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GLI1 is a transcription factor protein that has recently gained recognition in a morphologically distinct group of epithelioid soft tissue tumors characterized by GLI1 fusions or amplifications. The head and neck region, particularly the tongue, is a common location for GLI1-altered tumors. DDIT3 break apart fluorescence in situ hybridization (FISH), commonly used to identify translocations in myxoid/round cell liposarcoma, has been used as a surrogate test to detect both fusions and amplifications of the 12q13.3 region encompassing DDIT3 and GLI1 gene loci.We herein report 5 cases of GLI1-altered soft tissue tumors. Three arose in the oropharynx (base of tongue/vallecula, tonsil) and two arose in the tongue. Given the frequent oropharyngeal location and epithelioid morphology, p16 immunohistochemistry was performed on cases with available material. Commercially available DDIT3 break apart FISH, custom GLI1 specific FISH, and RNA sequencing were performed on select cases.Two cases showed amplification using DDIT3 FISH which was confirmed using GLI1 specific FISH. The remaining cases harbored ACTB::GLI1, one of which showed rearrangement of the 12q13.3 region by DDIT3 FISH with absence of amplification by GLI1 specific FISH. STAT6 immunoexpression was positive in the GLI1-amplified cases and negative in the GLI1-rearranged cases while MDM2 expression was positive in the 4 cases tested. CDK4 expression was strong and diffuse in the GLI1-amplified cases. p16 immunohistochemistry showed strong nuclear and cytoplasmic staining in 50-70% of tumor cells in all four tested cases.Here we show that GLI1-altered soft tissue tumors are frequently positive for p16 and can occur in tonsillar regions of the oropharynx. As such, positive p16 immunohistochemistry alone cannot be used as evidence for the diagnosis of HPV-related squamous cell carcinoma as strong and diffuse p16 expression may also occur in GLI1-altered soft tissue tumors. Commercially available DDIT3 break apart FISH, which is readily available in many cytogenetic laboratories, may be useful as a sensitive surrogate test for GLI1 fusions and amplifications.

Supplementary data

Representative video is shown of open-field imaging during intraoperative neck dissection from the 25mg/m2 dose cohort.

Purpose: Comprehensive cervical lymphadenectomy can be associated with significant morbidity and poor quality of life. This study evaluated the sensitivity and specificity of cetuximab-IRDye800CW to identify metastatic disease in patients with head and neck cancer.Experimental Design: Consenting patients scheduled for curative resection were enrolled in a clinical trial to evaluate the safety and specificity of cetuximab-IRDye800CW. Patients (n = 12) received escalating doses of the study drug. Where indicated, cervical lymphadenectomy accompanied primary tumor resection, which occurred 3 to 7 days following intravenous infusion of cetuximab-IRDye800CW. All 471 dissected lymph nodes were imaged with a closed-field, near-infrared imaging device during gross processing of the fresh specimens. Intraoperative imaging of exposed neck levels was performed with an open-field fluorescence imaging device. Blinded assessments of the fluorescence data were compared to histopathology to calculate sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV).Results: Of the 35 nodes diagnosed pathologically positive, 34 were correctly identified with fluorescence imaging, yielding a sensitivity of 97.2%. Of the 435 pathologically negative nodes, 401 were correctly assessed using fluorescence imaging, yielding a specificity of 92.7%. The NPV was determined to be 99.7%, and the PPV was 50.7%. When 37 fluorescently false-positive nodes were sectioned deeper (1 mm) into their respective blocks, metastatic cancer was found in 8.1% of the recut nodal specimens, which altered staging in two of those cases.Conclusions: Fluorescence imaging of lymph nodes after systemic cetuximab-IRDye800CW administration demonstrated high sensitivity and was capable of identifying additional positive nodes on deep sectioning. Clin Cancer Res; 23(16); 4744–52. ©2017 AACR.

MEIS1::NCOA1/2 sarcomas are a newly recognized group of exceedingly rare low-grade spindle cell sarcomas that often involve the genitourinary and gynecologic tracts. Due to its deceptively low-grade morphology and the non-specific immunoprofile, these neoplasms may pose a diagnostic challenge by histologically mimicking other entities such as endometrial stromal sarcoma, smooth muscle tumor, or uterine perivascular epithelioid cell tumor (PEComa). Histologically, MEIS1::NCOA1/2 sarcomas typically show spindle cell proliferation with hyperchromatic nuclei and a generalized cytologic uniformity, arranged in short fascicles and exhibiting alternating zones of hypo- and hypercellularity. Among the previously reported cases, molecular analysis revealed the MEIS1::NCOA2 fusion as the most commonly detected fusion gene, whereas the MEIS1::NCOA1 fusion gene has been reported in only a single case that involved kidney. Herein we report the first case of uterine sarcoma harboring the MEIS1::NCOA1 fusion gene that was initially misclassified as low-grade endometrial stromal sarcoma, demonstrating its clinicopathologic features, and highlighting the essential role of molecular pathology to arrive at the accurate diagnosis that may alter disease classification and inform therapy.

Nuclear protein of testis (NUT), a protein product of the NUTM1 gene (located on the long arm of chromosome 15) with highly restricted physiologic expression in post-meiotic spermatids, is the oncogenic driver of a group of emerging neoplasms when fused with genes involved in transcription regulation. Although initially identified in a group of lethal midline carcinomas in which NUT forms fusion proteins with bromodomain proteins, NUTM1-rearrangement has since been identified in tumors at non-midline locations, with non-bromodomain partners and with varied morphology. The histologic features of these tumors have also expanded to include sarcoma, skin adnexal tumors, and hematologic malignancies that harbor various fusion partners and are associated with markedly different clinical courses varying from benign to malignant. Most of these tumors have nondescript primitive morphology and therefore should be routinely considered in any undifferentiated neoplasm. The diagnosis is facilitated by the immunohistochemical use of the monoclonal C52 antibody, fluorescence in situ hybridization (FISH), and, recently, RNA-sequencing. The pathogenesis is believed to be altered expression of oncogenes or tumor suppressor genes by NUT-mediated genome-wide histone modification. NUTM1-rearranged neoplasms respond poorly to classical chemotherapy and radiation therapy. Targeted therapies such as bromodomain and extraterminal domain inhibitor (BETi) therapy are being developed. This current review provides an update on NUTM1-rearranged neoplasms, focusing on the correlation between basic sciences and clinical aspects.

