VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Evolving approaches in paediatric thyroid cytopathology: A review.

The guidelines for the workup of thyroid nodules have been established in adult populations and secondarily applied to paediatric populations. In particular, The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is commonly applied to both adult and paediatric thyroid nodules. However, as paediatric nodules have distinct molecular drivers and behavioural trajectories, there is renewed interest in diagnostic and management strategies that are paediatric specific. Here, we review key differences between paediatric and adult thyroid cancer and recent literature evaluating the use of TBSRTC in paediatric populations.

Impact of comorbidities on immediate post-operative complications in oral cavity free flap patients.

PURPOSE: To examine the relationship between comorbidities and the development of immediate post-operative complications in patients undergoing oral cavity composite resection (OCCR) with free flap (FF) reconstruction. MATERIALS AND METHODS: Retrospective analysis was completed on all consecutive OCCRs with FF reconstruction performed at a single quaternary care facility between 1999 and 2020. Comorbidities, immediate post-operative complications, patient demographics, and tumor characteristics were collected. Odds ratios (OR) with 95 % confidence intervals were calculated for associations between comorbidities and immediate post-operative complications. RESULTS: 320 patients who underwent OCCR with FF reconstruction were included. One hundred twenty-one (37.8 %) patients developed a post-operative complication during their initial hospital admission. The most common complications were non-pneumonia cardiopulmonary events (14.1 %), pneumonia (9.4 %), and wound infection (8.4 %). Other complications included flap compromise, bleeding, and fistula. On multivariate analysis, patients without comorbid conditions were less likely to develop a post-operative complication (OR 0.64; 0.41-0.98). Atrial fibrillation (OR 2.94; 1.17-7.39) and cerebrovascular disease (OR 2.28; 1.08-4.84) were associated with increased odds of developing any complications. Furthermore, cerebrovascular disease (OR: 2.33; 1.04-5.39) and peripheral vascular disease (OR: 2.7; 1.2-6.08) were independently associated with pneumonia. CONCLUSION: In this retrospective review of patients undergoing OCCR with FF reconstruction for oral cavity SCC, lack of identifiable comorbidities appeared to be protective for post-operative complications while atrial fibrillation and cerebrovascular disease were associated with increased odds of any complication. Pre-existing vascular disease was also associated with an increased risk of pneumonia.

Molecular signature incorporating the immune microenvironment enhances thyroid cancer outcome prediction.

Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.

Hashimoto's Thyroiditis and the Risk of Papillary Thyroid Cancer in Children.

The association between Hashimoto's thyroiditis (HT) and pediatric thyroid cancer is controversial. Most studies examining this connection have been based on adults, and larger studies in children are lacking. We performed a retrospective study of all sequential pediatric patients who underwent a thyroidectomy for a neoplasm at our institution over a twenty-year period in order to explore the link between HT and pediatric thyroid cancer. A total of 153 patients, median age 16.5 (interquartile range [IQR] 14.2-18.3) years, underwent thyroid surgery for a neoplasm. Patients were mainly female (80%) and White (84%). Median follow-up was 58.6 (IQR 20.7-105.4) months. Thirty-five (23%) patients had HT. Patients who underwent thyroid surgery and had HT were more likely to harbor a malignant neoplasm (p = 0.05); specifically, papillary thyroid carcinoma (PTC, p = 0.02). There was a difference in the distribution of HT among the subtypes of PTC (p = 0.03). Despite this, there was no difference in terms of survival between patients with/without HT. In conclusion, children with a thyroid malignancy, specifically, PTC, are more likely to have HT. The association between HT and pediatric PTC appears to be subtype-specific but does not seem to affect patient survival.

Engineering functional 3-dimensional patient-derived endocrine organoids for broad multiplatform applications.

