Image analysis reveals differences in tumor multinucleations in Black and White patients with human papillomavirus-associated oropharyngeal squamous cell carcinoma.

BACKGROUND: Understanding biological differences between different racial groups of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) patients, who have differences in terms of incidence, survival, and tumor morphology, can facilitate accurate prognostic biomarkers, which can help develop personalized treatment strategies. METHODS: This study evaluated whether there were morphologic differences between HPV-associated tumors from Black and White patients in terms of multinucleation index (MuNI), an image analysis-derived metric that measures density of multinucleated tumor cells within epithelial regions on hematoxylin-eosin images and previously has been prognostic in HPV-associated OPSCC patients. In this study, the authors specifically evaluated whether the same MuNI cutoff that was prognostic of overall survival (OS) and disease-free survival in their previous study, TTR , is valid for Black and White patients, separately. We also evaluated population-specific cutoffs, TB for Blacks and TW for Whites, for risk stratification. RESULTS: MuNI was statistically significantly different between Black (mean, 3.88e-4; median, 3.67e-04) and White patients (mean, 3.36e-04; median, 2.99e-04), with p = .0078. Using TTR , MuNI was prognostic of OS in the entire population with hazard ratio (HR) of 1.71 (p = .002; 95% confidence interval [CI], 1.21-2.43) and in White patients with HR of 1.72 (p = .005; 95% CI, 1.18-2.51). Population-specific cutoff, TW , yielded improved HR of 1.77 (p = .003; 95% CI, 1.21-2.58) for White patients, whereas TB did not improve risk-stratification in Black patients with HR of 0.6 (p = .3; HR, 0.6; 95% CI, 0.2-1.80). CONCLUSIONS: Histological difference between White and Black patient tumors in terms of multinucleated tumor cells suggests the need for considering population-specific prognostic biomarkers for personalized risk stratification strategies for HPV-associated OPSCC patients.

Neuroendocrine carcinomas of the head and neck: A small case series.

INTRODUCTION: Neuroendocrine tumors of the head and neck are rare and arise either from epithelial or neuronal origin. Debate continues over the classification systems and appropriate management of these pathologies. OBJECTIVE: By investigating a small set of cases of high grade epithelial-derived neuroendocrine tumors of the head and neck (neuroendocrine carcinomas or NEC) from one institution, we compare survival rates of NEC of the head and neck to pulmonary NEC. METHODS: We identified patients from pathology records with neuroendocrine carcinomas of the head and neck and retrospectively collected clinical data as well as immunohistochemical (IHC) staining data. RESULTS: We identified 14 patients with NEC, arising from the parotid (n = 5), nasal cavity (n = 4), larynx (n = 2), and other regions (n = 2). One additional patient had NEC arising in two sites simultaneously (parotid and nasal). Staining patterns using IHC were relatively consistent across specimens, showing reactivity to chromogranin and synaptophysin in 73% and 100% of specimens, respectively. Treatment courses varied across patients and included combinations of surgery, chemotherapy, and/or radiation. The overall survival rate at 1, 2, and 5 years of these patients was 56%, 56%, and 43% with a mean follow-up time of 2.12 years. CONCLUSION: Compared to NEC arising in the lung, this subset of patients had better survival rates, but worse survival rates than the more common squamous cell carcinoma of the head and neck.

Poorly differentiated thyroid carcinoma of childhood and adolescence: a distinct entity characterized by DICER1 mutations.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

Non-necrotizing granulomatous pneumonitis and chronic pleuritis in soldiers deployed to Southwest Asia.

