Establishment of a diverse head and neck squamous cancer cell bank using conditional reprogramming culture methods.

Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from the oral cavity, 4/16 were derived from the oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papillomavirus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, reverse transcription polymerase chain reaction (RT-PCR) of HPV16/18-specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of four oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity.

Features of endoscopic procedure site reaction associated with a recently approved submucosal lifting agent.

Endoscopic resection techniques, such as endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD), are frequently aided by injection of submucosal lifting solutions that create a plane for dissection and protect deeper mural layers. ORISE™ gel is a recently approved synthetic lifting solution that produces a localized inflammatory reaction associated with retained gel at the injection site. We describe a series of six cases of ORISE™-associated inflammatory lesions in patients who underwent endoscopic resections. Deposits comprised pale fibrillary or hyalinized eosinophilic material, depending on their age. All cases were associated with an inflammatory reaction composed of foreign-body giant cells and scattered eosinophils. ORISE™ gel extended laterally and deeply beyond residual tumors in all cases. Histochemically, the material proved to be negative for Congo Red, and mucicarmine, faint blue with Alcian blue, but positive for PAS and PAS-D. It stained blue with trichrome. Such deposits were absent in cases, wherein other widely-available lifting solutions were used. We compared ORISE™ deposits to histologically similar extracellular deposits, namely amyloid and pulse granulomata. Unlike ORISE™ material, amyloid deposits appear as waxy, more densely eosinophilic material, and stain positive with Congo Red. Amyloid demonstrated prominent intramucosal and perivascular distributions, features not seen in this series of ORISE™ deposits. Hyalinized pulse granulomata showed strong overlap with ORISE™ deposits, since they also comprise eosinophilic material associated with giant cell reaction. On the other hand, they form ribbons of glassy material in circumscribed lobules, unlike the ill-defined ORISE™ deposits. In summary, we describe the pathologic findings at injection sites in patients who underwent endoscopic procedures aided by the recently approved lifting agent, ORISE™. Pathologists should be aware of its appearance and associated reaction to avoid confusion with other common extracellular deposits seen in the gastrointestinal tract.

Characterization of a Diverse Set of Conditionally Reprogrammed Head and Neck Cancer Cell Cultures.

OBJECTIVE: To establish and characterize a diverse library of head and neck squamous cell cancer (HNSCC) cultures using conditional reprogramming (CR). METHODS: Patients enrolled on an IRB-approved protocol to generate tumor cell cultures using CR methods. Tumor and blood samples were collected and clinical information was recorded. Successful CR cultures were validated against banked reference tumors with short tandem repeat genotyping. Cell morphology was archived with photodocumentation. Clinical and demographic factors were evaluated for associations with successful establishment of CR culture. Human papilloma virus (HPV) genotyping, clonogenic survival, MTT assays, spheroid growth, and whole exome sequencing were carried out in selected cultures. RESULTS: Forty four patients were enrolled, with 31 (70%) successful CR cultures, 32% derived from patients who identified as Black and 61% as Hispanic. All major head and neck disease sites were represented, including 15 (48%) oral cavity and 8 (26%) p16-positive oropharynx cancers. Hispanic ethnicity and first primary tumors (vs. second primary or recurrent tumors) were significantly associated with successful CR culture. HPV expression was conserved in CR cultures, including CR-024, which carried a novel HPV-69 serotype. CR cultures were used to test cisplatin responses using MTT assays. Previous work has also demonstrated these models can be used to assess response to radiation and can be engrafted in mouse models. Whole exome sequencing demonstrated that CR cultures preserved tumor mutation burden and driver mutations. CONCLUSION: CR culture is highly successful in propagating HNSCC cells. This study included a high proportion of patients from underrepresented minority groups. LEVEL OF EVIDENCE: Not Applicable Laryngoscope, 134:2748-2756, 2024.

Ameloblastoma with adenoid features: Case report with cyto-histopathologic correlation and molecular findings.

Ameloblastomas are benign but locally aggressive odontogenic tumors that commonly present as expansile lesions in the tooth-bearing areas. Fine-needle aspiration (FNA) biopsies of ameloblastomas are rare in clinical practice, and only a handful of case reports and series have described their cytologic features. We present the case of a 70-year-old woman with a large and disfiguring maxillary sinus soft tissue mass sampled via transcutaneous FNA. Aspirate smears were composed of small clusters of cohesive and monotonous basaloid cells. The accompanying cellblock showed similar clusters of basaloid cells in gland-like, or "adenoid," configurations, eliciting a differential diagnosis that included sinonasal and salivary gland neoplasms. Excisional surgery material was consistent with ameloblastoma with adenoid morphology. Next-generation sequencing (NGS) analysis demonstrated FGFR2 and SMO pathogenic variants. This case exemplifies several uncommonly described features of ameloblastomas in cytology, including cyto-histologic correlation, adenoid morphology, and NGS findings. Awareness of the cytologic features of this neoplasm are important for cytopathologists confronted with maxillary sinus lesions.

