Head and neck squamous cell carcinomas of unknown primary: Can ancillary studies help identify more primary tumor sites?

A subset of head and neck squamous cell carcinomas present solely as metastatic disease in the neck and are of unknown primary origin (SCCUP). Most primary tumors will ultimately be identified, usually in the oropharynx. In a minority of cases, the primary site remains elusive. Here, we examine the role of ancillary testing, including mutational signature analysis (MSA), to help identify likely primary sites in such cases. Twenty-two cases of SCCUP in the neck, collected over a 10-year period, were classified by morphology and viral status; including human papillomavirus (HPV) testing by p16 immunohistochemistry (IHC) and RT-qPCR, as well as Epstein-Barr virus (EBV) testing by EBER-ISH. CD5 and c-KIT (CD117) IHC was done to evaluate for possible thymic origin in all virus-negative cases. Whole exome sequencing, followed by MSA, was used to identify UV signature mutations indicative of cutaneous origin. HPV was identified in 12 of 22 tumors (54.5%), favoring an oropharyngeal origin, and closely associated with nonkeratinizing tumor morphology (Fisher's exact test; p = 0.0002). One tumor with indeterminant morphology had discordant HPV and p16 status (p16+/HPV-). All tumors were EBV-negative. Diffuse expression of CD5 and c-KIT was identified in 1 of 10 virus-negative SCCUPs (10%), suggesting a possible ectopic thymic origin rather than a metastasis. A UV mutational signature, indicating cutaneous origin, was identified in 1 of 10 (10%) virus-negative SCCUPs. A cutaneous auricular primary emerged 3 months after treatment in this patient. Primary tumors became clinically apparent in 2 others (1 hypopharynx, 1 hypopharynx/larynx). Thus, after follow-up, 6 tumors remained unclassifiable as to the possible site of origin (27%). Most SCCUPs of the neck in our series were HPV-associated and thus likely of oropharyngeal origin. UV signature mutation analysis and additional IHC for CD5 and c-KIT for possible thymic origin may aid in further classifying virus-negative unknown primaries. Close clinical inspection of hypopharyngeal mucosa may also be helpful, as a subset of primary tumors later emerged at this site.

Standardised definitions and diagnostic criteria for extranodal extension detected on histopathological examination in head and neck cancer: Head and Neck Cancer International Group consensus recommendations.

Detection of extranodal extension on histopathology in surgically treated head and neck squamous cell carcinoma indicates poor prognosis. However, there is no consensus on the diagnostic criteria, interpretation, and reporting of histology detected extranodal extension, which has contributed to conflicting evidence in the literature, and likely clinical inconsistency. The Head and Neck Cancer International Group conducted a three-round modified Delphi process with a group of 19 international pathology experts representing 15 national clinical research groups to generate consensus recommendations for histology detected extranodal extension diagnostic criteria. The expert panel strongly agreed on terminology and diagnostic features for histology detected extranodal extension and soft tissue metastasis. Moreover, the panel reached consensus on reporting of histology detected extranodal extension and on nodal sampling. These consensus recommendations, endorsed by 19 organisations representing 34 countries, are a crucial development towards standardised diagnosis and reporting of histology detected extranodal extension, and more accurate data collection and analysis.

Overexpression of human DNA polymerase theta is a biomarker of aggressive and DNA repair-deficient papillary thyroid cancers.

