Supplemental Figure 3. Boxplot of NF- κB values across the three cluster subtypes. By unsupervised consensus clustering and self-organizing map (SOM) approaches, we identified three distinct biological subtypes within HPV-positive HNSCC: Cl1 (immune-related), Cl2 (epithelial-mesenchymal transition [EMT]-related), and Cl3 (proliferation-related). This classification holds prognostic significance, with Cl1 exhibiting the most favorable survival outcomes, Cl2 associated with the poorest prognosis, and Cl3 displaying an intermediate survival rate [Locati, 2019]. In contrast to recent studies that classified HPV-related HNSCC into immune and keratinocyte subtypes, our analysis confirmed the immune-associated subtype while further resolving the keratinocyte group into two distinct clusters with differing biological profiles and prognostic implications. NF-κB signature is significantly activated in Cl1-immune related cluster compared to the other 2 clusters: A) Metanalyisis-HPV267 cohort (1); B) BD2-HPV286 cohort (2).

Supplemental Figure 8A. UMAP dimensional reduction plots with marker gene validating cell type assignments. Marker genes related to epithelial cells, endothelial cells, fibroblasts, mast cells, dendritic cells, and myeloid cells. Supplemental Figure 8B. UMAP dimensional reduction plots with marker gene validating cell type assignments. Marker genes related to natural killer, B, T and cycling cells.

Supplemental Figure 9. Marker genes by cluster using Leiden method.

Supplemental Table 2. Association of NF-κB high and low groups with clinical parameters in both Metanalysis-HPV267 (1) and BD2-HPV286 (2). P-value assessed by χ2 test, with the exception of age (t-test). Abbreviations: CHT, chemotherapy; RT, radiotherapy.

Supplemental Figure 2. UMAP dimensional reduction plot with malignancy probabilities from scMalignantFinder. Low probability predictions (<0.5) were imputed with to a value of 0.5 for visual clarity.

Supplemental Figure 6. Survival Plot. Prognostic Value of NanoString NAC Classifier Measurements. In Figure 4, PFS was shown for (E) all patients and (F) de-escalation candidates in UNC and E1308 cohorts (non-smoker,

Supplemental Table 4. Association of NF-κB high and low groups with clinical parameters in combined UNC &E1308 cohorts (1) as pictured in Figure 4D-E. P-value assessed by χ2 test, except for age (assessed with t-test).

Supplemental Figure 4. Comprehensive comparison of all gene sets and potential summary metrics for the NF-κB signature using RNAseq from the UNC cohort. UNC Cohort originally presented in Schrank and colleagues PNAS 2023 (1). Correlation plot with scatter and Pearson correlation values. Nano str – the gene signature utilized in the nanostring assay including 232 NF-κB related genes, which are compared to 27 control genes, these genes were identified in our prior publications as outlined below and represent the combination of the PNAS and Onco large gene sets as defined below. Onco small – Top 50 NF-κB related genes identified in prior work using machine learning improved differential expression, prioritized by p-value in Schrank et at, Oncotarget 2022 (2), 49 of which are included (one excluded by probe design) in the NanoString panel. 29 of these genes are also identified as NF-κB related genes with an orthogonal approach in our prior work using WGCNA (1). Onco large – Top 75 NF-κB related genes identified in prior work using machine learning improved differential expression, prioritized by p-value in Schrank et at, Oncotarget 2022 (2), 74 of which are included (one excluded by probe design) in the NanoString panel 45 of these genes are also identified as NF-κB related genes with an orthogonal approach in our prior work using WGCNA (1). Pnas – 203 NF-κB related genes identified in our prior work using WGCNA, a network-based approach in Schrank and colleagues PNAS 2023 (3). Onco only – NF-κB genes identified by differential expression but not by WGCNA. Pnas only – NF-κB genes identified by WGCNA but not by differential expression. Mean – mean expression of signature genes. Median – median expression of signature genes. PCA – sample value of PC1, for PCA performed on signature genes only after expression normalization. ssGSEA – Signature sized normalized enrichment score (NES) calculated by the single sample gene set enrichment method.