AIMS: Reports of respiratory illnesses among soldiers returning from Southwest Asia have been described. During deployment to Southwest Asia, soldiers are exposed to various respiratory hazards, including dust storms, smoke from burn pits and industrial air pollutants. A few studies have reported increased rates of constrictive bronchiolitis and asthma in these patients. We sought to expand upon the pathological findings in this cohort. METHODS AND RESULTS: Lung biopsies from veterans of Southwest Asia were identified and re-reviewed. All patients had undergone pulmonary function tests and chest high-resolution CT imaging with no significant findings. Overall, 59 patients with a history of inhalational exposure to at least one of the following were identified: smoke from burn pit, dust storm and sulphur plant fire. Samples included video-assisted thoracoscopic lung biopsies (57 of 59, 96.6%) and cryobiopsies (two of 59, 3.4%). Patients were predominantly male (54 of 59, 91.5%) with an age range of 24-55 years (mean and median = 35). Non-necrotising, poorly formed granulomas were identified in 22 cases (22 of 59, 37.2%). The granulomas were mainly bronchiolocentric and were associated with chronic lymphoplasmacytic bronchiolitis, similar to hypersensitivity pneumonitis (HP). Pleural reaction in the form of focal chronic lymphocytic pleuritis and/or focal pleural adhesions were seen in 43 of 57 (75.4%) biopsies. CONCLUSIONS: To our knowledge, this is the first study to report pleural reaction as well as features of HP in this population, suggesting that pleural reaction and HP may be part of the spectrum of Southwest Asia deployment-related lung diseases.

KRAS mutation is predictive of outcome in patients with pulmonary sarcomatoid carcinoma.

AIMS: Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated non-small-cell lung carcinoma (NSCLC) with aggressive behaviour. This study aimed to evaluate the prognostic clinicopathological and genetic characteristics of PSCs. METHODS AND RESULTS: Fifty-three cases of surgically treated PSCs were selected, 23 of which were subjected to mutation and copy number variation analysis using the 50-gene Ion AmpliSeq Cancer Panel. The majority of the patients were male (32 of 53, 60.3%) and smokers (51 of 53, 96.2%). Overall, 25 (47.1%) patients died within 2-105 months (mean = 22.7 months, median = 15 months) after diagnosis, and 28 were alive 3-141 months (mean = 38.7 months, median = 21.5 months) after diagnosis. Five-year overall survival was 12.5%. KRAS codon 12/13 mutation in adenocarcinomas (P = 0.01), age more than 70 years (P = 0.008) and tumour size ≥4.0 cm (P = 0.02) were associated strongly with worse outcome. TP53 (17 of 23, 74.0%) and KRAS codon 12 of 13 mutations (10 of 23, 43.4%) were the most common genetic alterations. Potentially actionable variants were identified including ATM (four of 23, 17.3%), MET, FBXW7 and EGFR (two of 23, 8.7%), AKT1, KIT, PDGFRA, HRAS, JAK3 and SMAD4 (one of 23, 4.3%). MET exon 14 skipping and missense mutations were identified in two (11.1%) cases with adenocarcinoma histology. Copy number analysis showed loss of RB1 (three of 23, 13%) and ATM (two of 23, 8.7%). Copy number gains were seen in EGFR (two of 23, 13.0%) and in one (4.3%) of each PIK3CA, KRAS, MET and STK11. CONCLUSIONS: Potentially targetable mutations can be identified in a subset of PSC, although most tumours harbour currently untargetable prognostically adverse TP53 and KRAS mutations.

Recent Advances in the Diagnosis of Malignant Mesothelioma: Focus on Approach in Challenging Cases and in Limited Tissue and Cytologic Samples.

Mesothelial proliferations can be diagnostically challenging in small specimens, such as body fluid cytology and small tissue biopsies. A great morphologic challenge for pathologists is the separation of benign reactive mesothelial proliferations from malignant mesotheliomas. Reactive mesothelial proliferations may have histologic features that resemble malignancy including increased cellularity, cytologic atypia, and mitoses. Recent advances in mesothelioma genetics resulted in identification of BAP1 mutations and p16 deletions as features of malignant mesotheliomas. Hence, BAP1 immunohistochemistry and fluorescence in situ hybridization for p16 emerged as 2 most common diagnostically helpful ancillary studies used on limited samples when the question is whether the proliferation is malignant or benign. In contrast, separation of mesothelioma from other malignancies is relatively straight forward using morphology and immunohistochemical stains. The choice of antibody panel to be applied in an individual case is driven by morphology, either epithelioid or sarcomatoid. This brief review will focus on morphology and ancillary testing of mainly pleural mesothelial proliferations.

Assessing the feasibility of a multimodal liquid biopsy for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma.

