A fragment based competitive 19F LB-NMR platform for hotspot directed ligand profiling.

Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment-based competitive 19F Ligand Based-NMR (LB-NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand-binding hotspots. As a proof of concept, the method was applied to 4'-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X-ray crystallographic characterisation of the hits from a 960-member fragment screen identified three ligand-binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19F LB-NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X-ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment-based competitive 19F LB-NMR screening platform for hotspot-directed ligand profiling.

Granulomatous Lung Diseases: A Practical Approach and Review of Common Entities.

Granulomas are frequently encountered by pathologists in all types of lung specimens and arise from diverse etiologies. They should always be reported as necrotizing or non-necrotizing, with microorganism stains performed to evaluate for infection. With attention to distribution, quality (poorly vs well-formed), associated features, and correlation with clinical, radiologic, and laboratory data, the differential diagnosis for granulomatous lung disease can usually be narrowed to a clinically helpful "short list." This review describes a practical approach to pulmonary granulomas and reviews the clinicopathological aspects of common entities, including infectious (mycobacteria, fungi) and noninfectious (hypersensitivity pneumonitis, sarcoid, and vasculitis) causes.

Prognostic Immunohistochemistry for Ki-67 and OTP on Small Biopsies of Pulmonary Carcinoid Tumors: Ki-67 Index Predicts Progression-free Survival and Atypical Histology.

Prognostic stratification of pulmonary carcinoids into "typical" and "atypical" categories requires examination of large tissue volume. However, there is a need for tools that provide similar prognostic information on small biopsy samples. Ki-67 and OTP immunohistochemistry have shown promising prognostic value in studies of resected pulmonary carcinoids, but prognostic value when using biopsy/cytology specimens is unclear. Ki-67 immunohistochemistry was performed on small biopsy/cytology specimens from pulmonary carcinoid tumors (n=139), and labeling index was scored via automated image analysis of at least 500 cells. OTP immunohistochemistry was performed on 70 cases with sufficient tissue and scored as positive or negative (<20% tumor nuclei staining). Higher Ki-67 index was associated with worse disease-specific progression-free survival (ds-PFS), with 3% and 4% thresholds having similarly strong associations with ds-PFS ( P <0.001, hazard ratio ≥11). Three-year ds-PFS was 98% for patients with Ki-67 <3% and 89% for patients with Ki-67≥3% ( P =0.0006). The optimal Ki-67 threshold for prediction of typical versus atypical carcinoid histology on subsequent resection was 3.21 (AUC 0.68). Negative OTP staining approached significance with atypical carcinoid histology ( P =0.06) but not with ds-PFS ( P =0.24, hazard ratio=3.45), although sample size was limited. We propose that Ki-67 immunohistochemistry may contribute to risk stratification for carcinoid tumor patients based on small biopsy samples. Identification of a 3% hot-spot Ki-67 threshold as optimal for prediction of ds-PFS is notable as a 3% Ki-67 threshold is currently used for gastrointestinal neuroendocrine tumor stratification, allowing consideration of a unified classification system across organ systems.

Pulmonary gangliocytic paraganglioma: An under-recognized mimic of carcinoid tumor.

Gangliocytic paragangliomas are rare neoplasms occurring almost exclusively in the ampullary region of the gastrointestinal tract. Although these tumors are not typically considered in the differential diagnosis of primary pulmonary neoplasia, 5 cases of primary pulmonary gangliocytic paragangliomas have been previously reported. Herein we report our experience with 3 additional examples, all referred to our Anatomic Pathology Consultation service. The patients (a 32-year-old man, a 69-year-old woman and a 55-year-old man) each presented with an endobronchial (2 cases) or upper lobe lung mass, ranging from 1.5 to 2.5 cm in maximum dimension. Biopsy and endobronchial debulking specimens demonstrated the classic triphasic morphology of gangliocytic paraganglioma, with epithelial, spindled and ganglion-like cells. By immunohistochemistry, the tumors were positive for keratin, synaptophysin and chromogranin A in the epithelial component, S100 protein and glial fibrillary acidic protein (GFAP) in the Schwannian spindled cells, and synaptophysin in ganglion cells. TTF1 expression was seen in the epithelial components of 2 cases. The Ki-67 labelling index was low (<2%). Primary pulmonary gangliocytic paragangliomas should be distinguished from carcinoid tumors, given the different natural histories and risk stratification approaches for these morphologically similar tumors. Awareness that gangliocytic paraganglioma may occur in the lung and appropriate immunohistochemical studies are key to correct diagnosis.

