Thyroid poorly differentiated carcinoma metastatic to pancreas diagnosed by fine-needle aspiration and demonstrating a novel BRAF fusion.

Differentiating pancreatic duct adenocarcinoma from metastasis can be challenging by morphology alone. Metastasis from a classic papillary thyroid carcinoma can present as a poorly differentiated carcinoma and mimic pancreatic ductal adenocarcinoma's morphology.

Approach to small biopsies in the larynx.

Small biopsies in the larynx can make a definitive diagnosis challenging due to the sampling or tangential sectioning. The differential diagnosis can be divided into mucosal lesions (squamous papillomas, intraepithelial dysplasia, and invasive squamous cell carcinoma) or submucosal lesions (vocal cord polyps/nodules, amyloidosis, granular cell tumor, rhabdomyoma, neuroendocrine neoplasms, salivary gland tumors, and cartilaginous tumors). Diagnostic criteria (both morphologic and immunohistochemical) are reviewed to arrive at a diagnosis even on small biopsy.

Classic polymorphous adenocarcinoma: Fine needle aspiration cytopathology of eight cases.

INTRODUCTION: The cytopathology and diagnostic accuracy of salivary gland (SG) polymorphous adenocarcinoma (PAC) is the subject of a limited number of reports. We undertook a review of our experience with fine needle aspiration (FNA) biopsy and PAC. MATERIALS AND METHODS: A search was made of our cytopathology files for PAC cases that also had histopathological confirmation. FNA biopsy smears and cell-blocks were performed and examined using standard techniques. RESULTS: Eight FNA biopsy cases of histologically proven PAC from 7 patients [F:M = 1.3:1, age 39-75 years, mean = 58] met study inclusion. Metastatic aspirates were most common (4), followed by 3 primary cases and 1 locally recurrent neoplasm. Primary FNA sites included hard palate (1 case), lip (1), and lateral tongue (1); all metastatic sites were in the neck. A precise cytologic diagnosis was made in 38% of cases; however, when applying the Milan classification system, 100% could be categorised as either malignant or of uncertain malignant potential. Ancillary immunohistochemical testing performed in 44% of the cases was non-specific. Cytologic smears showed cellular uniformity and structural variety of cell groups with tubular, branching, cribriform, and convex patterns as well as variable, but occasionally abundant globular myxoid stroma leading to confusion with adenoid cystic carcinoma. CONCLUSION: The imitative cytopathology of PAC with other SG neoplasms as well as its infrequency in routine FNA biopsy practice makes specific interpretation difficult, but using a classification system allows for appropriate patient management. Molecular testing in future specimens holds promise for enhancing diagnostic accuracy.

Lobular neoplasia diagnosed on breast Core biopsy: frequency of carcinoma on excision and implications for management.

The appropriate follow-up and treatment for patients with a core biopsy diagnosis of lobular neoplasia (atypical lobular hyperplasia or lobular carcinoma in situ) remains controversial. Several studies have attempted to address this issue, with recommendations ranging from close clinical follow-up or surveillance to mandatory surgical excision in all cases. We report the findings at our institution, where virtually every core needle biopsy diagnosis of lobular neoplasia results in follow-up excision. The goal of the study was to identify potential predictors of upgrade to a more significant lesion. We identified 76 patients over a 15-year period with a core biopsy diagnosis of pure lobular neoplasia and no other high-risk lesions. Subsequent surgical excision identified 10 cases (13%) that were upgraded to carcinoma. Upgrade diagnoses included invasive ductal carcinoma (n=1), invasive lobular carcinoma (n=4), ductal carcinoma in situ (n=3), and pleomorphic lobular carcinoma in situ (n=2). All 10 upgraded cases had imaging findings suspicious for malignancy including irregular masses, asymmetric densities, or pleomorphic calcifications. Of the 10 upgraded cases, 7 were diagnosed as lobular carcinoma in situ on core biopsy. The data support a role for radiologic-pathologic correlation in the evaluation of suspicious breast lesions and suggest that the extent of lobular neoplasia in core biopsy specimens may be an indicator of the likelihood of upgrade to carcinoma.

