The Impact of Venous Anastomosis Technique on Outcomes of Free Tissue Transfer to the Head and Neck.

Objectives: To evaluate the impact of different techniques of microvascular venous anastomosis on the outcome of free tissue transfer to the head and neck. Methods: Retrospective case series of patients undergoing microvascular free tissue transfer (MFTT) from January 2006 to September 2021. Chi-square tests and t-tests were utilized to identify differences in flap outcomes by technique, and log-binomial regression analyses were utilized to identify differences in flap outcomes by technique. Results: A total of 1055 consecutive MFTTs were analyzed. One hundred four cases required a return to the operating room for any reason, and 19 were attributed to venous compromise (18.0%). Ultimately, there were 22 FTT failures requiring complete revision (2.1%). In total, 1055 MFTTs involved 1352 venous anastomoses, ranging from 1 to 3 anastomoses in each case. End-to-end (ETE) was used 1040 times (76.9%) and end-to-side (ETS) 204 times (15.0%). The calculated risk ratio for venous complication for ETS compared with ETE was 1.17 (0.34-3.98). A microvascular coupler was used in 355 cases (33.6%). The calculated risk ratio for coupler compared with suture anastomoses was 0.92 (0.35-2.39). Conclusions: There were no significant difference in regard to outcomes of MFTT when comparing ETE with ETS, nor when comparing coupler with suture anastomoses.

Associations between demographic factors and pediatric otolaryngology access disparities in the COVID-19 era.

OBJECTIVES: To assess the impact of demographic factors and telehealth on access to pediatric otolaryngology care during the COVID-19 pandemic, as measured by attendance. METHODS: Retrospective, observational study of all referrals to pediatric otolaryngology at a single, tertiary care pediatric hospital system in the US. All referrals placed to pediatric otolaryngology from March through December 2020 were compared with referrals between March and December 2019. Data on patient demographics, date of referral, duration between referral and appointment, appointment type, and diagnosis acuity were collected. A multivariate linear regression was used to evaluate the impact of the patient age, ethnicity, language, insurance, diagnosis acuity, time to appointment, and appointment type on attendance. RESULTS: This study included 1988 referrals placed between March 16th-December 31st, 2020 and 3704 referrals placed between March 16th-December 31st, 2019. In 2020, attendance proportions were significantly higher at 72% compared to 62% during 2019 (p < 0.001). In 2020, there was a significantly shorter duration between referral and appointment, averaging 10 days as compared to 26 days in 2019 (p < 0.001). Overall, Black and Hispanic patients, children over the age of one, publicly insured patients, and those with longer wait times were less likely to attend their appointments. Primary language and use of telehealth did not predict attendance. CONCLUSION: Early evidence has found significant healthcare access and outcome disparities across ethnicities during the COVID pandemic. However, there is limited data evaluating the effect of demographic factors or telehealth on access to pediatric otolaryngology care. This study identifies age, race and insurance type as predictors of access to pediatric otolaryngologic care, as measured by attendance.

Cochlear Implantation and Risk of Falls in Older Adults.

OBJECTIVE: To examine whether cochlear implantation (CI) increases the risk of clinically significant falls in older adults. STUDY DESIGN: Retrospective analysis of deidentified administrative claims from a US commercial insurance database. SETTING: Nationwide deidentified private insurance claims database (Clinformatics Data Mart; Optum). METHODS: Patients undergoing CI were identified through Current Procedural Terminology codes. Number of days with falls resulting in health care expenditure were counted 1 year pre- and post-CI. Generalized estimating equation Poisson regression was used to determine medical and sociodemographic predictors for fall days, including age, sex, race, and income, with pre- vs post-CI status. RESULTS: Between 2003 and 2019, 3773 patients aged >50 years underwent CI. An overall 139 (3.68%) patients recorded at least 1 fall diagnosis a year pre-CI, and 142 (3.76%) recorded at least 1 fall diagnosis post-CI. The average number of days with fall diagnoses per patient with a recorded fall was 3.12 pre-CI and 2.04 post-CI. In bivariate analysis, age (P < .0001) and Charlson Comorbidity Index (P < .0001) were predictive of falls, but sex (P < .10), race (P < .72), and income (P < .51) were not. Poisson regression demonstrated a statistically significant association between Charlson Comorbidity Index and days with fall diagnoses (risk ratio, 1.39 [95% CI, 1.30-1.49]; P < .0001]). No statistically significant difference in falls was seen pre- vs post-CI (risk ratio, 0.67 [95% CI, 0.34-1.33]; P < .25]). Age also was not predictive of falls in multivariate analysis. CONCLUSIONS: CI does not appear to increase the risk of falls in older adults. Patient comorbidities correlate most strongly with fall risk and should be considered in patient selection for CI.

