Tonsil Biopsy Research Articles

Tonsillectomy and tonsil biopsy in the search for primary oropharyngeal tumor in patients with CUP

The purpose of the study. To study the role of tonsillectomy and palatine tonsil biopsy in the identification of primary oropharyngeal tumor in patients with CUP. Material and methods. The study included the diagnostic findings of 69 patients with squamous cell cancer (SCC) metastases to the neck lymph nodes from an unknown primary site. Inclusion criteria for the study: the presence of morphologically verified SCC metastases in the cervical lymph nodes based on the FNA (fine needle aspiration biopsy) cytology findings, the pathology findings (cervical lymph node biopsy, core biopsy), no evidence of cervical lymph node metastases from another primary site, and no primary tumor detected upon the initial clinical examination. Thirteen (19%) patients out of 69 with no evidence of a primary tumor after clinical examination and instrumental investigations underwent transoral microsurgical palatine tonsillectomy using a Carl Zeiss operating microscope with cervical lymph node dissection. Biopsy of the tonsil was performed in 7 (10%) out of 69 patients. Results. According to the results of the study, the primary tumor localized in the palatine tonsil was detected in 11 cases (78%) of microsurgical palatine tonsillectomy, and in 2 cases (28%) of the palatine tonsil biopsy. Moreover, according to the IHC results, the verified tonsil cancer was associated with HPV in 62% of cases. Conclusion. Tonsillectomy is a valuable diagnostic option in the search for a primary oropharyngeal tumor in patients with CUP. It allows for targeted treatment (surgical treatment using minimally invasive microsurgical techniques, conformal radiation therapy), reducing the likelihood of developing adverse reactions that alter the patient’s quality of life. Цель исследования. Изучить роль тонзиллэктомии и биопсии небной миндалины в идентификации первичной опухоли ротоглотки у пациентов с CUP-синдромом. Материал и методы. В основу исследования положены результаты диагностики 69 пациентов с метастазами плоскоклеточного рака (ПКР) в лимфоузлы шеи из неизвестного первичного очага. Критерии включения в исследование: наличие морфологически верифицированных метастазов ПКР в лимфатические узлы шеи, полученным по результатам цитологического исследования ТАБ (тонкоигольная аспирационная биопсия), гистологического исследования (биопсии лимфоузлов шеи, трепан-биопсии), отсутствие данных за метастатическое поражение лимфоузлов шеи другой локализации, отсутствие первичной опухоли после первичного клинического обследования. Тринадцати (19%) пациентам из 69 с отсутствием данных о наличии первичной опухоли после клинического обследования, инструментальной диагностики была выполнена трансоральная микрохирургическая небная тонзиллэктомия с использованием операционного микроскопа Carl Zeiss, шейная лимфодиссекция. У 7 (10%) пациентов из 69 была выполнена биопсия небной миндалины. Результаты. По результатам проведенного исследования, первичный опухолевый очаг с локализацией в небной миндалине при микрохирургической небной тонзиллэктомии удалось выявить в 11 (78%) случаях, при биопсии небной миндалины – в 2 (28%) случаях. При этом в 62% случаев, по результатам иммуногистохимического исследования, верифицированный рак небной миндалины был ассоциирован с вирусом папилломы человека. Заключение. Выполнение небной тонзиллэктомии является ценной диагностической опцией в поиске первичной опухоли ротоглотки у пациентов с CUP-синдромом. Позволяет провести целенаправленное лечение (хирургическое лечение с использованием малоинвазивных микрохирургических методик, лучевую терапию в конформном режиме), снижая вероятность развития токсических реакций, ухудшающих качество жизни пациента.