Doing more with less: integrating small biopsies in cytology practice.

Cytopathologists are at the forefront of specimen acquisition during many different procedures while providing rapid on site evaluation (ROSE). This has added pressure to cytopathologists as more and more ancillary testing is being requested on smaller amounts of tissue. By focusing on the most common organ sites: lung, head and neck, and pancreas, there is a discussion of what the cytopathologist needs to know to triage tissue successfully. Finally, there is a discussion of the logistical aspects of integrating small biopsies into everyday practice.

Top 10 Nested Pattern Head and Neck Lesions to Notice.

BACKGROUND: Nested is defined as "cellular clusters arranged in small groupings with intervening vascular or stromal networks, lacking lumens or glandular formation." Using this definition, multiple neoplastic and non-neoplastic lesions of the head and neck come into the differential. We have broadly organized the differential diagnosis of "nested" tumors into entities with neuroendocrine differentiation, squamous differentiation, thyroid follicular cell differentiation, and other lesions. METHODS: Review. RESULTS: Many different entities have a nested appearance and the morphologic, immunohistochemical, clinical, and radiographic features contribute to the differential diagnosis. The different tumors covered in this review include neuroendocrine neoplasms, paraganglioma, middle ear neuroendocrine tumor (formerly known as middle ear adenoma), medullary thyroid carcinoma, poorly differentiated thyroid carcinoma, olfactory neuroblastoma, ectopic pituitary neuroendocrine tumor, hyalinizing trabecular tumor, solid subtype of papillary thyroid carcinoma, solid cell nests/C-cell hyperplasia, necrotizing sialometaplasia, and meningioma. CONCLUSION: In this review, we discuss the morphologic and immunohistochemical features of the covered entities as a guide to differential diagnosis when nested-patterned head and neck lesions are encountered.

Sinonasal Adenosquamous Carcinoma - Morphology and Genetic Drivers Including Low- and High-Risk Human Papillomavirus mRNA, DEK::AFF2 Fusion, and MAML2 Rearrangement.

BACKGROUND: Sinonasal adenosquamous carcinoma is rare, and there are almost no studies detailing morphology or characterizing their genetic driver events. Further, many authors have termed sinonasal tumors with combined squamous carcinoma and glands as mucoepidermoid carcinoma but none have analyzed for the presence of MAML2 rearrangement. METHODS: Cases from 2014 to 2020 were collected and diagnosed using World Health Organization criteria. They were tested for p16 expression by immunohistochemistry (70% cut-off), DEK::AFF2 fusion by fluorescence in situ hybridization (FISH) and AFF2 immunohistochemistry, MAML2 rearrangement by FISH, and low- and high-risk HPV by RNA ISH and reverse transcription PCR, respectively. Detailed morphology and clinical features were reviewed. RESULTS: There were 7 male (64%) and 4 female (36%) patients with a median age of 69 years, most Caucasian (10 of 11 or 91%). Most had tobacco exposure (8/11, 73%) and most presented with epistaxis, a visible nasal mass, and/or facial pain. Several had a precursor papillomas (3 of 11, 27%). The squamous component had variable keratinization, 5 of 11 (46%) of which would be described as keratinizing, 3 non-keratinizing, and 2 with mixed features. All had gland formation, by definition, and 2 of 11 (18%) had ciliated tumor cells. None of the 11 cases had MAML2 rearrangement and one had DEK::AFF2 fusion with associated positive nuclear AFF2 protein immunostaining. Most were p16 positive (7 of 11, 64%) and all 7 of these were hrHPV positive either by RNA ISH or RT-PCR. Two of the p16-negative tumors were positive for lrHPV by RNA ISH. Treatment included surgery alone (4 of 11, 36%), surgery with adjuvant radiation (5 of 11, 45%), and surgery with radiation and chemotherapy (2 of 11, 18%). Four of 11 patients (36%) suffered disease recurrence, two requiring re-operation and who were disease free at last follow-up, one receiving additional chemotherapy and who was alive with disease. The other elected to undergo palliative therapy and died of disease. CONCLUSION: Sinonasal adenosquamous carcinoma is a somewhat heterogeneous tumor not infrequently arising ex papilloma and having various drivers including high- and low-risk HPV and rarely DEK::AFF2 fusion. The prognosis appears favorable when proper treatment is possible.

