Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression.

Metastatic disease is a primary cause of cancer-related death, and factors governing tumor cell metastasis have not been fully elucidated. Here, we address this question by using tumor cell lines derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53. Despite having widespread somatic genetic alterations, the metastasis-prone tumor cells retained a marked plasticity. They transited reversibly between epithelial and mesenchymal states, forming highly polarized epithelial spheres in three-dimensional culture that underwent epithelial-to-mesenchymal transition (EMT) following treatment with transforming growth factor-beta or injection into syngeneic mice. This transition was entirely dependent on the microRNA (miR)-200 family, which decreased during EMT. Forced expression of miR-200 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize, and conferred transcriptional features of metastasis-incompetent tumor cells. We conclude that tumor cell metastasis is regulated by miR-200 expression, which changes in response to contextual extracellular cues.

Factors associated with successful electrolarynx use after total laryngectomy, a multi-institutional study.

Objective: To identify characteristics associated with successful electrolarynx (EL) use after total laryngectomy (TL). Methods: Records of 196 adults who underwent TL from 03/15/2012 to 03/15/2022 at the University of Washington and Puget Sound Veterans Affairs were reviewed. Characteristics included age, Charlson Comorbidity Index, social support, pre-operative radiation (RT) and chemoradiation (CRT), and 6-month post-TL swallow status. EL success was evaluated using pre-defined criteria of intelligibility, reliability, and independence with use. Poisson regressions and robust standard error estimates were used to estimate unadjusted risk ratios for each characteristic. Statistically significant characteristics were included in multivariate analysis (MVA) to estimate adjusted risk ratios. Results: Median age was 64, median Charlson Comorbidity Index was 5, 170 (87%) were male, 159 (81%) had high social support, and 159 (81%) attained post-TL full-oral diet. Pre-operatively, 110 (56%) had RT, including 55 (28%) with CRT. Ninety-three (47%) met our criteria for EL success. Characteristics significantly associated with EL success included social support (p = .037) and post-TL full-oral diet (p = .037); both approached significance on MVA. EL success varied by pre-operative treatment on univariate (p = .005) and MVA (p = .014). Compared to no prior RT or CRT, the probability of EL success was 29% higher with prior RT and 29% lower with prior CRT in MVA, although these associations did not reach significance. Conclusions: In this retrospective review, EL success correlated with high social support, post-TL full-oral diet, and pre-operative treatment history. These results warrant validation in a larger prospective study to help guide the choice of voice rehabilitation modalities or intensified speech therapy. Level of Evidence: 4.

Peripheral lymphocytes and lactate dehydrogenase correlate with response and survival in head and neck cancers treated with immune checkpoint inhibitors.

BACKGROUND: Little is known regarding associations between peripheral blood biomarkers (PBBMs) and survival, response, and toxicity in recurrent/metastatic head and neck squamous cell carcinomas (R/M HNSCC) treated with immune checkpoint inhibitors (ICIs). METHODS: In this single-institution retrospective cohort study, a dataset of patients with R/M HNSCC treated with ICIs between 08/2012-03/2021 was established, including demographic and clinicopathologic characteristics. Pretreatment PBBMs were collected and evaluated for associations with grade ≥3 adverse events (G ≥ 3AE) by CTCAEv5, objective response (ORR) by RECIST 1.1, overall survival (OS), and progression-free survival (PFS). Multivariable models for each outcome were created using elastic net variable selection. RESULTS: Our study included 186 patients, with 51 (27%) demonstrating complete or partial response to immunotherapy. Multivariable models adjusted for ECOG performance status (PS), p16, and smoking demonstrated that pretreatment higher LDH and absolute neutrophils, as well as lower percent lymphocytes correlated with worse OS and PFS. Higher LDH and lower % lymphocytes also correlated with worse ORR. CONCLUSIONS: In the largest study to date examining PBBMs in ICI-treated R/M HNSCCs, our variable selection method revealed PBBMs prognostic for survival and response to immunotherapy. These biomarkers warrant further investigation in a prospective study along with validation with CPS biomarker.