Unveiling the etiology of peritonsillar abscess using next generation sequencing

BackgroundPeritonsillar abscess (PTA) is a severe deep neck space infection with an insufficiently characterized bacterial etiology. We aimed to reveal the bacteria associated with PTA applying next generation sequencing (NGS). Tonsil biopsies and pus samples of 91 PTA patients were analysed applying NGS method.ResultsOver 400 genera and 800 species belonging to 34 phyla were revealed. The most abundant species in both sample types were Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. When present, S. pyogenes was normally a predominant species, although it was recovered as a minor population in some samples dominated by F. nucleatum and occasionally F. necrophorum. S. pyogenes and F. necrophorum were the predominant species (> 10% in a community) in 28 (31%) pus samples, while F. nucleatum in 21 (23%) and S. anginosus in 8 (9%) pus samples. We observed no substantial differences between the microbial findings in pus and tonsil biopsies.ConclusionsThe most probable causative agents of PTA according to our NGS-study include Streptococcus pyogenes, Fusobacterium necrophorum and Fusobacterium nucleatum. Some other streptococci (S. anginosus) and anaerobes (Prevotella, Porphyromonas) may contribute to the infection as well. Pus of the peritonsillar abscess is more representative specimen for microbiological examination than the tonsillar tissue. Our results are important in the context of optimizing the handling of the PTA patients.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Longitudinal detection of prion shedding in nasal secretions of CWD-infected white-tailed deer.

Chronic wasting disease (CWD) is an emergent prion disease spreading in cervid populations in North America, South Korea and Scandinavia. Rapid detection of CWD prions shed by live animals using minimally invasive methods remains an important need. Previous studies in deer, elk and hamsters have demonstrated prion replication in the nasal olfactory mucosa, yet the temporal profile of CWD prion shedding in nasal secretions has not been well characterized. Here we report nasal prion shedding in 18 deer orally exposed to low doses of CWD prions and monitored longitudinally by several parameters. Serially collected nasal swabs were assayed for CWD prion seeding activity using iron oxide magnetic extraction and real-time quaking-induced conversion (IOME RT-QuIC). These findings were correlated with the results from longitudinal tonsil biopsies, terminal tissues and PRNP genotype. We detected nasal prion shedding 3-16 months after the first positive tonsil biopsy in ten of the 18 deer; detectable shedding persisted thereafter in nine of the ten animals. Surprisingly, nasal swabs were negative in eight deer, even though all were CWD-infected as determined by tonsil biopsies and terminal tissue assays. Nasal shedding was detected more often in deer that were homozygous for glycine at codon 96, and those that were near or demonstrating symptoms of clinical disease shed earlier and more frequently, irrespective of prion exposure dose. The results of this study demonstrate nasal shedding of CWD prions that can be detected using minimally invasive nasal swab sampling and RT-QuIC analysis.

A two-centre experience of tonsil biopsies in the investigation of patients with tonsillar asymmetry.

We aim to evaluate our experience of tonsil biopsies in the investigation of patients presenting with asymmetrical tonsils. A two-centre retrospective analysis of all patients who underwent histology sampling of the palatine tonsils between 1 January 2013 and 31 December 2018 was completed. Data collected included patient demographics, method of obtaining tonsil tissue, histological diagnosis and need for repeat tissue sampling. A follow-up period of 36 months was allowed to establish whether any patients re-presented with missed diagnoses. In total, 937 patients were included for analysis: 375 (40.0%) had a biopsy, of which 191 (50.9%) were performed in clinic. The mean duration from initial appointment with the ear, nose and throat clinic to tissue sample collection was 17.6 days (range 0-327 days) for all biopsies, reducing to 0.2 days (range 0-17 days) for biopsies performed in clinic. This was significantly shorter than for tonsillectomies (mean 38.9 days, range 0-444 days; p<0.05). Of the patients who underwent tonsil biopsy, six (1.6%) had malignancy that was not unequivocally diagnosed on initial biopsy. In all six patients, prior clinical suspicion was high, and repeat tissue sampling was undertaken on receipt of negative histology results. Tonsil biopsy is a viable alternative to tonsillectomy for histology in the assessment of tonsil asymmetry. Tonsil biopsy in the outpatient setting has reduced surgical morbidity, significantly less delay in diagnosis, less inconvenience for patients and lower healthcare costs compared with formal tonsillectomy. Although tonsil biopsies should not be used in isolation, they can be useful in the investigation of patients presenting with tonsillar asymmetry.