BACKGROUND: DNA polymerase theta (POLQ) is an enzyme that repairs double-strand DNA breaks. POLQ is overexpressed in several cancer types, and increased expression is associated with a poor prognosis. Ablating POLQ function in vitro increases drug sensitivity to agents that cause double-strand DNA breaks, including chemotherapies and ionizing radiation. POLQ's role in thyroid cancer remains poorly understood. METHODS: Expression of POLQ and other genes of interest were analyzed in 513 papillary thyroid cancers (505 primary tumors and 8 metastatic lesions) and 59 normal thyroid samples available in the Cancer Genome Atlas. The Cancer Genome Atlas RNA and DNA sequencing data were queried with the Xena platform. The Recombination Proficiency Score was calculated to assess DNA repair efficiency. Other signaling events associated with thyroid tumorigenesis and clinical outcomes were analyzed. Univariate and multivariate analyses were performed. Treatment with the POLQ inhibitors ART558 and Novobiocin tested the effect of POLQ inhibition on in vitro thyroid cancer growth. RESULTS: POLQ expression was increased in papillary thyroid cancers compared to normal thyroid tissue (P < .05). POLQ expression levels were inversely correlated with Recombination Proficiency Score levels (P < .05). POLQ expression was highest in tall cell papillary thyroid cancers and in metastases. Higher POLQ expression was also associated with dedifferentiation, BRAF signaling, and shorter progression-free intervals (P < .05). Treatment with POLQ inhibitors decreased in vitro thyroid cancer growth (P < .05). CONCLUSION: These findings suggest that increased POLQ expression could serve as a valuable clinical marker and a potential therapeutic target in the treatment of thyroid cancer.

Seven years of Non-invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP): Rate of Acceptance and Variation of Diagnostic Approaches Across Different Continents.

CONTEXT: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was introduced as a new entity replacing the diagnosis of noninvasive encapsulated follicular variant of papillary thyroid carcinoma (PTC). Significant variability in the incidence of NIFTP diagnosed in different world regions has been reported. OBJECTIVE: To investigate the rate of adoption of NIFTP, change in practice patterns, and uniformity in applying diagnostic criteria among pathologists practicing in different regions. METHODS: Two surveys distributed to pathologists of the International Endocrine Pathology Discussion Group with multiple-choice questions on NIFTP adoption into pathology practice and whole slide images of 5 tumors to collect information on nuclear score and diagnosis. Forty-eight endocrine pathologists, including 24 from North America, 8 from Europe, and 16 from Asia/Oceania completed the first survey and 38 the second survey. RESULTS: A 94% adoption rate of NIFTP by the pathologists was found. Yet, the frequency of rendering NIFTP diagnosis was significantly higher in North America than in other regions (P = .009). While the highest concordance was found in diagnosing lesions with mildly or well-developed PTC-like nuclei, there was significant variability in nuclear scoring and diagnosing NIFTP for tumors with moderate nuclear changes (nuclear score 2) (case 2, P < .05). Pathologists practicing in North America and Europe showed a tendency for lower thresholds for PTC-like nuclei and NIFTP than those practicing in Asia/Oceania. CONCLUSION: Despite a high adoption rate of NIFTP across geographic regions, NIFTP is diagnosed more often by pathologists in North America. Significant differences remain in diagnosing intermediate PTC-like nuclei and respectively NIFTP, with more conservative nuclear scoring in Asia/Oceania, which may explain the geographic differences in NIFTP incidence.

Prognostic Factors for Survival Using a Clinical Severity Staging System Among Patients With Acute Invasive Fungal Sinusitis.

Importance: Despite the aggressive progression of fulminant acute invasive fungal sinusitis (AIFS), data on prognostic factors have been disparate, hindering the development of a staging system. A composite staging system may improve prognostication for patient counseling and conduct of clinical research. Objective: To identify prognostically important factors in AIFS and to incorporate the factors into a comprehensive Functional Severity Staging System and Clinical Severity Staging System. Design, Setting, and Participants: This retrospective cohort study included adult patients diagnosed with pathology-proven AIFS from June 1, 1992, to December 31, 2022, at Washington University Medical Center and Barnes-Jewish Hospital, a tertiary care center in St Louis, Missouri. Data were analyzed from April to July 2023. Main Outcome and Measures: Sequential sequestration and conjunctive consolidation was used to develop a composite staging system to predict 6-month overall survival. Results: Of 71 patients with pathology-proven AIFS over the 30-year period, the median (range) age of the cohort was 56 (19-63) years, and there were 47 (66%) male patients. The median (range) follow-up time was 2 (0-251) months. There were 28 patients alive within 6 months, for a 39% survival rate. Symptoms, comorbidity burden, and presence and duration of severe neutropenia were associated with 6-month survival and were consolidated into a 3-category Clinical Severity Staging System with 6-month survival of 75% for stage A (n = 16), 41% for stage B (n = 27), and 18% for stage C (n = 28). The discriminative power of the composite staging system was moderate (C statistic, 0.63). Conclusion and Relevance: This cohort study supports the clinical importance of symptomatology, comorbidity burden, and prolonged severe neutropenia at the time of AIFS presentation. The composite clinical staging system may be useful for clinicians when counseling patients with AIFS and conducting clinical research.