NUTM1 gene rearrangements were originally identified in NUT carcinoma. Recently, NUTM1 has been discovered to rearrange with a variety of gene partners in malignancies of diverse location and type. Only one NUTM1-rearranged tumor occurring in the colon has been reported. Herein we report five such tumors. The five tumors occurred in four females and one male, ranging from 38 to 67 years of age (median 51 years). The masses occurred in the colon (cecum, descending, sigmoid) and ileocecal valve region, measuring 2.5-20 cm in size (median 7 cm). Four patients had metastases at presentation (liver, n = 4; lymph nodes, n = 3). Histologically, the lesions arose in the submucosa, infiltrating into the mucosa and muscularis propria, and grew in fibrosarcoma-like fascicles and sheets of epithelioid or rhabdoid cells, with foci of hyalinized to vaguely osteoid-like matrix. The tumors were composed of relatively monomorphic, spindled to epithelioid cells with focal rhabdoid morphology, hyperchromatic nuclei, and small nucleoli. Mitotic activity was usually low (range 1-14/10 HPF; median 5/10 HPF); necrosis was present in two cases. Variable keratin expression and uniform nuclear NUT expression was present; KIT/DOG1 were negative and SMARCB1/SMARCA4 were retained. Next-generation sequencing identified MXD4-NUTM1 rearrangement in all cases (breakpoints: MXD4 exon 5, NUTM1 exons 2 or 3). Follow-up showed one of the four patients who presented with metastases to be dead of disease at 30 months; the other three patients were alive with metastatic disease. The final patient is disease-free, 5 months after diagnosis. NUTM1-rearranged colorectal sarcomas have characteristic morphologic, immunohistochemical, and molecular genetic features, suggesting that they represent a distinct entity within the family of NUTM1-rearranged neoplasia. A NUTM1-rearranged tumor should be considered for any difficult-to-classify submucosal spindle cell neoplasm of the gastrointestinal tract, in particular keratin-positive tumors showing an unusual combination of fibrosarcomatous, epithelioid to rhabdoid and hyalinized morphologies. Recognition of MXD4-NUTM1 rearranged sarcomas may be therapeutically important, even though best treatment is currently elusive/unknown.

Background Adnexal skin tumors are diagnostically challenging with few known molecular signatures. Recently, however, YAP1-MAML2 and YAP1-NUTM1 fusions were identified in poroid adnexal skin tumors. Methods Herein, we subjected eight poroid adnexal skin tumors (three poromas and five porocarcinomas) to fusion gene analysis by whole transcriptome sequencing and next-generation DNA sequencing analysis. Results YAP1 fusions were identified in six cases. YAP1-NUTM1 fusions were identified in two poromas and three porocarcinomas. A single case of porocarcinoma harbored a YAP1-MAML2 fusion. Two cases were negative for gene fusion. All cases that harbored YAP1-NUTM1 fusions showed nuclear protein in testis (NUT) expression by immunohistochemistry, with NUT being negative in the YAP1-MAML2-positive case. In this case series, we provide a detailed histopathologic description of six YAP1-fused poroid skin tumors, which we show harbor reproducible histopathologic features, to include broad, bulbous tumor tongues with admixtures of basaloid, poroid cells punctuated by squamatized cuticles and ductules, with uniform tumor nuclei featuring frequent grooves and pseudonuclear inclusions. Conclusions Awareness of the characteristic histopathologic features of YAP1-fused poroid adnexal skin tumor is a step toward a more reproducible classification of adnexal skin tumors as well as a step toward targeted therapy for metastatic and/or unresectable examples of this poroid group of neoplasms.

Purpose Fluorescently labeled epidermal growth factor receptor (EGFR) antibodies have successfully identified microscopic tumors in multiple in vivo models of human cancers with limited toxicity. The present study sought to demonstrate the ability of fluorescently labeled anti-EGFR, cetuximab-IRDye800, to localize to ameloblastoma (AB) tumor cells in vitro and in vivo. Material and Methods EGFR expression in AB cells was confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Primary AB cells were labeled in vitro with cetuximab-IRDye800 or nonspecific IgG-IRDye800. An in vivo patient-derived xenograft (PDX) model of AB was developed. The tumor tissue from 3 patients was implanted subcutaneously into immunocompromised mice. The mice received an intravenous injection of cetuximab-IRDye800 or IgG-IRDye800 and underwent imaging to detect infrared fluorescence using a Pearl imaging system (LI-COR Biosciences, Lincoln, NE). After resection of the overlying skin, the tumor/background ratios (TBRs) were calculated and statistically analyzed using a paired t test. Results EGFR expression was seen in all AB samples. Tumor-specific labeling was achieved, as evidenced by a positive fluorescence signal from cetuximab-IRDye800 binding to AB cells, with little staining seen in the negative controls treated with IgG-IRDye800. In the animal PDX model, imaging revealed that the TBRs produced by cetuximab were significantly greater than those produced by IgG on days 7 to 14 for AB-20 tumors. After skin flap removal to simulate a preresection state, the TBRs increased with cetuximab and were significantly greater than the TBRs with the IgG control for PDX tumors derived from the 3 patients with AB. The excised tissues were embedded in paraffin and examined to confirm the presence of tumor. Conclusions Fluorescently labeled anti-EGFR demonstrated specificity for AB cells and PDX tumors. The present study is the first report of tumor-specific, antibody-based imaging of odontogenic tumors, of which AB is one of the most clinically aggressive. We expect this technology will ultimately assist surgeons treating AB by helping to accurately assess the tumor margins during surgery, leading to improved long-term local tumor control and less surgical morbidity.