Abstract 2029: Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide

Abstract The GeoMx࣪ Digital Spatial Profiler (DSP) enables high-plex, high-throughput spatial profiling and quantification from a single slide for either protein or RNA. To fully understand the interplay between RNA and protein in tissue, we have developed a novel multi analyte assay that allows for the profiling of both analytes from the area of interest (AOI) on a single slide. Here we describe the development and performance of the multi analyte assay on cell pellet array (CPA) and various tissues including tonsil and colorectal cancer (CRC) using a high plex GeoMx Protein Panel and the GeoMx Human Whole Transcriptome Atlas (WTA). We successfully identified conditions to detect RNA and protein simultaneously, with comparable sensitivity and specificity to the single analyte conditions. The data accurately matched cell lines in an unbiased correlation analysis between GeoMx WTA and the entire CCLE RNAseq database. Furthermore, the assay performance was maintained in tissue samples. Tonsil and colorectal cancer biopsies were profiled for RNA (whole transcriptome) and protein from the same AOI, allowing simultaneous measurements of transcriptional and translational regulation of key biological pathways. This multi-analyte profiling enables deeper characterization of precious biological samples that are available in limited quantities. Citation Format: Shilah Bonnett, Nicholas Confuorto, Kit Fuhrman, Giang Ong, Alyssa Rosenbloom, John Lyssand, Gary Geiss, Joseph Beechem. Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2029.

Cutting Edge: SARS-CoV-2 Infection Induces Robust Germinal Center Activity in the Human Tonsil.

Understanding the generation of immunity to SARS-CoV-2 in lymphoid tissues draining the site of infection has implications for immunity to SARS-CoV-2. We performed tonsil biopsies under local anesthesia in 19 subjects who had recovered from SARS-CoV-2 infection 24-225 d previously. The biopsies yielded >3 million cells for flow cytometric analysis in 17 subjects. Total and SARS-CoV-2 spike-specific germinal center B cells, and T follicular helper cells, were readily detectable in human tonsils early after SARS-CoV-2 infection, as assessed by flow cytometry. Responses were higher in samples within 2 mo of infection but still detectable in some subjects out to 7 mo following infection. We conclude the tonsils are a secondary lymphoid organ that develop germinal center responses to SARS-CoV-2 infection and could play a role in the long-term development of immunity.

Sleep Disordered Breathing and Recurrent Tonsillitis Are Associated With Polymicrobial Bacterial Biofilm Infections Suggesting a Role for Anti-Biofilm Therapies.

BackgroundThe underlying pathogenesis of pediatric obstructive sleep disordered breathing (SDB) and recurrent tonsillitis (RT) are poorly understood but need to be elucidated to develop less invasive treatment and prevention strategies.MethodsChildren aged between 1- and 16-years undergoing adenoidectomy, tonsillectomy or adenotonsillectomy for SDB (n=40), RT alone (n=18), or both SDB and RT (SDB+RT) (n=17) were recruited with age-matched healthy controls (n=33). Total bacterial load and species-specific densities of nontypeable Haemophilus influenzae (NTHi), Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Moraxella catarrhalis were measured by qPCR in nasopharyngeal swabs, oropharyngeal swabs, adenoid and tonsillar tissue from children with SDB, SDB+RT and RT, and in naso- and oro- pharyngeal swabs from healthy children. A subset of tonsil biopsies were examined for biofilms using 16S rRNA FISH (n=3/group).ResultsThe 5 bacterial species were detected in naso- and oro- pharyngeal samples from all children. These species were frequently detected in adenotonsillar tissue (except S. aureus, which was absent in adenoids) from children with SDB, SDB+RT and RT. NTHi and S. aureus were observed in tonsils from 66.7-88.2% and 33.3-58.8% of children respectively. Similar total and species-specific bacterial densities were observed in adenotonsillar tissue from children with SDB, SDB+RT or RT. Nasopharyngeal and oropharyngeal swabs were more likely to have multiple bacterial species co-detected than adenotonsillar tissue where one or two targeted species predominated. Polymicrobial biofilms and intracellular bacteria were observed in tonsils from children with adenotonsillar disease.ConclusionsAntimicrobials, particularly anti-biofilm therapies, may be a strategy for managing children with SDB.

Characterization of Immune Cells in Oral Tissues of Non-human Primates.