OBJECTIVES: In this feasibility study, we explored the combined use of circulating tumor human papillomavirus (HPV) DNA (ctHPVDNA) and HPV serology as diagnostic tests for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). METHODS: Among patients with research-banked serum or plasma at diagnosis, IgG antibodies to oncoproteins from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 were detected with multiplex serology. Positivity for HPV 16 was defined based on detection of combinations of anti-E6, E1, E2, and E7 and for other high-risk types on detection of anti-E6 and anti-E7. Circulating tumor HPV DNA was detected by custom digital droplet polymerase chain reaction (ddPCR) assays for HPV types 16, 18, 33, 35, and 45. p16 immunohistochemistry and high-risk HPV RNA in situ hybridization (ISH) using a cocktail of 18 high-risk HPV types were performed on tissue. RESULTS: Of 75 patients, 67 (89.3%) were HPV-associated (p16 and HPV RNA ISH positive) and 8 (10.7%) were HPV-independent. All 8 HPV-independent patients were seronegative and negative for ctHPVDNA (100% specificity). Serology was positive in 53 (79.1%) of 67 HPV-associated patients, while ddPCR was positive for ctHPVDNA in 59 (88.6%) of 67 HPV-associated patients. Requiring both tests to be positive resulted in a sensitivity of 50 (74.6%) of 67 while combining assays (either positive) improved sensitivity to 62 (92.6%) of 67. CONCLUSIONS: Compared to HPV RNA ISH, HPV serology and ctHPVDNA are sensitive and highly specific biomarkers for HPV-associated OPSCC at the time of presentation.

Virtual 3D Specimen Mapping in Head & Neck Oncologic Surgery.

OBJECTIVES: Virtual 3D specimen mapping of oncologic surgical specimens provides a visual record of the specimen and margin sampling sites which can be utilized in a variety of cancer care settings. Our objective was to perform a retrospective review of head and neck surgical oncology cases where the specimen was mapped post-operatively and to evaluate the utility of these 3D specimen maps amongst the multidisciplinary cancer care team. METHODS: A retrospective review of our 3D specimen model biorepository was performed. Surgical specimens were 3D scanned and then graphically annotated (or "mapped") during routine pathologic processing. The resulting 3D specimen maps were distributed to the multidisciplinary oncologic care team. Final margin status and any use of the 3D specimen maps were recorded. RESULTS: A total of 28 cases were included. Virtual 3D specimen maps were utilized by the cancer care team in 8 cases (29%), including 2 positive margin cases, 2 close margin cases, and 4 indeterminate margin cases. 3D specimen maps were used to visualize positive margin sites for pathologist-surgeon communication as a visual reference during tumor board discussions and to inform radiation treatment planning. CONCLUSION: Post-operative virtual 3D specimen mapping of oncologic specimens creates a permanent visual record of the specimen and the margins sampled and may serve as a beneficial tool for communication amongst the multidisciplinary cancer care team. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:191-197, 2024.

Patterns of Major Frozen Section Interpretation Error: An In-Depth Analysis From a Complex Academic Surgical Pathology Practice.

OBJECTIVES: To establish baseline error rates due to misinterpretation and to identify scenarios in which major errors were most common and potentially preventable. METHODS: Our database was queried over a 3-year period for major discrepancies due to misinterpretation. These were stratified by histomorphologic setting, service, availability/type of prior material, and years of experience and subspecialization of the interpreting pathologist. RESULTS: The overall discordance rate between frozen section (FS) and final diagnoses was 2.9% (199/6,910). Seventy-two errors were due to interpretation, of which 34 (47.2%) were major. Major error rates were highest on the gastrointestinal and thoracic services. Of major discrepancies, 82.4% were rendered in subdisciplines outside those of the FS pathologist. Pathologists with fewer than 10 years' experience made more errors than those with more experience (55.9% vs 23.5%, P = .006). Major error rates were greater for cases without previous material compared to those with a prior glass slide (47.1% vs 17.6%, P = .009). Common histomorphologic scenarios in which disagreements were made involved discriminating mesothelial cells from carcinoma (20.6%) and accurately recognizing squamous carcinoma/severe dysplasia (17.6%). CONCLUSIONS: To improve performance and decrease future misdiagnoses, monitoring discordances should be a continuous component of surgical pathology quality assurance programs.

The computer-aided design margin: Ex vivo 3D specimen mapping to improve communication between surgeons and pathologists.

