Supernatant fluid from endobronchial ultrasound-guided transbronchial needle aspiration for rapid next-generation sequencing.

INTRODUCTION: There is an increasing demand to optimize the workflow and maximize tissue available for next-generation sequencing (NGS) for non-small cell carcinoma. We looked at transbronchial needle endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration samples and evaluated the performance of supernatant (SN) fluid processed from a dedicated aspirate collected for NGS testing. MATERIALS AND METHODS: Nineteen samples were collected and processed using a new workflow. Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to formalin for cell block (CB) processing. DNA and RNA were extracted from concentrated SN for targeted testing using the Oncomine Precision Assay (Thermo Scientific, Waltham, MA). NGS results from the corresponding CB samples were used as "controls" for comparison. RESULTS: Thirty-one mutations were detected in SN (Table 1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). There was 100% concordance between the mutations detected in SN and corresponding CBs with comparable variant allele frequencies. Turnaround time of NGS results was 1 day for SN compared to 4-10 days for CB. CONCLUSIONS: We were able to demonstrate the usefulness of SN for reliable rapid molecular results. We successfully incorporated the workflow for tissue handling and processing among our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.

Uncommon molecular alterations in follicular-derived thyroid carcinoma: A single institution study.

Thyroid carcinomas are the most common endocrine malignancy and commonly have alterations in the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathways in well-differentiated tumors. Alternative molecular alterations driving thyroid carcinomas have been identified rarely in the literature and are more likely to occur in poorly differentiated or anaplastic cases. In this study, uncommon genetic alterations such as MLH1, MSH2, NSD3::NUTM1, RET::SPECC1L, and G3BP2::FGFR2 were identified in patients with papillary thyroid carcinoma, poorly differentiated thyroid carcinoma, and differentiated high-grade thyroid carcinoma. Most of these tumors demonstrated an aggressive biological behavior. Atypical driver mutations in thyroid carcinomas can occur in patients with cancer predisposition syndromes as demonstrated by an NTRK1::TPM3 fusion in a patient with Li Fraumeni syndrome. In these settings of more aggressive disease, molecular testing targeting actionable fusions and mutations is important. As demonstrated in our case cohort, 100% of cases diagnosed as high-grade follicular-derived thyroid carcinoma had a mutation or fusion that is associated with worse prognosis, has a germline syndrome association requiring further work up, or an actionable mutation. This high yield seen in this cohort for molecular testing in patients with high-grade follicular-derived thyroid carcinoma suggests more routine molecular testing in this population would be a beneficial clinical practice.

Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.

BRAF Exon 15 Mutations in the Evaluation of Well-Differentiated Epithelial Nephroblastic Neoplasms in Children: A Report From the Children's Oncology Group Study AREN03B2.

CONTEXT.­: The distinction between well-differentiated epithelial favorable-histology Wilms tumor (EFHWT) and metanephric adenoma (MA) in children has historically been determined by the required absence of both a fibrous pseudocapsule and mitotic activity in MA. More recently these features have been allowed in adult MA. Mutations in exon 15 of the BRAF gene are reported in up to 88% of MAs but have not been reported in EFHWTs. OBJECTIVE.­: To clarify the pathologic and molecular features used to distinguish between pediatric MA and EFHWT. DESIGN.­: Stage I epithelial tumors classified as EFHWT on central review (36 patients) were identified from the Children's Oncology Group AREN03B2 study. Thirteen tumors had morphologic features overlapping those of MA and 23 lacked such features; 35 of 36 had tissue available for sequencing of BRAF. RESULTS.­: Patients with EFHWTs with MA features (13) were older (mean, 8.4 versus 1.9 years; P < .001), had smaller tumor diameters (mean, 6.0 versus 9.7 cm; P < .001), and had fewer mitoses (mean, 1 versus 48 mitoses per 10 high-power fields; P < .001) than patients with EFHWT lacking MA features (23). All EFHWTs with MA features contained at least a partial fibrous pseudocapsule; 7 of 12 (58%) had BRAF exon 15 mutation. No BRAF exon 15 mutations were identified in 23 EFHWTs lacking MA features. None of the 13 EFHWT patients with MA features have experienced relapse (median follow-up 5.9 years). CONCLUSIONS.­: Pediatric epithelial neoplasms with features of MA that show partial encapsulation and/or modest mitotic activity may be classified as MAs. Although BRAF mutation supports the diagnosis of MA, it is not required for the diagnosis.

ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

Agminated presentation of fusion-driven melanocytic neoplasms.

BACKGROUND: The conventionally understood pathogenesis of agminated Spitz nevi includes a mosaic HRAS mutation followed by copy number gains in 11p. However, we have recently observed agminated presentations of fusion-driven melanocytic neoplasms. METHODS: We retrieved cases from our database of benign fusion-induced melanocytic neoplasms with an agminated presentation. Both the primary lesion and the secondary lesion were sequenced. TERT-promoter mutational testing and the melanoma fluorescence in situ hybridization assay were also performed. RESULTS: Three cases were included. Two had a PRKCA fusion (partners ATP2B4 and MPZL1) and one had a ZCCHC8::ROS1 fusion. None of the cases met morphologic or molecular criteria for malignancy. There was no evidence of tumor progression in secondary lesions. The same fusion was identified in the primary and secondary lesions. None of the patients developed evidence of nodal or systemic metastasis. CONCLUSIONS: We present accumulating evidence that fusion-driven melanocytic neoplasms can present with an agminated presentation. The differential diagnosis of an agminated presentation versus a locally recurrent or potentially locally metastatic tumor is critical, and accurate diagnosis has significant prognostic and therapeutic consequences for the patient. As with HRAS mutations, fusion-driven melanocytic tumors may have an agminated presentation.

Clinical and histopathologic characterization of SETD2-mutated colorectal cancer.

With the advent of next-generation sequencing (NGS), identifying and better understanding genetic mutations in cancer pathways has become more feasible. A mutation now commonly reported in NGS panels is the SETD2 gene (H3K36 trimethyltransferase). However, its contributions to colorectal cancer (CRC) are not well described. In this study, we describe the clinicopathologic characteristics of SETD2-mutated CRC, determine common mutation sites on the SETD2 gene, and correlate these mutations with the loss of H3K36 trimethylation and the aberrant expression of beta-catenin. By searching pathology reports at our institution which included the 161-gene NGS panel from 2019 to 2021, we identify 24 individuals with SETD2-mutated CRC. All samples were evaluated for microsatellite status, H3K36 trimethylation, and beta-catenin via immunohistochemistry. In this cohort of 24 SETD2-mutated CRC individuals (a median age of 62.4 years [interquartile range: 49.1-73.6]), 10 (41.7%) patients presented at American Joint Committee on Cancer (AJCC) tumor stage II, seven (29.2%) at stage III, six (25%) at stage IV, and one (4.2%) at stage I. Most tumors studied were adenocarcinomas with no further specification (22, 92%), and most tumors were microsatellite stable (18, 82.5%). Thirty-three mutation locations were represented by 24 patients, with one patient having six mutations in the SETD2 gene and two patients having three mutations. The dominant mutation type is missense mutations (N = 29, 87.9%), and no mutation hotspots were found. Only two samples lost trimethylation of histone H3K36, both from individuals with multiple SETD2 mutations and aberrant nuclear beta-catenin expression. SETD2-mutated CRC is similar in clinical and histologic presentation to other commonly reported CRC. SETD2 mutations were missense dominantand showed no hotspots, and multiple mutations are likely necessary for loss of H3K36 trimethylation. These results warrant further study on determining a role of SETD2-histone H3K36 pathway in CRC.

Normalization of NPM1 mutant transcript to the wild-type transcript.

Current guidelines recommend that Acute Myeloid Leukemia (AML) patients with NPM1 mutations should be monitored for measurable residual disease by quantifying the transcripts and normalizing them to ABL1 transcripts. In this short report, a simple and highly accurate method to quantify the NPM1 mutant transcript normalized to the wild-type NPM1 transcript is presented. The percent mutant transcript correlates very well to the corresponding mutant allele frequency as determined by DNA-based methods allowing direct comparison of investigational studies that use RNA-based or DNA-based methods for monitoring NPM1 mutations.