The archetypal solitary fibrous tumor (SFT) features fibroblastic cells with varying cellularity without any particular architectural arrangement in a collagenous matrix, with staghorn vessels, CD34 and STAT6 expression, and NAB2::STAT6. To date, this fusion is thought to be specific for SFT. With more routine use of fusion gene panels, the histologic diversity of NAB2::STAT6-positive tumors is increasingly appreciated. Here we present four head and neck tumors harboring NAB2::STAT6 but exhibiting remarkably unusual morphologic features for SFT. All cases were pulled from the authors’ consultation files. Immunohistochemistry was performed, along with targeted RNA sequencing in all cases, plus DNA next-generation sequencing on two. The cases arose in the nasal cavity (n = 2), retromolar trigone (n = 1) and parapharynx (n = 1), in patients ranging from 39 to 54 (mean, 44). Both nasal cases were biphasic, with a variably cellular collagenized stroma that resembled SFT but also interspersed malignant epithelial and neuroepithelial nests. One of the nasal cases also exhibited overt rhabdomyoblastic differentiation within both components. The two non-nasal cases were comprised of plump, epithelioid cells that were diffusely positive for pan-cytokeratin. One of these cases had prominent cystic change lined by overtly squamous epithelium. STAT6 immunostaining was positive in all cases, although the epithelial/neuroepithelial nests in the sinonasal cases were negative. All cases were confirmed to harbor NAB2::STAT6 by RNA sequencing. The two sinonasal cases were also found to harbor oncogenic mutations. The presented cases highlight a much broader histologic diversity than previously known for neoplasms with NAB2::STAT6. The biphasic nasal cases closely resemble teratocarcinosarcoma, while the epithelioid, cytokeratin-positive cases could be conceptualized as “adamantinoma-like,” to borrow terminology already in use for Ewing sarcomas with complex epithelial differentiation. To identify similar cases, pathologists should have a low threshold for using STAT6 immunohistochemistry on any difficult-to-characterize head and neck tumor.

Although low-grade non-intestinal-type sinonasal adenocarcinoma (SNAC) is formally a diagnosis of exclusion defined by the absence of salivary or intestinal differentiation, most tumors in this category comprise a distinctive histologic group that are increasingly thought to derive from seromucinous glands. However, the molecular underpinnings of SNAC remain poorly understood, and it is unclear if diverse genetic alterations recently reported in isolated cases should delineate separate subgroups. This study aims to perform comprehensive evaluation of gene fusions and mutations and their histologic correlates in low-grade SNAC to clarify its pathogenesis and classification. We identified 18 non-intestinal-type SNAC that all displayed characteristic tubulopapillary architecture and low-grade cytology, although several cases had other unique histologic features and 3 showed intermixed high-grade areas. Among tumors stained with S100 protein, SOX10, and DOG1, 86% expressed at least one of these seromucinous markers. Of 17 cases with sufficient RNA or DNA available for analysis, likely oncogenic molecular alterations were identified in 76% of cases, most notably including CTNNB1 p.S33F mutations in 2 cases, concomitant BRAF p.V600E and AKT1 p.E17K mutations in 2 cases, and ETV6::NTRK3, PRKAR1A::MET, FN1::NRG1, and DNAJB1::PRKACA fusions in 1 case each. While tumors with most genetic alterations were histologically indistinguishable, cases with CTNNB1 mutations had intermixed squamoid morules and cases with BRAF and AKT1 mutations showed a myoepithelial cell population and prominent papillary to micropapillary architecture. Overall, these findings confirm previous reports of frequent seromucinous differentiation in low-grade SNAC. However, these tumors display striking molecular diversity with involvement of multiple kinase fusions, leading to frequent activation of signaling cascades including the MAPK pathway. While most genetic alterations are not associated with sufficiently distinctive histologic features to suggest separate classification, biphasic tumors with BRAF p.V600E mutations are more unique and may represent a distinctive subgroup.

Spindle cell squamous cell carcinoma (SpC-SCC) is rare, accounting for 0.4%–4% of head and neck (HN) SCCs. Better understanding of HN SpC-SCC clinicopathologic characteristics, especially features that predict outcome, is needed. We present a clinicopathologic review of 71 HN mucosal SpC-SCC from three tertiary centers. The patient population showed a median age of 63 years (range 20–91), slight male predominance (M:F=1.6:1) and a preponderance of smokers/ex-smokers (45/71, 64%). Most lesions involved oral cavity (42/71, 59%), especially oral tongue (n=18), and larynx (n=20, 28%). Polypoid/exophytic growth and surface ulceration were seen in 60% and 86% of cases, respectively. Histologically, most tumors showed sarcoma-like pattern (65/70, 93%), the remaining exhibiting granulation tissue-like or fibromatosis-like patterns, 5 lesions showed osteosarcomatous/chondrosarcomatous elements. Most tumors (53/71, 74%) showed a conventional SCC (C-SCC) component, keratinizing (86%) or non-keratinizing/basaloid (14%). Nodal metastases, seen in 22 (31%) of resection specimens, showed SpC-SCC and/or C-SCC histomorphology. By immunohistochemistry, 76% of lesions showed immunoreactivity for Keratin and 62%/60% of lesions were p40/p63 positive. Ki-67 proliferation index ranged from 5% to 70%. Follow-up was available on 69 patients, median of 1.1 years from the time of SpC-SCC diagnosis. The 3-year, 5-year, and 10-year disease specific survival (DSS) was 62%, 37%, and 12%, respectively. AJCC pN stage was an independent prognostic factor for DSS and distant metastasis free survival (DMFS), whereas the presence of C-SCC was independently associated with improved DMFS. HN SpC-SCC is rare and might be diagnostically challenging. AJCC pN stage and co-existing C-SCC component appear to be prognostically relevant.

