Programmed Death-Ligand 1 Expression and Tumor-Infiltrating Lymphocytes in Temporal Bone Squamous Cell Carcinoma.

OBJECTIVES/HYPOTHESIS: The tumor immune microenvironment in temporal bone squamous cell carcinoma (TBSCC), including the programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs), has not been established. STUDY DESIGN: Retrospective cohort study. METHODS: We performed immunohistochemistry analyses to retrospectively analyze 123 TBSCC cases for PD-L1 expression and TILs and their prognostic significance. We also evaluated the prognostic correlations between these immunomarkers and the therapeutic responses to chemoradiotherapy (CRT). RESULTS: PD-L1 expression (≥1%) was detected in 62 (50.4%) TBSCC cases and was significantly associated with worse prognosis: progression-free survival (PFS), P < .0001; overall survival (OS), P = .0009. A high density of CD8+ TILs was significantly associated with better prognosis (PFS, P = .0012; OS, P = .0120). In contrast, a high density of Foxp3+ TILs tended to be associated with an unfavorable prognosis (PFS, P = .0148; OS, P = .0850). With regard to the tumor microenvironment subtypes defined by CD8+ TILs and PD-L1 expression, the CD8low /PD-L1+ group showed significantly worse prognosis. Among the 36 neoadjuvant CRT-treated cases, PD-L1 expression was significantly associated with worse OS (P = .0132). Among the 32 CRT-treated cases without surgery, a high density of CD8+ TILs tended to be more highly associated with complete response to CRT compared to a low density of CD8+ TILs (P = .0702). CONCLUSIONS: These results indicate that the evaluation of the tumor immune microenvironment may contribute to the prediction of prognoses and the selection of an individualized therapeutic strategy for patients with TBSCC. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2674-2683, 2021.

Cellular immunologic responses to cochlear implantation in the human.

A cochlear implant array consists of biomaterials, including metal and polymeric in type which are biocompatible, but not necessarily bio-inert. Histologic evidence of a foreign body reaction has been described in temporal bones in patients who in life had undergone cochlear implantation. In the current study, the cellular immune response was characterized using immunohistochemical stains for B-cell lymphocytes (CD20), T-cell lymphocytes (CD3), and macrophages (CD68). In addition, energy dispersive spectroscopy by scanning electron microscopy (EDS-SEM) was performed to characterize the nature of particulate foreign material seen near the electrode array. Infiltrations of B-cell and Tcell lymphocytes and macrophages were identified immunohistochemically. The track of the electrode array was frequently lined by multi-nucleated foreign body giant cells. Energy dispersive X-ray spectroscopy identified the particulate material found in the fibrous sheeth surrounding the cochlear implant to be consistent with platinum. In conclusion, a cochlear implant generates a vigorous cellular immune response consisting of B and T lymphocytes, foreign body giant cells, and macrophages. Platinum was identified as one of the antigens likely responsible for this cellular response. This foreign body response may in certain cases result in migration or even extrusion of an implant device.

Dual immunofluorescence staining of proteoglycans in human temporal bones.

OBJECTIVES/HYPOTHESIS: Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section. STUDY DESIGN: Retrospective study of proteoglycan codistribution in archival human temporal bone tissues. METHODS: The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics. RESULTS: The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis. CONCLUSIONS: The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes.

Spindle cell carcinoma of the external auditory meatus with intracranial extension: histological, immunohistochemical and electron microscopic evaluation.

We present a case of squamous spindle cell carcinoma of the external auditory meatus in a 38-year-old man. The tumour was extended to the inner ear, the temporal bone, the middle cranial fossa and the meningo-cerebral tissue. The surgical intervention of temporo-occipital craniotomy removed most of the neoplasia. At pathologic examination, the tumour showed an undifferentiated spindle cell pattern. Immunohistochemistry with a large antibody panel found a weak positivity only to EMA. The diagnosis was made when the electron microscopy showed rare junctional structures and tonofilaments.

Some remarks on the histopathology of otosclerosis.

The histopathology of otosclerosis is described in detail in classical textbooks like Schuknecht's Histopathology of the Ear or Friedmann and Arnold's Pathology of the Ear. In this article, some of the important and new facts will be summarized which might affect the understanding of the pathomechanism of this unique measles-virus-associated inflammatory disease of the temporal bone.

Anti-inflammatory effect of erythromycin on histamine-induced otitis media with effusion in guinea pigs.