Validation Study of a Direct Real-Time PCR Protocol for Detection of Monkeypox Virus.

Monkeypox has recently been described as a public health emergency of international concern by the World Health Organization and a public health emergency by the United States. If the outbreak continues to grow, rapid scalability of laboratory testing will be imperative. During the early days of the coronavirus disease 2019 (COVID-19) pandemic, laboratories improved the scalability of testing by using a direct-to-PCR approach. To improve the scalability of monkeypox testing, a direct real-time PCR protocol for the detection of monkeypox virus was validated. The assay retains the sensitivity and accuracy of the indirect assay while eliminating the need for nucleic acid extraction kits, reducing laboratory technologist time per sample and decreasing exposure to an infectious agent. The direct method will make it easier for laboratories across the world to rapidly develop, validate, and scale testing for monkeypox virus.

The effect of human immunodeficiency virus and human papillomavirus strain diversity on the progression of anal squamous intraepithelial lesions.

Anal squamous cell carcinoma (SCC) is a human papillomavirus (HPV)-mediated malignancy with increasing incidence. Human immunodeficiency virus (HIV) infection is a significant risk factor for anal SCC; however, it is unknown if HIV infection alters anal lesion progression and HPV strain profile. This study aims to determine whether HIV coinfection is associated with progression of HPV-mediated anal lesions and on their HPV strain diversity. This is a retrospective cohort study of adults with anal squamous intraepithelial lesion (SIL) who presented for anorectal sampling between 2010 and 2019. Using the full cohort, we performed clinicopathologic epidemiologic analysis of HIV coinfection on lesion progression. Using a subset of patients, we conducted molecular analysis of HPV strain diversity as related to HIV status and progression. Our cohort included 2203 individuals, of which 940 (43%) were HIV+. HIV+ status was associated with faster progression at all levels of dysplasia. Our molecular cohort included 329 adults, of which 190 (57.8%) were HIV+. HIV+ status was associated with higher HPV strain diversity (median: 7 [5-9] versus median: 4 [4-6], P < .001). Latent class analysis identified specific HPV strain signatures associated with progression. We demonstrate that HIV+ individuals had faster rates of anal SIL progression and that almost all HPV strains were more prevalent in anal samples from HIV+ adults. Our results imply that HIV+ adults with anal SIL should undergo more frequent screening and obtain HPV genotyping at initial presentation, as it shows value as a biomarker of lesion progression.

Validation of Whole Genome Methylation Profiling Classifier for Central Nervous System Tumors.

The 2021 WHO Classification of Tumors of the Central Nervous System includes several tumor types and subtypes for which the diagnosis is at least partially reliant on utilization of whole genome methylation profiling. The current approach to array DNA methylation profiling utilizes a reference library of tumor DNA methylation data, and a machine learning-based tumor classifier. This approach was pioneered and popularized by the German Cancer Research Network (DKFZ) and University Hospital Heidelberg. This research group has kindly made their classifier for central nervous system tumors freely available as a research tool via a web-based portal. However, their classifier is not maintained in a clinical testing environment. Therefore, the Northwestern Medicine (NM) classifier was developed and validated. The NM classifier was validated using the same training and validation data sets as the DKFZ group. Using the DKFZ validation data set, the NM classifier's performance showed high concordance (92%) and comparable accuracy (specificity 94.0% versus 84.9% for DKFZ, sensitivity 88.6% versus 94.7% for DKFZ). Receiver-operator characteristic curves showed areas under the curve of 0.964 versus 0.966 for NM and DKFZ classifiers, respectively. In addition, in-house validation was performed and performance was compared using both classifiers. The NM classifier performed comparably well and is currently offered for clinical testing.

Maternal vs Fetal Origin of Placental Intervillous Thrombi.