Primary Pulmonary Myxoid Sarcoma and Thoracic Angiomatoid Fibrous Histiocytoma: Two Sides of the Same Coin?

Primary pulmonary myxoid sarcoma (PPMS) and thoracic angiomatoid fibrous histiocytoma (AFH) are rare neoplasms with EWSR1 fusions and overlapping morphology. Both tumor types often show epithelial membrane antigen expression, but AFH characteristically co-expresses desmin. We encountered a case of PPMS with the unexpected finding of patchy, strong anaplastic lymphoma kinase (ALK) (previously reported in AFH) and synaptophysin expression. We evaluated a cohort of PPMS and thoracic AFH with systematic morphologic comparison and surveyed for aberrant expression of ALK and synaptophysin. Medical records and slides were reviewed for 16 molecularly confirmed cases of PPMS (n=5) and thoracic AFH (n=11). Each case was scored for morphologic characteristics typical of PPMS and/or AFH. ALK, synaptophysin, chromogranin, desmin, and epithelial membrane antigen immunostains were performed on cases with available tissue. AFH and PPMS cases showed similar age at presentation and long-term tumor behavior. Almost all cases of PPMS and AFH had a fibrous pseudocapsule and lymphoid rim. All PPMS had myxoid stroma and reticular growth pattern, but these features were also present in a subset of AFH. Synaptophysin expression was present in 6 of 11 AFH and 1 of 5 PPMS; all tested cases were negative for chromogranin (n=15). One case of AFH and 1 case of PPMS showed focally strong coexpression of synaptophysin and ALK. AFH and PPMS show considerable clinicopathologic overlap. When supportive, the immunohistochemical findings described may aid in diagnosis before molecular confirmation. PPMS and AFH may be morphologic variants of the same clinicopathologic entity, which can show more immunophenotypic variability than previously reported.

Clinicopathologic and Molecular Characteristics of Resected Thoracic Mass Lesions in the Pediatric Population: A 25-Year Institutional Experience From a Tertiary Care Center.

CONTEXT.­: Primary thoracic neoplasms are rare in children, whereas nonneoplastic mass lesions or cysts and metastases are more common, and there is a relative paucity of comprehensive histopathologic and molecular data. OBJECTIVE.­: To define the clinicopathologic spectrum of neoplastic and nonneoplastic diseases observed in resected mass lesions in the chest of pediatric patients, and to identify somatic alterations observed in primary neoplasms. DESIGN.­: Clinicopathologic features of thoracic mass lesions (n = 385) resected from 373 patients aged ≤21 years in a 25-year period (1993-2018) were included. Primary neoplasms having sufficient material were tested by a laboratory-developed comprehensive genomic profiling assay that assesses tumor mutational burden, microsatellite instability, somatic sequence variants, gene amplifications, fusions, and specific transcript variants. RESULTS.­: The most commonly resected space-occupying lesions were nonneoplastic mass lesions and cysts or malformations, resected in 117 (31.4%) and 58 of 373 patients (15.5%) respectively. Metastatic neoplasms were observed in 169 of 373 patients (45.3%; mean age 14.4 years, range 1-21 years); the most common was osteosarcoma (68 of 169; 40.2% of metastases). Primary lung neoplasms occurred in 24 of 373 patients (6.4%; mean age 14.5 years, range 6 months-21 years), and 16 patients had primary extrapulmonary thoracic tumors. Carcinoid tumor was the most common primary lung neoplasm (7 typical, 3 atypical). Molecular testing showed a prevalence of somatic pathogenic or likely pathogenic mutations and copy-number alterations. No fusions or splice variants were identified. Tumors were microsatellite-stable with low tumor mutational burden. CONCLUSIONS.­: Resected pediatric thoracic mass lesions are more likely to be metastatic lesions, congenital cysts or malformations, or nonneoplastic lesions compared to primary thoracic neoplasms, which are encountered at a low frequency and tend to have relatively simple genetic profiles.

What's new in benign lung tumours?

While lung cancer is one of the most common malignancies routinely encountered by pathologists, benign pulmonary neoplasms are quite rare. However, it is important for pathologists to be familiar with the typical diagnostic features of benign lung tumors to avoid confusing them with malignant morphological mimics. There have also been intriguing discoveries in the genetics of benign pulmonary neoplasms in the past decade. This review will cover several of the most common benign lung tumors, including the diagnostic categories of pulmonary adenomas, bronchial papillomas, and benign mesenchymal tumors, with discussion of the current classification, differential diagnosis, and current knowledge regarding genetic drivers.