Radiation therapy for low- and high-risk perineural invasion in head and neck cutaneous squamous cell carcinoma: Clinical outcomes and patterns of failure.

BACKGROUND: Perineural invasion (PNI) in head and neck squamous cell carcinoma (HNSCC) portends poor prognosis. Extent of treatment of nerve pathways with varying degrees of PNI and patterns of failure following elective neural radiotherapy (RT) remain unclear. METHODS: Retrospective review of HNSCC patients with high-risk (clinical/gross, large-nerve, extensive) or low-risk (microscopic/focal) PNI who underwent curative-intent treatment from 2010 to 2021. RESULTS: Forty-four patients (mean follow-up 22 months; 59% high-risk, 41% low-risk PNI) were included. Recurrence following definitive treatment occurred in 31% high-risk and 17% low-risk PNI patients. Among high-risk patients, 69% underwent surgery with post-operative RT and 46% underwent elective neural RT. Local control (83% low-risk vs. 75% high-risk), disease-free, and overall survival did not differ between groups. CONCLUSIONS: High local control rates were achieved in high-risk PNI patients treated with adjuvant or primary RT, including treatment of both involved and uninvolved, communicating cranial nerves, with few failures in electively treated regions.

A Biofabrication Strategy for a Custom-Shaped, Non-Synthetic Bone Graft Precursor with a Prevascularized Tissue Shell.

Critical-sized defects of irregular bones requiring bone grafting, such as in craniofacial reconstruction, are particularly challenging to repair. With bone-grafting procedures growing in number annually, there is a reciprocal growing interest in bone graft substitutes to meet the demand. Autogenous osteo(myo)cutaneous grafts harvested from a secondary surgical site are the gold standard for reconstruction but are associated with donor-site morbidity and are in limited supply. We developed a bone graft strategy for irregular bone-involved reconstruction that is customizable to defect geometry and patient anatomy, is free of synthetic materials, is cellularized, and has an outer pre-vascularized tissue layer to enhance engraftment and promote osteogenesis. The graft, comprised of bioprinted human-derived demineralized bone matrix blended with native matrix proteins containing human mesenchymal stromal cells and encased in a simple tissue shell containing isolated, human adipose microvessels, ossifies when implanted in rats. Ossification follows robust vascularization within and around the graft, including the formation of a vascular leash, and develops mechanical strength. These results demonstrate an early feasibility animal study of a biofabrication strategy to manufacture a 3D printed patient-matched, osteoconductive, tissue-banked, bone graft without synthetic materials for use in craniofacial reconstruction. The bone fabrication workflow is designed to be performed within the hospital near the Point of Care.

Case of Esophageal Perforation and Repair with a Supraclavicular Artery Island Fascial Flap 15 Years After Anterior Spine Surgery.

BACKGROUND: Esophageal perforation represents a rare but potentially life-threatening complication of an anterior cervical diskectomy and fusion (ACDF). Delayed presentations of esophageal perforation more than 10 years following surgery are exceedingly rare and difficult to diagnose. Here, we discuss the case of an 80-year-old man who presented to the emergency department with progressive dysphagia 15 years after his ACDF. CASE DESCRIPTION: While prior outpatient workup was suggestive of a diverticulum, there was no evidence of esophageal perforation. Progressive symptoms and repeat imaging on admission were suggestive of retropharyngeal phlegmon. Operative esophagoscopy revealed that the spinal hardware had eroded through the posterior wall of the esophagus, creating a traction diverticulum. The hardware was removed, and the esophageal perforation was closed primarily and buttressed with vascularized tissue from a supraclavicular artery island fascial flap. CONCLUSIONS: This case emphasizes the importance of considering an esophageal perforation in patients who present with dysphagia at any interval following an ACDF, even in the extremely delayed setting. Furthermore, this is the first report, to the best of our knowledge, using a supraclavicular artery island fascial flap to reconstruct an esophageal perforation following an ACDF, and we introduce a novel strategy for managing these complicated injuries.