The oral mucosa contains distinct tissue sites with immune niches capable of either immunogenic or tolerogenic responses. However, immune cell compositions within oral mucosal tissues at homeostasis have not been well-characterized in human relevant tissues. Non-human primates (NHP) are a major model for the human immune system and oral anatomy, and therefore improved understanding of NHP oral immune cell populations can provide important insights for studying disease pathologies and developing therapies. Herein, we characterize immune cell types of three sites within the oral cavity (buccal, sublingual, lingual tonsil) sampled by biopsy and cytobrush in pigtail macaques. Tonsil biopsies had more T-cells, dendritic cells (DCs), DC subtypes, and CD4+ T-cells than buccal or sublingual biopsies when normalized by tissue mass. Biopsy proved to collect more immune cells than cytobrushes, however frequencies of CD45+ subpopulations were comparable between methods. Live cells isolated from biopsied tonsils had greater CD45+ leukocyte frequencies (mean 31.6 ± SD 20.4%) than buccal (13.8 ± 4.6%) or sublingual (10.0 ± 5.1%) tissues. T-cells composed more than half of the CD45+ population in sublingual tissue (60.1 ± 9.6%) and the tonsil (54.6 ± 7.5%), but only 31.9 ± 7.2% in buccal samples. CD20+ B-cells composed a greater percentage of CD45+ leukocytes in the tonsil (12.8 ± 9.1%) than buccal (1.2 ± 1.0%) or sublingual tissues (0.8 ± 1.2%). Immune population comparisons are also made between sex and age. These results present an important step for understanding the oral immune environment, oral disease, and site-specific therapy development.

Delayed metastatic melanoma to the pharyngeal tonsil in a Chinese male

Background: Melanoma is a relatively uncommon malignant neoplasm, either primary or secondary. We present a case of delayed metastatic melanoma to the pharyngeal tonsil. Case report: A 70-year-old Chinese male presented for half a year due to vague speech and snoring and then found tonsil enlargement. Three years prior to presentation, he had undergone primary resection and systemic therapy for cutaneous melanoma on the sole of his foot. Tonsil biopsy confirmed the diagnosis of metastatic melanoma. Currently the patient is undergoing chemotherapy. Conclusion: Metastatic melanoma is a rare disease, and metastatic lesions may occur at any location. For the long-term survival of patients, careful examination should be done during the follow-up period, including head and neck. Keywords: metastatic melanoma; tonsillar neoplasms.

A Challenging and Rare Case of Metastatic Follicular Dendritic Cell Sarcoma Originating from a Tonsil

Abstract Introduction/Objective Follicular dendritic cell sarcoma (FDCS) is a rare malignant tumor of follicular dendritic cells. Most cases of FDCS are described in lymph nodes, but it can involve extra-nodal locations such as the tonsil. Methods/Case Report We report a case of a 55-year-old male with a 6-month history of a slow growing left neck mass. The patient had a poorly differentiated carcinoma of the left tonsil diagnosed based on morphologic features eight years ago, followed by chemoradiation, and subsequent left tonsillectomy without tumor identified. Imaging reported a 4.0 cm mass in the left neck level III lymph node location. Biopsy of the mass revealed a spindle cell neoplasm. It was difficult to reach a definitive diagnosis despite an extensive work-up. Review of the previous left tonsil biopsy showed a pleomorphic epithelioid cell proliferation. On the radical resection specimen of the left neck mass, a 4.5 cm, tan-gray, fleshy, necrotic mass was present. Microscopically, it demonstrated a neoplasm with spindle cell and epithelioid cell proliferation, with extensive necrosis. A wide panel of immunostains was performed; the tumor cells showed positivity for CD21, CD23, CD35, and focally weak positivity for cytokeratin OSCAR and Synaptophysin. Squamous cell markers were negative. These findings are consistent with FDCS. Further work-up on the original tonsil tumor displayed immunostaining profile of FDCS. This case was consulted with the National Institute of Health (NIH) for confirmation of the diagnosis. Results (if a Case Study enter NA) NA Conclusion Morphologically, FDCS can present with various histologic patterns, which can lead to diagnostic pitfalls, and common confusion with poorly differentiated carcinoma or soft tissue tumors, especially in needle biopsies. Generally, patients present with a prolonged clinical course, with a 50% and 25% chance of recurrence and metastasis respectively, hence the awareness and accurate diagnosis of this entity is important in biopsies of lymph nodes or of extranodal locations such as a tonsil.

