An In-House Computer-Aided Design and Computer-Aided Manufacturing Workflow for Maxillofacial Free Flap Reconstruction is Associated With a Low Cost and High Accuracy.

PURPOSE: In-house computer-aided surgical design and computer-aided manufacturing (CAD/CAM) can be used in oral and maxillofacial surgery for virtual surgical planning and 3-dimensional printing of patient-specific models. The purpose of this study was to measure the cost and accuracy of an in-house CAD/CAM workflow for maxillofacial free flap reconstruction. MATERIALS AND METHODS: A retrospective cohort study of patients undergoing mandibular resection and free flap reconstruction was performed between July 2017 and March 2018 in which in-house CAD/CAM was used. The predictor variable was the in-house CAD/CAM workflow. The outcome variables were in-house workflow cost, as measured by the material expenses, and accuracy, as measured by comparative distance, osteotomy angle, and surfaced overlay measurements and the root mean square (RMS) between the preoperative virtual reconstructive plan and the postoperative computed tomography scan. Additional variables evaluated were time required for in-house CAD/CAM workflow, and clinical and radiographic outcomes. RESULTS: In-house CAD/CAM was used for 26 patients undergoing mandibular resection for benign or malignant disease and free flap reconstruction with fibula (n = 24) or scapula free flap (n = 2). Overall flap success rate was 95%. The mean in-house workflow cost per case was $3.87 USD. There were no significant differences between the mean comparative distance and osteotomy angle measurements between the planned and actual mandibular reconstructions with an RMS ranging from 5.11 to 9.00 mm for distance measurements and 17.41° for the osteotomy angle measurements. The mean surface overlay difference was 1.90 mm with an RMS of 3.72 mm. CONCLUSIONS: The in-house CAD/CAM workflow is a low cost and accurate option for maxillofacial free flap reconstruction. The in-house workflow should be considered as an alternative to current practices using proprietary systems in select cases.

Imaging modalities for drug-related osteonecrosis of the jaw (3), Positron emission tomography imaging for the diagnosis of medication-related osteonecrosis of the jaw.

Medication-related osteonecrosis of jaws (MRONJ) is one of the most complicated inflammatory conditions in oral and maxillofacial region. It is very difficult to correctly evaluate the degree and extent of necrosis and infection. This refractory osteonecrosis often needs extended surgery, leading to impaired quality-of-life. We have performed hyperbaric oxygen therapy (HBO) combined with conservative surgery for advanced cases. We have appraised the value of FDG-PET and 3-phase bone scintigraphy in the diagnosis and management of this condition. MRONJ showed significantly higher SUVmax on FDG-PET than the others. Although the 3 phase pool bone images did not change significantly, perfusion and static bone image as well as PET showed remarkable response to HBO for MRONJ. SUVmax after HBO was significantly lower than those of before HBO. These preliminary results indicate that FDG-PET is useful for monitoring the effect of HBO for MRONJ.

Intraoperative Depth of Invasion Is Accurate in Early-Stage Oral Cavity Squamous Cell Carcinoma.

PURPOSE: Depth of invasion (DOI) is one predictor of nodal metastasis in oral cavity squamous cell carcinoma (OCSCC) and can facilitate the decision to complete an elective neck dissection (END) in early-stage disease with a clinically negative neck. The purpose of this study was to investigate the accuracy of DOI in intraoperative frozen specimens for T1N0 oral OCSCC. MATERIALS AND METHODS: To compare the accuracy of DOI in frozen versus permanent specimens, we completed a prospective, blinded study of 30 patients with cT1N0 OCSCC who presented between October 2016 and December 2017. RESULTS: DOI in frozen specimens was 96.8% accurate in predicting the need for END with a sensitivity of 90.9%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 95.2%. A strong correlation was found between DOIs in frozen and permanent specimens measured by head and neck (HN) pathologists (r = 0.96; 95% confidence interval [CI], 0.93 to 0.97), between HN pathologists using frozen specimens (r = 0.98; 95% CI, 0.95 to 0.99) and permanent specimens (r = 0.95; 95% CI, 0.91 to 0.98), and in DOIs in frozen specimens communicated intraoperatively versus measured by HN pathologist 1 (r = 0.93; 95% CI, 0.86 to 0.97) and HN pathologist 2 (r = 0.95; 95% CI, 0.89 to 0.98). Only 1 patient who did not undergo an END based on frozen specimens was undertreated owing to upgrading of the DOI in permanent specimens. CONCLUSIONS: DOI in intraoperative frozen sections has an accuracy of 96.8% and may be reliably used as a clinical tool to determine the need for END in early-stage OCSCC.