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Biphenotypic Sinonasal Sarcoma with a Novel PAX7::PPARGC1 Fusion: Expanding the Spectrum of Gene Fusions Beyond the PAX3 Gene.

Biphenotypic sinonasal sarcoma (BSNS) is a rare low-grade malignancy occurring in the sinonasal tract that is characterized by dual neural and myogenic differentiation. Rearrangements involving the PAX3 gene, usually with MAML3, are a hallmark of this tumor type and their identification are useful for diagnosis. Rarely, a MAML3 rearrangement without associated PAX3 rearrangement has been described. Other gene fusions have not been previously reported. Herein, we report a 22 year-old woman with a BSNS harboring a novel gene fusion involving the PAX7 gene (specifically PAX7::PPARGC1A), which is a paralogue of PAX3. The histologic features of the tumor were typical with two exceptions: a lack of entrapment of surface respiratory mucosa and no hemangiopericytoma-like vasculature. Immunophenotypically, the tumor was notably negative for smooth muscle actin, which is usually positive in BSNS. However, the classic S100 protein-positive, SOX10-negative staining pattern was present. In addition, the tumor was positive for desmin and MyoD1 but negative for myogenin, a pattern that is common among BSNS with variant fusions. Awareness of the possibility of PAX7 gene fusions in BSNS is important as it may aid in the diagnosis of PAX3 fusion negative tumors.

Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma.

Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.

Mucoepidermoid carcinoma may be devoid of squamoid cells by immunohistochemistry: expanding the histologic and immunohistochemical spectrum of MAML2- rearranged salivary gland tumours.

Mucoepidermoid carcinoma (MEC) is historically defined by a mix of squamoid, intermediate, and mucous cells, but we have recently encountered several cases lacking immunoreactivity for squamous markers p40, p63, and CK5/6 despite MAML2 fusions. This study will characterise these unique tumours. Ten MEC were collected arising from the parotid gland (n = 4), submandibular gland (n = 2), nasopharynx (n = 1), base of tongue (n = 1), bronchus (n = 1), and trachea (n = 1). Six tumours were low-grade, two intermediate-grade, one high-grade, and one demonstrated low-grade areas with high-grade transformation. Four cases were oncocytic, four had clear-cell features, two had spindle cell features, and one high-grade MEC had prominent solid, cord-like, and micropapillary features. The tumours were negative for p40 (10/10), p63 (10/10), and CK5/6 (9/9). Targeted RNA sequencing demonstrated CRTC1::MAML2 in five cases, CRTC3::MAML2 in two, and a novel MAML2::CEP126 in the unusual high-grade case. In two cases with insufficient RNA, MAML2 fluorescence in situ hybridisation (FISH) showed rearrangement. Genetically-confirmed MEC may lack overt squamous differentiation by histology and immunohistochemistry. While most cases harboured canonical fusions and fit within the spectra of MEC variants with oncocytic, clear cell, and/or spindle cell features, one had a novel MAML2::CEP126 fusion and unusual morphology. In MEC without squamoid cells, the use of immunohistochemistry may hinder, rather than aid, the correct diagnosis. In such cases, MAML2 analysis is most useful. The historical definition of MEC as a carcinoma with squamoid, intermediate and mucous cells should be revisited.

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.

Nuclear expression of AFF2 C-terminus is a sensitive and specific ancillary marker for DEK::AFF2 carcinoma of the sinonasal tract.

