CYLD mutation characterizes a subset of HPV-positive head and neck squamous cell carcinomas with distinctive genomics and frequent cylindroma-like histologic features.

Mutations in the tumor suppressor CYLD, known to be causative of cylindromas, were recently described in a subset of high-risk (hr) HPV-positive head and neck squamous cell carcinomas (HNSCC). Pathologic and genetic characterization of these CYLD-mutant carcinomas, however, remains limited. Here, we investigated whether CYLD mutations characterize a histopathologically and genomically distinct subset of hrHPV-positive HNSCC. Comprehensive genomic profiling via hybrid capture-based DNA sequencing was performed on 703 consecutive head and neck carcinomas with hrHPV sequences, identifying 148 unique cases (21%) harboring CYLD mutations. Clinical data, pathology reports, and histopathology were reviewed. CYLD mutations included homozygous deletions (n = 61/148; 41%), truncations (n = 52; 35%), missense (n = 26; 18%) and splice-site (n = 9; 6%) mutations, and in-frame deletion (n = 1; 1%). Among hrHPV-positive HNSCC, the CYLD-mutant cohort showed substantially lower tumor mutational burden than CYLD-wildtype cases (n = 555) (median 2.6 vs. 4.4 mut/Mb, p < 0.00001) and less frequent alterations in PIK3CA (11% vs. 34%, p < 0.0001), KMT2D (1% vs. 16%, p < 0.0001), and FBXW7 (3% vs. 11%, p = 0.0018). Male predominance (94% vs. 87%), median age (58 vs. 60 years), and detection of HPV16 (95% vs. 89%) were similar. On available histopathology, 70% of CYLD-mutant HNSCC (98/141 cases) contained hyalinized material, consistent with basement membrane inclusions, within crowded aggregates of tumor cells. Only 7% of CYLD-wildtype cases demonstrated this distinctive pattern (p < 0.0001). Histopathologic patterns of CYLD-mutant HNSCC lacking basement membrane inclusions included nonkeratinizing (n = 22, 16%), predominantly nonkeratinizing (nonkeratinizing SCC with focal maturation; n = 10, 7%), and keratinizing (n = 11, 8%) patterns. The latter two groups showed significantly higher frequency of PTEN alterations compared with other CYLD-mutant cases (38% [8/21] vs. 7% [8/120], p = 0.0004). Within our cohort of hrHPV-positive HNSCCs, CYLD mutations were frequent (21%) and demonstrated distinctive clinical, histopathologic, and genomic features that may inform future study of prognosis and treatment.

Pan-sarcoma genomic analysis of KMT2A rearrangements reveals distinct subtypes defined by YAP1-KMT2A-YAP1 and VIM-KMT2A fusions.

Sarcomas are driven by diverse pathogenic mechanisms, including gene rearrangements in a subset of cases. Rare soft tissue sarcomas containing KMT2A fusions have recently been reported, characterized by a predilection for young adults, sclerosing epithelioid fibrosarcoma-like morphology, and an often aggressive course. Nonetheless, clinicopathologic and molecular descriptions of KMT2A-rearranged sarcomas remain limited. In this study, we identified by targeted next-generation RNA sequencing an index patient with KMT2A fusion-positive soft tissue sarcoma. In addition, we systematically searched for KMT2A structural variants in a comprehensive genomic profiling database of 14,680 sarcomas interrogated by targeted next-generation DNA and/or RNA sequencing. We characterized the clinicopathologic and molecular features of KMT2A fusion-positive sarcomas, including KMT2A breakpoints, rearrangement partners, and concurrent genetic alterations. Collectively, we identified a cohort of 34 sarcomas with KMT2A fusions (0.2%), and YAP1 was the predominant partner (n = 16 [47%]). Notably, a complex rearrangement with YAP1 consistent with YAP1-KMT2A-YAP1 fusion was detected in most cases, with preservation of KMT2A CxxC-binding domain in the YAP1-KMT2A-YAP1 fusion and concurrent deletions of corresponding exons in KMT2A. The tumors often affected younger adults (age 20-66 [median 40] years) and histologically showed variably monomorphic epithelioid-to-spindle shaped cells embedded in a dense collagenous stroma. Ultrastructural evidence of fibroblastic differentiation was noted in one tumor examined. Our cohort also included two sarcomas with VIM-KMT2A fusions, each harboring concurrent mutations in CTNNB1, SMARCB1, and ARID1A and characterized histologically by sheets of spindle-to-round blue cells. The remaining 16 KMT2A-rearranged sarcomas in our cohort exhibited diverse histologic subtypes, each with unique novel fusion partners. In summary, KMT2A-fusion-positive sarcomas most commonly exhibit sclerosing epithelioid fibrosarcoma-like morphology and complex YAP1-KMT2A-YAP1 fusions. Cases also include rare spindle-to-round cell sarcomas with VIM-KMT2A fusions and tumors of diverse histologic subtypes with unique KMT2A fusions to non-YAP1 non-VIM partners.