BACKGROUND: Recent advancements in 3-dimensional patient-derived organoid models have revolutionized the field of cancer biology. There is an urgent need for development of endocrine tumor organoid models for medullary thyroid carcinoma, adrenocortical carcinoma, papillary thyroid carcinoma, and a spectrum of benign hyperfunctioning parathyroid and adrenal neoplasms. We aimed to engineer functionally intact 3-dimensional endocrine patient-derived organoids to expand the in vitro and translational applications for the advancement of endocrine research. METHODS: Using our recently developed fine needle aspiration-based methodology, we established patient-derived 3-dimensional endocrine organoid models using prospectively collected human papillary thyroid carcinoma (n = 6), medullary thyroid carcinoma (n = 3), adrenocortical carcinoma (n = 3), and parathyroid (n = 5). and adrenal (n = 5) neoplasms. Multiplatform analyses of endocrine patient-derived organoids and applications in oncoimmunology, near-infrared autofluorescence, and radiosensitization studies under 3-dimensional in vitro conditions were performed. RESULTS: We have successfully modeled and analyzed the complex endocrine microenvironment for a spectrum of endocrine neoplasms in 3-dimensional culture. The endocrine patient-derived organoids recapitulated complex tumor microenvironment of endocrine neoplasms morphologically and functionally and maintained cytokine production and near-infrared autofluorescence properties. CONCLUSION: Our novel engineered endocrine patient-derived organoid models of thyroid, parathyroid and adrenal neoplasms represent an exciting and elegant alternative to current limited 2-dimensional systems and afford future broad multiplatform in vitro and translational applications, including in endocrine oncoimmunology.

Wnt Signaling in the Phenotype and Function of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) play an important role in supporting tumor growth and suppressing antitumor immune responses, and TAM infiltration has been associated with poor patient prognosis in various cancers. TAMs can be classified as pro-inflammatory, M1-like, or anti-inflammatory, M2-like. While multiple factors within the tumor microenvironment affect the recruitment, polarization, and functions of TAMs, accumulating evidence suggests that Wnt signaling represents an important, targetable driver of an immunosuppressive, M2-like TAM phenotype. TAM production of Wnt ligands mediates TAM-tumor cross-talk to support cancer cell proliferation, invasion, and metastasis. Targeting TAM polarization and the protumorigenic functions of TAMs through inhibitors of Wnt signaling may prove a beneficial treatment strategy in cancers where macrophages are prevalent in the microenvironment.

BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis.

Cancer therapies trigger diverse cellular responses, ranging from apoptotic death to acquisition of persistent therapy-refractory states such as senescence. Tipping the balance toward apoptosis could improve treatment outcomes regardless of therapeutic agent or malignancy. We find that inhibition of the mitochondrial protein BCL-xL increases the propensity of cancer cells to die after treatment with a broad array of oncology drugs, including mitotic inhibitors and chemotherapy. Functional precision oncology and omics analyses suggest that BCL-xL inhibition redirects the outcome of p53 transcriptional response from senescence to apoptosis, which likely occurs via caspase-dependent down-modulation of p21 and downstream cytostatic proteins. Consequently, addition of a BCL-2/xL inhibitor strongly improves melanoma response to the senescence-inducing drug targeting mitotic kinase Aurora kinase A (AURKA) in mice and patient-derived organoids. This study shows a crosstalk between the mitochondrial apoptotic pathway and cell cycle regulation that can be targeted to augment therapeutic efficacy in cancers with wild-type p53.

Racial, ethnic, and socioeconomic disparities in the presentation and management of pediatric thyroid cancer.

OBJECTIVES: Disparities across race and socioeconomic status (SES) in cancer treatment exist for many malignancies. Disadvantaged groups have repeatedly been shown to receive sub-optimal treatment. This study intends to analyze racial and SES disparities in the presentation and management of pediatric thyroid cancer. METHODS: A retrospective national database study of children who underwent thyroidectomy for thyroid papillary, medullary, and follicular carcinoma between 2007 and 2016 was conducted using the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database. Multivariable logistic regression was conducted to identify predictors of 1) tumor stage/size at diagnosis and 2) treatment modalities. RESULTS: 1942 children were analyzed. The average tumor size at presentation was 20 mm for White patients, 26 mm for Non-White patients, and 27 mm for Hispanic patients (p < 0.001). Stage of disease differed significantly by race/ethnicity (p < 0.001) with Non-White and Hispanic patients having more distant disease than White patients at presentation. On multivariable regression, Hispanic patients (OR 1.41, 95%CI 1.06-1.87, p = 0.017) were more likely to be diagnosed at later stages. Non-White (OR 2.03, 1.50-2.73, p < 0.001) and Hispanic patients (OR 1.57, 1.19-2.07, p = 0.002) had larger tumors at diagnosis than White patients after controlling for other SES factors. CONCLUSIONS: SES disparities exist in pediatric thyroid cancer. Non-White and Hispanic patients are more likely to present with larger tumors and distant disease as compared to White patients. Understanding and intervening on these SES disparities is essential to improve outcomes.

Molecular testing of cytology specimens: overview of assay selection with focus on lung, salivary gland, and thyroid testing.