DEK::AFF2 carcinoma of the sinonasal tract is an emerging entity. The tumor is typically characterized by papillary proliferation of non-keratinizing squamous epithelial cells with monotonous cytologic features, which may mimic other sinonasal tumors. The confirmation of this gene fusion has thus far relied solely on next-generation sequencing, fluorescence in situ hybridization (FISH), or reverse transcription polymerase chain reaction (RT-PCR). This current study aimed to validate an immunohistochemical assay for AFF2 C-terminus as an ancillary marker. We first analyzed publicly available RNA sequencing data of sinonasal tumors from the national center for biotechnology information (NCBI) sequence read archive and identified 3 DEK::AFF2 carcinomas out of 28 sinonasal tumors. The gene expression of AFF2 was significantly higher in the fusion-positive cases compared to the wild-type tumors (p < 0.001), while DEK was not. We then optimized an immunohistochemical assay with an anti-AFF2 C-terminus antibody for ancillary diagnosis. Seventeen DEK::AFF2 carcinomas, including 11 cases with predominantly low-grade morphology and one showing glandular differentiation, as well as 78 DEK FISH-negative sinonasal tumors were evaluated by AFF2 immunohistochemistry (IHC). Sixteen of the 17 DEK::AFF2 carcinomas showed nuclear AFF2 expression in ≥30% of tumor cells, including one decalcified case that failed FISH and RT-PCR confirmation. The one case that was negative for AFF2 IHC in the tumor cells also lacked expression in the internal positive control. It was thus considered a failure of the IHC rather than a truly negative case and was excluded from the statistical analysis. All DEK FISH-negative sinonasal tumors were negative for nuclear AFF2 expression. The nuclear expression of AFF2 IHC showed 100% sensitivity and specificity for DEK::AFF2 carcinoma. Accordingly, AFF2 IHC is a highly sensitive and specific ancillary marker that distinguishes DEK-AFF2 carcinoma from the other sinonasal tumors with overlapping morphological features and may be an especially useful alternative for decalcified specimens.

Unclassified Neuroendocrine Tumor with a Novel CHD4::AFF2 Fusion: Expanding the Family of AFF2-Rearranged Head and Neck Malignancies.

The past decade has seen a dramatic increase in the number of new head and neck tumor entities, most of which are genetically defined. DEK::AFF2 carcinoma is one of the most recently defined neoplasms; it shows a non-keratinizing squamous morphology and occurs in the sinonasal region. We present an unusual neoplasm that was found to harbor a novel fusion involving AFF2. The case was encountered in our clinical practice. Immunohistochemistry was performed along with targeted next generation sequencing (NGS). The case presented as a metastasis to a cervical lymph node from an unknown primary, in a 49-year-old man. The tumor consisted of sheets of primitive round cells which were strongly positive for synaptophysin and chromogranin but negative for cytokeratins, S-100 protein, WT-1, desmin, and many other markers. NGS uncovered CHD4::AFF2. We found a CHD4::AFF2 fusion in a high-grade neuroendocrine tumor. Although it is just a single case, the presence of a novel fusion in a neoplasm that is otherwise not classifiable suggests that it could be a distinct entity within a possible family of AFF2-rearranged tumors. Molecular analysis should be considered for any unclassified round cell tumor in the head and neck, as additional cases will be needed to further elucidate this area.

Comparison of gene fusion detection methods in salivary gland tumors.

Salivary gland neoplasms may pose diagnostic difficulties due to overlapping morphologic features. Recently, specific gene fusions have been discovered that correspond to particular tumor types, and can aid in accurate diagnosis. Gene rearrangements are commonly assessed by fluorescence in situ hybridization (FISH), although use of next-generation sequencing is increasing. However, there is no "gold standard" for fusion detection. We determined the concordance between FISH and a targeted RNA sequencing panel in gene fusion detection across twenty-two salivary gland tumors, including five mucoepidermoid carcinomas, four acinic cell carcinomas, four pleomorphic adenomas, two adenoid cystic carcinomas, two NUT carcinomas, and one each of basal cell adenoma, salivary duct carcinoma ex-pleomorphic adenoma, salivary duct carcinoma, clear cell carcinoma, and secretory carcinoma. Directed FISH testing based on the diagnosis was performed on cases that did not already have FISH conducted during clinical workup. Targeted RNA sequencing of 507 genes and their partners (using the Illumina TruSight Fusion Panel) was completed. Six of twenty-two (27.3%) cases had discordant results. In three cases, FISH results were negative while RNA sequencing results found fusion transcripts, which were all confirmed with RT-PCR and Sanger sequencing. In three cases, RNA sequencing results were negative while FISH results were positive for a gene rearrangement. Thus, if fusion analysis results are conflicting with the morphologic impression, a second mode of fusion detection may be warranted. Although both methods have advantages and drawbacks, RNA sequencing provides additional information about novel fusion partners and fusions that may not have been originally considered.

