Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Inter- and intra-tumor heterogeneity of SMAD4 loss in head and neck squamous cell carcinomas.

Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.

Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

The programmed death ligand 1 interactome demonstrates bidirectional signaling coordinating immune suppression and cancer progression in head and neck squamous cell carcinoma.

BACKGROUND: The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. METHODS: We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. RESULTS: PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. CONCLUSIONS: Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

Approach to the intraoperative consultation for neurosurgical specimens.

Intraoperative consultation remains an invaluable tool in the initial evaluation of surgically excised specimens. Good communication is required between the pathologist and surgeon to obtain the best care for their mutual patient. Intraoperative consultation (frozen section, FS) provides a preliminary diagnosis for the surgeon and aids in guiding his/her subsequent surgical approach. For the pathologist, it serves to assess tissue adequacy in the context of the clinical and imaging features of the patient. FS can guarantee that the surgeon is in the desired anatomic location, but most often serves to ensure that adequate amounts of abnormal, and likely diagnostic, tissue will be available to the pathologist to render a final diagnosis on permanent sections. The preliminary evaluation of tissue at the time of intraoperative FS also guides the pathologist in the ordering of ancillary studies, some of which need to be performed on fresh or frozen tissues and must be sent at the time of the intraoperative consultation. This brief review will specifically focus on the role of the pathologist who is called to perform a FS for a neurosurgical specimen. We will discuss (1) the goals of the neurosurgeon for the intraoperative consultation, (2) how to achieve optimal communication between neurosurgeon and pathologist at the time of the FS, (3) what constitutes reasonable and unreasonable expectations by the neurosurgeon for the FS, (4) choices of techniques that can be used by the pathologist, (5) what tissue should be triaged, and (6) common discrepancies between FS and permanent section diagnoses in central nervous system disorders. The published literature on FS and permanent section discrepancies will be briefly reviewed so that pathologists will understand that some difficulties are inherent in neurosurgical specimens and are not specific to their practice, or to a given pathologist. Hopefully, this knowledge will enhance pathologists' confidence as they negotiate how best to handle this time-sensitive, and sometimes angst-producing, task.

Studying Immunotherapy Resistance in a Melanoma Autologous Humanized Mouse Xenograft.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Osteochondroma of the convexity: pathologic-neuroimaging correlates of a lesion that mimics high-grade meningioma.

Intracranial chondromas are uncommon benign lesions usually attached to dura and located over the convexity of the skull. Osteochondromas are even rarer and additionally contain a benign bony component. Both lesions are reportedly difficult to distinguish from meningiomas on pre-operative neuroimaging studies, although few detailed pathologic-neuroimaging correlation studies have appeared in the literature, particularly for intracranial osteochondromas. A 33-year-old woman with a 4-year history of headaches presented with recent onset of left-sided muscle spasms and weakness. Two days prior to admission to our hospital, neuroimaging studies had shown a large right convexity mass with unusual multifocal bright signal intensities throughout an otherwise isointense mass. The bright signals were interpreted as showing multifocal hemorrhage and the mass was felt to be a convexity meningioma. However, subsequent catheter angiography characterized the lesion as being avascular. The mass was resected en bloc. Extensive histological sectioning revealed a benign osteochondroma predominantly composed of lobules of hypocellular cartilage. Microdissection of the different components revealed that the multifocal, spicule-like bright foci interpreted as hemorrhage on neuroimaging studies were instead foci of benign bone containing metaplastic bone marrow with trilineage hematopoietic cell populations and adipose tissue. Centrally, the hilum of the lesion contained avascular loose connective tissue. No recent or remote hemorrhage was identified anywhere in the lesion. Rare convexity osteochondromas may be mistaken for high-grade meningiomas on neuroimaging studies; their avascular nature, coupled with their complex signal pattern can serve as clues to the correct pre-operative diagnosis.

Leptomeningeal Metastasis from Adrenocortical Carcinoma: A Case Report.