Recurrent oral squamous cell carcinoma-incorporating advance care planning in education and practice.

This reflection describes a life-limiting case of oral squamous cell carcinoma (SCC) that required thoughtful management facilitated by an advance care plan (ACP). A 70-year-old female was diagnosed with a T4aN2bM0 biopsy-proven invasive, well-differentiated keratinizing SCC. Surgical wide-local excision included teeth #11-16 with left unilateral neck dissection, levels I-V. She was rehabilitated with maxillary obturator prosthesis and underwent chemoradiation therapy. Her course was complicated by dysphagia and trismus. She experienced multiple recurrences. At a certain point, negative margins could not be achieved without facial disfigurement. The patient, her husband, and providers decided together that further management would be palliative. Before the additional surgical procedures, she communicated a thorough ACP with her husband and providers who were prepared to facilitate difficult care decisions on her behalf. The patient passed away at home with hospice care at the age of 74. This motivated patient with oral SCC and impactful postmanagement complications appreciated the clarity of an ACP. Her values and goals of care were incorporated with ongoing communication and documentation of this plan, which was instrumental in facilitating her person-centered care. The providers apply lessons learned here in future practice and education of residents and students.

Ablation of Cancer Stem Cells by Therapeutic Inhibition of the MDM2-p53 Interaction in Mucoepidermoid Carcinoma.

PURPOSE: Unique cells characterized by multipotency, self-renewal, and high tumorigenic potential have been recently discovered in mucoepidermoid carcinomas. These cells are defined by high aldehyde dehydrogenase activity and high CD44 expression (ALDHhighCD44high) and function as cancer stem cells (CSC). It has been recently shown that p53 regulates cell differentiation, suggesting that induction of p53 by therapeutic blockade of the MDM2-p53 interaction may constitute a novel strategy to ablate CSCs. Here, we evaluated the effect of a small-molecule inhibitor of MDM2-p53 interaction (MI-773) on the fraction of CSCs in mucoepidermoid carcinoma. EXPERIMENTAL DESIGN: Human mucoepidermoid carcinoma cells (UM-HMC-1,-3A,-3B) were used to assess the effect of MI-773 on cell survival, cell cycle, fraction of CSCs, and expression of p53, p21, MDM2, and Bmi-1 (key regulator of self-renewal). Mice bearing xenograft tumors generated with these mucoepidermoid carcinoma cells were treated with MI-773 to determine the effect of MDM2-p53 inhibition on CSCs in vivo. RESULTS: MDM2 is highly expressed in human mucoepidermoid carcinoma tissues. MI-773 induced expression of p53 and its downstream targets p21 and MDM2, caused G1 cell-cycle arrest, and induced mucoepidermoid carcinoma tumor cell apoptosis in vitro. Importantly, a marked decrease in expression of Bmi-1 and in the fraction of ALDHhighCD44high (CSCs) was caused by MI-773 in vitro and in mice harboring mucoepidermoid carcinoma xenografts. CONCLUSIONS: Collectively, these data demonstrate that MI-773 reduces the fraction of CSCs, suggesting that patients with mucoepidermoid carcinoma might benefit from therapeutic inhibition of the MDM2-p53 interaction.

Increased Presence of Perineural Invasion in the Tongue and Floor of the Mouth: Could It Represent a More Aggressive Oral Squamous Cell Carcinoma, or Do Larger Aggressive Tumors Cause Perineural Invasion?