In this study, the anti-inflammatory effect of erythromycin was investigated in a model of histamine-induced otitis media with effusion (OME). OME was induced in guinea pigs by the transtympanic injection of histamine solution into the middle-ear cavity. Guinea pigs were randomly assigned to one of three groups: control, erythromycin treatment, or methylprednisolone treatment. After histamine injection, the animals were treated with intraperitoneal medication for five days consecutively. Afterwards, the animals were sacrificed and the temporal bones were removed. The samples were examined stereologically. In the erythromycin-treated group, it was observed that neutrophil infiltration was significantly inhibited when compared to the control group. This result shows that erythromycin may produce a significant anti-inflammatory effect in this model of OME.

Cellular proliferation of mucosa-associated lymphoid tissue with otitis media: a preliminary study.

Histopathologic and immunohistochemical analyses were used to investigate the cellular proliferation of mucosa-associated lymphoid tissue (MALT) in human temporal bones with and without evidence of otitis media (OM). Anti-proliferating cell nuclear antigen (PCNA) antibody (clone PC10) was applied after the antigen retrieval procedure. Positive PCNA expression was observed in temporal bones that had been stored for 10 to 31 years in 80% ethanol. In specimens with purulent OM, the MALT had faint germinal centers (GCs). Positive PCNA expression in the MALT was moderate and scattered. In specimens with mucous OM, the MALT had complete GCs. Positive PCNA expression in the MALT was moderate to strong, and the distribution of PCNA-positive cells was associated predominantly in the GCs, the mucosal epithelial layer, and/or the subepithelial layer. In specimens with serous OM, the MALT also had complete GCs. However, the PCNA expression was weak and scattered, and appeared to be similar to that of the MALT in the temporal bones without OM. These results indicate that the cellular proliferation of MALT in the temporal bone might reflect the activity that produces secretory IgA against invasion of foreign antigens. However, further studies are needed to elucidate whether the PCNA expression within MALT in the eustachian tube and middle ear is associated with a mucosal immune response to inflammation as in OM.

Growth of postoperative remnants of unilateral vestibular nerve schwannoma: role of the vestibular ganglion.

Histopathological examination of seven temporal bones from patients who underwent a removal of vestibular nerve schwannomas by the translabyrithine or middle fossa approaches has demonstrated small tumor remnants that failed to grow as long as 25 years after surgery. In spite of the high incidence of residual tumors, the clinical recurrence rate of tumors operated at our institution by the translabyrinthine or middle fossa approaches is low (0.3%). Immunohistochemical labeling of dividing cells demonstrated that segments of tumor adjacent to the vestibular nerve and ganglion contained more dividing cells than were present in areas of the tumor at a distance from them.

Application of labeling techniques to archival temporal bone sections.

Pathology of the human inner ear has traditionally been studied in celloidin-embedded, hematoxylin and eosin-stained sections of the temporal bone. Although the traditional histologic approach has yielded valuable information, it is now possible to extend these studies to include analysis of molecules using immunohistochemical and histochemical staining techniques. Fourteen antibodies and 6 lectins have been applied to 420 archival, celloidin-embedded human temporal bone sections. Tissues had been fixed in 10% formalin, embedded in celloidin, sectioned, and stored for as many as 40 years. The staining intensities varied among sections, so they were ranked from 'no label" to "dense label." To investigate the relationships between the extent of postmortem changes (PMCs), storage time, and staining intensity for each antibody, the sections were graded according to their PMCs, which ranged from good preservation of the temporal bone histologic structure to severe postmortem autolysis. Although statistical analysis indicated that both extent of PMCs and storage time in general decrease the staining intensity, both poorly fixed tissue and sections stored for a long time can yield good immunostaining results with some antibodies.

[Pathogenesis of otosclerosis. "State of the art"]. / Zur Pathogenese der Otosklerose. "State of the Art".