Impact of human papillomavirus on the tumor microenvironment in oropharyngeal squamous cell carcinoma.

Increasing evidence has elucidated the clinicopathological significance of tumor microenvironment (TME) cells. However, TME differences associated with human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC) have not been well characterized. In our study, we comprehensively determined the TME infiltration patterns in 315 OPSCC patients, and systematically correlated the TME phenotypes with genomic characteristics and clinical features of OPSCCs. In this way, we observed the enrichment of high endothelial cells and adaptive immune cells in HPV-positive (HPV+) OPSCCs, in contrast to the enrichment of fibroblasts and capillary endothelial cells in HPV- negative (HPV-) OPSCCs. By focusing on immune checkpoint genes, we constructed a coexpression network using genes that were differentially expressed between HPV+ and HPV- OPSCCs. Functional analysis of the network indicated that HPV+ OPSCCs had elevated immune activities by promoting adaptive immune response and suppressing activities related to extracellular matrix organization. Subsequently, clinical analysis showed that identified TME-relevant genes were closely associated with the prognosis and therapy response in OPSCC. Importantly, results from the TME analysis were further validated using an independent OPSCC cohort.

Computed Tomography as a Predictor of Sinonasal Inverted Papilloma Origin, Skull Base Involvement, and Stage.

Objective To investigate the diagnostic performance of computed tomography (CT) to determine the origin, skull base involvement, and stage of sinonasal inverted papilloma (IP). Design This is a retrospective cohort study. Setting This is set at a tertiary care medical center. Participants Patients with preoperative CT imaging who underwent extirpative surgery for histologically confirmed sinonasal IP between January 2005 and October 2019. Main Outcome Measures The likely sites of tumor origin, skull base involvement, and radiographic tumor stage were determined by two board-certified neuroradiologists after re-reviewing preoperative CT imaging. These radiologic findings were then compared with intraoperative and pathologic findings. Results Of 86 patients, 74% (64/86) had IP lesions with correctly classified sites of origin on CT. CT was not sensitive for diagnosing ethmoid sinus origin (48%, 52%), frontal sinus origin (80%, 40%), and skull base origin (17%, 17%). CT was not sensitive (62%, 57%) but specific (86%, 98%) for identifying any skull base involvement. There was substantial-to-near perfect agreement between radiographic and pathologic Cannady stages (weighted κ = 0.61 for rater 1; weighted κ = 0.81 for rater 2). Interrater agreement was substantial for identifying tumor origin (κ = 0.75) and stage (weighted κ = 0.62) and moderate for identifying skull base involvement (κ = 0.43). Conclusion Interrater agreement on CT findings was substantial except on skull base involvement. CT correctly predicted site of tumor origin in up to 74% of subjects. CT was not sensitive for diagnosing skull base involvement but had substantial-to-near perfect agreement with pathologic tumor staging. CT is a useful but albeit limited adjunct for tumor localization and surgical planning for sinonasal IP.

DEK-AFF2 fusion-associated papillary squamous cell carcinoma of the sinonasal tract: clinicopathologic characterization of seven cases with deceptively bland morphology.