Mandibular Intraosseous Angiofibroma-A Rare Clinical Entity.

Angiofibromas located in the maxillofacial region are rare and almost exclusively occur in adolescent males. These benign tumors are highly vascular, locally invasive, and commonly found in the nasopharyngeal space. In the present report, we describe a very rare case of an intraosseous mandibular angiofibroma in a 23-year-old male patient with histomorphologic and molecular confirmation. This type of tumor occurring in the mandible has been reported previously only once, to the best of our knowledge.

ADAM10-Dependent Signaling Through Notch1 and Notch4 Controls Development of Organ-Specific Vascular Beds.

RATIONALE: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. OBJECTIVE: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. METHODS AND RESULTS: Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. CONCLUSIONS: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

Gastroblastoma with a novel ACTB::GLI1 gene fusion in a 19-year-old male.

Gastroblastoma is a rare gastric biphasic tumor composed of mesenchymal and epithelial elements in variable proportions. These tumors usually arise in the gastric antrum of children and young adults and are reported to harbor a recurrent MALAT1::GLI1 fusion. Herein we report a case of gastroblastoma in a 19-year-old male who presented with intermittent epigastric abdominal discomfort. Antrectomy revealed a 5.6-cm multi-lobulated, tan-pink mass with solid and focally cystic areas involving the submucosa, muscularis propria, and subserosa. All tumor cells demonstrated immunoreactivity for GLI-1, CD56, and vimentin; epithelial elements expressed pancytokeratins (AE1/AE3 and Oscar), and mesenchymal cells demonstrated focal positivity for CD10. Next generation sequencing revealed a novel ACTB::GLI1 fusion without evidence of the recurrent MALAT1::GLI1 fusion. Nine months after surgery, the patient is well without evidence of recurrence or metastases. To our knowledge, this is the first case of gastroblastoma harboring this novel ACTB::GLI1 fusion.

Deletion of Adam10 in endothelial cells leads to defects in organ-specific vascular structures.

During vertebrate angiogenesis, Notch regulates the cell-fate decision between vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored ligands triggers proteolytic processing, first by Adam10 and then presenilins. This liberates the Notch intracellular domain, allowing it to enter the nucleus and activate Notch-dependent genes. Here we report that conditional inactivation of Adam10 in endothelial cells (A10ΔEC) recapitulates the increased branching and density of the retinal vasculature that is also caused by interfering with Notch signaling. Moreover, A10ΔEC mice have additional vascular abnormalities, including aberrant subcapsular hepatic veins, enlarged glomeruli, intestinal polyps containing endothelial cell masses, abnormal endochondral ossification, leading to stunted long bone growth and increased pathologic neovascularization following oxygen-induced retinopathy. Our findings support a model in which Adam10 is a crucial regulator of endothelial cell-fate decisions, most likely because of its essential role in canonical Notch signaling.

Intraoperative assessment of parathyroid pathology in sporadic primary hyperparathyroidism: an institutional experience.