Women suffer from otosclerosis 1.6 times more often than males. Histologically, otosclerotic foci can be found in temporal bones of females 1.9 times more often than in those of males. Characteristic topographic regions are the oval window, round window niche and promontory. Otosclerosis can also occur principally in any area of the enchondral/periosteal layer of the otic capsule. Evidence is presented that otosclerosis is an inflammatory tissue reaction associated with macrophages, T- and B-lymphocytes, HLA-DR positive cells and plasma cells. Dependent on the stage of the osteolytic bone disease present deposits of complement and immunoglobulins (IgG, IgA) can be found. These immunoglobulins have been identified as antibodies to measles virus proteins. Using the polymerase chain reaction we were successful in demonstrating RNA sequences of measles viruses in otosclerotic bone from footplates removed during stapes surgery. Since most of the otosclerotic lesions were in direct contact to the perilymphatic space, it may be expected that the endolymphatic sac--as the immune competent organ of the inner ear--specifically reacts to antigens delivered from the otosclerosis focus into the perilymph. Perilymph samples from patients were collected during stapes surgery and their antibody titers against measles were compared with that in corresponding blood serum. All samples revealed a significantly elevated-specific anti-measles IgG amount which was significantly higher than in the corresponding serum. In contrast, antibody titer in the perilymph against herpes simplex or cytomegalo viruses did not differ from that of the serum. These findings indicate that otosclerosis is a measles virus associated inflammatory osteolytic disease of the temporal bone. Since women suffer from severe measles virus infections more often than males, it can be hypothesized that females have a higher susceptibility of their cochleo-vestibular tissues to these infections (organotropism). In addition, estrogens are well-known stimulators of osteocytic activity and may play a dominant role during ossification of an otospongeotic bone lesion. This may explain the onset of a conductive hearing loss due to otosclerosis during pregnancy.

Localization of cytokines in cholesteatoma tissue.

Acquired cholesteatoma is associated with an intense inflammatory reaction with resultant tissue and bone destruction. Cytokines are molecules released by inflammatory cells at the site of infection and are potent mediators of inflammation and the immune response. Five cytokines, tumor necrosis factor-alpha, transforming growth factor-beta 1 and 2, and interleukin-1 and 6, were immunolocalized in human cholesteatoma epithelium and subepithelial stroma, with greater intensity of staining compared with noninflamed external auditory canal skin. Increased interleukin-6 activity in cholesteatoma epithelium and stroma correlated significantly with the presence of ossicular and bony erosion and granulation tissue noted intraoperatively. Transforming growth factor-beta 2 activity in cholesteatoma epithelium correlated significantly with bony erosion at surgery. Additionally, transforming growth factor-beta 1 activity in cholesteatoma epithelium correlated significantly with increased length of disease. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 appear to be involved in the inflammation and resultant remodeling associated with cholesteatoma. We hypothesized a protective function of transforming growth factor-beta 1 and 2 in the presence of cholesteatoma. The antiinflammatory and osteoclast and keratinocyte inhibitory actions of the transforming growth factor-beta s could potentially slow the proliferation and resultant tissue destructiveness associated with cholesteatoma.

Fibronectin-like immunoreactivity of the basilar membrane of celloidin-embedded human temporal bone sections.

Dysfunction of the mechanical properties of the basilar membrane is a potential cause of presbycusis. In cases of minimal sensorineural or strial degeneration it is believed to play a major role. The membrane has been shown to be partly composed of fibronectin. Fibronectin immunoreactivity is diminished in aged rats. Mesothelial cell line the perilymphatic surface of the membrane and are reduced in number in the aged rat cochlea. Fibronectin immunoreactivity was examined in human temporal bone sections (6 months to 92 years old). Hematoxylin and eosin stained section (17 to 97 years) were immunoreactivity was demonstrable in the human cochlea, but was not reduced, even in the eldest cases examined The number of mesothelial cells was reduced, however, and was related to the age of the individual, but not to the clinical diagnosis or audiogram shape. These two factors do not, therefore, appear to give rise to hearing losses associated with presbycusis.

Murine model of otitis media with effusion: immunohistochemical demonstration of IL-1 alpha antigen expression.

Recent studies have suggested that cytokines likely play a central role in the formation and maintenance of otitis media with effusion (OME). Currently, there is no immunologically defined animal model for the study of cytokines as they contribute to the formation of OME. In the present study, a murine model of OME, using eustachian tube blockage via an external surgical approach, was developed. The murine model temporal bone histology appears to mimic the histology found in chronic otitis media with effusion in humans. Additionally, using this murine model, interleukin-1 alpha (IL-1 alpha) expression was detected in the middle ear using standard immunohistochemical techniques. IL-1 alpha seemed localized to the epithelial lining of the middle ear as well as 5% to 10% of inflammatory cells. This model should provide the necessary tool to further study the immunologic aspects of OME.

Inner ear inflammation and round window otosclerosis.

