0128) and metastasis-free time (p = 0.0038). CONCLUSION: There is correlation between p53 overexpression and metastatic potential and there is a significant relationship between SPF and PF value and prognosis (metastasis-free and overall survival) of head and neck cancer.

Reoperations after cochlear implantation.

During the 14-year history of cochlear implantation at Semmelweis University, 95 implantations were performed. There were 15 reoperations, and 2 patients had been operated 3 times. All of the reoperated patients received round window or intracochlear MedE1 devices. Successful reinsertion of the electrode was performed without significant difficulty, though in some cases osteoneogenesis was found in the round window or in the scala tympani. None of the patients suffered any inner ear abnormalities due to the first implant, and even the patient with a third implant in the same ear had excellent results and obtained better discrimination scores with the new device. In the authors' opinion, on the basis of the accumulated experience of nearly 100 implantations and 15 reoperations, replacement of the early generation extracochlear implants is most advisable and can be accomplished without adverse effects.

Electronystagmographic analysis of optokinetic nystagmus for the evaluation of ocular symptoms in myasthenia gravis.

Ocular symptoms of 17 myasthenia gravis (MG) patients were examined by electronystagmographic registration of optokinetic nystagmus. The aim of this study was to replace the subjective methods used previously with a more reliable quantitative technique and thus assess ophthalmoplegia and diplopia, important initial symptoms in MG. Slow phase angular speed values of foveolar type optokinetic nystagmus in the horizontal plane at 10, 20 and 30 degrees/s target speed were determined. Measurements were performed before and after administration of Mestinon, a reversible cholinesterase inhibitor. Twelve healthy volunteers were examined as controls under standard conditions. Results showed significant differences between MG patients and control group. Slow-phase angular speed was significantly larger after Mestinon administration (p < 0.001). It is concluded, that the exhaustion of external ocular muscles in MG can be well characterized by the determination of the slow phase angular speed values of optokinetic nystagmus (OKN). The examination of OKN was also recommended for the evaluation of ocular symptoms in other neurological disorders.

Cochlear implantation influences contralateral hearing and vestibular responsiveness.

A cochlear implant program has been carried out at the ORL Clinic of Semmelweis University in Budapest since 1985. Different devices and techniques have been used in pre- and postlingual children and adults. Over the last 6 years contralateral hearing improvement has been observed in 18 patients. This phenomenon can usually be demonstrated 6 months after the operation. Since our first observation several other authors have confirmed this phenomenon. However, the underlying mechanism is still obscure; both the efferent innervation and the plasticity of the brain may be important factors. The vestibular function of cochlear implant candidates was checked routinely pre- and postoperatively and changes in vestibular responsiveness were observed. Over the last 3 years air-caloric stimulation has been performed by means of a computer-based ENG system. In most patients the vestibular function was unchanged after the implantation. but in some cases a significant improvement in vestibular responsiveness was noted. Here we briefly describe cases of re-implantation carried out at the clinic. All the patients obtained better speech discrimination scores after having been implanted with intracochlear devices. None of the patients suffered any inner ear abnormality due to the first implant.

Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx.

Extravascular fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumor growth. The distribution of fibrin deposits was investigated in squamous cell carcinomas representing different stages of tumor progression of the larynx (n = 25) and hypopharynx (n = 9) by immunofluorescent techniques. Double and treble labelings were used to detect fibrinogen and fibrin in combination with marker antigens for tumor cells (cytokeratin), endothelial cells (von Willebrand factor), macrophages (recognized by KiM7), as well as factor XIII subunit A (FXIIIA) and tenascin (an embryonic extracellular matrix protein newly expressed during tumorigenesis). All tissue samples showed specific staining for fibrinogen/fibrin. Fibrin deposition was localized almost exclusively in the connective tissue compartment of tumors with characteristic accumulation at the interface of connective tissue and the tumorous parenchyma. In certain tumor samples showing highly invasive characteristics, fibrin deposits were observed in close association with tumor blood vessels in the tumor cell nodules. The overlapping reactions with polyclonal antibody to fibrinogen/fibrin and monoclonal antibody to fibrin indicate the activation of the coagulation cascade resulting in in situ thrombin activation and fibrin formation. Fibrin was crosslinked and stabilized by FXIIIA as revealed by urea insolubility test. Accumulation of phagocytozing macrophages detected by Ki M7 monoclonal antibody could be seen in areas of fibrin deposition. The blood coagulation factor XIIIA was detected in and around the cells labeled with Ki M7 antibody. Tenascin and fibrin deposits were found in the same localization in the tumor stroma and in association with tumor blood vessels within the tumor cell nodules. Neither fibrin nor tenascin were detected in the histologically normal tissue adjacent to tumors. The close association between fibrin deposits and macrophage accumulation strongly suggests the active participation of tumor-associated macrophages in the formation of stabilized intratumoral fibrin that facilitates tumor matrix generation and tumor angiogenesis.