OBJECTIVES: To determine maternal vs fetal origin for blood in placental intervillous thrombi (IVTs). METHODS: We used comparative analysis of microsatellites (short tandem repeats [STRs]), sex chromosome fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC) for fetal (ɑ-fetoprotein [AFP]) and maternal (immunoglobulin M [IgM]) serum proteins to distinguish the origin of IVTs. Using an informatics approach, we tested the association between IVTs and fetomaternal hemorrhage (FMH). RESULTS: In 9 of 10 cases, the preponderance of evidence showed that the thrombus was mostly or entirely maternal in origin. In 1 case, the thrombus was of mixed origins. STR testing was prone to contamination by entrapped fetal villi. FISH was useful but limited only to cases with male fetuses. IgM showed stronger staining than AFP in 9 cases, supporting maternal origin. By informatics, we found no association between IVTs and FMH. CONCLUSIONS: Evidence supports a maternal origin for blood in IVTs. IHC for IgM and AFP may be clinically useful in determining maternal vs fetal contribution to IVTs.

Kikuchi-Fujimoto disease presenting in a patient with SARS-CoV-2: a case report.

BACKGROUND: We present a yet to be described association of SARS-CoV-2 infection with Kikuchi-Fujimoto disease. CASE PRESENTATION: A 32-year-old physician with history of SARS-CoV-2 infection presented to the emergency department with 2 weeks of fever, chills, and right sided cervical lymphadenopathy. He was treated empirically for presumed folliculitis with worsening of symptoms leading to repeat presentation to the emergency department. Extensive workup was unrevealing of an infectious cause and needle biopsy of the lesion was unrevealing. An excisional lymph node biopsy revealed follicular hyperplasia with necrotic foci showing abundance of histiocytes at the edge of necrosis with CD8 predominance of T-cells. Final diagnosis was deemed to be Kikuchi-Fujimoto disease. Antibiotic therapy was discontinued, and the patient's symptoms resolved with steroid therapy and expectant management. CONCLUSIONS: This is the first report of a patient developing Kikuchi-Fujimoto disease following SARS-CoV-2 infection. Clinicians should be aware of Kikuchi-Fujimoto disease as a possibility when approaching patients with hyper-inflammatory states who present with cervical lymphadenopathy.

Solitary fibrous tumor of thoracic cavity, extra-thoracic sites and central nervous system: Clinicopathologic features and association with local recurrence and metastasis.

Published risk stratification models of solitary fibrous tumor (SFT) have been associated with distant metastases outside the central nervous system (CNS), but have not been studied for tumors occurring in the CNS. In a retrospective review, we identified 72 cases of solitary fibrous tumor or hemangiopericytoma (HPC) diagnosed between January 2011 and December 2020 at our institution. The tumors involved the central nervous system (N = 17), thoracic cavity (N = 28), and extrathoracic sites (N = 27). The risk of local recurrence, distant metastasis, or death at 5 years was 57% (95% CI 23%, 76%) in the CNS, 24% (95% CI 2%, 41%) in the thoracic cavity, and 13% (95% CI 0%, 25%) in extrathoracic sites. By contrast, the risk of distant metastasis or death at 5 years was 13% (95% CI 0%, 29%) in CNS primaries, 5% (95% CI 0%, 14%) in thoracic primaries, and 14% (95% CI 0%, 27%) in extrathoracic primaries. Using the published 3- and 4-variable risk stratification models by Demicco et al., we retrospectively assessed our cases for risk of local recurrence, distant metastasis, and death. For tumors outside the CNS, we show that three- and four-variable risk stratification models were associated with recurrence-free survival in addition to the previously known association with distant metastasis (all P < 0.05). In contrast, inside the CNS, we show that neither risk model is a significantly associated with clinical behavior, and that WHO grade is likely the best available prognostic tool, though none of the differences were significant. The lack of significant differences can be likely explained by the younger median age (47 years vs 61 years) and smaller median tumor size (3.5 cm vs 5.6 cm), downgrading the risk stratification scores in CNS compared to non-CNS primaries. In conclusion, existing risk stratification models of SFT are not associated with clinical behavior for tumors arising inside the CNS, but are associated with local recurrence in addition to distant metastasis outside the CNS.

Paired-like homeobox gene (PHOX2B) nonpolyalanine repeat expansion mutations (NPARMs): genotype-phenotype correlation in congenital central hypoventilation syndrome (CCHS).