PURPOSE: Despite data showing worse outcomes and aggressive disease behavior, perineural invasion (PNI) has not been well characterized in terms of tumor location, histopathologic features, or cervical lymph node status. The specific aims of this study were to measure correlations between PNI, tumor location, and other known histopathologic characteristics used to define aggressive disease. MATERIALS AND METHODS: This was a retrospective cohort study of adult patients with primary squamous cell carcinoma of the oral cavity who underwent neck dissection. We excluded patients whose neck was previously treated with surgery or radiation therapy. Demographic and histopathologic variables of interest were obtained from patient charts. The primary outcome of interest was PNI, and the predictors of interest included tumor location, histopathologic tumor characteristics, and cervical lymph node status. For continuous variables, mean differences were compared by t tests. For categorical variables, the differences in the distribution of the proportions were analyzed with the χ2 test. All variables were entered simultaneously into a multivariate logistic regression model to control for possible confounding. Statistical significance for the study was set at P < .05. RESULTS: Three hundred sixty-eight patients met the study criteria. PNI showed statistically significant correlations with lymph node status, tumor depth, and specific primary tumor location. PNI was more likely to be seen in tumors located in the tongue or floor of the mouth. Tumors with PNI had a deeper depth of invasion: 15.9 ± 10.9 mm versus 10.2 ± 10.0 mm (P < .001). PNI tumors had a higher mean total number of positive nodes: 2.85 ± 5.23 versus 0.83 ± 1.80 (P < .001). CONCLUSIONS: PNI is statistically correlated with tongue and floor-of-the-mouth subsites within the oral cavity, as well as larger tumors, deeper tumors, and disease that has progressed to the lymph nodes. Whether this correlation represents causation in either direction remains unknown.

Correlating the depth of invasion at specific anatomic locations with the risk for regional metastatic disease to lymph nodes in the neck for oral squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the critical primary tumor depth of invasion in oral squamous cell carcinoma that would lead to a 20% or greater risk of nodal metastasis. METHODS: An institutional review board approved retrospective review of our head and neck database was performed from 2009 to 2014 and the data were statistically analyzed. RESULTS: Two hundred eighty-six patients with a diagnosis of oral squamous cell carcinoma who met our inclusion criteria underwent primary excision and neck dissection. For a depth of invasion of 1 mm or less, there were no patients with a positive node. From 1.1 mm to 2 mm of depth of invasion, there was 1 of 11 patients (9%) who had at least 1 positive node. At 2.1 mm to 3 mm, 5 of 25 patients (20%) had at least 1 positive node. CONCLUSION: Depth of invasion and the location of the tumor are 2 important variables to consider when making treatment recommendations to patients with clinical N0 disease. © 2017 Wiley Periodicals, Inc. Head Neck 39: 974-979, 2017.

Therapeutic Inhibition of the MDM2-p53 Interaction Prevents Recurrence of Adenoid Cystic Carcinomas.

Purpose: Conventional chemotherapy has modest efficacy in advanced adenoid cystic carcinomas (ACC). Tumor recurrence is a major challenge in the management of ACC patients. Here, we evaluated the antitumor effect of a novel small-molecule inhibitor of the MDM2-p53 interaction (MI-773) combined with cisplatin in patient-derived xenograft (PDX) ACC tumors.Experimental Design: Therapeutic strategies with MI-773 and/or cisplatin were evaluated in SCID mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in low passage primary human ACC cells (UM-HACC-2A, -2B, -5, -6) in vitro The effect of therapy on the fraction of cancer stem cells (CSC) was determined by flow cytometry for ALDH activity and CD44 expression.Results: Combined therapy with MI-773 with cisplatin caused p53 activation, induction of apoptosis, and regression of ACC PDX tumors. Western blots revealed induction of MDM2, p53 and downstream p21 expression, and regulation of apoptosis-related proteins PUMA, BAX, Bcl-2, Bcl-xL, and active caspase-9 upon MI-773 treatment. Both single-agent MI-773 and MI-773 combined with cisplatin decreased the fraction of CSCs in PDX ACC tumors. Notably, neoadjuvant MI-773 and surgery eliminated tumor recurrences during a postsurgical follow-up of more than 300 days. In contrast, 62.5% of mice that received vehicle control presented with palpable tumor recurrences within this time period (P = 0.0097).Conclusions: Collectively, these data demonstrate that therapeutic inhibition of MDM2-p53 interaction by MI-773 decreased the CSC fraction, sensitized ACC xenograft tumors to cisplatin, and eliminated tumor recurrence. These results suggest that patients with ACC might benefit from the therapeutic inhibition of the MDM2-p53 interaction. Clin Cancer Res; 23(4); 1036-48. ©2016 AACR.