Ancillary and molecular testing of cytopathology specimens has emerged as a reliable and useful tool to provide diagnostic information and treatment-related biomarker status for the management of cancer patients. The cytology specimens obtained through minimally invasive means have proven suitable testing substrates for a variety of ancillary tests, including immunohistochemistry, fluorescence in situ hybridization, as well as polymerase chain reaction and next generation sequencing molecular techniques. By focusing specifically on the cytology specimen, this review provides an overview of basic testing considerations and assay selection in addition to updates on the ancillary testing of cytologic tumor specimens from the lung, salivary gland, and thyroid.

Cytology and LGBT+ health: establishing inclusive cancer screening programs.

There are substantial disparities in cancer screening for sexual minorities and gender non-conforming patients. In additional to patients having trauma due to negative experiences with the healthcare system, disparities may be heightened due to heteronormative and cisnormative design of screening programs and electronic medical record systems. Furthermore, there are morphologic challenges specific to certain specimen types from the LGBT + population, such as anal cytology samples, cervical cytology from transgender men taking testosterone, and neovaginal cytology samples. Men who have sex with men are at increased risk for anal cancer compared with the general population. While early detection of anal dysplasia decreases the risk of invasive carcinoma, screening programs are not widespread. Cervical cancer screening may be psychologically and physically challenging for transgender men and non-binary patients. The use of exogenous testosterone therapy causes atrophic changes in cervical cytology samples which mimic high-grade dysplasia. The rate of unsatisfactory samples are also increased in this population. Although HPV driven cancers have been reported in patients with neovaginas, there are currently no guidelines about appropriate screening for transgender women and intersex patients who have neovaginas. Cytopathologists can optimize the health of LGBT + patients in many ways including advocating for inclusive screening guidelines, validating self-collection for HPV and cytology samples, updating requisition forms to better capture the spectrum of gender expression, and recognizing the morphologic changes in cytology samples due to exogenous hormone use.

Evaluation of the Molecular Landscape of Pediatric Thyroid Nodules and Use of a Multigene Genomic Classifier in Children.

Importance: Definitive diagnosis of a thyroid nodule in a child is obtained through diagnostic surgery. This is problematic because pediatric thyroid surgery is associated with higher rates of complications. In adults, preoperative molecular testing improves the management of thyroid nodules, but this has not been validated in children. Objective: To determine whether the molecular landscape of pediatric thyroid nodules is amenable to detection by a multigene genomic classifier (GC) test (ThyroSeq v3; Sonic Healthcare USA). Design, Setting, and Participants: This was a retrospective consecutive case series and GC testing of fine-needle aspiration (FNA) and formalin-fixed paraffin-embedded (FFPE) tissues from sequential pediatric thyroidectomies performed between January 2003 and December 2019 at a single tertiary academic medical center. The study included 95 patients (median [range] age, 16.3 [4.8 to 21.1] years; 75 [79%] female) who underwent surgery for a thyroid nodule. Interventions: A total of 118 thyroid nodule samples (95 FFPE, 23 companion FNAs) yielded informative next-generation sequencing data and multigene GC. Main Outcomes and Measures: The primary outcome was the determination of the pediatric thyroid molecular landscape. The secondary outcome was the diagnostic accuracy of the GC test for pediatric thyroid nodules. Results: Of the 95 patients, 75 (79%) were female, and the median (IQR) age was 16.3 (14.0-17.3) years. Next-generation sequencing confirmed the unique molecular landscape of malignant pediatric thyroid nodules (compared with adults), which is dominated by gene fusions (most commonly RET and NTRK), rare BRAF/RAS alterations, and no TP53 or TERT promoter pathogenic variants. Several poorly differentiated thyroid cancers harbored DICER1 variants. Benign nodules appeared to be almost exclusively associated with TSHR and DICER1 alterations. The test demonstrated a 96% sensitivity (95% CI, 87%-99%) and 78% specificity (95% CI, 64%-88%). The negative predictive value was 95% (95% CI, 88%-98%) and the positive predictive value was 83% (95% CI, 74-89%). The concordance of GC between 23 pairs of matched FFPE and FNA tissues was 96%. Conclusions and Relevance: The study results of this retrospective consecutive case series suggest that the molecular landscape of pediatric nodules is unique but remains amenable to molecular classification. The multigene GC test, with high sensitivity and reasonably high specificity, represents a potential addition to the diagnostic workup of children with thyroid nodules and may decrease the use of diagnostic surgery.