Adrenocortical carcinoma (ACC) is an uncommon endocrine malignancy with limited treatment options. While the overall 5-year survival rate in patients with ACC is 35%, the disease is often rapidly progressive with long-term survival in only 5% of patients. Although tumor stage, grade, and excess hormonal activity predict unfavorable prognosis, additional biomarkers are needed to identify patients with aggressive disease. A 23-year-old woman presented with rapidly progressing signs and symptoms of Cushing's syndrome, with associated abdominal pain and fullness. Evaluation revealed a large left adrenal mass which had developed over 8 months. En bloc surgical resection was performed by an endocrine surgeon, and pathology revealed adrenocortical carcinoma with Ki67 of 60%. Despite adjuvant treatment with mitotane and etoposide-doxorubicin-carboplatin chemotherapy, the patient had rapid disease progression with metastatic spread to liver, lung, bone, brain, and leptomeningies, and she died 11 months after the initial diagnosis. Subsequent analysis of the patient's tumor revealed mutations in TP53 and MEN1. RNA sequencing was compared against the the Cancer Genome Atlas data set and clustered with the high steroid, proliferative subtype, associated with the worst prognosis. The tumor also demonstrated a low BUB1B/PINK1 ratio and G0S2 hypermethylation, both predictive of very aggressive ACC. This case represents a subset of ACC characterized by rapid and fatal progression. Clinically available predictors as well as recently reported molecular signatures and biomarkers correlated with this tumor's aggressiveness, suggesting that development and validation of combinations of biomarkers may be useful in guiding personalized approaches to patients with ACC.

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

Development of an Adrenocortical Cancer Humanized Mouse Model to Characterize Anti-PD1 Effects on Tumor Microenvironment.

CONTEXT: Although the development of immune checkpoint inhibitors has transformed treatment strategies of several human malignancies, research models to study immunotherapy in adrenocortical carcinoma (ACC) are lacking. OBJECTIVE: To explore the effect of anti-PD1 immunotherapy on the alteration of the immune milieu in ACC in a newly generated preclinical model and correlate with the response of the matched patient. DESIGN, SETTING, AND INTERVENTION: To characterize the CU-ACC2-M2B patient-derived xenograft in a humanized mouse model, evaluate the effect of a PD-1 inhibitor therapy, and compare it with the CU-ACC2 patient with metastatic disease. RESULTS: Characterization of the CU-ACC2-humanized cord blood-BALB/c-Rag2nullIl2rγnullSirpaNOD model confirmed ACC origin and match with the original human tumor. Treatment of the mice with pembrolizumab demonstrated significant tumor growth inhibition (60%) compared with controls, which correlated with increased tumor infiltrating lymphocyte activity, with an increase of human CD8+ T cells (P < 0.05), HLA-DR+ T cells (P < 0.05) as well as Granzyme B+ CD8+ T cells (<0.001). In parallel, treatment of the CU-ACC2 patient, who had progressive disease, demonstrated a partial response with 79% to 100% reduction in the size of target lesions, and no new sites of metastasis. Pretreatment analysis of the patient's metastatic liver lesion demonstrated abundant intratumoral CD8+ T cells by immunohistochemistry. CONCLUSIONS: Our study reports the first humanized ACC patient-derived xenograft mouse model, which may be useful to define mechanisms and biomarkers of response and resistance to immune-based therapies, to ultimately provide more personalized care for patients with ACC.

Inhibiting Translation Elongation with SVC112 Suppresses Cancer Stem Cells and Inhibits Growth in Head and Neck Squamous Carcinoma.

Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.

Response to Immunotherapy in Combination With Mitotane in Patients With Metastatic Adrenocortical Cancer.