PURPOSE: CCHS is an extremely rare congenital disorder requiring artificial ventilation as life support. Typically caused by heterozygous polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene, identification of a relationship between PARM length and phenotype severity has enabled anticipatory management. However, for patients with non-PARMs in PHOX2B (NPARMs, ~10% of CCHS patients), a genotype-phenotype correlation has not been established. This comprehensive report of PHOX2B NPARMs and associated phenotypes, aims at elucidating potential genotype-phenotype correlations that will guide anticipatory management. METHODS: An international collaboration (clinical, commercial, and research laboratories) was established to collect/share information on novel and previously published PHOX2B NPARM cases. Variants were categorized by type and gene location. Categorical data were analyzed with chi-square and Fisher's exact test; further pairwise comparisons were made on significant results. RESULTS: Three hundred two individuals with PHOX2B NPARMs were identified, including 139 previously unreported cases. Findings demonstrate significant associations between key phenotypic manifestations of CCHS and variant type, location, and predicted effect on protein function. CONCLUSION: This study presents the largest cohort of PHOX2B NPARMs and associated phenotype data to date, enabling genotype-phenotype studies that will advance personalized, anticipatory management and help elucidate pathological mechanisms. Further characterization of PHOX2B NPARMs demands longitudinal clinical follow-up through international registries.

Comprehensive evaluation of bronchoalveolar lavage from patients with severe COVID-19 and correlation with clinical outcomes.

Information on bronchoalveolar lavage (BAL) in patients with COVID-19 is limited, and clinical correlation has not been reported. This study investigated the key features of BAL fluids from COVID-19 patients and assessed their clinical significance. A total of 320 BAL samples from 83 COVID-19 patients and 70 non-COVID-19 patients (27 patients with other respiratory viral infections) were evaluated, including cell count/differential, morphology, flow cytometric immunophenotyping, and immunohistochemistry. The findings were correlated with clinical outcomes. Compared to non-COVID-19 patients, BAL from COVID-19 patients was characterized by significant lymphocytosis (p < 0.001), in contrast to peripheral blood lymphopenia commonly observed in COVID-19 patients and the presence of atypical lymphocytes with plasmacytoid/plasmablastic features (p < 0.001). Flow cytometry and immunohistochemistry demonstrated that BAL lymphocytes, including plasmacytoid and plasmablastic cells, were composed predominantly of T cells with a mixture of CD4+ and CD8+ cells. Both populations had increased expression of T-cell activation markers, suggesting important roles of helper and cytotoxic T-cells in the immune response to SARS-CoV-2 infection in the lung. More importantly, BAL lymphocytosis was significantly associated with longer hospital stay (p < 0.05) and longer requirement for mechanical ventilation (p < 0.05), whereas the median atypical (activated) lymphocyte count was associated with shorter hospital stay (p < 0.05), shorter time on mechanical ventilation (p < 0.05) and improved survival. Our results indicate that BAL cellular analysis and morphologic findings provide additional important information for diagnostic and prognostic work-up, and potential new therapeutic strategies for patients with severe COVID-19.

Liver Pathology and SARS-CoV-2 Detection in Formalin-Fixed Tissue of Patients With COVID-19.

OBJECTIVES: The novel coronavirus, severe acute respiratory syndrome coronavirus 2, causing coronavirus disease 2019 (COVID-19) remains a global health threat and a significant source of human morbidity and mortality. While the virus primarily induces lung injury, it also has been reported to cause hepatic sequelae. METHODS: We aimed to detect the virus in formalin-fixed tissue blocks and document the liver injury patterns in patients with COVID-19 compared with a control group. RESULTS: We were able to detect viral RNA in the bronchioalveolar cell blocks (12/12, 100%) and formalin-fixed, paraffin-embedded tissue of the lung (8/8, 100%) and liver (4/9, 44%) of patients with COVID-19. Although the peak values of the main liver enzymes and bilirubin were higher in the patients with COVID-19 compared with the control group, the differences were not significant. The main histologic findings were minimal to focal mild portal tract chronic inflammation (7/8, 88%, P < .05) and mild focal lobular activity (6/8, 75%, P = .06). CONCLUSIONS: We found that most patients who died of COVID-19 had evidence of mild focal hepatitis clinically and histologically; however, the virus was detected in less than half of the cases.