Photoacoustic Molecular Imaging for the Identification of Lymph Node Metastasis in Head and Neck Cancer Using an Anti-EGFR Antibody-Dye Conjugate.

The presence of lymph node (LN) metastases is an essential prognostic indicator in patients with head and neck squamous cell carcinoma (HNSCC). This study assessed photoacoustic molecular imaging (PAMI) of the antiepidermal growth factor receptor antibody (panitumumab) conjugated to a near-infrared fluorescent dye, IRDye800CW (panitumumab-IRDye800CW; pan800), for the identification of occult metastatic LNs in patients with HNSCC (n = 7). Methods: After in vitro photoacoustic imaging characterization of pan800, PAMI was performed on excised neck specimens from patients infused with pan800 before surgery. Freshly obtained neck specimens were imaged with 3-dimensional, multiwavelength spectroscopic PAMI (wavelengths of 680, 686, 740, 800, 860, 924, and 958 nm). Harvested LNs were then imaged with a closed-field near-infrared fluorescence imager and histologically examined by the pathologist to determine their metastatic status. Results: In total, 53 LNs with a maximum diameter of 10 mm were analyzed with photoacoustic and fluorescence imaging, of which 4 were determined to be metastatic on the final histopathologic report. Photoacoustic signals in the LNs corresponding to accumulated pan800 were spectrally unmixed using a linear least-square-error classification algorithm. The average thresholded photoacoustic signal intensity corresponding to pan800 was 5-fold higher for metastatic LNs than for benign LNs (2.50 ± 1.09 arbitrary units [a.u.] vs. 0.53 ± 0.32 a.u., P < 0.001). Fluorescence imaging showed that metastatic LNs had a 2-fold increase in fluorescence signal compared with benign LNs ex vivo (P < 0.01, 0.068 ± 0.027 a.u. vs. 0.035 ± 0.018 a.u.). Moreover, the ratio of the average of the highest 10% of the photoacoustic signal intensity over the total average, representative of the degree of heterogeneity in the pan800 signal in LNs, showed a significant difference between metastatic LNs and benign LNs (11.6 ± 13.4 vs. 1.8 ± 0.7, P < 0.01) and an area under the receiver-operating-characteristic curve of 0.96 (95% CI, 0.91-1.00). Conclusion: The data indicate that PAMI of IRDye800-labeled tumor-specific antibody may have the potential to identify occult LN metastasis perioperatively in HNSCC patients.

Co-administered antibody improves penetration of antibody-dye conjugate into human cancers with implications for antibody-drug conjugates.

Poor tissue penetration remains a major challenge for antibody-based therapeutics of solid tumors, but proper dosing can improve the tissue penetration and thus therapeutic efficacy of these biologics. Due to dose-limiting toxicity of the small molecule payload, antibody-drug conjugates (ADCs) are administered at a much lower dose than their parent antibodies, which further reduces tissue penetration. We conducted an early-phase clinical trial (NCT02415881) and previously reported the safety of an antibody-dye conjugate (panitumumab-IRDye800CW) as primary outcome. Here, we report a retrospective exploratory analysis of the trial to evaluate whether co-administration of an unconjugated antibody could improve the intratumoral distribution of the antibody-dye conjugate in patients. By measuring the multiscale distribution of the antibody-dye conjugate, this study demonstrates improved microscopic antibody distribution without increasing uptake (toxicity) in healthy tissue when co-administered with the parent antibody, supporting further clinical investigation of the co-administration dosing strategy to improve the tumor penetration of ADCs.

Tumour-specific fluorescence-guided surgery for pancreatic cancer using panitumumab-IRDye800CW: a phase 1 single-centre, open-label, single-arm, dose-escalation study.