18FDG-PET/CT Specificity for the Detection of Lymphoma Recurrence in the Tonsils.

ObjectiveIn patients with a history of lymphoma who demonstrate palatine tonsil uptake on posttreatment PET/CT (positron emission tomography/computed tomography), tonsillectomy is often performed to evaluate for lymphoma recurrence. However, predictive clinical and imaging factors for true tonsil recurrence in this setting are not well established; this will be explored herein.Study DesignRetrospective case series.SettingPatients treated at a tertiary medical center from January 2008 to May 2020.MethodsChart review was performed on all patients with a history of treated lymphoma in clinical remission who presented for evaluation of abnormal PET/CT imaging findings and subsequently underwent tonsillectomy.ResultsAmong 15 patients who met inclusion criteria, 14 had benign findings on surgical pathology, yielding a false-positive rate of 93%. The patient with malignancy was identified on biopsy after inconclusive surgical pathology and is the only documented case of recurrence in this specific patient population throughout the literature. The patient presented with B symptoms, irregularly shaped tonsils, increased lymph node activity on PET/CT, and uptrending bilateral tonsil activity but with one of the lowest maximum standardized uptake values of the cohort. The singular distinguishing feature for the patient with recurrent disease was a prior tonsil biopsy suspicious for recurrence, which prompted the otolaryngology referral.ConclusionPET/CT lacks specificity in identifying lymphoma recurrence in the oropharynx. Clinical and radiographic features that were previously considered concerning for recurrence are most likely not indicative of malignancy in this patient population. Our findings call into question whether tonsillectomy should be routinely performed in this patient population.

A 71-Year-Old Man Presenting with Anemia and a Solitary Skin Lesion Associated with Primary Cutaneous Marginal Zone Lymphoma

A 71-year-old man with a solitary red lesion on his left arm, which initially thought to be a hematoma, was admitted for inpatient evaluation of anemia, which after a positive direct Coombs test was qualified as autoimmune hemolytic anemia. The patient also presented with hoarseness and biopsies from the swelled nasopharynx and an enlarged right tonsil were obtained. Due to uncertain diagnosis and the patient’s previous history of malignancy, (low grade lymphoproliferative lymphoma) skin and bone marrow biopsies were performed. Unfortunately, the patient’s status rapidly deteriorated as a result of septic shock. The obtained skin, nasopharynx, and tonsil biopsies became available after the patient died and showed marginal zone lymphoma with no evidence of lymphoproliferative neoplasm in the bone marrow. &#x0D; This case demonstrates the diagnostic complexity of nodal marginal zone lymphoma complicated by severe life-threatening autoimmune hemolytic anemia in an elderly patient with multimorbidity, which presents management challenges for health care providers.

Actinomyces causing chronic tonsillitis: a retrospective study

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Actinomyces is often overlooked and hence reported as rare infection of palatine tonsils. It can often lead to chronic tonsillitis and can masquerade as oropharyngeal malignancy. Objectives were to estimate the proportion of actinomyces causing chronic tonsillitis using histopathological evidence and to describe the histopathology of tonsillectomy specimens.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Total 33 patients had undergone tonsillectomy in the study period of June 2018 to September 2020. Tonsil biopsy specimens stained with haematoxylin-Eosin stain and gram Stain were evaluated by light microscopy examination for the presence of actinomyces. &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; Total 4 Patients (8 tonsillectomy specimens) had evidence of actinomycosis on histopathological examination constituting 12.12 % of the total specimens examined, the mean age was 17 years (ranging from 5 to 42 years). One of the specimens had unilateral tonsillar cyst which revealed colonies of actinomyces.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Actinomycosis of tonsil is a common condition which is usually missed as routine histopathological examination of excised tonsils is not done. Hence, we recommend routine histopathological examination of all tonsillectomy specimens irrespective of age.&lt;/p&gt;

Abstract 2677: Sequential same slide multiplex immunofluorescence and H&amp;E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections

Abstract Multiplex immunofluorescence (mIF) has become an established method for characterizing the tumor immune microenvironment. Currently, hematoxylin and eosin (H&amp;E) staining is typically performed on a serial tissue section to provide morphologic context for the mIF data. Serial sections do not represent identical cell populations, however, and are not always possible to obtain when the amount of available tissue is limited. Consequently, a method to examine the same tissue section by both fluorescence and brightfield imaging would be advantageous. Here we present a streamlined method and system for serial mIF and H&amp;E staining on a single tissue slide for a comprehensive analysis to support high-throughput tissue immunophenotyping. The UltiMapper I/O PD-L1 kit was used for multiplex immunofluorescence staining of CD8, CD68, PD-L1, and pan-Cytokeratin in formalin-fixed, paraffin-embedded (FFPE) samples from human tonsil and primary tumor biopsies using the Leica Biosystems BOND RX autostainer. Stained tissues were imaged in five spectrally distinct fluorescence channels (DAPI, FITC, TRITC, Cy5, Cy7) on the RareCyte CyteFinder II HT Instrument. Slides were de-coverslipped and stained with H&amp;E, then imaged with brightfield using the CyteFinder II HT instrument. To segment the tumor and stroma tissue regions, a HALO AI classifier was created. Fluorescence images were analyzed using the HALO Highplex FL module to identify CD8+ cytotoxic T-cells, CD68+ macrophages, CD68+/PD-L1+ immuno-suppressive macrophages, pan-CK+ tumor cells, and pan-CK+/PD-L1+ immune-evading tumor cells within the tumor and stromal regions identified by the H&amp;E stain. This novel combination of fluorescence and brightfield analysis enables deep phenotyping of immune cells as well as assessment of cellular morphology and tumor architecture in the same tissue section. Citation Format: Katir K. Patel, Amanda Bares, Mael Manesse, Mark Burton, Bonnie Phillips, Kate Lillard, Anne Hellebust, Melinda Duplessis, Kyla Teplitz, Tad George. Sequential same slide multiplex immunofluorescence and H&amp;E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2677.

Tonsillar granuloma associated with hypogammaglobulinemia

BackgroundRare tonsillar granulomas may be caused for example by infections, malignancies or sarcoidosis. Granulomas also occur in inborn errors of immunity (IEI) such as common variable immunodeficiency (CVID) with B cell maturation defects and hypogammaglobulinemia. CVID shares various features with sarcoidosis and drug-induced secondary hypogammaglobulinemia; careful consideration of differential diagnosis between these conditions is warranted.Case presentationA 29-year-old female with epilepsy developed dysphagia, dyspnea and impaired exercise tolerance. Obstruction caused by swollen lingual tonsil and edema in the epiglottis and arytenoid mucosa were found. Lingual tonsil and epiglottis biopsies displayed non-necrotizing granulomas. There was no evidence of viral, bacterial, mycobacterial or fungal infections. Chest X-ray, computerized tomography of chest and ultrasound of neck and abdomen remained unremarkable. Positron emission tomography/computed tomography (PET/CT) showed laryngeal enhancement. Empiric antimicrobials combined with prednisolone were insufficient to control her disease. In immunological evaluation, the patient had normal counts of B and T cells. Proportions of CD27+ memory B cells (30.3%) and IgD−IgM−CD27+ switched memory B cells (7.2%; normal range 6.5–29.2%) were normal. Percentage of activated CD21low B cells was high (6.6%; normal range 0.6–3.5%). IgG (3.5 g/L; normal range 6.77–15.0 g/l) and all IgG subclass concentrations were low. Anti-polysaccharide responses were impaired, with 3/10 serotypes reaching a level of 0.35 µg/ml after immunization with Pneumovax®. The findings were consistent with hypogammaglobulinemia resembling CVID, possibly secondary to antiepileptic medication. Her dyspnea and dysphagia responded favorably to subcutaneous IgG and rituximab.ConclusionsTonsillar granulomas can be the presenting and only clinical feature of B cell deficiency, highlighting the diversity of symptoms and findings in primary or secondary immunodeficiencies.

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry.

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.