BACKGROUND: Numerous challenges exist in determining surgical margin status. Communication between surgeons and pathologists is crucial for specimen orientation and accurate margin assessment. METHODS: A prospective study to determine feasibility of incorporating three-dimensional (3D) scanning into surgical pathology workflow was performed. A structured-light 3D scanner captured the photorealistic surface topography of fresh surgical specimens. Computer-aided design (CAD) software was used to document sites of margin sampling and sectioning. Surveys were distributed among faculty and staff stakeholders to assess feasibility. RESULTS: A series of 40 cases were 3D-scanned. Median image acquisition time was 8 min. The majority of respondents agreed that the experimental 3D system helped achieve clearer communication. 3D specimen maps assisted in the communication of a focally positive or close margin in 4 of 17 cases. CONCLUSIONS: Routine 3D scanning and specimen mapping is feasible and represents an innovative approach to intraoperative and final pathology documentation, margin analysis, and surgeon-pathologist communication.

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.

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.

Sinonasal Bacteroma/Bacteria-related Concretions: A Distinct Pathologic Entity of the Sinonasal Tract.

Patients can be seen where "fungal debris," "mycetoma," or "mass-like obstruction" of the sinonasal tract is suspected clinically but lack fungus and instead have granular, eosinophilic debris and bacterial colonies. We report and characterize 15 such cases, tentatively termed "bacteromas," compared with randomly selected cases of mycetoma and allergic fungal sinusitis (AFS). Pathology reports from 2016 to 2021 were searched. All candidate cases were examined microscopically and included if they had granular, amorphous debris with negative Grocott methenamine silver staining and lacked diagnostic features of other entities. The 7 males and 8 females ranged from 21 to 78 years old. Imaging frequently revealed opacification of the paranasal sinuses. Operative reports showed all to have paranasal sinus involvement. Most were unilateral (13/15, 87%). The maxillary sinus was involved in 11/15 (73%) cases, sphenoid sinus in 2/15 (13%), and frontal and ethmoid sinuses in 1/15 (7%), each. Bacteroma patients frequently had a history of allergic rhinitis (8/15, 53%), more than mycetomas (1/15, 7%) and AFS (5/15, 33%) ( P =0.0142). Facial pain was a common presenting symptom (13/15, 87%) in bacteromas compared with mycetomas (5/15, 33%) or AFS (1/15, 7%). Morphologically, cases consisted of large aggregates of paucicellular to acellular debris with a characteristic densely eosinophilic granular appearance, commonly associated with bacteria. Four of the 10 cultured patients grew Pseudomonas aeruginosa . Course posttreatment ranged from symptom resolution 1 week postoperatively to recurrent infections and symptoms 23 months from the initial operation. In summary, "bacteroma" is a heretofore undescribed pathologic entity of the sinuses that appears to be related to chronic bacterial infection and is distinct from mycetoma, AFS, and rhinolithiasis.

CD163 Immunohistochemical "Circle Sign" Staining Pattern Differentiates Sinonasal Papillomas From Morphologically Similar Non-neoplastic Lesions.

Sinonasal papillomas are a diverse group of benign epithelial neoplasms of the sinonasal tract. Inverted papilloma, in particular, must be distinguished from other lesions with no malignant potential. The aim of this study was to distinguish sinonasal papillomas from morphologically similar lesions using CD163 immunostaining. Cases from a 19-year period were identified. These included 49 inverted, 10 exophytic, and 12 oncocytic papillomas, 21 chronic sinusitides with squamous metaplasia, 27 inflammatory polyps, 5 verrucae vulgares, 5 respiratory epithelial adenomatoid hamartomas, and 6 DEK::AFF2 carcinomas of the sinonasal tract. A subset of biopsy cases (8 inverted papillomas, 5 inflammatory polyps) was separately analyzed. CD163 immunohistochemistry (IHC) was performed. A unique "circle" staining pattern was identified in the surface epithelium. After locating a hotspot, circles were quantified in 10 consecutive high-power fields. Circles were present in 66/71 (93%) cases of sinonasal papilloma, with a mean of 35 circles/10 HPF (range: 0 to 160/10 HPF) and a median of 19 circles/10 HPF. Circles were present in 20/58 (34%) non-neoplastic cases, with a mean of 2 circles/10 HPF (range: 0 to 27/10 HPF) and a median of 0. Considering all resection and biopsy cases, performance for distinguishing papillomas from non-neoplastic lesions was best at a cutoff of 10 circles/10 HPF (2-tailed P <0.0001) with sensitivity, specificity, positive predictive value, and negative predictive value of 66.2%, 93.1%, 92.1%, and 69.2%, respectively. The results were similar in the biopsy subset. One other neoplastic entity, the DEK::AFF2 carcinomas, also showed prominent CD163 circle staining. In summary, sinonasal papillomas demonstrate extensive CD163 "circle" staining in the epithelium compared with the non-neoplastic lesions studied. As such, the "circle sign" on CD163 IHC may be helpful in distinguishing between diagnoses, particularly on small biopsies or equivocal specimens.