Poorly differentiated neoplasms lacking characteristic histopathologic features represent a significant challenge to the pathologist for diagnostic classification. Classically, NUT carcinoma (previously NUT midline carcinoma) is poorly differentiated but typically exhibits variable degrees of squamous differentiation. Diagnosis is genetically defined by NUTM1 rearrangement, usually with BRD4 as the fusion partner. In this multi-institutional next-generation sequencing and fluorescence in situ hybridization study, 26 new NUTM1-rearranged neoplasms are reported, including 20 NUT carcinomas, 4 sarcomas, and 2 tumors of an uncertain lineage. NUTM1 fusion partners were available in 24 of 26 cases. BRD4 was the fusion partner in 18/24 (75%) cases, NSD3 in 2/24 cases (8.3%), and BRD3 in 1/24 (4.2%) cases. Two novel fusion partners were identified: MGA in two sarcomas (myxoid spindle cell sarcoma and undifferentiated sarcoma) (2/24 cases 8.3%) and MXD4 in a round cell sarcoma in the cecum (1/24 cases 4.2%). Eleven cases tested for NUT immunoexpression were all positive, including the MGA and MXD4-rearranged tumors. Our results confirm that NUTM1 gene rearrangements are found outside the classic clinicopathological setting of NUT carcinoma. In addition, as novel fusion partners like MGA and MXD4 may not be susceptible to targeted therapy with bromodomain inhibitors, detecting the NUTM1 rearrangement may not be enough, and identifying the specific fusion partner may become necessary. Studies to elucidate the mechanism of tumorigenesis of novel fusion partners are needed.

PAX8 is used as a diagnostic aid in classifying retroperitoneal (RP) spindle cell tumors. PAX8 positivity in a spindled RP tumor is typically associated with sarcomatoid renal cell carcinoma (SRCC). However, PAX8 expression in solitary fibrous tumor (SFT), a tumor not uncommon to the RP, has not been extensively studied. We investigated the expression of PAX8 in SFTs and other spindle cell RP tumors. We collected 30 SFT, 23 SRCC, 11 gastrointestinal stromal tumors, 2 synovial sarcomas, 6 dedifferentiated liposarcomas (DDLS), 4 well differentiated liposarcomas (WDLS), and select other tumors. We identified nuclear PAX8 expression in 13 of 30 (43%) SFT, 0 of 6 (0%) DDLS, and 1 of 4 (25%) WDLS. Twenty-eight of 30 (93%) SFT, 0 of 23 (0%) SRCC, 2 of 6 (33%) DDLS, and 1 of 4 (25%) WDLS showed nuclear STAT6 expression. All gastrointestinal stromal tumors were negative for both PAX8 and STAT6. Of the 13 SFT showing PAX8 expression, 8 showed diffuse expression and 5 expressed PAX8 focally. Extrapleural SFTs were more likely to express PAX8 compared with pleural SFTs (10/13; 77% vs. 3/17; 18%, respectively; P=0.00117). Twenty of 23 (87%) SRCC expressed PAX8; the sarcomatoid component of all 23 SRCC was negative for STAT6. Of the other spindle cell tumors studied, 1 of 2 synovial sarcomas and 1 of 2 histiocytic sarcomas showed PAX8 expression. Pathologists should be aware of the potential pitfall of the relatively frequent expression of PAX8 by SFT and STAT6 expression in liposarcoma. PAX8 expression by a spindle cell lesion of RP would not allow distinction between SFT, SRCC, or sclerosing liposarcoma by itself. A STAT6/PAX8 phenotype excludes SRCC.

Adnexal skin tumors are diagnostically challenging with few known molecular signatures. Recently, however, YAP1-MAML2 and YAP1-NUTM1 fusions were identified in poroid adnexal skin tumors.Herein, we subjected eight poroid adnexal skin tumors (three poromas and five porocarcinomas) to fusion gene analysis by whole transcriptome sequencing and next-generation DNA sequencing analysis.YAP1 fusions were identified in six cases. YAP1-NUTM1 fusions were identified in two poromas and three porocarcinomas. A single case of porocarcinoma harbored a YAP1-MAML2 fusion. Two cases were negative for gene fusion. All cases that harbored YAP1-NUTM1 fusions showed nuclear protein in testis (NUT) expression by immunohistochemistry, with NUT being negative in the YAP1-MAML2-positive case. In this case series, we provide a detailed histopathologic description of six YAP1-fused poroid skin tumors, which we show harbor reproducible histopathologic features, to include broad, bulbous tumor tongues with admixtures of basaloid, poroid cells punctuated by squamatized cuticles and ductules, with uniform tumor nuclei featuring frequent grooves and pseudonuclear inclusions.Awareness of the characteristic histopathologic features of YAP1-fused poroid adnexal skin tumor is a step toward a more reproducible classification of adnexal skin tumors as well as a step toward targeted therapy for metastatic and/or unresectable examples of this poroid group of neoplasms.

We present a case of a malignant Ewing-like neoplasm of the parotid gland in a 20-year-old woman with an EWSR1-KLF15 gene fusion that presented with pulmonary metastasis. Despite the fact that the tumor was essentially immunohistochemically negative for keratins, p63, and p40, we interpret this neoplasm as an unusual form of a high-grade myoepithelial carcinoma based on its focal plasmacytoid cytology, chondromyxoid matrix, SOX10, S100 protein, and calponin expression, and the knowledge that the EWSR1-KLF15 gene fusion has, to date, only been identified in 2 tumors, both myoepithelial carcinomas of the kidney. We also present a cytogenetic analysis of this unusual tumor. This “Ewing-like myoepithelial carcinoma” initially did not respond to 2 cycles of ifosfamide and etoposide alternated with a cycle of cytoxan, adriamycin, and vincristine, a standard regimen for Ewing sarcoma. Subsequent oral pazopanib therapy did result in a reduction of the patient’s pulmonary and nodal disease.