Intraoperative consultation for assessment of parathyroid tissue is a controversial area of endocrine pathology. This assessment often follows historical institutional and individual surgical practitioner practices rather than documented utility data and adjuvant intraoperative testing data, including intraoperative parathyroid hormone level testing by clinical pathologists and the use of Oil Red O vital stain on frozen tissue sections by anatomic pathologists, as a means of conferring etiology of parathyroid disease. The American Association of Endocrine Surgeons (AAES), in 2016, proposed guidelines for the management of primary hyperparathyroidism, including recommendations for intraoperative consultation, recommending against the use of intraoperative frozen section to determine parathyroid functional status but in support for its use for parathyroid identification. In this series, we review a one-year, retrospective cohort of consecutive parathyroid surgeries at Massachusetts General Hospital, including over 200 cases meeting inclusion criteria for which primary hyperparathyroidism was the indication for surgery, discussing outcomes, compliance with AAES guidelines, and overall utility of intraoperative consultation and adjuvant testing.

Pan-Cancer Landscape Analysis Reveals Recurrent KMT2A-MAML2 Gene Fusion in Aggressive Histologic Subtypes of Thymoma.

PURPOSE: Thymomas are epithelial neoplasms that represent the most common thymic tumors in adults. These tumors have been shown to harbor a relatively low mutational burden. As a result, there is a lack of genetic alterations that may be used prognostically or targeted therapeutically for this disease. Here, we describe a recurrent gene rearrangement in type B2 + B3 thymomas. PATIENTS AND METHODS: A single index case of thymoma was evaluated by an RNA-based solid fusion assay. Separately, tissues from 255,008 unique advanced cancers, including 242 thymomas, were sequenced by hybrid capture-based next-generation DNA sequencing/comprehensive genomic profiling of 186 to 406 genes, including lysine methyltransferase 2A (KMT2A) rearrangements, and a portion were evaluated for RNA of 265 genes. We characterized molecular and clinicopathologic features of the pertinent fusion-positive patient cases. RESULTS: We identified 11 patients with thymomas harboring a gene fusion of KMT2A and mastermind-like transcriptional coactivator 2 (MAML2). Fusion breakpoints were identified between exon 8, 9, 10, or 11 of KMT2A and exon 2 of MAML2. Fifty-five percent were men, with a median age of 48 years at surgery (range, 29-69 years). Concurrent genomic alterations were infrequent. The 11 thymomas were of B2 or B3 type histology, with 1 case showing foci of thymic carcinoma. The frequency of KMT2A-MAML2 fusion was 4% of all thymomas (10 of 242) and 6% of thymomas of B2 or B3 histology (10 of 169). CONCLUSION: KMT2A-MAML2 represents the first recurrent fusion described in type B thymoma. The fusion seems to be specific to type B2 and B3 thymomas, the most aggressive histologic subtypes. The identification of this fusion offers insights into the biology of thymoma and may have clinical relevance for patients with disease refractory to conventional therapeutic modalities.

Association of Estrogen Receptor Alpha Expression With Survival in Oropharyngeal Cancer Following Chemoradiation Therapy.