A novel DEK-AFF2 fusion has been recently identified in four cases of basaloid to nonkeratinizing squamous cell carcinoma (SCC) in the sinonasal tract and middle ear with high-grade morphology. The exceptional response to immune checkpoint inhibitor in the first reported case highlights the potential clinical importance of identifying tumors with DEK-AFF2 fusions. We herein reported the first series of seven cases of DEK-AFF2 fusion-associated sinonasal SCC with deceptively bland morphology, including four cases of low-grade papillary Schneiderian carcinoma, which is a recently described tumor type with unknown molecular underpinnings. The DEK gene rearrangement was confirmed by DEK break-apart fluorescence in situ hybridization and DEK-AFF2 fusion transcripts were detected by reverse transcription polymerase chain reaction. In contrast to the previously reported DEK-AFF2 fusion-positive high-grade carcinomas, these tumors had a monotonous and bland morphology and were all initially diagnosed as sinonasal papilloma (SP) of various types, with or without dysplasia or carcinoma in situ. The tumor was characterized by mixed exophytic and inverted patterns, broad papillary fronds, acantholytic change, cellular monotony, dense neutrophilic infiltrates, and peripheral palisading. All tumors were diffusely positive for p40 or p63 and negative for NUT and p16. Molecular drivers associated with SP, including EGFR and KRAS mutations and both high and low-risk human papillomavirus infection, were negative in all cases. Although there was no overt stromal invasion or desmoplastic reaction in the initial specimens, these tumors tended to progress locoregionally through a prolonged clinical course and occasionally develop lymph node metastases, high-grade transformation, or extensively local destruction eventually leading to death. These justify more aggressive clinical management. Therefore, we propose the new terminology "DEK-AFF2 fusion-associated papillary SCC of the sinonasal tract" to better describe this clinicopathologically and molecularly distinct entity.

Assessment Criteria and Clinical Implications of Extranodal Extension in Head and Neck Cancer.

Tumor breaching the capsule of a lymph node is termed extranodal extension (ENE). It reflects aggressiveness of a tumor, creates anatomic challenges for disease clearance, and increases the risk of distant metastasis. Extranodal extension can be assessed on a pathology specimen, by radiology studies, and by clinical examination. Presence of ENE in a pathology specimen has long been considered a high-risk feature of disease progression and would ordinarily benefit from the addition of chemotherapy to adjuvant radiotherapy. Although the eighth edition of the Union for International Cancer Control/American Joint Committee on Cancer stage classification dichotomizes pathologic ENE according to its presence or absence, emerging evidence suggests that the extent of a pathologic ENE may provide additional value for risk stratification to guide adjuvant therapy. Recent data suggest that the prognostic importance of pathologic ENE is also applicable for HPV-associated head and neck squamous cell carcinoma. In addition, compelling data demonstrate that indisputable radiologic ENE is a powerful risk stratification tool to identify patients at high risk for treatment failure, especially distant metastasis, applicable for both HPV-positive and HPV-negative head and neck squamous cell carcinoma. However, the definition and taxonomy of radiologic ENE requires standardization. The goal of this review is to clarify the contemporary understanding of the prognostic implications of ENE in head and neck squamous cell carcinoma, present the nuances of what is presently known and unknown, and elucidate how to classify ENE pathologically and radiologically with an understanding of the strengths and weaknesses of each approach. Finally, with the development of several risk stratification methods, the relative role of ENE and other prognostic schema will be explored.

Low Grade Papillary Sinonasal (Schneiderian) Carcinoma: A Series of Five Cases of a Unique Malignant Neoplasm with Comparison to Inverted Papilloma and Conventional Nonkeratinizing Squamous Cell Carcinoma.

There have been a few case reports and one small series of low grade papillary sinonasal (Schneiderian) carcinomas (LGPSC) which mimic papillomas but have overtly invasive growth and which occasionally metastasize. We describe the morphologic, clinical, immunohistochemical, and molecular features of five patients with LGPSC compared with eight cases each of inverted papilloma (IP) and conventional nonkeratinizing squamous cell carcinoma (SCC) with papillary growth. All LGPSC were nested with predominantly pushing invasion, no stromal reaction, and frequent surface papillary growth. All consisted of one cell type only, with polygonal cells with round nuclei, no (or limited) cytologic atypia, low mitotic activity, and prominent neutrophilic infiltrate. One patient had slightly more infiltrative bone invasion, another lymphovascular, perineural, and skeletal muscle invasion, and a third nodal metastasis after 17 years. By comparison, IPs had bland cytology, neutrophilic microabscesses, mixed immature squamous, goblet cell, and respiratory epithelium, and extremely low mitotic activity. Nonkeratinizing SCCs had basaloid-appearing cells with nuclear pleomorphism, brisk mitotic activity, and apoptosis. All LGPSC were p63 positive. Mitotic activity and Ki67 indices were significantly higher for LGPSCs than IPs and significantly lower than NKSCCs, while p53 immunohistochemistry in LGPSC was identical to nonkeratinizing SCC and higher than for IP. Sequencing showed all five tumors to harbor a MUC6 mutation, one tumor to harbor CDKN2A and PIK3R1 mutations, and one tumor to harbor a NOTCH1 mutation. All LGPSC lacked EGFR and KRAS mutations and lacked copy number variations of any main cancer genes. At a median follow up of 12 months, two LGPSC recurred locally, and one patient died after massive local recurrences and nodal metastases. LGPSC is a distinct, de novo sinonasal carcinoma that can be differentiated from papillomas by morphology and selected immunohistochemistry.