Purpose: Comprehensive cervical lymphadenectomy can be associated with significant morbidity and poor quality of life. This study evaluated the sensitivity and specificity of cetuximab-IRDye800CW to identify metastatic disease in patients with head and neck cancer. Experimental Design: Consenting patients scheduled for curative resection were enrolled in a clinical trial to evaluate the safety and specificity of cetuximab-IRDye800CW. Patients (n = 12) received escalating doses of the study drug. Where indicated, cervical lymphadenectomy accompanied primary tumor resection, which occurred 3 to 7 days following intravenous infusion of cetuximab-IRDye800CW. All 471 dissected lymph nodes were imaged with a closed-field, near-infrared imaging device during gross processing of the fresh specimens. Intraoperative imaging of exposed neck levels was performed with an open-field fluorescence imaging device. Blinded assessments of the fluorescence data were compared to histopathology to calculate sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). Results: Of the 35 nodes diagnosed pathologically positive, 34 were correctly identified with fluorescence imaging, yielding a sensitivity of 97.2%. Of the 435 pathologically negative nodes, 401 were correctly assessed using fluorescence imaging, yielding a specificity of 92.7%. The NPV was determined to be 99.7%, and the PPV was 50.7%. When 37 fluorescently false-positive nodes were sectioned deeper (1 mm) into their respective blocks, metastatic cancer was found in 8.1% of the recut nodal specimens, which altered staging in two of those cases. Conclusions: Fluorescence imaging of lymph nodes after systemic cetuximab-IRDye800CW administration demonstrated high sensitivity and was capable of identifying additional positive nodes on deep sectioning. Clin Cancer Res; 23(16); 4744–52. ©2017 AACR.

Conventional acinic cell carcinoma (CACC) represents a prototypical low-grade salivary malignancy. Rarely, acinic cell carcinoma (ACC) can demonstrate aggressive features (zones of necrosis, apoptosis, varying nuclear atypia) warranting classification as "ACC with high-grade transformation" (HGT-ACC) or "dedifferentiated" ACC. This study reports ten new cases of HGT-ACC. There is potential for subtlety in recognizing high-grade transformation and distinguishing discrete nodules of necrosis from cytology aspiration changes. We compared immunohistochemical (IHC) profiles, specifically β-catenin (bCAT) and cyclin D1 expression, which have been touted as potentially helpful in this context. We quantified morphology (primary axis nucleus, nuclear area and perimeter) in HGT-ACC and CACC. Clinical outcome is known for eight HGT-ACC patients; three patients developed locoregional or distant metastases, five remained disease-free. Nine of ten HGT-ACC expressed strong, diffuse, membranous bCAT. CACC demonstrated lower intensity of membranous bCAT expression. Strong, diffuse nuclear cyclin D1 was seen in five of ten HGT-ACC whereas no CACC demonstrated cyclin D1 with distribution greater than 50 %. The quantified nuclear morphologic features of CACC and HGT-ACC demonstrated overlapping means values. Maximum values for nuclear primary axis, area, and perimeter were greater for HGT-ACC versus CACC, corresponding to a subpopulation of larger tumor cells in HGT-ACC. The poor outcome associated with HGT-ACC justifies its recognition, which should alter surgical approach with respect to elective neck dissection or possible facial nerve sacrifice. With respect to ancillary IHC studies, strong, diffuse membranous bCAT expression, with or without strong nuclear cyclin D1 ≥ 50 % distribution or Ki67 index ≥ 25 % supports this diagnosis.

Molecular diagnostics have become indispensable to the pathologist’s workup of salivary gland tumors and to the treating clinician. Molecular advances have led to the identification of previously unidentified entities and variants in salivary gland pathology and are increasingly used to help guide personalized therapy. In this chapter, the salient molecular signatures of salivary gland neoplasms will be presented as they relate to diagnosis and therapy.

Currently considered a variant of Ewing sarcoma, adamantinoma-like Ewing sarcoma is a rare malignancy that shows classic Ewing sarcoma-associated gene fusions but also epithelial differentiation. Here we present the 6th reported case of adamantinoma-like Ewing sarcoma involving the thyroid gland. Sections of the thyroid tumor from a 20-year old woman showed sheets, lobules and trabeculae of primitive, uniform, small round blue cells that diffusely expressed pankeratin, p40 and CD99. Fluorescent in situ hybridization revealed an EWSR1 gene rearrangement and an EWSR1-FLI1 fusion was detected by RT-PCR. Neck lymph nodes were not involved, and the patient was treated with a Ewing sarcoma chemotherapy protocol and radiation and is disease free 7 months after surgery. The unusual histology and immunohistochemical profile of adamantinoma-like Ewing sarcoma makes diagnosis and classification very challenging. We also present a literature review of adamantinoma-like Ewing sarcoma involving the thyroid.

Background With the increased use of molecular testing of thyroid fine-needle biopsies, the frequency and extent of thyroid resection for thyroid nodules has changed. Although the role of frozen-section analysis of the thyroid has been reduced markedly in recent years, many surgeons still routinely use it intraoperatively. We sought to determine the utility of frozen section during thyroidectomy in the era of molecular testing. Study Design We reviewed 236 consecutive patients who had thyroidectomy with intraoperative frozen-section analysis at our institution between November 2015 and October 2017. We re-reviewed the preoperative diagnosis, frozen-section diagnosis, final pathology, and whether operative management changed from the initial plan based on frozen section. Results Mean age of the patients was 55.6 ± 14.1 years, and 83% were female. Of the 236 patients, frozen section did not change the intraoperative management in 225 (95%). Of the 11 patients whose thyroid operation was modified, the operation was either too much or not enough in 6 patients. In only 5 (2.1%) patients, frozen-section analysis correctly changed the extent of thyroidectomy. Conclusions Thyroid frozen-section analysis adds cost and time to thyroid operations without notable benefit. In our cohort, only 2.1% of frozen sections accurately changed intraoperative management. We recommend against its routine use.

OBJECTIVE To discuss the current available techniques for intraoperative margin assessment in the surgical treatment of oral squamous cell carcinoma (OSCC) through a review of the available literature. METHODS A systematic review was undertaken of the available English literature between 2008 through 2018 regarding surgical margins in OCSS. A total of 893 relevant articles were returned; 144 met criteria for review; and 64 articles were included. RESULTS In this review, we discuss the data surrounding the use of frozen section in OCSS. Additionally, alternative techniques for margin assessment are discussed, including Mohs, molecular analysis, nonfluorescent dyes, fluorescent dyes, autofluorescent imaging, narrow-band imaging, optical coherence tomography, confocal microscopy, high-resolution microendoscopy, and spectroscopy. For each technique, particular emphasis is placed on the local recurrence, disease-free survival, and overall survival rates when available. CONCLUSION This review provides support for the practice of specimen-driven margin assessment when using frozen section analysis to improve the utility of the results. Finally, several alternatives for intraoperative margin assessment currently under investigation, including pathologic, wide-field imaging and narrow-field imaging techniques, are presented. We aim to fuel further investigation into methods for margin assessment that will improve survival for patients with OSCC through a critical analysis of the available techniques. LEVEL OF EVIDENCE NA Laryngoscope, 130:128-138, 2020.