BACKGROUND: Oropharyngeal squamous carcinoma (OPSC) continues to increase in incidence secondary to human papillomavirus (HPV) infection. Despite the good overall prognosis for these patients, treatment with chemoradiation is associated with morbidity and treatment failure. Better predictors for disease outcome are needed to guide de-intensification regimens. We hypothesized that estrogen receptor α (ERα), a prognostic biomarker in oncology with therapeutic implications, might have similar utility in OPSC. METHODS: To investigate associations among ERα and demographics, HPV status, and survival, we analyzed ERα mRNA expression of head and neck squamous carcinomas (HNSC) from The Cancer Genome Atlas (TCGA) and immunohistochemistry (IHC) of pretreatment biopsy specimens from an independent group of 215 OPSC patients subsequently treated with primary chemoradiation (OPSC-CR). Associations among variables were evaluated with Fisher exact tests and logistic regression; associations with survival were evaluated with log-rank tests and Cox proportional hazards regression. RESULTS: Among 515 patients in TCGA, ERα mRNA expression was highest in HPV-positive OPSC. High ERα mRNA expression was associated with improved survival among those receiving chemoradiation (hazard ratio adjusted for HPV status = 0.44, 95% confidence interval = 0.21 to 0.92). In OPSC-CR, ERα was positive by IHC in 51.6% of tumors and was associated with improved overall, disease-specific, progression-free, and relapse-free survival (log-rank tests: P < .001, P < .001, P = .002, P = .003, respectively); statistically significant associations of ERα positivity with improved survival were maintained after adjusting for clinical risk factors including HPV status. CONCLUSION: In two independent cohorts, ERα is a potential biomarker for improved survival that also may represent a therapeutic target in OPSC.

Metastatic Follicular Thyroid Carcinoma and the Primary Thyroid Gross Examination: Institutional Review of Cases from 1990 to 2015.

The diagnosis of follicular-patterned carcinomas, including follicular thyroid carcinoma, oncocytic (Hürthle cell) carcinoma, and the encapsulated follicular variant of papillary thyroid carcinoma, requires evidence of capsular and/or vascular invasion. With minimally invasive carcinomas classified often within less than a millimeter of tissue segregating them from adenomas and non-invasive follicular thyroid neoplasms with papillary-like nuclear features, opinions vary internationally over how much of the capsule to submit in order to deem it well enough represented, considering that even if grossly entirely submitted in microcassettes, without leveling through each tissue block, the capsule is truly never entirely examined microscopically. Here, we retrospectively examine submission practices and outcomes at a single, high-volume institution over a 25-year period. Our results indicate that the vast majority of lesions with poor outcomes are those with wide invasion, and tumors lacking gross evidence of capsular perturbation rarely lead to recurrence or metastasis, an unsurprising result that should prompt re-evaluation of our grossing methods and approach to follicular-patterned tumors in a time of cost restraint, molecular diagnostics, and low biological potential of encapsulated, circumscribed neoplasia of the thyroid.

Combined Targeted Therapy for BRAF-Mutant, Treatment-Related Acute Myeloid Leukemia.

A 44-year-old woman with a prior history of myxoid liposarcoma who was previously treated with radiation, chemotherapy, and resection, was admitted with syncope and pancytopenia. She was diagnosed with a high-grade therapy-related myelodysplastic syndrome and treated with decitabine. Her disease soon progressed to overt acute myeloid leukemia (AML). She was treated with cytotoxic chemotherapy including cytarabine and topotecan, but she was not a candidate for further anthracycline exposure given her prior treatment for sarcoma and concern for cardiotoxicity. Her AML was refractory to two sequential induction regimens. Given that targeted genomic sequencing had revealed a BRAF V600E mutation with a high allelic fraction, she was then placed on combined targeted BRAF/MEK therapy with dabrafenib and trametinib for her refractory disease. This resulted in a dramatic response with clearance of circulating myeloblasts, restoration of normal hematopoiesis, a significant decrease in marrow leukemic burden, and a concordant decrease in the BRAF V600E allelic burden. The response was transient, however, with a rapid increase in circulating blasts a few weeks later. At the time of subsequent progression, four separate KRAS mutations were identified. She died approximately 4 months after her diagnosis from rapidly progressive AML.

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus.

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (<24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the alpha-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.

Lack of ADAM10 in endothelial cells affects osteoclasts at the chondro-osseus junction.