Reproducibility of AJCC Criteria for Classifying Deeply Invasive Colon Cancers Is Suboptimal for Consistent Cancer Staging.

The eighth edition of the American Joint Committee on Cancer (AJCC) Staging Manual attempts to address ambiguity in the pT category assignment for colon cancer from prior editions. Despite modifications, the distinction between the pT3 and pT4a categories continues to be a source of diagnostic confusion. In this study, we assessed interobserver agreement among pathologists from different institutions in the application of AJCC eighth edition criteria for categorizing deeply invasive colonic adenocarcinomas. We identified morphologic patterns that produce diagnostic confusion. We assessed 47 colon cancers that closely approached the serosal surface. Six pathologists with interest in gastrointestinal pathology and 4 focused in other subspecialties classified each case as pT3 or pT4a, based on examination of low-magnification and high-magnification images of the most deeply invasive area. Interobserver agreement was assessed using Fleiss' κ. Cases displayed 3 morphologic patterns at the advancing tumor edge, namely, (1) continuous invasion through an inflammatory focus, (2) pushing border, and (3) infiltrative glands and cell clusters with serosal reaction. Gastrointestinal pathologists achieved slight (κ=0.21) or moderate (κ=0.46) and (κ=0.51) agreement in each category, whereas agreement among nongastrointestinal pathologist was fair (0.31) and (0.39), or moderate (0.57) for each category, respectively. In 10 (21%) cases, the distinction between pT3 and pT4a would have changed the overall clinical stage. We conclude that histologic criteria for serosal penetration is a persistent source of diagnostic ambiguity for gastrointestinal and general pathologists in the pT categorization of colon cancers. Clarification of these criteria will help ensure uniform reporting of pathologic and clinical stage.

Differentiating Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features from Classic Papillary Thyroid Carcinoma: Analysis of Cytomorphologic Descriptions Using a Novel Machine-Learning Approach.

BACKGROUND: Recent studies show various cytomorphologic features that can assist in the differentiation of classic papillary thyroid carcinoma (cPTC) from noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Differentiating these two entities changes the clinical management significantly. We evaluated the performance of support vector machine (SVM), a machine learning algorithm, in differentiating cases of NIFTP and encapsulated follicular variant of papillary thyroid carcinoma with no capsular or lymphovascular invasion (EFVPTC) from cases of cPTC with the use of microscopic descriptions. SVM is a supervised learning algorithm used in classification problems. It assigns the input data to one of two categories by building a model based on a set of training examples (learning) and then using that learned model to classify new examples. METHODS: Surgical pathology cases with the diagnosis of cPTC, NIFTP, and EFVPTC, were obtained from the laboratory information system. Only cases with existing fine-needle aspiration matching the tumor and available microscopic description were included. NIFTP cases with ipsilateral micro-PTC were excluded. The final cohort consisted of 59 cases (29 cPTCs and 30 NIFTP/EFVPTCs). RESULTS: SVM successfully differentiated cPTC from NIFTP/EFVPTC 76.05 ± 0.96% of times (above chance, P < 0.05) with the sensitivity of 72.6% and specificity of 81.6% in detecting cPTC. CONCLUSIONS: This machine learning algorithm was successful in distinguishing NIFTP/EFVPTC from cPTC. Our results are compatible with the prior studies, which show cytologic features are helpful in differentiating these two entities. Furthermore, this study shows the power and potential of this approach for clinical use and in developing data-driven scoring systems, which can guide cytopathology and surgical pathology diagnosis.

Refining the utility and role of Frozen section in head and neck squamous cell carcinoma resection.