Renal cell carcinoma (RCC) comprises a group of tumors that represent the most common malignant renal neoplasms. Several distinct subtypes of RCC exist, each characterized by unique morphologic, cytogenetic, molecular, and clinical characteristics. Aside from RCC, there are also several benign tumors that occur in the kidney, representing up to 20% of all renal cortical neoplasms. In this chapter we review the pathology of renal tumors.

Objective Compare human papillomavirus (HPV) status and outcomes in patients undergoing salvage surgical resection for a recurrent oropharyngeal squamous cell carcinoma (OPSCC). Methods Case series with chart review (2005–2013). Results Sixty-nine patients were identified who underwent salvage surgical resection for a recurrent OPSCC after primary radiation therapy. There was no difference in the incidence of HPV negative (52%; n = 36) and HPV positive (48%; n = 33) tumors. The mean time from completion of radiation therapy to salvage surgery was 2.4 years. At the time of salvage operation, there was no correlation with HPV status, as assessed by p16 immunohistochemistry, and lymph node metastases (p = 0.21), T classification (p = 0.22), tracheostomy dependence (p = 0.59), gastrostomy tube dependence (p = 0.82), or duration from radiation therapy (p = 0.63). The majority of patients were either current or former tobacco users (75%) and of the HPV positive patients, 66% were tobacco users. Development of a new recurrence after salvage surgical resection occurred in 33% of patients (n = 26), with a higher incidence in patients with HPV negative disease (52%, n = 17/33; p = 0.05). The overall 2- and 5-year survival rates were 0.47 and 0.23. There was no difference in overall survival rates when stratified by HPV status or tobacco use. Decreased overall 5-year survival rates did correlate with cervical lymph node metastases (p = 0.01), advanced tumor stage (p = 0.04) and dependence on gastrostomy tube postoperatively (p = 0.04). Conclusions This study found cervical lymph node metastases, clinical stage, and dependence on gastrostomy tube for nutrition to have the greatest impact on overall survival for patients with recurrent OPSCC.

The purpose of this study was to assess the potential of U.S. Food and Drug Administration-cleared devices designed for indocyanine green-based perfusion imaging to identify cancer-specific bioconjugates with overlapping excitation and emission wavelengths. Recent clinical trials have demonstrated potential for fluorescence guided surgery, but the time and cost of the approval process may impede clinical translation. To expedite this translation, we explored the feasibility of repurposing existing optical imaging devices for fluorescence-guided surgery. Methods: Consenting patients (n = 15) scheduled for curative resection were enrolled in a clinical trial evaluating the safety and specificity of cetuximab-IRDye800 (NCT01987375). Open-field fluorescence imaging was performed preoperatively and during the surgical resection. Fluorescence intensity was quantified using integrated instrument software, and the tumor-to-background ratio characterized fluorescence contrast. Results: In the preoperative clinic, the open-field device demonstrated potential to guide preoperative mapping of tumor borders, optimize the day of surgery, and identify occult lesions. Intraoperatively, the device demonstrated robust potential to guide surgical resections, as all peak tumor-to-background ratios were greater than 2 (range, 2.2-14.1). Postresection wound bed fluorescence was significantly less than preresection tumor fluorescence (P

Accurately identifying close or positive margins in real-time permits re-excision during surgical procedures. Intraoperative assessment of margins via gross examination and frozen section is a widely used tool to assist the surgeon in achieving complete resection. While this methodology permits diagnosis of freshly resected tissue, the process is fraught with misinterpretation and sampling errors. During fluorescence-guided surgery, an exogenous fluorescent agent specific for the target disease is imaged in order to navigate the surgical excision. As this technique quickly advances into the clinic, we hypothesize that the disease-specific fluorescence inherently contained within the resected tissues can be used to guide histopathological assessment. To evaluate the feasibility of fluorescence-guided pathology, we evaluated head and neck squamous cell carcinoma tumour specimens and margins resected from animals and patients after systemic injection of cetuximab-IRDye800CW. In a preclinical model of luciferase-positive tumour resection using bioluminescence as the gold standard, fluorescence assessment determined by closed-field fluorescence imaging of fresh resected margins accurately predicted the presence of disease in 33/39 positive margins yielding an overall sensitivity of 85%, specificity of 95%, positive predictive value (PPV) of 94%, and a negative predictive value (NPV) of 87%, which was superior to both surgical assessment (54%, 61%, 57%, and 58%) and pathological assessment (49%, 95%, 91%, and 66%), respectively. When the power of the technique was evaluated using human-derived tumour tissues, as little as 0.5mg (1mm(3)) of tumour tissue was identified (tumour-to-background-ratio:5.2). When the sensitivity/specificity of fluorescence-guided pathology was determined using traditional histological assessment as the gold standard in human tissues obtained during fluorescence-guided surgery, the technique was highly accurate with a sensitivity of 91%, specificity of 85%, PPV of 81%, and NPV of 93% for 90 human-derived samples. This approach can be used as a companion to the pathologist, eliminating confounding factors while impacting surgical intervention and patient management.