The Immune Micro-Environment in Diffuse Large B Cell Lymphoma Is Characterised By an Immunosuppressive Shift in T Cell Subsets

Diffuse large B-cell Lymphoma (DLBCL) is the most frequent non-Hodgkin's lymphoma with 3 molecularly distinct subtypes based on cell of origin. Genetic alterations in DLBCL, expression of checkpoint molecules and an immunosuppressive microenvironment (ME) all contribute to escape from host anti-lymphoma immunity. The clinical success of monoclonal antibodies that engage the immune system and CAR-T cellular therapy have further highlighted the importance and therapeutic potential of the immune ME in DLBCL. Here we present data from comprehensive phenotyping of cell suspensions from diagnostic DLBCL and reactive lymph node / tonsil (RLNT) biopsies by cytometry by time of flight (CyTOF), with a focus on the T-cell compartment. Cryopreserved samples from 6 DLBCL (5 LN, 1 spleen) at diagnosis and 5 RLNT (3 LN, 2 tonsil) were stained with a panel of metal-tagged antibodies and analysed by CyTOF2. Samples were acquired in 2 batches with the same RLNT (LN) sample with each to ensure staining consistency. Data were normalised, uploaded to Cytobank, gated to CD45+ CD3+ live single cells and exported for further analysis with Cytofkit in R. CD3+ events were gated further into CD4+ and CD8+ subsets, which demonstrated that CD4+ T cells were the predominant phenotype in all samples. However, there was a marked skewing of the CD4:CD8 ratio, with CD4+ T cells lower as a percentage of CD3+ T cells in the DLBCL samples (55.84 v 78.18, p=0.0173*). CD8+ T cells were higher as a percentage in DLBCL (36.22 v 16.75, p=0.03*) with no difference seen in double negative (DN) T cells. CD3+ T cells were then clustered with FlowSOM and visualised according to the tSNE algorithm. A heatmap of median marker expression intensity was generated to facilitate cluster identification. This revealed a number of differences in cluster abundance between the groups, with a significant shift in differentiation away from naïve and towards an effector memory (EM) phenotype in DLBCL. There were fewer cells in the CD27+ CD28+ CCR7+ CD45RA+ CD4+ naïve cluster in the DLBCL samples than the RLNT (p=0.0173*). Although the DLBCL samples showed an overall reduction in CD4+ T cells, the clusters of regulatory T cells (Treg: CD4+ CD25+ FOXP3+ and CD127-/low) consisted of more cells from these cases than the RLNT (p=0.0043**). Within the Treg population, the DLBCL patients had more Th1 polarised (T-bet+) Tregs and more PD-1 expressing Tregs. The Th1 Tregs predominantly secreted the suppressive cytokines IL-2, IL-10 and TGF-β on stimulation and may play a role in inhibiting Th1 responses. Conventional Th1 were not increased in DLBCL resulting in a higher Th1 Treg to Th1 ratio than in RLNT. There was a trend for RLNT samples to contribute more cells to the PD-1 high follicular helper T cell (TFH) cluster and DLBCL to the PD-1+ TIM-3+ DN cluster. The DLBCL ME had relatively more CD8+ T cells and contributed more to the CCR7- CD45RA- CD8+ EM clusters (p=0.0173*) but the CD8+ T cells in the RNLT samples tended to a naïve CCR7+ CD45RA+ PD-1- phenotype (p=0.0519). The CD8+ EM cells enriched in the DLBCL ME expressed the cytotoxic markers granzyme and perforin and responded to stimulation with degranulation (CD107a) and cytokine production (IFNγ, TNFα, TGFβ and IL-10), not suggestive of exhaustion. It is also notable that a cluster of PD-1+ TIM-3+ CD8+ EM with reduced markers of cytotoxicity, low CD107a expression and poor cytokine production after stimulation was predominantly made up of cells from DLBCL suspensions (p=0.002**). CyTOF analysis of the DLBCL ME has demonstrated a shift in the balance of T cell subsets and CD4:CD8 ratio with a relative abundance of immunosuppressive Tregs despite an overall reduction in the CD4+ population and a skew towards differentiation in CD4+ and CD8+ populations. The cytotoxic T cells in DLBCL tended to have an EM phenotype and express immune checkpoint molecules but remained capable of cytokine production. However, the production of IFNγ by these effector T cells may play a role in the development of inhibitory Tregs with a Th1 phenotype, which were enriched in these patients. A cluster of CD8+ EM cells expressing checkpoint molecules and displaying characteristics of exhaustion following stimulation was also seen in these DLBCL patients. These data provide new insights into the immunosuppressive nature of the DLBCL ME and provide a rationale for targeting the ME alongside existing therapeutic approaches, including CAR-T cells to improve outcomes. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