Apoptosis in the human inner ear. Detection by in situ end-labeling of fragmented DNA and correlation with other markers.

The aim of this study was to obtain baseline data on the recently described special form of single cell death, apoptosis, in normal human inner ears. For this purpose, in situ end-labeling of the fragmented DNA was applied, in conjunction with apoptosis-related markers, to detect cellular elements showing programmed cell death in decalcified and paraffin-embedded tissues. Over 20 specimens were analyzed which were obtained from autopsy cases with no history of acoustic lesions confirmed by histopathology. Based on staining results, we saw no apoptotic signs in the majority of normal adult inner ears. An apoptotic cell captured in the Reissner's membrane of the cochlea from an old patient may, however, indicate an age-related subtle cell loss with the process of apoptosis. Nevertheless, the fact that more apoptosis was not found in our cases suggests that this phenomenon does not contribute significantly to the tissue homeostasis in the adult inner ear under normal conditions. These data are in accordance with our immunohistochemical findings on the p53 nucleoprotein, and proliferating cell nuclear antigen expression since there was no staining in any of the cellular elements, including the mesenchymal cells. This reflects a stationary and stable condition of cells of the vestibular and the cochlear structures, probably to maintain their integrity and the fine sensory functions. As opposed to the above findings, during inner ear development, the epithelial cells lining the cochlear lumen, the ossifying cartilage of the temporal bone, and the mesenchymal cells show different degrees of proliferation in combination with single cell death as signs of maturation of the vestibular and the cochlear apparatus. In addition, apoptosis has been demonstrated in cells of the cochlear stria vascularis from an adult patient treated with high doses of cisplatin, vinblastine and bleomycin prior to death. Furthermore, a wide range of apoptosis could be induced experimentally in a normal ear by an external perfusion of actinomycin D (ActD), which is known to produce programmed cell death in many cell types of different origins. The potential role of cytostatic agents in the apoptotic process of the inner ear needs, however, to be confirmed in large-scale specimens from patients treated with genotoxins. The fact, however, that apoptotic cells are also seen in association with ActD indicates that the fine sensory structure of the cochlea may also be a target for certain chemotherapeutic agents when administered in high doses.

Expression of invasion markers CD44v6/v3, NM23 and MMP2 in laryngeal and hypopharyngeal carcinoma.

Twelve laryngeal squamous cell carcinoma cases (7 laryngeal and 5 hypopharyngeal cancer; 15 samples) were analysed by immunohistochemistry for the expression of invasion markers CD44v6/v3, NM23 and matrix metalloproteinase, MMP2. The laryngeal epithelium showed CD44v6+/v3+/NM23-/MMP2- phenotype. When tumors were grouped into TNM categories the phenotype of the T2 and T3 tumors was similar, characterised by decreased CD44v3+ and lack of MMP2 expressions. Meanwhile the NM23 expression was more frequent in T3 tumors. In T4 stage the frequency of NM23 and MMP2 positive cases increased (5/6 and 4/6, respectively) but there was no correlation with the appearence of lymph node metastasis. Comparison of the phenotype of laryngeal and hypopharyngeal tumors, irrespective of the TNM stages, revealed characteristic differences: T2 stage laryngeal tumors showed decreased CD44v3 and occasional NM23 and MMP2 positivity, while in T3 stage these tumors were characterised by increased frequency of NM23 positivity. The phenotype of the hypopharyngeal tumors was significantly different with a high frequency of MMP2 positive cases (5/6) and NM23+/low CD44v3+ phenotype. The sharp differences in the phenotypes of laryngeal and hypopharyngeal carcinomas were connected to the differences in their invasive capacity unlike to the size of the tumors, since the T4 stage hypopharyngeal tumors had a significantly smaller size than laryngeal ones, even at lower stages.

Otoacoustic emission in myasthenia gravis patients and the role of efferent activation.

We performed transient evoked otoacoustic emission (TEOAE) measurements on 29 ears of myasthenia gravis (MG) patients. The purpose of the study was to support the role of acetylcholine (ACh) in the efferent innervation of cochlear outer hair cells (OHCs). Another aim was to establish additional diagnostic tools for the early determination of MG. Initially, threshold audiometry and impedance measurements showed normal values on the ears examined. The main finding was that TEOAE values were significantly lower in MG patients than in healthy controls. Mestinon, a reversible cholinesterase inhibitor, resulted in a significant increase in mean values of TEOAEs, although these values were still lower than normal. The results suggest that in MG, acetylcholine receptor (AChR) autoantibodies inhibit the function of OHC AChRs. Thus, the TEOAE generated by the active movements of OHCs is decreased in MG. Mestinon prevents the degradation of ACh, and thus stimulates efferent function and increases TEOAE values. The results obtained in this study support the role of ACh in the efferent function of OHC, as well as the impaired function of hair cell AChRs in MG patients. Consequently, measuring TEOAEs may be useful in the early diagnosis of some forms of MG. These results reinforce the importance of collaboration between neurologists and otolaryngologists in the management of diseases with pathological neurotransmission.