Human Papillomavirus-Associated Oral Cavity Squamous Cell Carcinoma: An Entity with Distinct Morphologic and Clinical Features.

BACKGROUND: HPV-associated oral cavity squamous cell carcinoma (SCC) is not well-characterized in the literature, and also has a clinical significance that is poorly understood. METHODS: We gathered a cohort of oral cavity (OC) SCC with nonkeratinizing morphology, either in the invasive or in situ carcinoma (or both), tested for p16 by immunohistochemistry and high risk HPV E6/E7 mRNA by RTPCR (reference standard for transcriptionally-active high risk HPV) and gathered detailed morphologic and clinicopathologic data. RESULTS: Thirteen patients from two institutions were proven to be HPV-associated by combined p16 and high risk HPV mRNA positivity. All 13 patients (100%) were males, all were heavy smokers (average 57 pack/year), and most were active drinkers (9/11 or 81.8%). All 13 (100%) involved the tongue and/or floor of mouth. All had nonkeratinizing features, but maturing squamous differentiation varied widely (0-90%; mean 37.3%). Nonkeratinizing areas had high N:C ratios and larger nests, frequently with pushing borders, and minimal (or no) stromal desmoplasia. The carcinoma in situ, when present, was Bowenoid/nonkeratinizing with cells with high N:C ratios, full thickness loss of maturation, and abundant apoptosis and mitosis. HPV was type 16 in 11 patients (84.6%) and type 33 in two (15.4%). Nine patients had treatment data available. These underwent primary surgical resection with tumors ranging from 1.6 to 5.2 cm. Most had bone invasion (6/9-66.7% were T4a tumors), and most (6/9-66.7%) had extensive SCC in situ with all 6 of these patients having final margins positive for in situ carcinoma. CONCLUSIONS: HPV-associated OCSCC is an uncommon entity that shows certain distinct clinical and pathologic features. Recognition of these features may help pathologic diagnosis and could potentially help guide clinical management.

A Quality Analysis of Bony Specimens for Optimal Ethylenediaminetetraacetic Acid (EDTA) Decalcification.

Introduction: Weak acids, such as etheylenediaminetetraacetic acid (EDTA), chelate calcium ions from the surface of tissues, decalcifying slowly but preserving molecular structures for ancillary testing. There is a need for optimization of EDTA protocols for specimen decalcification. We studied the effects of EDTA on different types of bony specimens for quality assurance. Methods: Specimens included: proximal femur curettage (0.7 g), fibula shave (1 mm thick, 0.5 g), tibia shave (2 mm thick, 0.9 g), and femur 11-gauge core biopsies. Curettage and shave specimens were placed in formalin then EDTA (Newcomersupply, Middleton, WI, USA) with continuous stirring. Specimens were removed at 24, 48, 72, 96, and 120 hours. Core biopsies were processed in EDTA with heat at 4, 8, 12, and 16 hours. X-ray imaging (Kubtec Xpert 40, Stratford, CT, USA) was obtained. Histological processing was attempted and H&E slides compared to radiologic imaging. Results: Fibula and tibia sections showed appropriate radiolucency at 120 hours and 7 days, respectively. Curettage specimens showed radiolucency of medullary bone at 48 hours. Curettage and fibula sections showed quality histology at 96 hours and 120 hours, respectively. Quality tibia sections were obtained at 7 days, requiring one hour of additional block surface decalcification with 1% HCL solution. Medullary and cortical core biopsies showed quality histology at 12 and 16 hours with heat, respectively. Conclusion: Imaging can determine appropriate decalcification of bony specimens. Medullary bone undergoes more rapid decalcification in EDTA than cortical bone. EDTA decalcification is best used for curettage and core biopsy specimens.