Nuclear Protein in Testis (NUT) Carcinoma: A Comprehensive Immunohistochemical Analysis of 57 Cases With Consideration of Interpretation and Pitfall Recognition.

CONTEXT.­: Nuclear protein in testis (NUT) carcinoma is an aggressive carcinoma defined by NUTM1 gene rearrangement. Diagnostic challenges include morphologic overlap with poorly differentiated squamous cell carcinoma, small cell carcinoma, thoracic SMARCA4-deficient undifferentiated tumor, and other small round blue cell tumors. OBJECTIVE.­: To comprehensively study the immunohistochemistry (IHC) features of a large cohort of NUT carcinomas. DESIGN.­: Fifty-seven NUT carcinoma cases were identified from 2012-2022, including 38 thoracic/mediastinal, 13 head and neck/sinonasal, and 6 from other sites. Pathology reports and available slides were reviewed. Comprehensive IHC studies were performed on available cases. RESULTS.­: Keratin stains showed variable positivity and were entirely negative in 15% (8 of 55) of cases. p40 was only positive in 65% (24 of 37) of cases, implying inferior sensitivity when compared to p63 (87% sensitivity, 20 of 23 cases) and other squamous cell markers. Neuroendocrine markers were focally/weakly positive in few cases; however, INSM1 was positive in 54% (7 of 13) of cases, indicating a possible diagnostic pitfall. TTF-1 was mostly negative with focal positivity in 26% (10 of 38) of cases. Occasional CD34 (15%, 3 of 20 cases) and CD99 (21%, 3 of 14 cases) positivity could also cause potential diagnostic confusion. S100, desmin, CD45, and SALL4 were rarely positive. BRG1 and INI1 were retained in all cases. Ki-67 proliferative index was high (median, 60%). PD-L1 was negative in all tested cases. CONCLUSIONS.­: This comprehensive IHC study demonstrates the immunohistochemical spectrum of NUT carcinoma. The findings can help narrow the differential diagnosis and recognize potential pitfalls.

Fatal Infections Differentially Involve Allograft and Native Lungs in Single Lung Transplant Recipients.

CONTEXT.­: Respiratory infections complicate lung transplantation and increase the risk of allograft dysfunction. Allograft lungs may have different susceptibilities to infection than native lungs, potentially leading to different disease severity in lungs of single lung transplant recipients (SLTRs). OBJECTIVE.­: To study whether infections affect allograft and native lungs differently in SLTRs but similarly in double LTRs (DLTRs). DESIGN.­: Using an institutional database of LTRs, medical records were searched, chest computed tomography studies were systematically reviewed, and histopathologic features were recorded per lung lobe and graded semiquantitatively. A multilobar-histopathology score (MLHS) including histopathologic data from each lung and a bilateral ratio (MLHSratio) comparing histopathologies between both lungs were calculated in SLTRs and compared to DLTRs. RESULTS.­: Six SLTRs died of infection involving the lungs. All allografts showed multifocal histopathologic evidence of infection, but at least 1 lobe of the native lung was uninvolved. In all 5 DLTRs except 1, histopathologic evidence of infection was seen in all lung lobes. On computed tomography, multifocal ground-glass and/or nodular opacities were found in a bilateral distribution in all DLTRs but in only 2 of 6 SLTRs. In SLTRs, the MLHSAllograft was higher than MLHSNative (P = .02). The MLHSratio values of SLTR and DLTR were significantly different (P < .001). CONCLUSIONS.­: Allograft and native lungs appear to harbor different susceptibilities to infections. The results are important for the management of LTRs.

Two tumors with combined features of bronchiolar adenoma/ciliated muconodular papillary tumor and sclerosing pneumocytoma.

OBJECTIVES: Bronchiolar adenoma/ciliated muconodular papillary tumor (BA/CMPT) and sclerosing pneumocytoma (SP) are both rare and morphologically unique peripheral lung tumors with indolent behavior. These tumors have not been previously described as showing overlapping morphologic features and are generally genetically distinct. METHODS: Two cases were recently encountered that show hybrid morphologic features between BA/CMPT and SP, and the morphology and immunophenotype are described in detail. RESULTS: Both cases showed interstitial round cells typical of SP (TTF1+, EMA+), as well as areas more typical of BA/CMPT. One case showed BRAFV600E expression in the BA/CMPT areas but not in the SP-like cells. CONCLUSIONS: Although it is possible that these cases represent collision tumors or are examples of unusual metaplastic epithelial changes in SP, they also raise the possibility that these 2 entities could occasionally coexist in true hybrid tumors.