Supplemental Figure 1. Correlation plots for the: A) Metanalyisis-HPV267 (1) cohort; B) BD2-HPV286 (2) cohorts, comparing various summary metrics (mean, ssGSEA enrichment score and for the NF-κB RNA signature in microarray gene expression data. Values represent Pearson correlation coefficients. *** p-value of Pearson correlation <0.0005.

Supplemental Table 1. Association of NF-κB pathway variants with patient clinical features. P-value assessed by χ2 test, except for age (assessed with t-test).

Supplemental Figure 5. NF-κB pathway mutations are found in cases with high NF-κB scores. A) NanoString NF-κB expression scores and NF-κB pathway mutational data from UNC cohort where both metrics were available. B) Non-linear least squares fit of mono-exponential function relating NanoString and RNAseq data. This fit was used to transpose the threshold defined by NanoString to the RNAseq data, where 3 more cases were available.

Supplemental Figure 7. IF staining was performed at UNC Pathology Services Core. Patients in the UNC RNAseq cohort were stratified by RNA NF-κB Signature score as previously published. (1) The following antibodies were used: NIK antibody: Cat#SC-8417 (Santa Cruz) and Pan CK: Leica NCL-L-AE1/AE3-601 (Leica). High resolution images of IF slides were obtained performed with the Aperio Versa 200 scanner (Leica Biosystems Inc.) at an apparent magnification of 10X. Images were uploaded to the eSlideManager database (Aperio) at the Pathology Services Core at UNC. ** p-value <0.00005, Wilcox test.

Molecular profiling has revolutionized the diagnosis and management of patients with non-small cell lung cancer (NSCLC), enabling personalized treatment approaches that have significantly improved patient outcomes. Identifying specific gene mutations and protein expression profiles guides therapeutic decisions and has become standard in the management of patients with NSCLC. Multiple organizations have published guidelines for biomarker testing. However, differences in the timing, methodology, and scope of recommended targets create potential for inconsistencies in clinical practice. This article reviews the clinical importance of biomarker testing in NSCLC, compares major guidelines, and highlights the challenges and implications of guideline variability in formulating recommendations on biomarker testing for patients with NSCLC.

Supplemental Table 3. NF-κB high and low patient samples with clinical parameters in combined scRNAseq data from Kurten and colleagues (1) No recurrence or progression events were noted in these 6 patients.

A 37-year-old non-smoking female presented with a slowly growing, firm, nontender 1-cm exophytic tan lesion on the left lateral tongue, which had been present for three months and was initially suspected to be lingual adenoid tissue. An incisional biopsy was performed, and hematoxylin and eosin (H&E) stained slides revealed a biphasic neoplasm consisting of hyperparakeratotic and acanthotic stratified squamous epithelium, with underlying crowded glandular structures consisting of ducts lined by cuboidal to columnar epithelium. Both the squamous and glandular components displayed cytologic atypia with prominent nucleoli, and a diagnosis of adenosquamous carcinoma was made. Subsequently partial glossectomy and neck dissection were performed. H&E of the excised lesion showed a biphasic neoplasm composed of exophytic squamous epithelium with a polypoid and papillary architecture, along with an underlying endophytic glandular, cystic, and micropapillary proliferation. Immunohistochemical analysis showed less than 5% Ki-67 reactivity and BRAF V600E was positive in both components. The histopathological features, combined with the immunoprofile, supported a diagnosis of sialadenoma papilliferum. This case illustrates a rare occurrence of this benign salivary gland tumor in the oral tongue and emphasizes the importance of obtaining a properly sized incisional biopsy to ensure optimal patient care and prevent potential pitfalls.