Elective neck dissection during salvage laryngectomy: A systematic review and meta-analysis.

OBJECTIVES: The primary objective was to determine the rate of occult cervical nodal metastasis in patients undergoing elective neck dissection (END) during salvage laryngectomy. The secondary objective was to compare survival and postoperative complication rates between patients undergoing END versus observation. METHODS: A medical librarian performed a comprehensive search for END outcomes in laryngeal cancer patients undergoing salvage laryngectomy after primary chemoradiation therapy. Seventeen retrospective studies and 1 prospective study met inclusion criteria, with a total of 1,141 patients (799 END, 350 observed). RESULTS: The rate of nodal positivity was 11% among patients who underwent END during their salvage laryngectomy. Three studies and 155 patients were included in a 5-year overall survival (OS) analysis with no significant difference in OS (95% confidence interval [CI]: 0.82-2.22). After inclusion of six studies and 494 patients (249 END, 245 observed), the risk of fistula formation was not statistically different (95% CI: 0.61-2.56). Due to significant heterogeneity between studies and inadequate data, most patients could not be included in the meta-analysis of outcomes. CONCLUSION: Salvage laryngectomy patients undergoing END have an occult nodal positivity rate of 11%. Meta-analysis showed no statistically significant differences in 5-year OS between patients undergoing END versus observation. Laryngoscope, 130:899-906, 2020.

Nationwide analysis of unplanned 30-day readmissions after transsphenoidal pituitary surgery.

BACKGROUND: Transsphenoidal pituitary surgery has evolved into a safe procedure with shorter hospitalizations, yet unplanned readmissions remain a quality measure for which there is a paucity of data. We sought to examine rates, timing, etiologic factors, and costs surrounding readmission after transsphenoidal pituitary surgery. METHODS: The Nationwide Readmissions Database (NRD) was queried for patients who underwent transsphenoidal pituitary between January 2013 and November 2013. Patient, procedure, admission, and hospital-level characteristics were compared for patients with and without unplanned 30-day readmission. Multivariate logistic regression was used to identify readmission predictors. A total of 8546 patients were included in this retrospective study. RESULTS: A total of 8546 patients with a median age of 54 years and female predominance were identified, with 742 patients experiencing at least 1 unplanned readmission within 30 days of index admission. Hypertension, hypothyroidism, diabetes, and obesity were common comorbidities among readmitted patients. Readmission was most frequently because of nervous system complications, followed by neurohypophyseal or electrolyte disorders, cerebrospinal fluid leak, hemorrhage, and meningitis. Median length and cost of stay of index admission was greater in the readmission group (p < 0.001). Fluid and electrolyte disorders as well as neurologic disease (most commonly epilepsy or convulsions) present on initial admission were predictive of length of initial stay and readmission (p < 0.001). Median readmission cost was $7723 and was expected to occur within 7 days. CONCLUSION: Approximately 8.7% of patients undergoing transsphenoidal pituitary surgery experience an unplanned readmission within 30 days of discharge. Risk factors identified should be considered to reduce preventable readmissions and identify medically complex patients.

Treatment outcomes of patients with primary squamous cell carcinoma of the retromolar trigone.