[Detection of human papillomavirus gene sequences in laryngeal tumors and premalignant changes by polymerase chain reaction]. / Humán papillomavírus génszakaszok kimutatása laryngealis daganatokban és praemalignus elváltozásokban polimeráz láncreakcióval.

Human papillomavirus gene sequences have been detected in a number of malignant and benign tumours. Non-oncogenic types 6 and 11 are etiological factors of benign mucosal tumours. Types 16 and 18 can be detected in malignancies most often but their role in the etiopathogenesis of cancers is still unclear. In our study we examined formalin-fixed and paraffin-embedded archive laryngeal tissues containing squamous cell carcinoma, papilloma and precancerous lesions for the presence of human papillomavirus genes. As a control we also examined tissues harbouring laryngeal nodules which represented the normal larynx in our study. After DNA preparation from the paraffin blocks we performed polymerase chain reaction to detect the DNA of human papillomavirus types 6, 11, 16 and 18. In the squamous cell carcinomas, papillomas and precancerous lesions the presence of human papillomavirus gene sequences was significantly higher than in the control group. To verify the integrity of DNA we also amplified a sequence deriving from the cellular beta-globin gene. Based on the 100% positivity for this gene we declare that the combination of our DNA preparation and polymerase chain reaction is a reliable method for detecting DNA sequences from formalin-fixed and paraffin-embedded tissues.

A proposal for introducing a modified vestibular index in neurotology. The classification of vestibular neuronitis.

The parameters for vestibular dysfunction were modified after our own studies. This index includes the degree of vertigo present, spontaneous nystagmus, dysfunction of the vestibulospinal reflexes and caloric and postrotatory side differences. The index is applicable for defining the extent of a lesion, follow-up, defining its stage and the results of therapy. Introduction of the modified vestibular index is proposed for use in clinical diagnosis. Classification of vestibular neuronitis into groups A, B and C is suggested on the basis of the reversibility of spontaneous nystagmus after caloric stimulation.

A peptide-like putative marker substance of laryngeal cancer patients.

Acidic ethanol precipitation, gel-filtration chromatography (Sephadex G-25) and capillary isotachophoresis were performed on serum and tumor tissue samples from eight patients with stage III and IV laryngeal cancers. An anionic 0.3-5 kDA molecular mass substance that was probably peptide was recorded in both the serum and cancer tissue, but could not be shown in the serum of five control subjects. This substance disappeared from the sera of four patients after total laryngectomy and they have now lived without tumor recurrence for about 2 years. Three of the other four patients developed tumor recurrences while one patient has remained tumor-free. Our findings support a hypothesis that the peptide-like molecule is characteristic of laryngeal cancer and can possibly be used for monitoring the disease.

The barrier-like role of activated connective tissue against the spread of supraglottic laryngeal carcinoma.

Light microscopic investigations of whole organ serial sections of 15 laryngectomy specimens with supraglottic carcinomas and comparative electron microscopic studies of the upper and lower poles of the tumors were carried out. The ultrastructure of different poles of the same supraglottic tumors was investigated and showed a more progressive fibrillogenesis with an activation of the connective tissue elements of the border on the lower pole of the malignancy. A significant role is attributed to the barrier-forming feature of activated connective tissue with respect to the inhibition of the extension of tumor into the glottic region.

Depressed lectin-dependent and enhanced antibody-dependent cell-mediated cytotoxicity in patients with stage I cancer of the larynx.

Lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood mononuclear cells (PBMC) from patients with stage I cancer of the larynx (LC) was evaluated using human adherent 3H-TdR-prelabeled HEp-2 carcinoma cells as targets at 50:1 effector-target ratio with 25 micrograms/ml concanavalin A (Con A) in a 24-hour assay. Under these conditions, but without Con A, no considerable natural cell-mediated cytotoxicity (NCMC) was performed by PBMC either from control or from LC donors. Depressed levels of LDCC, but augmented ADCC to chicken red blood cells were detected in LC patients. Natural killer activity to K562 targets was not different from that of control subjects. In parallel studies, normal Con A-induced blastogenesis and B cell counts, low T, and active T cell counts, as well as high Leu-11a+ cell counts were detected in patients with LC. The relationship between depressed LDCC and low T, and active T cell counts, and enhanced ADCC and high Leu-11a+ cell counts is suggested in stage I LC patients.