The differential impact of COVID-19 across health service psychology students of color: An embedded mixed-methods study.

OBJECTIVES: Communities of color in the United States systematically experience inequities in physical and mental health care compared to individuals who identify as non-Hispanic White. The coronavirus disease 2019 (COVID-19) pandemic exacerbated these structural drivers of inequity to disproportionate and devastating effects for persons of color. In addition to managing the direct effects of COVID-19 risk, persons of color were also navigating increased racial prejudice and discrimination. For mental health professionals and trainees of color, the effects of COVID-19 racial health disparities and the increase in acts of racism may have been compounded by their work responsibilities. The current study used an embedded mixed-methods approach to examine the differential impact of COVID-19 on health service psychology (HSP) students of color as compared to their non-Hispanic White peers. METHOD: Using quantitative and qualitative data from the Epidemic-Pandemic Impacts Inventory, measures of perceived support and of discrimination, and open-ended questions about students' experiences with racism and microaggressions, we examined the extent to which different racial/ethnic HSP student groups experienced COVID-19-related discrimination, the impacts of COVID-19 felt by students of color, and how these experiences differed from those of their non-Hispanic White peers. RESULTS: HSP students of color endorsed greater impacts of the pandemic on both self and others in the home, perceived themselves as less supported by others, and reported more experiences of racial discrimination than non-Hispanic White HSP students. CONCLUSION: Throughout the graduate experience, HSP students of color and their experiences of discrimination need to be addressed. We provided recommendations to HSP training program directors and students both during and after the COVID-19 pandemic.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Allowing Surgical Pathology Fellows to Release Preliminary Reports Increases Learner Independence and Satisfaction.

CONTEXT.­: Progressive independence in medicine is critical to building confidence and decisiveness in trainees. However, this can be difficult to accomplish in the strict regulatory environment of pathology. OBJECTIVE.­: To pilot and adopt a process whereby surgical pathology fellows independently manage a subset of cases and release preliminary reports. DESIGN.­: Upon program approval, board-certified surgical pathology fellows were eligible for preliminary report sign-out at their discretion. Eligible cases were sent from outside institutions for confirmatory review. Preliminary reports were viewable in the electronic medical record. Safety measures were used to ensure timely release of final reports by attending pathologists. RESULTS.­: Fellows participating in the pilot (n = 4) released 59 preliminary reports out of 101 cases reviewed (58%), with 1 potentially significant discrepancy between preliminary and final report. Turnaround time was not affected. The process was endorsed by all participants and adopted as standard practice. During the first year, eligible fellows (n = 8) released 123 preliminary reports out of 1260 cases reviewed (9.8%). There were no major diagnostic discrepancies and no effects on turnaround time. The number of preliminary reports released by each fellow was variable (range, 2-48; median, 8), likely a reflection of both external factors (number of trainees on service, volume) and trainee-specific factors (confidence, efficiency). CONCLUSIONS.­: Fellows showed good judgment when independently managing cases, with just 1 potentially significant discrepancy out of 182 cases (<1%). No patients were adversely impacted. Use of this process varied widely among fellows and may require closer monitoring and encouragement for fellows who are tentative about releasing preliminary reports.

Financial stress and debt in clinical psychology doctoral students.

INTRODUTION: Few studies examine the financial burden of clinical psychology doctoral programs and its impact on achievements, stress, and mental health. OBJECTIVES: The current study sought to better understand students' financial stress and debt, and how financial stress may impact their mental health and the attainment of personal and professional milestones. METHOD: Students (N = 912) completed an online survey assessing demographics, sources of income and expenditures, mental health, and milestones. RESULTS: After accounting for yearly inflation, stipends have not kept pace with the average cost of living in the United States. Over one-third of students indicated that they had no expendable pretax income after paying for their education and typical living expenses. Additionally, over 80% reported acquiring additional debt in graduate school to offset their living expenses. Financial concerns were associated with delays in major life milestones (e.g., buying a car/house, getting married/starting a family, having children), as well as avoiding medical (34.2%) or mental (41.4%) health care, with 17.5% of participants experiencing a health crisis they could not afford while in graduate school. Financial stress was associated with an increase in time spent thinking about finances, higher rates of depression and anxiety symptoms, and decreased sleep. CONCLUSION: Many clinical psychology doctoral students experience financial stress and are often unable to afford basic educational, personal living, and health care expenses, likely worsening mental health. Academic programs and leadership are encouraged to increase student stipends, improve financial transparency, provide access to health care, and alleviate financial stress and debt.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Tumor Microenvironment CD14+ Cells Correlate with Poor Overall Survival in Patients with Early-Stage Lung Adenocarcinoma.