Clinical Presentation An 11-year-old male patient presented with a right-sided facial asymmetry and proptosis of the right eye with associated vision loss and neuropathy. Review of his health history was otherwise unremarkable. Computed tomography (CT) scan of the head revealed a well-demarcated, expansile, heterogeneous radiodense mass with central lucent zone that obliterated the right nasal cavity and ethmoid sinus spaces (Figure 1). While impinging on the optic nerve, the lesion displaced the globe of the right eye, deviated the nasal septum, and displaced the sphenoid bone posteriorly. Differential Diagnosis The differential diagnosis for sinonasal conditions associated with ocular proptosis is broad and includes a range of reactive processes along with benign and malignant neoplastic conditions arising in both soft tissues and hard tissues.1 Since this lesion demonstrates radiodensity comparable to those of the hard tissues, primary intraosseous pathologic conditions are favored in the differential diagnostic considerations with notable exception of sinonasal meningioma.Meningioma is a tumor of possible meningiothelial cell origin that represents 24%-30% of all intracranial neoplasms.2 It arises across a broad age range with rare occurrence in children and an overall female predilection.3 Primary extracranial meningiomas are relatively rare representing less than 1% of all cases which mostly occur in the orbit, middle ear, soft tissues, skin, and sinonasal tract.3,4 Clinical manifestations of sinonasal meningiomas are nonspecific including nasal obstruction, epistaxis, nasal discharge, proptosis, facial pain and deformity.2,3 On CT imaging, sinonasal meningioma presents as a homogenously enhancing mass associated with hyperostosis of the surrounding bone. While it generally demonstrates soft tissue density, speckled opacifications may also be observed especially in the psammomatoid variant of this tumor.3-5 The present case demonstrates a heterogeneous radiodensity comparable to that of hard tissues and therefore meningioma is not considered a likely diagnostic consideration.Osteomas are the most common primary bone tumors of the craniofacial skeleton which may arise in paranasal sinuses, maxilla, mandible, mastoid air cells, external auditory canal, and the cranial vault.6 While the exact pathogenesis of osteomas remain largely elusive, possible traumatic, infectious, and developmental etiologies have been suggested.6,7 In the paranasal sinuses, osteomas show a propensity for frontal and ethmoid sinuses.6 Arising across a broad age range, most sinonasal osteomas are diagnosed in 5th-6th decades of life.7,8 While most tumors are asymptomatic and incidentally discovered on imaging studies, depending on their location, sinonasal osteomas may induce symptoms such as headache, nasal congestion, local pain, ocular proptosis, and visual field disturbances.6-8 On CT imaging sinonasal osteoma is characterized by a well-delineated, homogenous radiodense mass that arises from the cortical surface of bone and occupies the sinus space. However, depending on the proportions of dense and cancellous bones, heterogeneous radiographic variants are also recognized.8 The present case is characterized by a heterogeneous well-delineated radiodense mass with a central lucent core that does not originate from cortical bone surface prompting exclusion of sinonasal osteoma from the diagnostic considerations.Osteoblastomas are rare mesenchymal neoplasms that comprise 1-4% of all primary bone tumors.9 Most commonly involving the vertebrae and long skeletal bones, sinonasal osteoblastomas are extremely rare.9,10 While the paucity of sinonasal osteoblastomas precludes a definitive clinicoradiographic characterization, differentiation of these tumors from the more common fibro-osseous sinonasal lesions is crucial from the therapeutic stand point.11 Most commonly observed in adolescents and young adults, sinonasal osteoblastomas are mostly asymptomatic. However, when impinging on the adjacent structures, sinonasal osteoblastomas may produce symptoms such as ocular proptosis, visual disturbance, and nasal obstruction.12 On CT imaging studies, sinonasal osteoblastoma is characterized by a well-defined expansile, heterogeneous lytic lesion that often exhibits an eccentric cap of radiodense mature bone.11 The absence of this characteristic feature in this current case disfavors sinonasal osteoblastoma as a likely diagnostic consideration.Fibrous dysplasia (FD) is a sporadic benign fibro-osseous lesion characterized by progressive replacement of bone with fibrous connective tissue.13 Characterized by postzygotic activating missense mutation of the GNAS gene, the timing of mutation impacts the extent of the disease involvement hence the subclassification of FD into monostotic and polyostotic forms. FD demonstrates a predilection for membranous bones of the skeleton such as femur and tibia.13,14 Polyostotic FD may rarely be accompanied with endocrinopathies and café-au-lait skin pigmentations (McCune-Albright syndrome) or soft tissue myxoma in the setting of Mazabraud syndrome (15). Between 50% and 100% of polyostotic FD and 10%-30% of monostotic cases may involve the craniofacial skeleton.14 Usually arising before the age of 30 years, craniofacial FD frequently affects the maxilla and mandible followed by frontal, parietal, and