OBJECTIVES/HYPOTHESIS: Previous studies report high-accuracy rates for intraoperative frozen sections, but reliability of frozen sections in predicting the ultimate final margin status is unknown. We compared frozen and permanent reads to identify risk factors for overall discrepancies between intraoperative and final margin status. STUDY DESIGN: Retrospective chart review. METHODS: Pathology reports of 437 surgical resections between 2010 and 2013 were retrospectively reviewed. A total of 253 cases, generating 1,109 individual specimens, met inclusion criteria. Patient demographics, treatment, recurrence, and survival, as well as pathology data pertaining to the specimen, were recorded. RESULTS: Frozen read accuracy was 96.7% (83.1% sensitivity, 97.9% specificity) relative to permanent evaluation. However, 4.3% of cases had a final positive margin not detected by frozen section; 17.8% had a close margin not detected by frozen section. In eight of 11 cases with missed positive margins, the involved margin was never sampled intraoperatively. Cases where intraoperative margins were only taken from surrounding tissue had a higher risk of missing a close or positive final margin when compared to cases where some or all margins were taken from the specimen (odds ratio = 5.05, 95% confidence interval [2.31, 11.07], P <0.0001). Disease subsite, risk score, prior radiation, staging, and p16 expression were not significantly associated with the likelihood of missing a close or positive final margin. CONCLUSION: Individual frozen section reads are highly accurate. However, negative intraoperative margins do not guarantee margin-negative resections. The process of selecting representative margins for intraoperative analysis, specifically the practice of sampling the resection bed, should be refined. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:1768-1775, 2016.

Fibroblasts in Head and Neck Squamous Cell Carcinoma Associated With Perineural Invasion Have High-Level Nuclear Yes-Associated Protein (YAP) Expression.

We retrospectively studied the expression of Yes-associated protein (YAP) using immunohistochemical staining in 10 cases of head and neck squamous cell carcinoma with associated perineural invasion. We find that fibroblasts in areas associated with perineural invasion show higher levels of nuclear YAP compared to fibroblasts in the stroma of normal mucosa, with a median cell count of 35.4 per high-power field in the former and 3.9 in the latter. No differences were observed between the expression of YAP phosphorylated at Ser127 in the tumoral stroma compared to that in the normal mucosa, with a median cell count expression of 4.9 in the former versus 5.0 in the latter. Therefore, a strong and increased nuclear YAP expression in fibroblasts associated with perineural invasion in head and neck squamous cell carcinoma suggests that YAP-mediated transcription programs in these fibroblasts may contribute to perineural invasion.

Medullary thyroid cancer with undetectable serum calcitonin.

CONTEXT: Calcitonin is a sensitive biomarker that is used for diagnosis and follow-up in medullary thyroid cancer (MTC). In patients with tumors > 1 cm, it is uncommon for preoperative serum calcitonin to be in the normal laboratory reference range in patients with MTC, and even more unusual for serum calcitonin to be undetectable. THE CASE: A 39-year-old woman was found to have a left thyroid nodule on magnetic resonance imaging done for neck pain. Ultrasound and fine-needle aspiration biopsy were performed, and cytopathology was positive for malignant cells. The cells also had features suggestive of a neuroendocrine tumor, and the specimen was immune-stained with calcitonin. There was positive immunoreactivity for calcitonin in isolated cells of the cytospin, highly favoring a diagnosis of MTC. Serum calcitonin was < 2 pg/mL (<6 pg/mL), and serum carcinoembryonic antigen was 3.1 ng/mL (<5.2 ng/mL). Given the low calcitonin levels, procalcitonin was also tested and was elevated at 0.21 ng/mL (< 0.1 ng/mL). The patient subsequently underwent a total thyroidectomy and central and ipsilateral lateral lymph node dissection. Histopathology confirmed a 2.6 × 2.0 × 1.2-cm MTC, with strong, diffuse immunostaining for calcitonin. Postoperatively, serum calcitonin has remained undetectable, carcinoembryonic antigen has remained within the reference range, and procalcitonin has become undetectable. CONCLUSIONS: We present a rare case of a patient with MTC with undetectable preoperative serum calcitonin, whose tumor demonstrated strong, diffuse immunohistochemical staining for calcitonin. We discuss the possible pathogenesis of calcitonin-negative MTC and the challenges in following patients with this condition.

Isolated splenic metastasis from rectal carcinoma: a rare occurrence.

The presence of isolated splenic metastasis in rectal carcinoma is uncommon and usually presents as an asymptomatic mass, noted incidentally on imaging. Splenectomy is usually performed with the goal of curing metastatic disease. It is unclear if adjuvant chemotherapy affords any benefit, and the prognosis is unknown. The case of a young woman is reported, in whom an isolated metastatic lesion in the spleen was discovered 9 months after adjuvant chemotherapy for stage III rectal adenocarcinoma. The patient has remained disease-free for nearly 5 years following splenectomy and chemotherapy. To our knowledge, this is the fourth reported case in the English literature of an isolated splenic metastatic lesion from rectal cancer. We discuss the unique presentation, the importance of post-treatment surveillance, and the implementation of multi-modality treatment strategies in this young patient.