Mice lacking ADAM10 in endothelial cells (Adam10ΔEC mice) have shorter femurs, tibiae, and humeri than controls, raising questions about how endothelial cells could control long bone growth. We performed a histopathological evaluation of the femur and tibia growth plates at different postnatal stages, and assessed the distribution of TRAP-positive osteoclasts and endothelial cells at the growth plate. The growth plates in Adam10ΔEC mice appeared normal at P7 and P14, but a thickened zone of hypertrophic chondrocytes and increased trabecular bone density were apparent by P21 and later. The number of TRAP+ cells at the COJ was normal at P7 and P14, but was strongly reduced at P21 and later. Moreover, the density of endomucin-stained endothelial cells at the COJ was increased starting at P7. The defects in long bone growth in Adam10ΔEC mice could be caused by a lack of osteoclastogenesis at the COJ. Moreover, ADAM10 appears to regulate endothelial cell organization in the developing bone vasculature, perhaps in a similar manner as in the developing retinal vascular tree, where ADAM10 is thought to control Notch-dependent endothelial cell fate decisions. This study provides evidence for the regulation of osteoclast function by endothelial cells in vivo.

Intravitreal injection of TIMP3 or the EGFR inhibitor erlotinib offers protection from oxygen-induced retinopathy in mice.

PURPOSE: Pathological neovascularization is a crucial component of proliferative retinopathies. Previous studies showed that inactivation of A disintegrin and metalloproteinase 17 (ADAM17), a membrane-anchored metalloproteinase that regulates epidermal growth factor receptor (EGFR) signaling, reduces pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Here, we tested how genetic inactivation of a physiological ADAM17 inhibitor, the tissue inhibitor of matrix metalloproteinases-3 (TIMP3), or intravitreal injection of TIMP3 or of the EGFR inhibitor erlotinib influenced the outcome of OIR. METHODS: Wild-type mice were subjected to OIR in a chamber with 75% oxygen for 5 days beginning at postnatal day 7 (P7). Upon removal from the oxygen chamber at P12, they received a single intravitreal injection of TIMP3, erlotinib, or control. The central avascular area and neovascular tufts were measured after 5 days in room air (21% oxygen) at P17. Moreover, OIR experiments were performed with Timp3-/- mice and littermate controls. RESULTS: Timp3-/- mice showed greater revascularization of the central avascular area and developed equal or fewer neovascular tufts compared to littermate controls, depending on the genetic background. Wild-type mice injected with TIMP3 or erlotinib developed fewer neovascular tufts when compared to untreated littermates. Moreover, vessel regrowth into the avascular area was reduced in TIMP3-injected mice, but not in erlotinib-injected mice. CONCLUSIONS: These studies demonstrate that TIMP3 and erlotinib inhibit pathological neovascularization in the mouse retina, most likely due to inactivation of ADAM17 and the EGFR, respectively. Thus, TIMP3 and erlotinib emerge as attractive candidate antiangiogenic compounds for prevention and treatment of proliferative retinopathies.

The effect of seawater composition and osmolality on hemolymph levels of methyl farnesoate in the green crab Carcinus maenas.

Green crabs, Carcinus maenas, exposed to dilute seawater (e.g., 5 ppt salinity, approximately 150 mOsm/kg) have hemolymph levels of methyl farnesoate (MF) that are up to 10-fold higher than animals in isosmotic seawater (27 ppt, approximately 800 mOsm/kg). In this paper, we examine aspects of osmotic and ionic stress to identify factors involved in elevating MF levels. MF levels did not rise after exposure to concentrated seawater, so only hypoosmotic stress elevates MF. MF levels rose in animals exposed to dilute seawater containing mannitol to make it isosmotic, indicating that the hypoosmotic rise in MF is due to decreased ion concentrations. Individual ions were investigated by exposing crabs either to isosmotic seawater with low concentrations of an ion or to dilute seawater with high concentrations of an ion. Ca(2+) and Mg(2+) in combination affected MF levels. Finally, we found that the increase in MF levels was accelerated when hemolymph osmolality was precociously lowered by partially replacing hemolymph with deionized water prior to transferring animals to dilute seawater. Thus, the 6-8 h delay between exposing crabs to dilute sea water and observing an increase in MF appears to reflect the time needed for specific hemolymph ions to decrease below a threshold concentration.