Adrenocortical carcinoma (ACC) is a rare orphan disease with a dismal prognosis. Surgery remains the first-line treatment, but most patients eventually develop metastatic disease. Mitotane is often used with chemotherapy with modest success. Little information is available concerning the efficacy of immunotherapy in combination with mitotane. We conducted a retrospective review of our initial six patients with metastatic ACC, for whom mitotane alone or with chemotherapy failed, and who were subsequently treated with a combination of pembrolizumab and mitotane, between July 2016 and March 2019. Imaging was analyzed per Response Evaluation Criteria in Solid Tumours 1.1 criteria. Two patients had a partial response and four patients had stable disease (8 to 19 months). One patient had grade 3 hepatitis and pembrolizumab was discontinued after 8 months. She died with disease progression 16 months after initiating pembrolizumab. One patient developed brain metastasis after 19 months of treatment and was transitioned to hospice. One patient had focal pneumonitis after 18 months of treatment, and pembrolizumab was discontinued. Three remaining patients continue pembrolizumab plus mitotane at the time of this writing. The current standard of care for ACC is a combination of etoposide, doxorubicin, cisplatin, and mitotane with an overall survival of 14.8 months. All six patients lived for at least 16 months after starting pembrolizumab added to mitotane therapy. The therapy appeared to be effective in both microsatellite instability-high and microsatellite stable tumors, suggesting some synergistic effect with mitotane. Combined immunotherapy and mitotane should be considered in future clinical trials in patients with ACC.

Targeting PDZ-binding kinase is anti-tumorigenic in novel preclinical models of ACC.

Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.

Association Between Lymph Node Ratio and Recurrence and Survival Outcomes in Patients With Oral Cavity Cancer.

Importance: Oral cavity squamous cell carcinoma (OCSCC) is associated with often-delayed clinical diagnosis, poor prognosis, and expensive therapeutic approaches. Prognostic accuracy is important in improving treatment outcomes of patients with this disease. Objectives: To assess lymph node ratio (LNR) and other factors in estimating response to treatment and provide prognostic information helpful for clinical decision making. Design, Setting, and Participants: A retrospective cohort study was conducted from January 1, 2000, to December 31, 2015, at an academic hospital in Denver, Colorado. Participants included 149 patients with primary OCSCC who received curative-intent surgery and/or postoperative adjuvant therapies. Analysis was performed from December 8, 2017, to August 15, 2018. Main Outcomes and Measures: Overall survival (OS), disease-free survival (DFS), locoregional disease-free survival (LRDFS), and distant metastasis-free survival (DMDFS) adjusted for known prognostic risk factors, as well as correlation of LNR with other histopathologic prognostic factors. Results: Of the 149 patients included in analysis, 105 were men (70.5%); the median age at diagnosis was 59 years (range, 28-88 years). Using the Kaplan-Meier method, the 5-year survival estimates for OS rate was 40.4% (95% CI, 31.3%-49.3%); DFS, 48.6% (95% CI, 38.6%-58.0%); LRDFS, 57.7% (95% CI, 46.6%-67.2%); and DMDFS, 74.7% (95% CI, 65.1%-82.0%). The median follow-up was 20 months for all patients and 34.5 months (range, 0-137 months) for surviving patients. Nonwhite race (hazard ratio [HR], 2.15; 95% CI, 1.22-3.81), T3-T4 category (HR, 1.99; 95% CI, 1.18-3.35), and LNR greater than 10% (HR, 2.71; 95% CI, 1.39-5.27) were associated with poorer OS. Nonwhite patients also had higher risk of locoregional failures (HR, 2.47; 95% CI, 1.28-4.79), whereas women were more likely to have distant metastasis (HR, 2.55; 95% CI, 1.14-5.71). Floor-of-mouth subsite had fewer locoregional recurrences than did other subsites (HR, 0.45, 95% CI, 0.21-0.99). An LNR greater than 10% independently was associated with worse OS (HR, 2.71; 95% CI, 1.39-5.27), DFS (HR, 2.48; 95% CI, 1.18-5.22), and DMDFS (HR, 6.05; 95% CI, 1.54-23.71). The LNR was associated with N-stage (Cramer V, 0.69; 95% CI, 0.58-0.78), extracapsular extension (Cramer V, 0.55; 95% CI, 0.44-0.66), lymphovascular invasion (Cramer V, 0.46; 95% CI, 0.27-0.61); number of excised lymph nodes (Cramer V, 0.24; 95% CI, 0.06-0.37), margin (Cramer V, 0.22; 95% CI, 0.05-0.38), and tumor thickness combined with depth of invasion (Cramer V, 0.25; 95% CI, 0.05-0.38). Conclusions and Relevance: Locoregional treatment failure remained the predominant pattern of failure. An advanced pathologic stage and nonwhite race were found to be associated with worse outcomes. The findings from this study suggest that LNR is the most robust prognostic factor and appears to have implications for risk stratification in this disease.