Oral/Head and Neck Oncologic and Reconstructive Surgery Fellowship Training Programs: Transformation of the Specialty From 2005 to 2015: Report from the AAOMS Committee on Maxillofacial Oncology and Reconstructive Surgery.

Oral and Maxillofacial Surgeons have increasing opportunities to train in head and neck oncologic and reconstructive surgery. The development of a fellowship training programs within our speciality has had a transformative effect on the speciality. This survey by the commitee on Maxillofacial Oncology and Reconstructive is aimed to evaluate the impact of fellowship training on the careers of the fellows and speciality from 2005-2015.

Correlation of Lymph Node Density With Negative Outcome Predictors in Oral and Maxillofacial Squamous Cell Carcinoma.

PURPOSE: Lymph node density is defined as the number of positive lymph nodes per total number of excised lymph nodes. In oral and maxillofacial cancer, there are recent data showing that increased lymph node density leads to worse outcomes for patients. However, data correlating lymph node density with other known risk parameters are lacking. This study investigated whether a direct correlation exists among cervical lymph node density, depth of invasion, perineural invasion, and extracapsular tumor spread. MATERIALS AND METHODS: A retrospective chart review was undertaken to include all patients who underwent neck dissection with resection of primary oral and maxillofacial squamous cell carcinoma from January 2009 through July 2014. After applying the exclusion criteria, 286 patients were identified. Primary tumor depth of invasion, perineural invasion, and lymph node status, including extracapsular spread, were obtained from the standard pathology report. Descriptive statistics were applied. The association between 2 continuous tumor characteristics was summarized with the Pearson correlation coefficient, and the association between a continuous and a binary tumor characteristic was summarized with 2-sample t test. Statistical significance for the study was set at a P value less than .05. RESULTS: Mean age at time of surgery was 63.9 ± 12.5 years. The final study included 169 men and 117 women (N = 286). The mean depth of invasion was 12.3 ± 11 mm (range, 1 to 69 mm). Mean lymph node density was 0.04 ± 0.1 (range, 0 to 0.81). There was a positive association between lymph node density and depth of tumor invasion (Pearson correlation coefficient, r = 0.21; P < .001). Tumors with perineural invasion had a statistically significant difference in mean lymph node density (0.074 for positive vs 0.024 for negative; P < .001). There also was a significant association in mean lymph node density with the presence of extracapsular spread (0.143 for positive and 0.010 for negative; P < .001). CONCLUSIONS: Statistically relevant positive linear relations among lymph node density, depth of invasion, perineural invasion, and extracapsular spread were identified. Lymph node density could have prognostic implications, because it is statistically correlated with other known prognostic features that lead to poor outcomes. Lymph node density could be an important feature to capture in future prospective trials. Pathology standards would be crucial in this endeavor.

Targeting MDM2 for Treatment of Adenoid Cystic Carcinoma.

PURPOSE: There are no effective treatment options for patients with advanced adenoid cystic carcinoma (ACC). Here, we evaluated the effect of a new small molecule inhibitor of the MDM2-p53 interaction (MI-773) in preclinical models of ACC. EXPERIMENTAL DESIGN: To evaluate the anti-tumor effect of MI-773, we administered it to mice harboring three different patient-derived xenograft (PDX) models of ACC expressing functional p53. The effect of MI-773 on MDM2, p53, phospho-p53, and p21 was examined by Western blots in 5 low passage primary human ACC cell lines and in MI-773-treated PDX tumors. RESULTS: Single-agent MI-773 caused tumor regression in the 3 PDX models of ACC studied here. For example, we observed a tumor growth inhibition index of 127% in UM-PDX-HACC-5 tumors that was associated with an increase in the fraction of apoptotic cells (P = 0.015). The number of p53-positive cells was increased in MI-773-treated PDX tumors (P < 0.001), with a correspondent shift in p53 localization from the nucleus to the cytoplasm. Western blots demonstrated that MI-773 potently induced expression of p53 and its downstream targets p21, MDM2, and induced phosphorylation of p53 (serine 392) in low passage primary human ACC cells. Notably, MI-773 induced a dose-dependent increase in the fraction of apoptotic ACC cells and in the fraction of cells in the G1 phase of cell cycle (P < 0.05). CONCLUSIONS: Collectively, these data demonstrate that therapeutic inhibition of the MDM2-p53 interaction with MI-773 activates downstream effectors of apoptosis and causes robust tumor regression in preclinical models of ACC. Clin Cancer Res; 22(14); 3550-9. ©2016 AACR.