occipital bones along with infrequent involvement of the sinonasal tract.13 Although most cases are clinically asymptomatic and discovered incidentally, craniofacial FD may be associated with bone deformity, headaches, visual defects, ocular proptosis, hearing loss, nasal obstructions, and anosmia depending on the anatomic location.14 The most characteristic finding in CT imaging with FD is bone expansion exhibiting a ground-glass appearance with ill-defined borders that blend with the surrounding bone. However, depending on the degree of metaplastic bone formation, radiographic appearance may range from radiolucency to a dense, homogenous radiopacity.16 Although the absence of a ground glass appearance excludes fibrous dysplasia as a likely diagnostic consideration of the present case, correlation with the clinical, intraoperative, and histopathologic features is required to ultimately rule out this entity.Ossifying fibromas (OF) are benign fibro-osseous neoplasms characterized by replacement of normal bone with a cellular fibrous stroma exhibiting bone or cementum-like calcifications.17 Two major variants of this entity are recognized with overlapping clinicopathologic features which include cemento-ossifying fibroma (COF) and juvenile ossifying fibroma (JOF). Furthermore, JOF is subdivided in psammomatoid and trabecular types. Of these, the psammomatoid variant of JOF demonstrates a predilection for ethmoid sinus and orbits.17,18 Arising primarily in patients younger than the age of 15 years, paranasal JOF often displays steady progressive growth leading to ocular proptosis, impaired vision, facial swelling, nasal obstruction, periorbital pain, and headache.19 The lesions arising in younger ages or those with concurrent development of an aneurysmal bone cyst may be associated with rapid growth and locally aggressive clinical behavior.20 Radiographically, JOF is well-delineated mixed-dense lesion that characteristically demonstrates peripheral ossification surrounding a radiolucent core.17,18,21 Although distinction between FD and JOF may be challenging when solely based on imaging studies, the present case exhibits characteristic and distinctive demographic, anatomic, clinical, and radiographic features which are in favor of the diagnosis of JOF. However, ultimately, a correlation with histopathologic and intraoperative findings is warranted for a definitive diagnosis. Diagnosis and Management A biopsy of the right sinonasal mass demonstrated a cellular uniform bland fibroblastic stroma composed of spindled to stellate cells (Figure 2). Interspersed between the fibroblastic cells were numerous spherical lamellated ossicles exhibiting varying degrees of calcification. These psammoma body-like ossicles were relatively acellular, with a concentric pattern of lamination, basophilic centers, and peripheral eosinophilic rims (Figure 3). Mitotic figures were not evident. The final diagnosis was juvenile psammomatoid ossifying fibroma. Complete surgical excision was performed (Figure 4) with no recurrence detected at 2 years follow up. Discussion Ossifying fibroma (OF) is a benign neoplasm that falls into the broader category of benign fibro-osseous lesions (BFOLs). Despite having distinct clinical features and treatment approaches, all BFOLs are characterized by the replacement of native bone with fibrous and mineralized tissues, and they are grouped together because of their histologic similarities.22 Because an ossifying fibroma may contain bone and/or cementum, the term cemento-ossifying fibroma (COF) can also be used interchangeably.22 OF is subdivided into conventional and juvenile clinicopathologic subtypes.25 By convention the term ossifying fibroma generally refers to the cemento-ossifying type, which is associated with tooth-bearing areas, whereas the term "juvenile" refers to juvenile ossifying fibroma. The juvenile ossifying fibromas are characterized by rapid and destructive growth, occur in younger patients relative to conventional OF, and are seen with considerably less frequency than their conventional counterpart.26 These neoplasms have been further categorized into juvenile trabecular ossifying fibroma (JTOF) and juvenile psammomatoid ossifying fibroma (JPOF) by the World Health Organization (WHO).23 While the trabecular variant is more prevalent in children under the age of 15 and prefers the maxilla, the psammomatoid variant is typically seen in the paranasal sinuses and in those 20 years and older. Nonetheless, older patients may occasionally be affected by both categories. Radiographically, the lesion presents as a well-delineated, expansile radiolucent lesion with variable focal calcifications.27 The JTOF is characterized histologically by cellular fibrous background comprising variable trabeculae of immature bone with osteoblastic rimming and collagenous rim. The JPOF has a variably cellular background with irregular spherical ossicles exhibiting varying degrees of calcification. These psammoma body-like ossicles are relatively acellular, with a concentric pattern of lamination.28 Furthermore, both variations frequently exhibit multinucleated giant cells and scattered mitotic figures. Without treatment, tumors continue to enlarge requiring complete surgical excision. Recurrence rates reach 58%, necessitating reconstruction surgery that may be potentially disfiguring.