Leading edge or tumor core: Intratumor cancer stem cell niches in oral cavity squamous cell carcinoma and their association with stem cell function.

OBJECTIVES: To describe differences in cancer stem cell (CSC) presence and behavior associated with their intratumor compartment of origin using a patient-derived xenograft (PDX) model of oral cavity squamous cell carcinoma (OCSCC). MATERIALS AND METHODS: Four HPV-negative OCSCC PDX cases were selected (CUHN004, CUHN013, CUHN096, CUHN111) and the percentage of CSCs (ALDH+CD44high) was measured in the tumor Leading Edge (LE) and Core compartments of each PDX tumor case via fluorescence activated cell sorting (FACS). The fraction of cells in the proliferative phase was measured by Ki-67 labelling index of paraffin embedded tissue. The proliferation and invasion of LE versus Core CSCs were compared using sphere and Matrigel invasion assays, respectively. RESULTS: Both CUHN111 and CUHN004 demonstrate CSC enrichment in their LE compartments while CUHN013 and CUHN096 show no intratumor difference. Cases with LE CSC enrichment demonstrate greater Ki-67 labelling at the LE. CSC proliferative potential, assessed by sphere formation, reveals greater sphere formation in CUHN111 LE CSCs, but no difference between CUHN013 LE and Core CSCs. CUHN111 CSCs do not demonstrate an intratumor difference in invasiveness while CUHN013 LE CSCs are more invasive than Core CSCs. CONCLUSION: A discrete intratumor CSC niche is present in a subset of OCSCC PDX tumors. The CSC functional phenotype with regard to proliferation and invasion is associated with the intratumor compartment of origin of the CSC: LE or Core. These individual functional characteristics appear to be modulated independently of one another and independently of the presence of an intratumor CSC niche.

Pancreatic Tumor Microenvironment Modulation by EphB4-ephrinB2 Inhibition and Radiation Combination.

PURPOSE: A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunologic adjuvant is radiotherapy. Radiation, however, has also been shown to induce immunosuppressive factors, which can contribute to tumor progression and formation of fibrotic tumor stroma. To capitalize on the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis.Experimental Design: On the basis of previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation can regulate the microenvironment response postradiation, leading to increased tumor control in PDAC. This hypothesis was explored using both cell lines and in vivo human and mouse tumor models. RESULTS: Our data show this treatment regimen significantly reduces regulatory T-cell, macrophage, and neutrophil infiltration and stromal fibrosis, enhances effector T-cell activation, and decreases tumor growth. Furthermore, our data show that depletion of regulatory T cells in combination with radiation reduces tumor growth and fibrosis. CONCLUSIONS: These are the first findings to suggest that in PDAC, ephrinB2-EphB4 interaction has a profibrotic, protumorigenic role, presenting a novel and promising therapeutic target.

Inhibition of EphB4-Ephrin-B2 Signaling Enhances Response to Cetuximab-Radiation Therapy in Head and Neck Cancers.

Purpose: The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4-ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers.Experimental Design: We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4-ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation.Results: Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4-ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis.Conclusions: Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4-ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. Clin Cancer Res; 24(18); 4539-50. ©2018 AACR.

Elucidating the Role of the Maternal Embryonic Leucine Zipper Kinase in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.