Recently, it has been suggested that otosclerosis represents the host's ongoing immunologic response to measles or other viral antigens. Documentation of past inflammation within the inner ear would serve as further evidence that this mechanism may be at play in the pathogenesis of the disease. Among the characteristic signs of prior inflammation in the inner ear is the presence of lamellar bone at the site of inflammation. This has been described in the temporal bone of a patient with immune-mediated deafness and with the temporal bones of experimental models of immune-mediated inner ear disease. Review of temporal bones with round window otosclerosis from the Eastern Temporal Bone Bank at the Massachusetts Eye and Ear Infirmary show that in four of ten cases there are characteristic signs of a prior severe inflammatory event centered in the scala tympani adjacent to the otosclerotic lesion. Otosclerosis, therefore, may have an inflammatory stage that is the consequence of a host response to an inciting event.

Técnica inmunohistoquímica en hueso temporal en enfermedad autoinmune de oido interno / Immunohistochemical technic in temporal bone in autoimmune disease of the inner ear

Diecisiete sueros de pacientes portadores de enfermedades del oído interno que ya habían sido sometidos a la búsqueda de anticuerpos anticocleares mediante la técnica de inmunofluorescencia indirecta, fueron estudiados mediante una técnica inmunohistoquímica usando peroxidasa de rábano picante. El objetivo fue el de precisar el lugar en el que el fenómeno antígeno anticuerpo tuvo lugar. Así mismo, se quiso determinar la especificidad y sensibilidad del nuevo método. Los resultados positivos demostraron que los complejos marcados con inmunoperoxidasa tuvieron afinidad por aquellas estructuras que tienen relación con el territorio arteriolar de la coclea, en especial con la estría vascular y la membrana bacilar. Comparado con la técnica de inmunofluorescencia indirecta se demostró que la especificidad se mantuvo, pero que la sensibilidad disminuyó. Esto último se atribuyó a que el procedimiento de fijación y descalcificación del hueso temporal obtenido, y que se usó como antígeno, pudo alterar la antigenicidad del mismo

Bovine temporal bones as a source of inner ear antigen.

Modern immunologic techniques of immunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in studies of development, function, and pathology of the ear. These techniques require a source of inner ear tissue for production of antigen extract. Human tissue is not readily available, and other mammalian species common in auditory research are small in size. Bovine temporal bones are readily available, and the membranous portions of the inner ear are abundant and easily accessible. Herein we report our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of inner ear antigen.

A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.

Development of endolymphatic hydrops following immune response in the endolymphatic sac of the guinea pig.

The present study investigated immune injury associated with endolymphatic hydrops (e.hydrops) following locally mounted immune reaction in the endolymphatic sac (e.sac) of guinea pigs. E.hydrops occurred, progressing rapidly within the first week post secondary Keyhole limpet hemocyanin (KLH) challenge in the e.sac and developed into two phases, acute and chronic. On the other hand, primary KLH challenge of the e.sac, PBS inoculation into the e.sac or intradural secondary KLH challenge were incapable of inducing e.hydrops. These results indicate that reversible and irreversible e.hydrops are induced by the immune response of the e.sac, suggesting that local immunological events of the e.sac may provide an animal model of Meniere's disease.

Preparation of human temporal bone for immunohistochemical investigation.

Successful treatment of progressive sensorineural hearing loss with corticosteroids and cyclophosphamide has promoted the notion of a new entity 'autoimmune hearing loss'. In this context, we have examined sera of patients suffering from idiopathic sensorineural hearing loss, in order to study the binding of humoral antibodies to structures of the normal human inner ear. Investigations were carried out with indirect fluorescence techniques on temporal bones obtained at autopsy. To minimize the possibility of artifacts, we suggested that indirect immunofluorescence with the patients' sera should yield a reproducible positivity on sections of at least three normal temporal bones. In this study, we describe a method for removal and preparation of human temporal bone which ensures reproducible immunohistochemical results.

The role of type II collagen autoimmunity in otosclerosis revisited.

A recent theory, suggesting that otosclerosis results from autoreactivity to type II collagen present in the fetal cartilaginous remnants of the human bony labyrinth, is based on two observations. Otosclerotic patients have increased concentrations of circulating antibody to type II collagen, and immunization of rodents with cartilage collagen induces 'otosclerosis-like' lesions. Independent researchers have been unable to confirm the first promising results. No significant abnormalities could be found in immunized animals. We report the result of type II collagen antibody recordings in a well described group of otosclerotic patients and controls, and the histological findings in temporal bones of MRL/1-mice with spontaneous type II collagen autoreactivity. Our results cannot support the view of autoreactivity to type II collagen as an etiopathogenetic factor in otosclerosis.