BACKGROUND: Complete surgical resection remains the primary curative option for pancreatic ductal adenocarcinoma, with positive margins in 30-70% of patients. In this study, we aimed to evaluate the use of intraoperative tumour-specific imaging to enhance a surgeon's ability to detect visually occult cancer in real time. METHODS: In this single-centre, open-label, single-arm study, done in the USA, we enrolled patients who had clinically suspicious or biopsy-confirmed pancreatic ductal adenocarcinomas and were scheduled for curative surgery. Eligible patients were 19 years of age or older with a life expectancy of more than 12 weeks and a Karnofsky performance status of at least 70% or an Eastern Cooperative Oncology Group or Zubrod level of one or lower, who were scheduled to undergo curative surgery. Patients were sequentially enrolled into each dosing group and 2-5 days before surgery, patients were intravenously infused with 100 mg of unlabelled panitumumab followed by 25 mg, 50 mg, or 75 mg of the near-infrared fluorescently labelled antibody (panitumumab-IRDye800CW). The primary endpoint was to determine the optimal dose of panitumumab-IRDye800CW in identifying pancreatic ductal adenocarcinomas as measured by tumour-to-background ratio in all patients. The tumour-to-background ratio was defined as the fluorescence signal of the tumour divided by the fluorescence signal of the surrounding healthy tissue. The dose-finding part of this study has been completed. This study is registered with ClinicalTrials.gov, NCT03384238. FINDINGS: Between April, 2018, and July, 2019, 16 patients were screened for enrolment onto the study. Of the 16 screened patients, two (12%) patients withdrew from the study and three (19%) were not eligible; 11 (69%) patients completed the trial, all of whom were clinically diagnosed with pancreatic ductal adenocarcinoma. The mean tumour-to-background ratio of primary tumours was 3·0 (SD 0·5) in the 25 mg group, 4·0 (SD 0·6) in the 50 mg group, and 3·7 (SD 0·4) in the 75 mg group; the optimal dose was identified as 50 mg. Intraoperatively, near-infrared fluorescence imaging provided enhanced visualisation of the primary tumours, metastatic lymph nodes, and small (<2 mm) peritoneal metastasis. Intravenous administration of panitumumab-IRDye800CW at the doses of 25 mg, 50 mg, and 75 mg did not result in any grade 3 or higher adverse events. There were no serious adverse events attributed to panitumumab-IRDye800CW, although four possibly related adverse events (grade 1 and 2) were reported in four patients. INTERPRETATION: To our knowledge, this study presents the first clinical use of panitumumab-IRDye800CW for detecting pancreatic ductal adenocarcinomas and shows that panitumumab-IRDye800CW is safe and feasible to use during pancreatic cancer surgery. Tumour-specific intraoperative imaging might have added value for treatment of patients with pancreatic ductal adenocarcinomas through improved patient selection and enhanced visualisation of surgical margins, metastatic lymph nodes, and distant metastasis. FUNDING: National Institutes of Health and the Netherlands Organization for Scientific Research.

Trends in Age of Cochlear Implant Recipients, and the Impact on Perioperative Complication Rates.

OBJECTIVE: The aim of the study is to examine trends in the age of patients receiving cochlear implants and to determine the effect of age on the rate of perioperative complications. STUDY DESIGN: Retrospective analysis of deidentified administrative claims data from a US commercial insurance database (Optum). PATIENTS: Individuals undergoing cochlear implantation between 2003 and 2016. SETTING: US hospital and outpatient facilities serving commercially insured patients. INTERVENTION: Cochlear implantation. MAIN OUTCOME MEASURES: Age at implantation, incidence of perioperative complications within 30 days identified by ICD9/10 codes including device problems, myocardial infarction, stroke, venous thromboembolism, local infection, meningitis, stroke, cerebrospinal fluid leak, and facial weakness. RESULTS: Between 2003 and 2016, 3420 patients underwent a total of 4154 cochlear implants. The number of implants per year increased annually from 171 in 2003 to 531 in 2016, with the greatest growth demonstrated in those aged 60 and older.The age of patients undergoing implantation increased annually from an average of 26.6-57.2 years (p < 0.001). The implantation rates from 2003 to 2016, per 100,000 enrollees, increased from 1.64 to 6.82 for patients 60-79 years of age, and 0 to 11.57 for patients greater than 80 years of age (p < 0.001). No significant differences in 30-day complication rates were found between patients when grouped by age in decades, except for device related problems, which was significantly higher in younger patients (<18 years). CONCLUSION: Over the past decade and a half, cochlear implantation is more frequently being performed, and in an increasingly aging population. This trend does not seem to alter the risk of perioperative complications.