Conventional acinic cell carcinoma (CACC) represents a prototypical low-grade salivary malignancy. Rarely, acinic cell carcinoma (ACC) can demonstrate aggressive features (zones of necrosis, apoptosis, varying nuclear atypia) warranting classification as “ACC with high-grade transformation” (HGT-ACC) or “dedifferentiated” ACC. This study reports ten new cases of HGT-ACC. There is potential for subtlety in recognizing high-grade transformation and distinguishing discrete nodules of necrosis from cytology aspiration changes. We compared immunohistochemical (IHC) profiles, specifically β-catenin (bCAT) and cyclin D1 expression, which have been touted as potentially helpful in this context. We quantified morphology (primary axis nucleus, nuclear area and perimeter) in HGT-ACC and CACC. Clinical outcome is known for eight HGT-ACC patients; three patients developed locoregional or distant metastases, five remained disease-free. Nine of ten HGT-ACC expressed strong, diffuse, membranous bCAT. CACC demonstrated lower intensity of membranous bCAT expression. Strong, diffuse nuclear cyclin D1 was seen in five of ten HGT-ACC whereas no CACC demonstrated cyclin D1 with distribution greater than 50 %. The quantified nuclear morphologic features of CACC and HGT-ACC demonstrated overlapping means values. Maximum values for nuclear primary axis, area, and perimeter were greater for HGT-ACC versus CACC, corresponding to a subpopulation of larger tumor cells in HGT-ACC. The poor outcome associated with HGT-ACC justifies its recognition, which should alter surgical approach with respect to elective neck dissection or possible facial nerve sacrifice. With respect to ancillary IHC studies, strong, diffuse membranous bCAT expression, with or without strong nuclear cyclin D1 ≥ 50 % distribution or Ki67 index ≥ 25 % supports this diagnosis.

ETV6 gene abnormalities are well described in tumor pathology. Many fusion partners of ETV6 have been reported in a variety of epithelial, mesenchymal, and hematological malignancies. In salivary gland tumor pathology, however, the ETV6-NTRK3 translocation is specific for (mammary analog) secretory carcinoma, and has not been documented in any other salivary tumor type. The present study comprised a clinical, histologic, and molecular analysis of 10 cases of secretory carcinoma, with typical morphology and immunoprofile harboring a novel ETV6-RET translocation.

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a malignant biphasic neoplasm of the thyroid or neck with propensity for late metastasis. Unlike synovial sarcoma, its main morphologic mimic, SETTLE lacks synovial sarcoma-associated translocations. A single case of SETTLE has shown a KRAS mutation but to date no comprehensive next generation sequencing studies of this rare neoplasm have been undertaken. Herein, we subjected 5 well defined cases of SETTLE to direct sequence analysis of 592 genes and fusion gene analysis of 52 genes frequently rearranged in human cancers. We identified one case with two pathogenic variants in the KMT2D gene, one being in an intron splice site (c.674-1A>G) and the other being a frameshift variant (p.M2829fs). This same case also had a pathogenic nonsense variant in the KMT2C gene (p.R1237*). A second case of SETTLE carried a pathogenic NRAS missense variant, Q61R. No other molecular alterations, microsatellite instability, gene fusions or amplifications were identified. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12105-018-0927-1) contains supplementary material, which is available to authorized users.

Acute kidney injury (AKI) occurs commonly in patients with advanced cirrhosis and negatively impacts pre- and post-transplant outcomes. Physiologic changes that occur in patients with decompensated cirrhosis with ascites, place these patients at high risk of AKI. The most common causes of AKI in cirrhosis include prerenal injury, acute tubular necrosis (ATN), and the hepatorenal syndrome (HRS), accounting for more than 80% of AKI in this population. Distinguishing between these causes is particularly important for prognostication and treatment. Treatment of Type 1 HRS with vasoconstrictors and albumin improves short term survival and renal function in some patients while awaiting liver transplantation. Patients with HRS who fail to respond to medical therapy or those with severe renal failure of other etiology may require renal replacement therapy. Simultaneous liver kidney transplant (SLK) is needed in many of these patients to improve their post-transplant outcomes. However, the criteria to select patients who would benefit from SLK transplantation are based on consensus and lack strong evidence to support them. In this regard, novel serum and/or urinary biomarkers such as neutrophil gelatinase-associated lipocalin, interleukins-6 and 18, kidney injury molecule-1, fatty acid binding protein, and endothelin-1 are emerging with a potential for accurately differentiating common causes of AKI. Prospective studies are needed on the use of these biomarkers to predict accurately renal function recovery after liver transplantation alone in order to optimize personalized use of SLK.

We report a case of a 53-year-old woman who presented with rectal bleeding and a 9.5 cm hemicircumferential ascending colon mass. Histology revealed adenosquamous carcinoma (ASC), a rare subtype comprised of malignant squamous and glandular elements. Immunohistochemistry revealed loss of MLH1/PMS2 expression and retained MSH2/MSH6 expression in squamous and glandular components, indicative of microsatellite instability (MSI). MSI is caused by loss-of-function defects in DNA mismatch repair genes, leading to increased susceptibility to a variety of neoplasms; the role of MSI in colorectal ASC is unknown. The tumor was negative for MLH1 gene promoter hypermethylation, the patient had a germline MLH1 mutation, and met criteria for Lynch syndrome. To our knowledge this is the first report of a MSI-high colorectal carcinoma (CRC) showing adenosquamous histology. Further evaluation of MSI status in colorectal ASC may be warranted as this may be another histologic type of CRC associated with MSI.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma is a distinct tumor entity with clinical, epidemiologic, genetic, histologic, prognostic, and treatment differences from smoking- and alcohol-related head and neck squamous cell carcinoma. This is now well known by the pathology and medical community. What is not yet widely known is that several emerging variants of HPV-related carcinoma of the head and neck exist apart from the prototypical non-keratinizing morphology. Further, there is currently considerable variation in methodologies used and clinical scenarios in which to test for HPV-related head and neck squamous cell carcinoma, and no standard approach has emerged. In this article, we will review the morphology of prototypical HPV-related squamous cell carcinoma of the oropharynx and other HPV-related variants of head and neck carcinoma with an emphasis on their differential diagnosis, grade, and prognosis, as well as outline the current best practices for testing for HPV in head and neck carcinomas.

Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined.

The above article, published online in Wiley Online Library as the Version of Record on March 28, 2017 (doi 10.1002/hed.24754), has been retracted by agreement between the Editor-in-Chief, Ehab Y. Hanna, and Wiley Periodicals, Inc. The retraction has been agreed owing to a dispute as to authorship and inclusion of some data in the analysis.Velosa, C., Shi, Q., Stevens, T. M., Chiosea, S. I., Purgina, B., Carroll, W., Rosenthal, E., Morlandt, A., Loree, T. and Brandwein-Weber, M. S. (2017), Worst pattern of invasion and occult cervical metastases for oral squamous carcinoma. Head Neck. doi:10.1002/hed.24754.