Utility of autopsy in uncovering unexpected neuropathology.

Autopsy rates have declined in the last several decades for a variety of reasons. The purpose of this study is to compare autopsy neuropathologic findings from 2 periods to assess the prevalence of unexpected neuropathologic findings and unexpected neuropathologic diagnoses determined to be the major cause of death. Retrospective review of autopsies with examination of the central nervous system was performed in 2007 to 2008 (n = 289) and 1984 to 1985 (n = 328). Unexpected neuropathologic diagnoses were found at autopsy in 42.4% of cases from 1984 to 1985 vs 38.8% of cases from 2007 to 2008. The neuropathology was felt to contribute to the cause of death in 22% of cases from 1984 to 1985 vs 19.7% of cases from 2007 to 2008. Unexpected neuropathologic findings were the cause of death in 5.2% of cases from 1984 to 1985 vs 3.1% of cases from 2007 to 2008. These findings underscore the continued use of brain and spinal cord examination at autopsy despite advances in "modern" medicine.

Fine needle aspiration cytopathology of eccrine spiradenoma.

INTRODUCTION: Eccrine spiradenoma (ES) is a rare benign cutaneous adnexal tumor. The aim of our study was to discuss the clinical presentation, cytomorphologic features, and differential diagnosis of a series of 3 cases of ES diagnosed by fine needle aspiration (FNA). MATERIALS AND METHODS: The pathology databases were searched for cases of ES diagnosed by FNA and confirmed by follow-up surgical excision. FNA smears, cell blocks, and histologic sections were examined. RESULTS: Three cases of ES that had presented as a soft tissue mass from 3 patients were reviewed. The sites included the left forearm, left leg, and left ankle. Cytology smears showed the presence of hypercellular 3-dimensional dense cell clusters and smaller loose cell aggregates, single cells, and bare nuclei. Most cells had round to oval nuclei, scant cytoplasm, and indistinct cell borders. A second population of cells had more spindled nuclei and were often dispersed as single cells. Scattered lymphocytes were present. Two cases showed the presence of pseudo-rosettes composed of hyaline globules of basement membrane-like material with a surrounding row of basaloid cells. None of the cases showed cytologic atypia, necrosis, or mitoses. Immunohistochemistry was used in 2 cases and showed positive staining with myoepithelial markers (smooth muscle actin, calponin, S100, and CK5). The cytology diagnoses were ES, basaloid cutaneous adnexal neoplasm, and suspicious for ES. CONCLUSIONS: FNA cytopathology of ES demonstrated banal basaloid and spindle cells, lymphocytes, and infrequent metachromatic-stained hyaline globules. A specific diagnosis requires immunohistochemistry testing to avoid confusion with other cutaneous basaloid neoplasms.