Predicting Therapeutic Antibody Delivery into Human Head and Neck Cancers.

PURPOSE: The efficacy of antibody-based therapeutics depends on successful drug delivery into solid tumors; therefore, there is a clinical need to measure intratumoral antibody distribution. This study aims to develop and validate an imaging and computation platform to directly quantify and predict antibody delivery into human head and neck cancers in a clinical study. EXPERIMENTAL DESIGN: Twenty-four patients received systemic infusion of a near-infrared fluorescence-labeled therapeutic antibody followed by surgical tumor resection. A computational platform was developed to quantify the extent of heterogeneity of intratumoral antibody distribution. Both univariate and multivariate regression analyses were used to select the most predictive tumor biological factors for antibody delivery. Quantitative image features from the pretreatment MRI were extracted and correlated with fluorescence imaging of antibody delivery. RESULTS: This study not only confirmed heterogeneous intratumoral antibody distribution in-line with many preclinical reports, but also quantified the extent of interpatient, intertumor, and intratumor heterogeneity of antibody delivery. This study demonstrated the strong predictive value of tumor size for intratumoral antibody accumulation and its significant impact on antibody distribution in both primary tumor and lymph node metastasis. Furthermore, this study established the feasibility of using contrast-enhanced MRI to predict antibody delivery. CONCLUSIONS: This study provides a clinically translatable platform to measure antibody delivery into solid tumors and yields valuable insight into clinically relevant antibody tumor penetration, with implications in the selection of patients amenable to antibody therapy and the design of more effective dosing strategies.

Optimal Dosing Strategy for Fluorescence-Guided Surgery with Panitumumab-IRDye800CW in Head and Neck Cancer.

PURPOSE: To identify the optimal dosing strategy for fluorescence-guided surgery in patients with head and neck squamous cell carcinoma, we conducted a dose-ranging study evaluating the anti-epidermal growth factor receptor (EGFR) therapeutic antibody, panitumumab, that was fluorescently labeled with the near-infrared dye IRDye800CW. PROCEDURES: Patients (n = 24) received either 0.5 or 1.0 mg/kg panitumumab-IRDye800CW in the weight-based dosing group or 25 or 50 mg panitumumab-IRDye800CW in the fixed dosing group. Following surgery, whole primary specimens were imaged in a closed-field device and the mean fluorescence intensity (MFI) and tumor-to-background ratio (TBR) were assessed. Clinical variables, including dose, time of infusion-to-surgery, age, unlabeled dose, gender, primary tumor site, and tumor size, were analyzed to evaluate the factors affecting the fluorescence intensity in order to identify the optimal dose for intraoperative fluorescence imaging. RESULTS: A total of 24 primary tumor specimens were imaged and analyzed in this study. Although no correlations between TBR and dose of panitumumab-IRDye800CW were found, there were moderate-strong correlations between the primary tumor MFI and panitumumab-IRDye800CW dose for fixed dose (mg) (R2 = 0.42) and for dose/weight (mg/kg) (R2 = 0.54). Results indicated that the optimal MFI was at approximately 50 mg for fixed dose and 0.75 mg/kg for dose/weight. No significant differences were found for the primary tumor MFI and TBRs between the weight-based dosing and the fixed dosing groups. MFIs significantly increased when the infusion-to-surgery window was reduced to within 2 days (vs. 3 days or more, p < 0.05). CONCLUSIONS: Antibody-based imaging for surgical resection is under investigation in multiple clinical trials. Our data suggests that a fixed dose of 50 mg is an appropriate diagnostic dose for successful surgical fluorescence imaging.

Endoscopic Fluorescence-Guided Surgery for Sinonasal Cancer Using an Antibody-Dye Conjugate.