Salivary gland-type tumors have been rarely described in the thyroid gland. Mammary Analog Secretory Carcinoma (MASC) is a recently defined type of salivary gland carcinoma characterized by a t(12;15)(p13;q25) resulting in an ETV6-NTRK3 fusion gene. We report 3 cases of MASC involving the thyroid gland without clinical evidence of a salivary gland or breast primary; the clinico-pathologic characteristics are reviewed. Assessment for rearrangement of the ETV6 (12p13) locus was conducted by fluorescence in situ hybridization (FISH) on representative FFPE sections using an ETV6 break apart probe (Abbott Molecular, Des Plaines, IL, USA). The patients were two females (52 and 55 years-old) and 1 male (74 years-old). The tumors were poorly circumscribed solid white tan nodules involving the thyroid. Histologically, they were invasive and showed solid, microcystic, cribriform, and tubular growth patterns composed of variably bland polygonal eosinophilic cells with vesicular nuclear chromatin and conspicuous nucleoli. All three cases showed metastasis to lymph nodes; one case showed lateral neck involvement. The tumor cells were positive for S100 and mammaglobin. GATA-3 and PAX-8 were positive in 2 cases, one of which only focally so. All three cases were negative for TTF-1 and thyroglobulin. Rearrangement of the ETV6 locus was confirmed in all cases and a diagnosis of MASC rendered for each case. A site of origin distinct from the thyroid gland was not identified, with a median follow up of 24 months. MASC may rarely involve the thyroid gland. The origin of these lesions is unknown; while an origin from ectopic salivary gland-type cells is entertained, a metastatic origin from an occult primary cannot be definitively excluded at this time. Given the histologic (follicular-like microcystic pattern with colloid-like secretions and papillary pattern), immunophenotypic (PAX-8), and even molecular overlap, MASC can be mistaken for papillary thyroid carcinoma and should be considered in the differential diagnosis of a thyroid mass.

To identify occult metastases within lymph nodes (LNs) reported as negative by routine histologic evaluation. In patients with high-grade, muscle-invasive urothelial carcinoma (UC) of the bladder, pelvic lymphadenectomy during radical cystectomy demonstrates a survival advantage, increasing with the number of LNs removed, even if negative for metastatic disease. This finding may potentially be explained by the presence of occult metastases.Radical cystectomy specimens with high-grade UC invading the perivesical tissue and negative LNs (pT3N0) between 2000 and 2014 were reviewed. Five levels were cut for each LN block. Two sections were cut per level: 1 stained for hematoxylin and eosin and 1 for AE1/AE3. Micrometastases were defined as tumor deposits0.2 mm but2 mm. Isolated tumor cells were defined as ≤0.2 mm. Medical records and survival data were reviewed.We identified 21 cases, consisting of 370 lymph nodes. Six of 21 patients (29%) had occult metastases, including 5 occult metastatic UC and 1 occult metastatic prostate adenocarcinoma. There were 10 positive LNs; 2 macrometastases, 2 micrometastases, and 6 with ITCs. Two of 6 patients (33%) had lymphovascular invasion identified in the primary tumor. Kaplan-Meier analysis showed no significant difference in overall survival between the group of patients who remained N0 versus those upstaged due to discovery of occult metastases (P-value = .42).In patients with pT3 UC undergoing cystectomy, we demonstrated the presence of occult metastases in 29% of patients. The high percentage of occult metastases present in these cases possibly explains the proven survival advantage of removing "negative" LNs. This finding might also have implications in the histologic evaluation of LNs.

Adenomatoid tumor is an uncommon benign neoplasm of mesothelial differentiation that distinctively arises in and around the genital organs. In rare instances, it has been described in extragenital locations. There have been only two reports documenting its occurrence in the anterior mediastinum, and no reports documenting its occurrence in the posterior mediastinum. We report the first case of posterior mediastinal adenomatoid tumor. A 37-year-old Caucasian woman presented with symptoms of bronchitis. Imaging studies identified a 2.0 cm posterior mediastinal mass abutting the T9 vertebral body, clinically and radiologically most consistent with schwannoma. Histologic sections revealed a lesion composed of epithelioid cells arranged in cords and luminal profiles embedded in a fibrotic to loose stroma and surrounded by a fibrous pseudocapsule. Lesional cells showed vacuolated eosinophilic cytoplasm and peripherally displaced nuclei with prominent nucleoli. There was focal cytologic atypia but no mitotic figures or necrosis was identified. The lesional cells expressed cytokeratin, calretinin, and nuclear WT1 but were negative for PAX8, TTF1, p53, chromogranin, CD31, and CD34, and Ki67 showed

Human papillomaviruses (HPVs) have been etiologically linked to a subset of head and neck squamous cell carcinomas (HNSCCs), generally arising in young patients without a history of tobacco smoking or alcohol use. These tumors typically lack mutations in TP53 and may show enhanced sensitivity to chemoradiation therapy with a correspondingly better overall prognosis. The determination of the HPV status in HNSCC therefore has therapeutic implications. We compared the Ventana ISH iView Blue Plus Detection Kit in situ hybridization (ISH) system and the Roche Linear Array HPV Genotyping Test for the detection of HPV in 98 formalin-fixed, paraffin-embedded HNSCC samples. A moderate concordance rate (70.4%) was observed between ISH and the Linear Array assays. ISH detected HPV in 39.8% of cases, whereas Linear Array detected HPV in 57.1% of cases. Sensitivity and specificity of ISH for detecting HPV in HNSCC specimens were determined to be 58.9% and 85.7%, respectively, using the Linear Array as the method of comparison (McNemar test, P=0.003). ISH offers the advantage of visual cell-type localization of viral infection but overall it is less sensitive than the polymerase chain reaction-based detection of HPV in HNSCC.