A multidisciplinary approach to the work up and management of pulmonary carcinoid tumors and DIPNECH: a narrative review.

Background and Objective: Low and intermediate grade neuroendocrine tumors of the lung are uncommon malignancies representing 2% of all lung cancers. These are termed typical and atypical pulmonary carcinoid tumors. These can arise in the setting of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH). The presentation, workup, management and outcomes of patients with these tumors can overlap with more common lung cancers but differ in that many of these patients have a prolonged clinical course. The objective of this narrative review is to summarize the literature and provide evidence and expert-based algorithms for work up and treatment of pulmonary carcinoids and DIPNECH. Methods: A search of PubMed and Web of Science databases ending April 15, 2022, with the following keywords "lung carcinoid", "DIPNECH", "lung neuroendocrine," and "bronchopulmonary carcinoid". Key Content and Findings: Pulmonary carcinoid tumors benefit from a multidisciplinary approach. Pre-treatment imaging with contrast-enhanced computed tomography, and DOTATATE positron emission tomography is required. Surgical resection is the gold standard for curative intent, and possibly including sublobar resections. Patients can recur or develop new primaries thus emphasizing the importance of surveillance; national guidelines recommend at least a 10-year follow up. A growing body of literature support the use of endobronchial therapy, with long responses documented. Systemic therapy consists of everolimus, somatostatin analogs, peptide receptor radionuclide therapy, and chemotherapy. Diffuse idiopathic pulmonary neuroendocrine tumor cell hyperplasia is rare, but series suggest somatostatin analogs may confer clinical benefit. Conclusions: Pulmonary carcinoid tumors and DIPNECH are rare. Despite lack of regulatory approvals for advanced disease, multiple options are available but should be sequenced according to the clinical status and disease biology. Each patient should be discussed in a multidisciplinary setting and clinical trials should be considered if available.

The Association between Exposures and Disease Characteristics in Familial Pulmonary Fibrosis.

Rationale: Heterogeneous characteristics are observed in familial pulmonary fibrosis (FPF), suggesting that nongenetic factors contribute to disease manifestations. Objectives: To determine the relationship between environmental exposures and disease characteristics of FPF, including the morphological characteristics on chest computed tomography (CT) scan, and timing of FPF symptom onset, lung transplantation, or death. Methods: Subjects with FPF with an exposure questionnaire and chest CT were selected from a prospective cohort at Vanderbilt. Disease characteristics were defined by lung parenchymal findings on chest CT associated with fibrotic hypersensitivity pneumonitis (fHP) or usual interstitial pneumonia (UIP) and by time from birth to symptom onset or a composite of lung transplantation or death. After assessing the potential for confounding by sex or smoking, adjusted logistic or Cox proportional hazards regression models identified exposures associated with fHP or UIP CT findings. Findings were validated in a cohort of patients with sporadic pulmonary fibrosis enrolled in the LTRC (Lung Tissue Research Consortium) study. Results: Among 159 subjects with FPF, 98 (61.6%) were males and 96 (60.4%) were ever-smokers. Males were less likely to have CT features of fHP, including mosaic attenuation (FPF: adjusted [for sex and smoking] odds ratio [aOR], 0.27; 95% confidence interval [CI], 0.09-0.76; P = 0.01; LTRC: aOR, 0.35; 95% CI, 0.21-0.61; P = 0.0002). Organic exposures, however, were not consistently associated with fHP features in either cohort. Smoking was a risk factor for honeycombing in both cohorts (FPF: aOR, 2.19; 95% CI, 1.12-4.28; P = 0.02; LTRC: aOR, 1.69; 95% CI, 1.22-2.33; P = 0.002). Rock dust exposure may also be associated with honeycombing, although the association was not statistically-significant when accounting for sex and smoking (FPF: aOR, 2.27; 95% CI, 0.997-5.15; P = 0.051; LTRC: aOR, 1.51; 95% CI, 0.97-2.33; P = 0.07). In the FPF cohort, ever-smokers experienced a shorter transplant-free survival (adjusted hazard ratio, 1.64; 95% CI, 1.07-2.52; P = 0.02), whereas sex was not associated with differential survival (male adjusted hazard ratio, 0.75; 95% CI, 0.50-1.14; P = 0.18). Conclusions: In FPF, smoking contributes to shortened transplant-free survival and development of honeycombing, a finding that is also likely applicable to sporadic pulmonary fibrosis. Females are more likely to manifest CT features of fHP (mosaic attenuation), a finding that was incompletely explained by sex differences in exposures. These findings may have implications for pulmonary fibrosis classification and management.