OBJECTIVE: Endoscopic resection of sinonasal squamous cell carcinoma has become the standard of care, but challenges remain in obtaining clear resection margins. The current study evaluated the feasibility of endoscopic fluorescence-guided surgery (FGS) to improve surgical resection in a human sinus surgical model. METHODS: A fluorescence endoscope optimized for near-infrared (NIR) fluorescence detection was evaluated in a phantom study. Various endoscope diameters (4 and 10 mm) and viewing angles (0, 30, and 45 degrees) were evaluated to determine the sensitivity of the system for IRDye800CW detection at various working distances (1-5 cm). Endoscopic FGS was then validated in a three-dimensional human sinus surgical model to which squamous cell tumors derived from mice were inserted. Mice had received intravenous panitumumab-IRDye800CW and upon fluorescence-guided tumor resection, mean fluorescence intensity (MFI) and tumor-to-background ratio (TBR) were calculated in in situ and ex vivo settings. RESULTS: A significantly higher fluorescence intensity was found when using the 10-mm diameter endoscope compared to the 4mm diameter endoscope (P < .001). No significant difference in MFI was found among the viewing angles of the 4-mm diameter endoscope. Using the human sinus model, the highest MFI and TBR were obtained at a 1-cm working distance compared to longer working distances. CONCLUSION: We demonstrate that clinically acceptable TBRs were obtained with several working distances to discriminate tumor tissue from adjacent normal tissue in a human sinus model, and that endoscopic FGS may have great potential in identifying residual tumor tissue regions during surgery. Laryngoscope, 2019.

Probe-based fluorescence dosimetry of an antibody-dye conjugate to identify head and neck cancer as a first step to fluorescence-guided tissue preselection for pathological assessment.

BACKGROUND: Despite the rapid growth of fluorescence imaging, accurate sampling of tissue sections remains challenging. Development of novel technologies to improve intraoperative assessment of tissue is needed. METHODS: A novel contact probe-based fluorescence dosimeter device, optimized for IRDye800CW quantification, was developed. After evaluation of the device in a phantom setup, its clinical value was defined ex vivo in patients with head and neck squamous cell carcinoma who received panitumumab-IRDye800CW. RESULTS: Ten patients were enrolled with a total of 216 data points obtained. Final histopathology showed tumor in 119 spots and normal tissue in 97 spots. Fluorescence-to-excitation ratios in tumor tissue were more than three times higher than those in normal tissue. The area under the curve was 0.86 (95% CI: 0.81-0.91) for tumor detection. CONCLUSIONS: Fluorescence-guided tissue preselection using a fluorescence dosimeter could have substantial impact on tissue sampling for frozen section analysis and potentially reduce sampling errors.

Optical molecular imaging can differentiate metastatic from benign lymph nodes in head and neck cancer.

Identification of lymph node (LN) metastasis is essential for staging of solid tumors, and as a result, surgeons focus on harvesting significant numbers of LNs during ablative procedures for pathological evaluation. Isolating those LNs most likely to harbor metastatic disease can allow for a more rigorous evaluation of fewer LNs. Here we evaluate the impact of a systemically injected, near-infrared fluorescently-labeled, tumor-targeting contrast agent, panitumumab-IRDye800CW, to facilitate the identification of metastatic LNs in the ex vivo setting for head and neck cancer patients. Molecular imaging demonstrates a significantly higher mean fluorescence signal in metastatic LNs compared to benign LNs in head and neck cancer patients undergoing an elective neck dissection. Molecular imaging to preselect at-risk LNs may thus allow a more rigorous examination of LNs and subsequently lead to improved prognostication than regular neck dissection.

Intraoperative Tumor Assessment Using Real-Time Molecular Imaging in Head and Neck Cancer Patients.

BACKGROUND: In head and neck cancer, surgical resection using primarily visual and tactile feedback is considered the gold standard for solid tumors. Due to high numbers of tumor-involved surgical margins, which are directly correlated to poor clinical outcomes, intraoperative optical imaging trials have rapidly proliferated over the past 5 years. However, few studies report on intraoperative in situ imaging data that could support surgical resection. To demonstrate the clinical application of in situ surgical imaging, we report on the imaging data that are directly (ie in real-time) available to the surgeon. STUDY DESIGN: Fluorescence intensities and tumor-to-background ratios (TBRs) were determined from the intraoperative imaging data-the view as seen by the surgeon during tumor resection-of 20 patients, and correlated to patient and tumor characteristics including age, sex, tumor site, tumor size, histologic differentiation, and epidermal growth factor receptor (EGFR) expression. Furthermore, different lighting conditions in regard to surgical workflow were evaluated. RESULTS: Under these circumstances, intraoperative TBRs of the primary tumors averaged 2.2 ± 0.4 (range 1.5 to 2.9). Age, sex, tumor site, and tumor size did not have a significant effect on open-field intraoperative molecular imaging of the primary tumors (p > 0.05). In addition, variation in EGFR expression levels or the presence of ambient light did not seem to alter TBRs. CONCLUSIONS: We present the results of successful in situ intraoperative imaging of primary tumors alongside the optimal conditions with respect to both molecular image acquisition and surgical workflow. This study illuminates the potentials of open-field molecular imaging to assist the surgeon in achieving successful cancer removal.