An Evaluation of Clinical Efficacy of Immune Checkpoint Inhibitors for Patients with Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid malignancy that is associated with poor prognosis. Current treatment options include surgery, radiation, cytotoxic chemotherapy, and multikinase inhibitor therapy. The role of immunotherapy in ATC is an area of active interest and recent evidence suggests that it may be a potentially effective treatment option. Methods: We report a case series of 13 patients with locally advanced or metastatic unresectable ATC who received immune checkpoint inhibitor therapy (pembrolizumab or nivolumab) at a single institution. Results: The patients' median age was 70 years, 54% (7/13) were male, and 85% (11/13) had stage IVC disease with lungs and lymph nodes being the most common sites of metastases. Ten patients had tumor tissue available for programmed death-ligand 1 (PD-L1) expression testing, all of which were positive for PD-L1, and seven of these patients also had a BRAFV600E mutation. The median progression-free survival was 1.9 months and median overall survival (OS) was 4.4 months. The objective response rate was 16% (2/13). Two patients had partial response (PR), and three patients had durable stable disease. Among patients with a clinical benefit, after a median follow-up of 13.5 months, median OS had not been reached (range 4+ to 29+ months). Responses were ongoing in four subjects. The one-year survival rate was 38% (5/13). Six patients (46%) experienced an immune-related adverse event, and 15% (2/13) experienced a grade 3 or higher adverse event, including one patient with grade 5 immune checkpoint-related thyroiditis. Conclusions: Immune checkpoint blockade was well tolerated with a toxicity profile consistent with published literature on PD-1/PD-L1-targeting therapies. For ATC patients, immune checkpoint inhibition may represent an effective treatment option with robust sustained responses seen in a subset of patients.

Near-infrared Autofluorescence Features of Parathyroid Carcinoma.

Introduction: Parathyroid carcinoma is very rare, and intraoperative definitive diagnosis can be elusive with currently available diagnostics. Near-infrared (NIR) autofluorescence is an emerging tool that identifies parathyroid glands in real time. It is not known whether NIR autofluorescence can detect parathyroid carcinoma intraoperatively. Methods: Patients with preoperative suspicion for parathyroid carcinoma were identified from ongoing studies examining parathyroid autofluorescence with a NIR camera and probe. Specimens from these patients were examined intraoperatively to determine their autofluorescence patterns. Results: Three patients with suspected parathyroid carcinoma were identified preoperatively. Intraoperative NIR autofluorescence imaging showed a relative lack of autofluorescence for all cases, in contrast to parathyroid adenomas and normal parathyroid glands, which typically exhibit significant autofluorescence. Final pathology confirmed parathyroid carcinoma in all cases. Conclusion: Parathyroid carcinoma can be difficult to confirm prior to final pathology review. Our 3 cases suggest that absence of NIR autofluorescence may suggest the likelihood of parathyroid carcinoma, but more studies are needed to investigate this experience.

Cytopathology of acinic cell carcinoma: A study of 50 cases, including 9 with high-grade transformation.

BACKGROUND: Although largely readily recognizable in tissue sections, acinic cell carcinoma (ACC) remains diagnostically problematic in fine-needle aspiration (FNA) cytopathology. The authors undertook an analysis of a large series of ACC aspirates, including acinic cell carcinoma with high-grade transformation (ACC-HGT). METHODS: The authors searched their cytopathology files for ACC cases with histopathologic confirmation. FNA biopsy was performed according to standard techniques. RESULTS: Fifty FNA biopsy cases of ACC (including 36 of parotid origin [72%]) from 41 patients (female to male ratio, 1.4:1; age range, 23-84 years; average, 54 years) met the study inclusion requirements. Primary neoplasm aspirates were most common (72%), and they were followed by recurrent tumors (16%) and metastases (12%). A precise cytologic diagnosis was made for 64%. Three of 9 ACC-HGT cases (33%) were correctly interpreted as such; 98% of conventional ACC cases were correctly graded as low-grade. With the Milan classification system, 74% fit into the malignant category. Ancillary testing was performed for only 36%. Conventional ACC had moderately to highly cellular smears; monotonous cells in aggregates and single forms; rounded nuclei; and microvacuolated, finely granular, oncocyte-like, or nonspecific cytoplasm. ACC-HGT smears contained larger nuclei, high nuclear to cytoplasmic ratios, coarse nuclear chromatin, and a loss of cytoplasmic granules/vacuoles. CONCLUSIONS: A correct diagnosis of ACC via FNA biopsy was made in almost two-thirds of the cases. With the Milan classification, 84% of the cases would have been classified as malignant or suspicious for malignancy. An absence of conventional serous acinar cell morphology in some cases as well as an absence of ancillary immunohistochemistry testing in almost two-thirds of the cases prevented even better diagnostic performance.