THE ROLE OF PARASTERNAL MEDIASTINOTOMY IN THE DIAGNOSIS OF MEDIASTINAL TUMORS

Parasternal mediastinotomy is a surgical method of morphological verification of mediastinal tumors, widely performed in oncological clinics. The article provides information about the method of implementation and the results of parasternal mediastinotomy for malignant tumors of the mediastinum.Purpose of the study. Evaluation of the results of parasternal mediastinotomy for morphological verification of mediastinal tumors.Patients and methods. The study included 77 patients who for the period from 2008 to 2018. 80 parasternal mediastinotomies were performed with a biopsy of a mediastinal tumor in the conditions of the Department of Thoracic Surgery of the P. Hertsen Moscow Oncology Research Institute. At the stage of preoperative examination, all patients underwent standard diagnostic studies: computed tomography of the chest, abdominal cavity, and a comprehensive ultrasound. In the case of the presence of the endobronchial component of the tumor, fibrobronchoscopy was performed. MRI of the brain was performed in patients with neurological symptoms. 12-lead ECG, echocardiography, and a study of the function of external respiration were performed to assess the functional status of patients.Results. The final histological diagnosis was made in 76 of 77 (99%) patients. The clinical diagnosis of lymphoma was set in 66, thymoma — in 6 patients, lung cancer with affection of mediastinal lymph nodes — in 4 patients, mediastinal tumor — in 4 patients. In 24 patients (31%), operations were preceded by other attempts at morphological verification. In 12 patients — mediastinal tumor biopsy under ultrasound control, in 3 — parasternal mediastinotomy, in 2 — transthoracic mediastinal tumor biopsy combined with biopsy of supraclavicular lymph node under ultrasound control, 2 — biopsy of supraclavicular lymph node under ultrasound control, in 1 — mediastinum tumor biopsy under control. — chest wall biopsy, in 1 — open biopsy with thoracotomic access, in 1 — tonsil biopsy, in 1 — trachea biopsy. In all cases, the indication for performing a parasternal mediastinotomy was an insufficient amount of material for carrying out a complete immunohistochemical study.Conclusion. Parasternal mediastinotomy is a safe and reliable method of morphological verification of the formations of the anterior mediastinum and para-aortic region. In case of the ineffectiveness of transthoracic biopsy under the control of ultrasound or CT navigation, the performing of parasternal mediastinotomy allows to establish a morphological diagnosis and to begin a specific treatment in the shortest possible time.

The interplay between local immune response and Epstein-Barr virus-infected tonsillar cells could lead to viral infection control.

Epstein Barr virus (EBV) gains access to the host through tonsillar crypts. Our aim was to characterize microenvironment composition around EBV+ cells in tonsils from pediatric carriers, to disclose its role on viral pathogenesis. LMP1 expression, assessed by immunohistochemistry (IHC), was used to discriminate EBV + and - zones in 41 tonsil biopsies. Three regions were defined: Subepithelial (SE), interfollicular (IF) and germinal center (GC). CD8, GrB, CD68, IL10, Foxp3, PD1, CD56 and CD4 markers were evaluated by IHC; positive cells/100 total cells were counted. CD8+, GrB+, CD68+ and IL10+ cells were prevalent in EBV+ zones at the SE region (p < 0.0001, p = 0.03, p = 0.002 and p = 0.002 respectively, Wilcoxon test). CD4+ and CD68+ cell count were higher in EBV + GC (p = 0.01 and p = 0.0002 respectively, Wilcoxon test). Increment of CD8, GrB and CD68 at the SE region could indicate a specific response that may be due to local homing at viral entry, which could be counterbalanced by IL10, an immunosuppressive cytokine. Additionally, it could be hypothesized that CD4 augment at the GC may be involved in the EBV-induced B-cell growth control at this region, in which macrophages could also participate.