NDN and CD1A are novel prognostic methylation markers in patients with head and neck squamous carcinomas.

BACKGROUND: HPV-associated HNSCCs have a distinct etiologic mechanism and better prognosis than those with non-HPV associated HNSCCs. However, even within the each group, there is heterogeneity in survival time. Here, we test the hypothesis that specific candidate gene methylation markers (CCNA1, NDN, CD1A, DCC, p16, GADD45A) are associated with tumor recurrence and survival, in a well-characterized, prospective, cohort of 346 HNSCC patients. METHODS: Kaplan-Meier curves were used to estimate survival time distributions. Multivariable Cox Proportional Hazards models were used to test associations between each methylation marker and OST/RPFT after adjusting for known or identified prognostic factors. Stratified Cox models included an interaction term between HPV and methylation marker to test for differences in the associations of the biomarker with OST or RPFT across HPV status. RESULTS: Methylation markers were differentially associated with patient characteristics. DNA hypermethylation of NDN and CD1A was found to be significantly associated with overall survival time (OST) in all HNSCC patients (NDN hazard ratio (HR): 2.35, 95% CI: 1.40-3.94; CD1A HR: 1.31, 95% CI: 1.01-1.71). Stratification by HPV status revealed hypermethylation of CD1A was associated with better OST and recurrence/persistence-free time (RPFT) (OST HR: 3.34, 95% CI: 1.88-5.93; RPFT HR: 2.06, 95% CI: 1.21-3.49), while hypomethylation of CCNA1 was associated with increased RPFT in HPV (+) patients only (HR: 0.31, 95% CI: 0.13-0.74). CONCLUSIONS: This study is the first to describe novel epigenetic alterations associated with survival in an unselected, prospectively collected, consecutive cohort of patients with HNSCC. DNA hypermethylation of NDN and CD1A was found to be significantly associated with increased overall survival time in all HNSCC patients. However, stratification by the important prognostic factor of HPV status revealed the immune marker, CD1A, and the cell cycle regulator, CCNA1 to be associated with prognosis in HPV (+) patients, specifically. Here, we identified novel methylation markers and specific, epigenetic molecular differences associated with HPV status, which warrant further investigation.

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas.

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

BH3-mimetic small molecule inhibits the growth and recurrence of adenoid cystic carcinoma.

OBJECTIVES: To evaluate the anti-tumor effect of BM-1197, a new potent and highly specific small molecule inhibitor of Bcl-2/Bcl-xL, in preclinical models of human adenoid cystic carcinoma (ACC). METHODS: Low passage primary human adenoid cystic carcinoma cells (UM-HACC-2A,-2B,-5,-6) and patient-derived xenograft (PDX) models (UM-PDX-HACC) were developed from surgical specimens obtained from 4 patients. The effect of BM-1197 on cell viability and cell cycle were evaluated in vitro using this panel of low passage ACC cells. The effect of BM-1197 on tumor growth, recurrence and tumor cell apoptosis in vivo was evaluated with the PDX model of ACC (UM-PDX-HACC-5). RESULTS: Exposure of low passage primary human ACC cells to BM-1197 mediated an IC50 of 0.92-2.82 µM. This correlated with an increase in the fraction of apoptotic cells (p<0.0001) and an increase in caspase-3 activity (p<0.0001), but no noticeable differences in cell cycle (p>0.05). In vivo, BM-1197 inhibited tumor growth (p=0.0256) and induced tumor cell apoptosis (p=0.0165) without causing significant systemic toxicities, as determined by mouse weight over time. Surprisingly, weekly BM-1197 decreased the incidence of tumor recurrence (p=0.0297), as determined by Kaplan-Meier analysis. CONCLUSION: These data demonstrated that single agent BM-1197 induces apoptosis and inhibits tumor growth in preclinical models of adenoid cystic carcinoma. Notably, single agent BM-1197 inhibited tumor recurrence, which is considered a major clinical challenge in the clinical management of adenoid cystic carcinoma. Collectively, these results suggest that patients with adenoid cystic carcinoma might benefit from therapy with a BH3-mimetic small molecule.