Utilizing Three-Dimensional Culture Methods to Improve High-Throughput Drug Screening in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most aggressive endocrine neoplasm, with a median survival of just four to six months post-diagnosis. Even with surgical and chemotherapeutic interventions, the five-year survival rate is less than 5%. Although combination dabrafenib/trametinib therapy was recently approved for treatment of the ~25% of ATCs harboring BRAFV600E mutations, there are no approved, effective treatments for BRAF-wildtype disease. Herein, we perform a screen of 1525 drugs and evaluate therapeutic candidates using monolayer cell lines and four corresponding spheroid models of anaplastic thyroid carcinoma. We utilize three-dimensional culture methods, as they have been shown to more accurately recapitulate tumor responses in vivo. These three-dimensional cultures include four distinct ATC spheroid lines representing unique morphology and mutational drivers to provide drug prioritization that will be more readily translatable to the clinic. Using this screen, we identify three exceptionally potent compounds (bortezomib, cabazitaxel, and YM155) that have established safety profiles and could potentially be moved into clinical trial for the treatment of anaplastic thyroid carcinoma, a disease with few treatment options.

Adrenal gland cytology reporting: a multi-institutional proposal for a standardized reporting system.

BACKGROUND: With the development of new technologies and the changing patient profiles, cytopathology departments receive increasing numbers of adrenal gland cytology specimens. In this study, the authors analyzed archival adrenal gland cytology cases and attempted to implement a diagnostic reporting system. DESIGN: Retrospective electronic medical record search was performed for adrenal gland cytology specimens in seven tertiary care centers. The cytology diagnoses were grouped in 7 categories: nondiagnostic, nonneoplastic, benign adrenal cortical elements (BACE), primary neoplasm of noncortical origin (NONC), atypia of undetermined significance (AUS), suspicious for malignancy (SM), and malignant (MAL). If available, histopathology results of concurrent and/or follow-up biopsies and/or resections were documented. RESULTS: A total of 473 adrenal gland cytology cases were included. BACE cases comprised 21.8%, whereas MAL cases were 57.5% of all cases. For BACE and MAL categories, there were 100% and 98.9% correlation, respectively, in the cases with histopathology follow-up. Six of 10 NONC cases had histopathology diagnoses and there were 3 pheochromocytomas and 3 schwannomas. Twenty-one AUS cases had histology follow-up and 10 (47.6%) of them were malignant. Six cases of SM had histopathology follow-up, and all of them were malignant on the follow-up. CONCLUSIONS: The authors propose a 7-tier diagnostic scheme for adrenal gland cytology. The risk of malignancy was 98.9% in MAL cases (87/88) in the cohort. The only case with discordance was reported as "adrenal cortical adenoma with marked atypia"' on resection. There was no difference between endoscopic ultrasound-guided and percutaneous methods. Further studies are needed to validate and make this approach universal.

Proximity of immune and tumor cells underlies response to BRAF/MEK-targeted therapies in metastatic melanoma patients.

Acquired resistance to BRAF/MEK-targeted therapy occurs in the majority of melanoma patients that harbor BRAF mutated tumors, leading to relapse or progression and the underlying mechanism is unclear in many cases. Using multiplex immunohistochemistry and spatial imaging analysis of paired tumor sections obtained from 11 melanoma patients prior to BRAF/MEK-targeted therapy and when the disease progressed on therapy, we observed a significant increase of tumor cellularity in the progressed tumors and the close association of SOX10+ melanoma cells with CD8+ T cells negatively correlated with patient's progression-free survival (PFS). In the TCGA-melanoma dataset (n = 445), tumor cellularity exhibited additive prognostic value in the immune score signature to predict overall survival in patients with early-stage melanoma. Moreover, tumor cellularity prognoses OS independent of immune score in patients with late-stage melanoma.

Immunofluorescent staining of cancer spheroids and fine-needle aspiration-derived organoids.

Our organoid generation technique has allowed for the development of downstream organoid applications. Here, we detail an accessible, straightforward protocol for immunofluorescent staining and imaging of thyroid cancer organoids, particularly those with tumor de-differentiation. Immunofluorescence is a powerful tool to help understand the localization of cell types within organoids and determine the interactions between those cells. As organoids have been shown to recapitulate patient tumor morphology, immunofluorescent staining and imaging of organoids allows for enhanced understanding of near in vivo structures. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Salivary Gland NUT Carcinoma with Prolonged Survival in Children: Case Illustration and Systematic Review of Literature.