Merkel Cell Carcinoma of the Maxillary Sinus: An Unusual Presentation of a Common Tumor.

Merkel cell carcinoma is most commonly seen in the skin of sun exposed areas, particularly the head and neck and is associated with Merkel cell polyomavirus. Merkel cell carcinoma at an extracutaneous mucosal site of the head and neck is rare. We report a case of a 74-year-old women who presented with an enlarging thyroid mass found to be neuroendocrine carcinoma consistent with Merkel cell carcinoma (positive for synaptophysin, chromogranin, CK20). Subsequent work up revealed a maxillary sinus mass with extension into the nasal cavity. Biopsy was diagnostic for Merkel cell carcinoma (positive for synaptophysin, chromogranin, CK20 and Merkel cell polyomavirus). There are only case reports and small case series of Merkel cell carcinoma arising in the mucosal sites of the head and neck most commonly in the oral cavity, rarely the sinonasal mucosa. Merkel cell carcinoma metastasizing to the thyroid has only been reported in three other case reports, all from skin primaries. In addition to our case, we review the literature of extracutaneous sinonasal Merkel cell carcinoma and metastases to the thyroid.

Thyroid sclerosing mucoepidermoid carcinoma with eosinophilia in conjunction with parotid basal cell adenoma: Cytologic, histologic, and molecular features.

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMECE) is a rare malignancy in the thyroid: only 56 cases with histologic descriptions are reported in the literature and fewer reports describe the cytomorphology. Given the rarity of SMECE, data on the cytomorphologic and molecular features are limited. We report a case of a 53-year-old woman with a 5 cm left thyroid mass. Fine-needle aspiration (FNA) revealed atypia of undetermined significance and pathology of left thyroid lobectomy specimen showed SMECE. Additionally, a left pre-auricular lump was noted and FNA followed by left superficial parotidectomy showed basal cell adenoma. Next-generation sequencing showed point mutations in NTRK3 and NF1. Unlike salivary gland mucoepidermoid carcinoma, MAML2 translocations are not present in SMECE. Even though it is a rare entity, awareness of SMECE of the thyroid is important. In this case report we review the cytomorphologic, histologic, and molecular features.

The utility of high-risk human papillomavirus E6/E7 mRNA in situ hybridization in assessing HPV status on cell block.

INTRODUCTION: Assessment of human papillomavirus (HPV) status is critical to the treatment and prognosis of patients with oropharyngeal squamous cell carcinoma. Patients often present with enlarged cervical lymph nodes which are amenable to fine needle aspiration (FNA) and cell block creation. The most widely used method for assessing HPV status is the surrogate marker p16. Other HPV specific methods such as high-risk HPV E6/E7 mRNA in situ hybridization (ISH) have been shown to perform as well as p16 and are easier to interpret. Our study evaluates the utility of high-risk HPV mRNA ISH in cell block specimens. METHODS: Thirty-six cases of metastatic squamous cell carcinoma in cervical neck lymph node FNAs were identified over a 3-year period. All cases had p16 immunohistochemistry (IHC) performed on cell block. HR HPV mRNA ISH was performed on the cell block and compared to the p16 results. Additionally, p16 and HR HPV mRNA ISH status was assessed in those cases with corresponding surgical resections. RESULTS: HR HPV mRNA ISH confirmed the p16 IHC (either positive or negative) in 24 of the 36 cases (66.7%). Six false negative cases were p16 negative/HR HPV mRNA ISH positive. HR HPV mRNA ISH was positive in 75% of the four p16 equivocal cases. Two cases were p16 positive/HR HPV mRNA ISH negative. CONCLUSIONS: HR HPV mRNA ISH is no more difficult to perform in the IHC lab and is easier to interpret than p16 IHC. HR HPV mRNA ISH is a useful alternative to p16 in cell block specimens.