Significantly Decreased Recurrence Rates in Keratocystic Odontogenic Tumor With Simple Enucleation and Curettage Using Carnoy's Versus Modified Carnoy's Solution.

PURPOSE: A variety of modalities has been suggested for treatment of keratocystic odontogenic tumor (KOT), including Carnoy's solution (CS) and modified Carnoy's (without chloroform) solution (MC). The purpose of the present study was to investigate the effect of CS versus MC as it relates to the KOT recurrence rates when used in conjunction with simple enucleation and curettage (E&C) for treatment of KOT. MATERIALS AND METHODS: A retrospective cohort study of patients with a pathologic diagnosis of KOT treated with E&C and application of CS or MC by 3 surgeons at a single center from January 1996 to April 2014 was completed. The demographic, clinical, radiographic, and histologic data were collected for each patient. All disease recurrences were confirmed by biopsy. The primary outcome variable of the study was the interval to recurrence, with the predictor of CS versus MC. Other variables included in the analysis were gender, age, surgeon, and lesion location. Multivariate analysis, including the Wilcoxon test and χ(2) test of associations, was performed. RESULTS: A total of 210 patient medical records were reviewed, with 80 patients meeting the final study criteria. Of the 80 patients, 44 were in the CS arm and 36 in the MC arm. The median age was 47 years (range 10 to 89) in the CS group and 50 years (range 14 to 72) in the MC group (P = .70). Women accounted for 43% (19 of 44) and 44% (16 of 36) of the patients in the CS and MC treatment arms, respectively (P = .91). The lesions were found in the mandible in 26 of the 44 patients (59%) treated with CS and 22 of the 36 patients (61%) treated with MC (P = .85). Surgeon 1 treated 37 of the 44 patients (84%) and 21 of 36 patients (58%) in the CS and MC groups, respectively (P = .01). The recurrence rate was 10% for the CS arm and 35% for the MC arm (P = .027; hazard ratio 6.9). CONCLUSION: In the present retrospective study, the recurrence rate of KOTs treated by E&C with application of CS is significantly lower than that of MC. The data provided could be considered by the Food and Drug Administration for a clinical trial of CS in patients with KOT.

Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling.

Cisplatin induces Bmi-1 and enhances the stem cell fraction in head and neck cancer.

Recent evidence has unveiled a subpopulation of highly tumorigenic, multipotent cells capable of self-renewal in head and neck squamous cell carcinomas (HNSCCs). These unique cells, named here cancer stem cells (CSCs), proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that CSCs are found in perivascular niches and rely on endothelial cell-secreted factors [particularly interleukin-6 (IL-6)] for their survival and self-renewal in HNSCC. Here, we hypothesized that cisplatin enhances the stem cell fraction in HNSCC. To address this hypothesis, we generated xenograft HNSCC tumors with University of Michigan-squamous cell carcinoma 22B (UM-SCC-22B) cells and observed that cisplatin treatment increased (P = .0013) the fraction of CSCs [i.e., aldehyde dehydrogenase activity high and cluster of differentiation 44 high (ALDH(high)CD44(high))]. Cisplatin promoted self-renewal and survival of CSCs in vitro, as seen by an increase in the number of orospheres in ultralow attachment plates and induction in B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and octamer-binding transcription factor 4 expression. Cisplatin-resistant cells expressed more Bmi-1 than cisplatin-sensitive cells. IL-6 potentiated cisplatin-induced orosphere formation generated when primary human HNSCC cells were sorted for ALDH(high)CD44(high) immediately after surgery and plated onto ultralow attachment plates. IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in UM-SCC-22B cells, whereas IL-6-induced extracellular signal-regulated kinase (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Notably, cisplatin-induced Bmi-1 was inhibited by interleukin-6 receptor blockade in parental and cisplatin-resistant cells. Taken together, these results demonstrate that cisplatin enhances the fraction of CSCs and suggest a mechanism for resistance to cisplatin therapy in head and neck cancer.