Key Clinical MessagePericardial sarcoidosis is an uncommon cause of chest pain to consider, and it requires a heightened level of suspicion and thorough history gathering. If there is suspicion of inflammatory disease, pursuing advanced imaging and biopsies is crucial, as early immunosuppressive treatment can enhance outcomes.AbstractPericardial involvement in sarcoidosis is a rare condition with limited research. This case study discusses a 52‐year‐old African American woman who presented with subacute chest pain and was diagnosed with pericardial sarcoidosis. Diagnostic evaluation revealed extensive lymphadenopathy and pericardial effusion, and a pericardial biopsy confirmed non‐caseating granulomatous inflammation. Treatment with steroids and methotrexate resulted in clinical improvement. Eight months follow‐up showed near resolution of pericardial disease. This case emphasizes the importance of considering cardiac sarcoidosis in sarcoidosis patients, utilizing advanced imaging for accurate diagnosis, and tailoring treatment to the level of cardiac involvement.

Solitary plasmacytoma is a rare neoplasm characterized by localized proliferation of monoclonal plasma cells and is classified as solitary bone or solitary extramedullary plasmacytoma. Here, we present two rare cases of plasmacytoma of the head and neck. The first is a 78-year-old male who presented with a 3-month history of epistaxis and progressive obstruction of the right nasal passage. Computerized tomography (CT) imaging revealed a mass in the right nasal cavity with destruction to the maxillary sinus. An excisional biopsy was performed revealing anaplastic plasmacytoma. The second is a 64-year-old male with a past medical history significant for prostate cancer who presented with a 2-month history of left ear pain and progressive non-tender temporal swelling. A PET/CT revealed a highly avid, destructive, and lytic left temporal mass with no other evidence of distant disease. A left temporal craniectomy and infratemporal fossa dissection revealed plasma cell dyscrasia with monoclonal lambda in situ hybridization. Although plasmacytomas are uncommon tumors of the head and neck, they may mimic other entities that require different treatment. Prompt and accurate diagnosis is critical for appropriate therapeutic decisions and prognosis.

•Echocardiography is essential in diagnosing cardiogenic shock secondary to acute MR.•TMVEER can stabilize these patients who present as prohibitive risks for surgery.•Careful monitoring is warranted after TMVEER.

Acute Chagas disease reactivation (CDR) after cardiac transplantation is a well-known phenomenon in endemic countries of Central and South America and Mexico, but is rare outside of those countries. In this report, we describe a case of a 49-year-old male who presented 25 weeks after heart transplant with clinical features concerning for acute rejection, including malaise, anorexia, weight loss, and fever. His immunosuppression therapy included tacrolimus, mycophenolate, and prednisone. An endomyocardial biopsy revealed lymphocytic and eosinophilic inflammation, myocyte damage, and rare foci of intracellular organisms consistent with Trypanosoma cruzi amastigotes. The patient had no known history of Chagas disease. Upon additional questioning, the patient endorsed bites from reduviid bugs during childhood in El Salvador. Follow-up serum PCR testing was positive for T. cruzi DNA. Tests for other infectious organisms and donor specific antibodies were negative. This case illustrates the striking clinical and histologic similarities between acute cellular rejection and acute CDR with cardiac involvement in heart transplant patients, and thus emphasizes the importance of pre-transplant testing for Chagas in patients with epidemiologic risk factors.