ROC analysis of p16 expression in cell blocks of metastatic head and neck squamous cell carcinoma.

BACKGROUND: Oropharyngeal squamous cell carcinoma is associated with human papillomavirus (HPV) and often presents with early metastasis to cervical neck lymph nodes that are amenable to fine-needle aspiration (FNA). The most common method of HPV status determination is p16 immunohistochemistry (IHC). The literature suggests that a lower threshold is needed for p16 positivity on cell block. We examined and quantified p16 IHC staining on cell block and used receiver operating characteristics (ROC) curve analysis to determine an optimal cutoff value with high sensitivity and specificity. METHODS: Thirty-six FNAs of metastatic squamous cell carcinoma from cervical lymph nodes with p16 IHC were evaluated. The p16 stain was quantified in 5% increments and high-risk HPV mRNA in situ hybridization was performed as a gold standard test. Statistical analysis was performed. RESULTS: Interobserver variability was evaluated and was shown to be low with an intraclass correlation coefficient of 0.857. ROC analysis was performed and showed that a cell block p16 IHC cutoff of 15% yielded the highest sensitivity (80%) and specificity (81.8%). CONCLUSION: Our data show that a threshold of 15% p16 staining in cell block maximizes sensitivity and specificity.

Interobserver agreement in programmed cell death-ligand 1 immunohistochemistry scoring in nonsmall cell lung carcinoma cytologic specimens.

BACKGROUND: The evaluation of PD-L1 expression in nonsmall cell lung carcinoma (NSCLC) is becoming increasingly important given the effectiveness of PD-L1 inhibitors. Although cytologic specimens have been shown to be compatible with surgical specimens to evaluate PD-L1 immunohistochemistry (IHC), evidence of the reproducibility of PD-L1 in cytologic specimens is lacking. The aim of this study is to evaluate interobserver agreement in PD-L1 IHC in cytologic specimens. METHODS: PD-L1 IHC was performed on 86 NSCLC cytology specimens using Dako PD-L1 IHC 22C3 pharmDx. The digitally scanned whole slide images (WSI) were read by five pathologists. Each case was given a Tumor Proportion Score (TPS) and the results were compared between the observers. The interobserver concordance was assessed using 1% and 50% as cutoffs. RESULTS: TPSs were highly correlated among observers (Spearman correlation coefficient, 0.86-0.94). Using greater than 1% as a cutoff, interobserver agreement measured by Fleiss Kappa was 0.74 for all pathologists and Cohen's Kappa coefficient ranged from 0.49 to 0.83, consistent with moderate to substantial agreement. With a cutoff of greater than 50%, Fleiss Kappa was 0.79 for all pathologists and the kappa values ranged from 0.63 to 0.90, consistent with substantial to almost perfect agreement. Several pitfalls were identified by reviewing discordant cases, including staining in macrophages, stromal cells, and intratumoral heterogeneity. CONCLUSION: Our data suggest that TPS of PD-L1 IHC on cytology specimens is reproducible, with a better agreement when using 50% as the cutoff value. However, special attention is required when the TPS is near the 1% cutoff.

Mediastinal sarcomas: experience using fine needle aspiration cytopathology.

Fine needle aspiration (FNA) cytology is a sparsely used diagnostic method in the evaluation of mediastinal sarcomas in most medical centers worldwide with most literature citations regarding this category of malignancies consisting of small series and individual case reports. Most of these published studies highlight vascular sarcomas such as epithelioid hemangioendothelioma, and angiosarcoma, various subtypes of liposarcoma including well-differentiated liposarcoma, myxoid liposarcoma, and pleomorphic liposarcoma, malignant peripheral nerve sheath tumor, and sarcomas of uncertain differentiation, primary synovial sarcoma and the Ewing sarcoma family of tumors. This paucity of cytopathology reports regarding mediastinal sarcomas is in marked contrast to the almost daily application of endobronchial ultrasound (EBUS)-guided FNA biopsy for sampling mediastinal lymph nodes and mediastinal masses for primary and metastatic carcinomas which, of course, are considerably more common that any type of sarcoma in this location. EBUS, endoscopic ultrasound-guided (EUS) needle biopsy, and percutaneous image-guided biopsy using either core needle, fine-needle, or both can serve a potentially useful role for diagnostic sampling of mediastinal sarcomas, be they primary or metastatic. This review catalogues much of the published data regarding FNA cytopathology and its application to mediastinal sarcomas. An attempt is made to primarily highlight case series rather than individual case reports; however, due to the paucity of these, case reports are cited and discussed where appropriate.