NUT (midline) carcinoma is a rare, highly aggressive, poorly differentiated carcinoma that characteristically harbors a rearrangement of the NUTM1 gene. Most of these tumors occur in adolescents and young adults, arise from the midline structures of the thorax, head, and neck, and are associated with extremely poor outcomes. Rare cases originating from salivary glands have been reported with clinicopathologic features comparable to NUT carcinoma of other sites. Outcome studies regarding this subgroup are currently lacking. We report a case of NUT carcinoma arising in a submandibular gland of a 12-year-old boy. Diagnosis was confirmed by fluorescence in situ hybridization demonstrating fusion of the BRD4 (19p13.12) and NUTM1 (15q14) gene loci. A systematic review of all previously reported salivary gland NUT carcinomas (n = 15) showed exclusive occurrence of pediatric cases (n = 6) in males compared to adult patients (n = 9, male: female = 1:2; p < 0.05). The median survival was 24 and 4 months for pediatric and adult patients, respectively (95% confidence interval was 8-24 and 1-7 months, respectively; p < 0.01). The 1-year overall survival was 67% for pediatric and 11% for adult patients. Among all NUT carcinomas, pediatric salivary gland tumors may represent a distinct clinical subset associated with male predilection and comparatively prolonged survival.

Instrument Gauge and Type in Uveal Melanoma Fine Needle Biopsy: Implications for Diagnostic Yield and Molecular Prognostication.

PURPOSE: To systematically evaluate and compare the effects of using small-gauge needles and vitrectors on the ability to obtain adequate diagnostic and prognostic uveal melanoma biopsy specimens. DESIGN: Comparative evaluation of biopsy instruments. METHODS: Survival of uveal melanoma cells was evaluated in vitro following needle aspiration. Five therapeutically enucleated eyes were sampled in triplicate for ex vivo diagnostic biopsy experiments with 25 gauge (25 G) needle, 27 gauge (27 G) needle, and 27 G vitrector. During surgery in 8 patients, paired diagnostic transscleral fine needle aspiration biopsies were performed using both 25 G and 27 G needles. A review of cytologic specimens was performed by a panel of 3 expert cytopathologists. A retrospective chart review was performed to evaluate 100 consecutive tumors undergoing prognostic biopsy for gene expression profiling to assess the relationship between needle gauge and prognostic adequacy. RESULTS: No significant cell shearing of uveal melanoma cells occurred in vitro with 25 G, 27 G, or 30 G needles. For ex vivo biopsy samples, diagnostic yield was 100% using 25 G needle (5/5) or 27 G vitrector (5/5) but 60% using a 27 G needle (3/5). For in vivo samples, no difference in diagnostic yield was found between 25 G (75%, 6/8) or 27 G (75%, 6/8) needle sizes. Of 100 molecular prognostic biopsy samples evaluated, 65 were obtained using 27 G needles; for these biopsies, the prognostic yield was 65/65 (100%). CONCLUSIONS: For diagnostic biopsy of uveal melanoma, a larger-gauge needle or a 27 G vitrector may have better overall cellularity and diagnostic yield when compared to a 27 G needle. However, for much more common molecular prognostic testing, a 27 G needle provided adequate sample in 100% (65/65) of cases, and a larger needle provided no additional benefit.

Targeted education as a method for reinforcing Paris System criteria and reducing urine cytology atypia rates.

INTRODUCTION: The Paris System for Urine Cytology (TPS) provides well-defined diagnostic criteria for the category of atypical urothelial cells (AUC). The current study compares the rate of AUC diagnoses at a large academic medical center before and after an educational intervention (EI) by a urine cytology expert. MATERIALS AND METHODS: An expert in TPS delivered an educational intervention consisting of an interactive microscope session and a didactic session that focused on the AUC diagnostic category. The number of urine cytology cases, the AUC rate, and the false-negative percentage were calculated before and after the EI, using the electronic medical records and cytologic-histologic correlation records. RESULTS: A total of 4026 urine cytology cases were signed out in the 25 months prior to the educational intervention and 1585 cases were signed out in the 10 months after the intervention. EI had a significant impact on diagnostic categorization, including a reduction in AUC (19.6% versus 12.5%) and suspicious for high-grade urothelial carcinoma (3.9% versus 3.1%) diagnoses. The cytotechnologists also placed fewer cases into the AUC category during primary screening (27.6% versus 23.0%). Although a higher percentage of cases was reported as negative for high-grade urothelial carcinoma, the false-negative rate did not significantly change after the intervention (1.8% versus 2.0% of negative cases, P = 0.65). CONCLUSIONS: Focused educational sessions for pathologists and cytotechnologists on the diagnostic criteria for AUC as defined by TPS can significantly reduce the rate of atypical diagnoses without a significant increase in the rate of false negatives.