Patients with early-stage lung adenocarcinoma have a high risk of recurrent or metastatic disease despite undergoing curative intent therapy. We hypothesized that increased CD14+ cells within the tumor microenvironment (TME) could stratify patient outcomes. Immunohistochemistry for CD14 was performed on 189 specimens from patients with lung adenocarcinoma who underwent curative intent surgery. Outcomes and associations with clinical and pathologic variables were determined. In vitro studies utilized a coculture system to model the lung cancer TME containing CD14+ cells. Patients with high levels of TME CD14+ cells experienced a median overall survival of 5.5 years compared with 8.3 and 10.7 years for those with moderate or low CD14 levels, respectively (p < 0.001). Increased CD14+ cell tumor infiltration was associated with a higher stage at diagnosis and more positive lymph nodes at the time of surgery. This prognostic capacity remained even for patients with early-stage disease. Using an in vitro model system, we found that CD14+ cells reduced chemotherapy-induced cancer cell death. These data suggest that CD14+ cells are a biomarker for poor prognosis in early-stage lung adenocarcinoma and may promote tumor survival. CD14+ cell integration into the lung cancer TME can occur early in the disease and may be a promising new therapeutic avenue.

Malignant rhabdoid tumor: Cyto-histologic correlation and immunohistochemical characterization of a rare pediatric malignancy and its differential diagnoses.

Malignant rhabdoid tumor of the kidney (MRTK) is a rare aggressive pediatric renal tumor which can be diagnosed via fine-needle aspiration (FNA) cytology and core biopsy. The diagnosis of MRTK is challenging, and requires morphologic, immunohistochemical and clinical correlation to distinguish it from other entities. The differential diagnosis includes Wilms tumor, desmoplastic small round cell tumor, rhabdomyosarcoma, synovial sarcoma, renal medullary carcinoma, and epithelioid sarcoma. Here we describe a case of MRTK diagnosed on renal cytology and core biopsy with immunohistochemistry and follow by nephrectomy with gross and morphologic findings.

Late Complications of COVID-19.

CONTEXT.­: Studies of lungs in patients with COVID-19 have focused on early findings. OBJECTIVE.­: To systematically study histopathologic and imaging features and presence of SARS-CoV-2 RNA in lung tissue from patients in later stages of COVID-19. DESIGN.­: Autopsies, explants, surgical lung biopsies, transbronchial biopsies, cryobiopsies, and needle biopsies from patients with COVID-19 whose onset of symptoms/confirmed diagnosis was more than 28 days before the procedure were studied. Available images were reviewed. Reverse transcription droplet digital polymerase chain reaction for SARS-CoV-2 RNA was performed on lung tissue. RESULTS.­: Of 44 specimens (43 patients; median age, 59.3 years; 26 [60.5%] male) features of acute lung injury (ALI) were seen in 39 (88.6%), predominantly organizing pneumonia and diffuse alveolar damage, up to 298 days after onset of COVID-19. Fibrotic changes were found in 33 specimens (75%), most commonly fibrotic diffuse alveolar damage (n = 22) and cicatricial organizing pneumonia (n = 12). Time between acquiring COVID-19 and specimen was shorter in patients with diffuse ALI (median, 61.5 days) compared with patients with focal (140 days) or no ALI (130 days) (P = .009). Sixteen (of 20; 80%) SARS-CoV-2 reverse transcription droplet digital polymerase chain reaction tests were positive, up to 174 days after COVID-19 onset. Time between COVID-19 onset and most recent computed tomography in patients with consolidation on imaging was shorter (median, 43.0 days) versus in patients without consolidation (87.5 days; P = .02). Reticulations were associated with longer time to computed tomography after COVID-19 onset (median, 82 versus 23.5 days; P = .006). CONCLUSIONS.­: ALI and SARS-CoV-2 RNA can be detected in patients with COVID-19 for many months. ALI may evolve into fibrotic interstitial lung disease.