OBJECTIVES/HYPOTHESIS: Squamous cell carcinoma of the retromolar trigone (RMT SCC) is a relatively uncommon primary site for oral cavity malignancy. However, given its proximity to the mandible and buccal mucosa, RMT SCC typically exhibits early invasion and generally presents at an advanced stage. Large-sample studies are needed to assess the epidemiology and clinical outcomes of this tumor. Our aim was to describe the determinants of survival in patients with RMT SCC. STUDY DESIGN: Retrospective cohort study. METHODS: Retrospective, population-based cohort study of patients in the Surveillance, Epidemiology, and End Results tumor registry who were diagnosed with RMT SCC from 1973 to 2012. Primary endpoints were overall survival (OS) and disease-specific survival (DSS). RESULTS: A total of 4,022 cases of RMT SCC were identified. The mean age at diagnosis was 65 years. Thirty-nine percent of cases presented with stage IV disease. The median OS by stages I to IV were 73.7, 52.4, 27.5, and 23.4 months, respectively (P < .05). Overall, 34.3% of patients underwent surgery, 23.5% received radiation therapy, and 34.1% had both surgical and radiation therapy. On multivariate analysis, advanced age, greater tumor size, and advanced stage were associated with worse OS and DSS (P < .05), surgery predicted improved OS and DSS (P < .05), and radiation therapy predicted improved OS only (P < .05). CONCLUSIONS: RMT SCC is an aggressive malignancy that portends a poor prognosis, though early-stage tumors (stages I and II) have significantly improved survival. Any surgical intervention independently predicted higher survival outcomes. There may be a role of dual modality approaches, particularly for larger tumors. LEVEL OF EVIDENCE: 4 Laryngoscope, 128:2740-2744, 2018.

The microRNA-200/Zeb1 axis regulates ECM-dependent ß1-integrin/FAK signaling, cancer cell invasion and metastasis through CRKL.

Tumor cell metastasis is a complex process that has been mechanistically linked to the epithelial-mesenchymal transition (EMT). The double-negative feedback loop between the microRNA-200 family and the Zeb1 transcriptional repressor is a master EMT regulator, but there is incomplete understanding of how miR-200 suppresses invasion. Our recent efforts have focused on the tumor cell-matrix interactions essential to tumor cell activation. Herein we utilized both our Kras/p53 mutant mouse model and human lung cancer cell lines to demonstrate that upon miR-200 loss integrin ß1-collagen I interactions drive 3D in vitro migration/invasion and in vivo metastases. Zeb1-dependent EMT enhances tumor cell responsiveness to the ECM composition and activates FAK/Src pathway signaling by de-repression of the direct miR-200 target, CRKL. We demonstrate that CRKL serves as an adaptor molecule to facilitate focal adhesion formation, mediates outside-in signaling through Itgß1 to drive cell invasion, and inside-out signaling that maintains tumor cell-matrix contacts required for cell invasion. Importantly, CRKL levels in pan-cancer TCGA analyses were predictive of survival and CRKL knockdown suppressed experimental metastases in vivo without affecting primary tumor growth. Our findings highlight the critical ECM-tumor cell interactions regulated by miR-200/Zeb1-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis.

A synthetic matrix with independently tunable biochemistry and mechanical properties to study epithelial morphogenesis and EMT in a lung adenocarcinoma model.

Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)-dependent epithelial-mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase-degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-ß induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology.

Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells.

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.

ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression.

Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient's likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients.

Targets of the tumor suppressor miR-200 in regulation of the epithelial-mesenchymal transition in cancer.

The microRNA-200 (miR-200) family restricts epithelial-mesenchymal transition (EMT) and metastasis in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma. To determine the mechanisms responsible for EMT and metastasis regulated by this microRNA, we conducted a global liquid chromatography/tandem mass spectrometry analysis to compare metastatic and nonmetastatic murine lung adenocarcinoma cells which had undergone EMT because of loss of miR-200. An analysis of syngeneic tumors generated by these cells identified multiple novel proteins linked to metastasis. In particular, the analysis of conditioned media, cell surface proteins, and whole-cell lysates from metastatic and nonmetastatic cells revealed large-scale modifications in the tumor microenvironment. Specific increases were documented in extracellular matrix (ECM) proteins, peptidases, and changes in distribution of cell adhesion proteins in the metastatic cell lines. Integrating proteomic data from three subproteomes, we defined constituents of a multilayer protein network that both regulated and mediated the effects of TGFß. Lastly, we identified ECM proteins and peptidases that were directly regulated by miR-200. Taken together, our results reveal how expression of miR-200 alters the tumor microenvironment to inhibit the processes of EMT and metastasis.