Oral lichenoid reactions may possibly be associated with abatacept: A case report and literature update.

Oral lichenoid reactions (OLRs) comprise a group of conditions with a common clinical appearance and histopathologic pattern that may be induced by several conditions or medications. This report describes an OLR possibly induced by a biologic agent. A 69-year-old woman with rheumatoid arthritis presented with a chief complaint of oral pain. The patient retroactively reported of skin lesions as well. Clinically, she had mixed red-white mucosal lesions and ulcers suggestive of an OLR. This diagnosis was supported by histopathologic findings. Withholding the putative etiologic agent, abatacept, resulted in immediate alleviation of both oral and skin lesions. Abatacept and other biologics are thought to help treat inflammation and are becoming more commonly prescribed to treat rheumatoid arthritis. However, the clinicians should explore these medications as a causative factor for OLR.

Prevalence of High-Risk Human Papillomavirus in Tonsil Tissue in Healthy Adults and Colocalization in Biofilm of Tonsillar Crypts.

IMPORTANCE: The pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma is currently an important topic of elucidation. The presence of latent HPV infection in tonsil tissue of healthy adults may provide an explanation for a component of this process and contribute to the understanding of HPV-associated squamous cell carcinoma oncogenesis of the oropharynx. OBJECTIVE: To determine the prevalence of oropharyngeal HPV and to determine the spatial relationship between the virus and crypt biofilm in tonsil tissue. DESIGN, SETTING, AND PARTICIPANTS: A retrospective, cross-sectional study was carried out using samples obtained from tonsils that were archived at a university hospital following elective nononcologic tonsillectomy from 2012 to 2015. Samples consisted of formalin-fixed paraffin embedded samples of tumor-free tonsil tissue from 102 adults between the ages of 20 and 39 years. EXPOSURES: Human papillomavirus status was assessed by polymerase chain reaction, and high-risk subtypes 16 and 18 were assessed with quantitative polymerase chain reaction assay. Samples that demonstrated presence of HPV were then analyzed by in situ hybridization to localize the viral capsid protein. These samples were then stained with concanavalin A to establish biofilm presence and morphology. These samples were also stained with diamidino-phenylindole (DAPI) to visualize location of the virus in relation to cell nuclei. These data were then assembled for aggregate analysis to colocalize HPV in the biofilm of the tonsillar crypts. MAIN OUTCOMES AND MEASURES: Outcome measurements were determined prior to data collection and include prevalence of high-risk HPV types 16 and 18 in tonsil tissue of otherwise healthy adults, as well as demonstration with immunohistochemistry of HPV in tonsillar crypt biofilm. RESULTS: In 102 otherwise healthy adults (55 [53.9%] female; age range, 20-39 years), the overall prevalence of HPV in tonsils was 4.9% (n = 5); and high-risk type 16 or 18, 3.9% (n = 4). In this sample population, in situ hybridization colocalized HPV virus to the biofilm of the tonsillar crypts. CONCLUSIONS AND RELEVANCE: Biofilm is present in the tonsillar crypts in a considerable proportion of tonsil tissues and may be reproducibly identified. Human papillomavirus is demonstrated to colocalize to the crypt biofilm. This has important implications with respect to the determination of HPV prevalence rates in the oropharynx. It may also play a role in the pathogenesis of HPV-related oropharyngeal carcinoma.