An Imaging Biomarker of Tumor-Infiltrating Lymphocytes to Risk-Stratify Patients With HPV-Associated Oropharyngeal Cancer.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has excellent control rates compared to nonvirally associated OPSCC. Multiple trials are actively testing whether de-escalation of treatment intensity for these patients can maintain oncologic equipoise while reducing treatment-related toxicity. We have developed OP-TIL, a biomarker that characterizes the spatial interplay between tumor-infiltrating lymphocytes (TILs) and surrounding cells in histology images. Herein, we sought to test whether OP-TIL can segregate stage I HPV-associated OPSCC patients into low-risk and high-risk groups and aid in patient selection for de-escalation clinical trials. METHODS: Association between OP-TIL and patient outcome was explored on whole slide hematoxylin and eosin images from 439 stage I HPV-associated OPSCC patients across 6 institutional cohorts. One institutional cohort (n = 94) was used to identify the most prognostic features and train a Cox regression model to predict risk of recurrence and death. Survival analysis was used to validate the algorithm as a biomarker of recurrence or death in the remaining 5 cohorts (n = 345). All statistical tests were 2-sided. RESULTS: OP-TIL separated stage I HPV-associated OPSCC patients with 30 or less pack-year smoking history into low-risk (2-year disease-free survival [DFS] = 94.2%; 5-year DFS = 88.4%) and high-risk (2-year DFS = 82.5%; 5-year DFS = 74.2%) groups (hazard ratio = 2.56, 95% confidence interval = 1.52 to 4.32; P < .001), even after adjusting for age, smoking status, T and N classification, and treatment modality on multivariate analysis for DFS (hazard ratio = 2.27, 95% confidence interval = 1.32 to 3.94; P = .003). CONCLUSIONS: OP-TIL can identify stage I HPV-associated OPSCC patients likely to be poor candidates for treatment de-escalation. Following validation on previously completed multi-institutional clinical trials, OP-TIL has the potential to be a biomarker, beyond clinical stage and HPV status, that can be used clinically to optimize patient selection for de-escalation.

The impact of tissue block sampling on the detection of p53 signatures in fallopian tubes from women with BRCA 1 or 2 mutations (BRCA+) and controls.

The tubal p53 signature is a putative precursor to pelvic serous carcinoma, but its frequencies in women with inherited mutations in the BRCA1 or BRCA2 genes (BRCA+) and controls has been controversial. An initial section and two levels (100-200 µm) from every block in BRCA+ (24) and control tubes (40) were stained for p53. The frequency of p53 signatures was computed between the populations and across the three levels from each block, and analyzed by Fisher exact test. A total of 17 (71%) BRCA+ and 20 (50%) control tubes were p53 signature positive (P=0.12); 21 and 16% of all tissue blocks sectioned harbored signatures (P=0.29), and 76 and 67% were found in the fimbria. In 49 and 32% of p53 signature positive cases in the two groups, the p53 signatures were not discovered until the second or third round of sectioning. In all, 38 and 40% of BRCA+ and control subjects harbored p53 signatures in more than one focus in a single block. In one case (BRCA+), a highly atypical proliferation was identified in one serial section. The p53 signatures are more common than previously reported and the frequency of detection increases as a function of sectioning through the tissue block, both in absolute frequency and in numbers of p53 signatures detected in a given block. There is a trend for a higher absolute frequency of p53 signatures (71 vs 50%; P=0.12) in BRCA+ subjects, but this is not reflected in a greater average number of p53 signatures or positive blocks per case. This study underscores the importance of systematic immunohistochemical examination of fallopian tubes when conducting epidemiological studies that compare the frequency of p53 signatures in different populations. Attention to this detail is critical when exploring risk factors germane to early serous carcinogenesis.