Oropharyngeal Squamous Cell Carcinoma With Discordant p16 and HPV mRNA Results: Incidence and Characterization in a Large, Contemporary United States Cohort.

Early studies estimate that 5% to 10% of oropharyngeal squamous cell carcinomas overexpress p16 but are unassociated with transcriptionally-active high-risk human papillomavirus (HPV). Patients with discordant HPV testing may experience clinical outcomes that differ from traditional expectations. To document the rate of p16 and HPV mRNA positivity, characterize patients with discordant testing, and identify features that may warrant selective use of HPV-specific testing after p16 IHC, a multi-institutional, retrospective review of oropharyngeal squamous cell carcinoma patients with p16 IHC and HPV mRNA testing by reverse transcriptase polymerase chain reaction was performed. Of the 467 patients, most had T1 or T2 tumors (71%), 82% were p16 positive, and 84% were HPV mRNA positive. Overall, most tumors were nonkeratinizing (378, 81%), which was strongly associated with p16 and HPV positivity (93% and 95%, respectively). Overall, 81% of patients were double positive, 14% double negative, and 4.9% discordant (3.4% p16 negative/HPV mRNA positive and 1.5% p16 positive/HPV mRNA negative). The survival rates of these discordant patient groups fell squarely between the 2 concordant groups, although in multivariate analysis for both disease-free survival and overall survival, discordant patients were not found to have statistically significantly different outcomes. Reclassifying patients by applying HPV mRNA testing when p16 results and morphology do not match, or when p16 results are equivocal, improved prognostication slightly over p16 or HPV mRNA testing alone. Patients with discordant testing demonstrate a borderline significant trend toward survival differences from those with concordant tests. When evaluated independently, patients who were p16 negative but HPV mRNA positive had a prognosis somewhat closer to double-positive patients, while those who were p16 positive, but HPV mRNA negative had a prognosis closer to that of double-negative patients. We suggest an algorithm whereby confirmatory HPV mRNA testing is performed in patients where p16 status is not consistent with tumor morphology. This captures a majority of discordant patients and improves, albeit modestly, the prognostication.

Acute Invasive Fungal Sinusitis: A 30-Year Review of Pathology Practice and Possible Utility of the DiffQuik® Stain.

Acute invasive fungal rhinosinusitis (AIFRS) is a fulminant disease with a high mortality rate. Here, we review the clinical and pathologic features of AIFRS over a 30-year period at a tertiary-care academic center focusing on diagnostic practice, especially the use of intra-operative consultation (IOC). A 1-year trial of intraoperative staining with DiffQuik® (DQ) was also assessed. There were 202 cases from 104 patients with AIFRS. The incidence of AIFRS increased over time (6.8 per year in the past decade versus 1.9 before 2009) as did the number of IOCs per case (2.4 per case in the past decade versus 0.6 before 2009). Disagreement between final diagnosis and IOC was seen in 8.3 % of patients, and the block-by-block error rate was 14.9 %. Fusarium was the most common fungus identified. An attempt to categorize the fungal organism based on histopathology was performed in 85.6 % of patients, with 91 % agreement with microbial cultures or PCR. Fungal hyphae were subjectively easier to identify on DQ stained cryostat sections compared to on routine hematoxylin and eosin stained sections and hyphae were identified in all 5 blocks in which organisms were present. The increasing incidence of AIFRS and expanding use of IOC indicate a need to improve and standardize the diagnostic protocol. The use of DQ as visual aid in IOC for AIFRS may be useful.