Fluorescence molecular imaging for identification of high-grade dysplasia in patients with head and neck cancer.

OBJECTIVE: High-grade dysplasia is associated with a risk of malignant transformation, and it is necessary to distinguish from normal epithelium or low-grade dysplasia, especially in the intraoperative setting. We hypothesize that an anti-epidermal growth factor receptor (EGFR) contrast agent can be used to differentiate high-grade dysplasia from low-grade dysplasia and normal epithelium. MATERIALS AND METHODS: Patients with biopsy proven head and neck squamous cell carcinoma (HNSCC) were enrolled in a clinical trial using systemically injected fluorescently labeled anti-EGFR antibody (panitumumab-IRDye800CW) (NCT02415881). Paraffin embedded tumor specimens from 11 patients were evaluated by fluorescence histopathology. Hematoxylin and eosin (H&E) slides were reviewed by a board-certified pathologist, and regions of invasive squamous cell carcinoma, high-grade dysplasia and low-grade dysplasia were delineated. EGFR expression was assessed for each patient by way of immunohistochemistry. RESULTS: 11 patients were included in the study with a total of 219 areas on tissue sections analyzed; 68 normal epithelium, 53 low-grade dysplasia, 48 high-grade dysplasia, and 50 malignant regions. The signal-to-background ratio (SBR) increased proportionally with increasing grade of dysplasia; normal epithelium (1.5 ±â€¯0.1), low-grade dysplasia (1.8 ±â€¯0.1), high-grade dysplasia: (2.3 ±â€¯0.2). High-grade dysplasia had a significantly higher SBR when compared to normal or low-grade dysplasia (p < 0.05). Fluorescence histopathology positively correlated with EGFR expression by immunohistochemistry, which also increased proportionally with increasing degree of dysplasia. CONCLUSION: Molecular imaging with an anti-EGFR agent can successfully discriminate high-grade dysplastic lesions from low-grade dysplasia and normal epithelium.

The Sentinel Margin: Intraoperative Ex Vivo Specimen Mapping Using Relative Fluorescence Intensity.

PURPOSE: Despite major advancements in surgical oncology, the positive margin rate for primary head and neck cancer resection remains around 15%-30%. In particular, the deep surface margin is the most challenging to adequately assess. Inadequate margins are directly correlated to poor survival, and as such, mitigation of these rates is critical to improve patient outcomes. We have developed an ex vivo imaging strategy that utilizes fluorescence intensity peaks (relative to background signal) of an injected anti-EGFR antibody conjugated to a fluorescent probe to locate potential close or positive margins on the deep surface of the resected tumor specimen. EXPERIMENTAL DESIGN: Twelve patients with head and neck cancer scheduled for surgery received systemic administration of a tumor-specific contrast-agent (panitumumab-IRDye800CW). After surgical resection, the tumor specimen was imaged using a fluorescence imager. The three highest fluorescence intensity-peaks on the deep surface of the specimen were isolated and correlated to histology to determine the margin distance at these regions. RESULTS: Relative fluorescence peak intensities identified the closest margin on the deep surface of the specimen within 2.5 minutes. The highest intensity peak consistently (100%) detected the closest margin to the tumor. The difference in tumor margin distance between the first and second highest fluorescence intensity peak averaged 2.1 ± 1.4 mm. The tumor-margin difference between the second and third highest peak averaged 1.6 ± 0.6 mm. CONCLUSIONS: Fluorescence intensity peaks can identify the region on the specimen where tumor is closest to specimen's edge on the deep surface. This technique could have broad applications in obtaining adequate margins in oncological surgery.

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

PURPOSE: Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. EXPERIMENTAL DESIGN: Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. RESULTS: GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. CONCLUSIONS: Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

The Clinical Application of Fluorescence-Guided Surgery in Head and Neck Cancer.

Although surgical resection has been the primary treatment modality of solid tumors for decades, surgeons still rely on visual cues and palpation to delineate healthy from cancerous tissue. This may contribute to the high rate (up to 30%) of positive margins in head and neck cancer resections. Margin status in these patients is the most important prognostic factor for overall survival. In addition, second primary lesions may be present at the time of surgery. Although often unnoticed by the medical team, these lesions can have significant survival ramifications. We hypothesize that real-time fluorescence imaging can enhance intraoperative decision making by aiding the surgeon in detecting close or positive margins and visualizing unanticipated regions of primary disease. The purpose of this study was to assess the clinical utility of real-time fluorescence imaging for intraoperative decision making. Methods: Head and neck cancer patients (n = 14) scheduled for curative resection were enrolled in a clinical trial evaluating panitumumab-IRDye800CW for surgical guidance (NCT02415881). Open-field fluorescence imaging was performed throughout the surgical procedure. The fluorescence signal was quantified as signal-to-background ratios to characterize the fluorescence contrast of regions of interest relative to background. Results: Fluorescence imaging was able to improve surgical decision making in 3 cases (21.4%): identification of a close margin (n = 1) and unanticipated regions of primary disease (n = 2). Conclusion: This study demonstrates the clinical applications of fluorescence imaging on intraoperative decision making. This information is required for designing phase III clinical trials using this technique. Furthermore, this study is the first to demonstrate this application for intraoperative decision making during resection of primary tumors.

Intraoperative Molecular Imaging for ex vivo Assessment of Peripheral Margins in Oral Squamous Cell Carcinoma.

Objective: Complete surgical resection is the standard of care for treatment of oral cancer although the positive margin rate remains 15-30%. Tissue sampling from the resected specimen and from the wound bed for frozen section analysis (FSA) remains the mainstay for intraoperative margin assessment but is subject to sampling error and can require the processing of multiple samples. We sought to understand if an ex vivo imaging strategy using a tumor-targeted fluorescently labeled antibody could accurately identify the closest peripheral margin on the mucosal surface of resected tumor specimen, so that this "sentinel margin" could be used to guide pathological sampling. Materials and Methods: Twenty-nine patients with oral squamous cell carcinoma scheduled for surgical resection were consented for the study and received systemic administration of a tumor-targeted fluorescently labeled antibody (Panitumumab IRDye800CW). After surgical resection, the tumor specimen was imaged using a closed-field fluorescent imaging device. Relevant pathological data was available for five patients on retrospective review. For each of these five patients, two regions of highest fluorescence intensity at the peripheral margin and one region of lowest fluorescence intensity were identified, and results were correlated with histology to determine if the region of highest fluorescence intensity along the mucosal margin (i.e., the sentinel margin) was truly the closest margin. Results: Imaging acquisition of the mucosal surface of the specimen immediately after surgery took 30 s. In all of the specimens, the region of highest fluorescence at the specimen edge had a significantly smaller margin distance than other sampled regions. The average margin distance at the closest, "sentinel," margin was 3.2 mm compared to a margin distance of 8.0 mm at other regions (p < 0.0001). Conclusions: This proof-of-concept study suggests that, when combined with routine FSA, ex vivo fluorescent specimen imaging can be used to identify the closest surgical margin on the specimen. This approach may reduce sampling error of intraoperative evaluation, which should ultimately improve the ability of the surgeon to identify the sentinel margin. This rapid sentinel margin identification improves the surgeon's orientation to areas most likely to be positive in the surgical wound bed and may expedite pathology workflow.

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

Heat shock protein vaccines against glioblastoma: from bench to bedside.

Current adjuvant treatment regimens available for the treatment of glioblastoma are widely ineffective and offer a dismal prognosis. Advancements in conventional treatment strategies have only yielded modest improvements in overall survival. Immunotherapy remains a promising adjuvant in the treatment of GBM through eliciting tumor specific immune responses capable of producing sustained antitumor response while minimizing systemic toxicity. Heat shock proteins (HSP) function as intracellular chaperones and have been implicated in the activation of both innate and adaptive immune systems. Vaccines formulated from HSP-peptide complexes, derived from autologous tumor, have been applied to the field of immunotherapy for glioblastoma. The results from the phase I and II clinical trials have been promising. Here we review the role of HSP in cellular function and immunity, and its application in the treatment of glioblastoma.