HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E.

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

Challenges and opportunities for early phase clinical trials of novel drug-radiotherapy combinations: recommendations from NRG Oncology, the American Society for Radiation Oncology (ASTRO), the American College of Radiology (ACR), the Sarah Cannon Research Institute, and the American College of Radiation Oncology (ACRO).

NRG Oncology's Developmental Therapeutics and Radiation Therapy Subcommittee assembled an interdisciplinary group of investigators to address barriers to successful early phase clinical trials of novel combination therapies involving radiation. This Policy Review elucidates some of the many challenges associated with study design for early phase trials combining radiotherapy with novel systemic agents, which are distinct from drug-drug combination development and are often overlooked. We also advocate for potential solutions that could mitigate or eliminate some of these barriers, providing examples of specific clinical trial designs that could help facilitate efficient and effective evaluation of novel drug-radiotherapy combinations.

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): A pilot, first-in-human study of interferon gamma-stimulated marrow mesenchymal stromal cells for treatment of radiation-induced xerostomia.

BACKGROUND AIMS: Xerostomia, or the feeling of dry mouth, is a significant side effect of radiation therapy for patients with head and neck cancer (HNC). Preliminary data suggest that mesenchymal stromal/stem cells (MSCs) can improve salivary function. We performed a first-in-human pilot study of interferon gamma (IFNγ)-stimulated autologous bone marrow-derived MSCs, or MSC(M), for the treatment of radiation-induced xerostomia (RIX). Here we present the primary safety and secondary efficacy endpoints. METHODS: A single-center pilot clinical trial was conducted investigating the safety and tolerability of autologous IFNγ-stimulated MSC(M). The study was conducted under an approved Food and Drug Administration Investigational New Drug application using an institutional review board-approved protocol (NCT04489732). Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated, cultured, stimulated with IFNγ and cryopreserved for later use. Banked cells were thawed and allowed to recover in culture before patients received a single injection of 10 × 106 MSC(M) into the right submandibular gland under ultrasound guidance. The primary objective was determination of safety and tolerability by evaluating dose-limiting toxicity (DLT). A DLT was defined as submandibular pain >5 on a standard 10-point pain scale or any serious adverse event (SAE) within 1 month after injection. Secondary objectives included analysis of efficacy as measured by salivary quantification and using three validated quality of life instruments. Quantitative results are reported as mean and standard deviation. RESULTS: Six patients with radiation-induced xerostomia who had completed radiation at least 2 years previously (average 7.8 years previously) were enrolled in the pilot study. The median age was 71 (61-74) years. Five (83%) patients were male. Five patients (83%) were treated with chemoradiation and one patient (17%) with radiation alone. Grade 1 pain was seen in 50% of patients after submandibular gland injection; all pain resolved within 4 days. No patients reported pain 1 month after injection, with no SAE or other DLTs reported 1 month after injection. The analysis of secondary endpoints demonstrated a trend of increased salivary production. Three patients (50%) had an increase in unstimulated saliva at 1 and 3 months after MSC(M) injection. Quality of life surveys also showed a trend toward improvement. CONCLUSIONS: Injection of autologous IFNγ-stimulated MSC(M) into a singular submandibular gland of patients with RIX is safe and well tolerated in this pilot study. A trend toward an improvement in secondary endpoints of salivary quantity and quality of life was observed. This first-in-human study provides support for further investigation into IFNγ-stimulated MSC(M) injected in both submandibular glands as an innovative approach to treat RIX and improve quality of life for patients with HNC.

Autophagy awakens-the myriad roles of autophagy in head and neck cancer development and therapeutic response.

Autophagy is an evolutionarily conserved cell survival mechanism that degrades damaged proteins and organelles to generate cellular energy during times of stress. Recycling of these cellular components occurs in a series of sequential steps with multiple regulatory points. Mechanistic dysfunction can lead to a variety of human diseases and cancers due to the complexity of autophagy and its ability to regulate vital cellular functions. The role that autophagy plays in both the development and treatment of cancer is highly complex, especially given the fact that most cancer therapies modulate autophagy. This review aims to discuss the balance of autophagy in the development, progression, and treatment of head and neck cancer, as well as highlighting the need for a deeper understanding of what is still unknown about autophagy.

Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): Study protocol for a phase 1 dose-escalation trial of patients with xerostomia after radiation therapy for head and neck cancer: MARSH: Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction.

BACKGROUND: Xerostomia, or dry mouth, is a common side effect of head and neck radiation. Current treatment options for radiation-induced xerostomia are generally supportive in nature. Adult stem cells are the ultimate source for replenishment of salivary gland tissue. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a viable cell-based therapy for xerostomia. We have undertaken studies enabling U.S. Food and Drug Administration Investigational New Drug status, demonstrating the normal phenotype, intact functionality, and pro-growth secretome of interferon-γ (IFNγ)-stimulated BM-MSCs taken from patients with head and neck cancer who have undergone radiation ± chemotherapy. Here we present the protocol of MARSH, a first-in-human clinical trial of bone marrow-derived, IFNγ-activated BM-MSCs for the treatment of radiation-induced xerostomia. METHODS: This single-center phase 1 dose-escalation with expansion cohort, non-placebo-controlled study will assess the safety and tolerability of BM-MSCs for the treatment of radiation-induced xerostomia in patients who had head and neck cancer. The phase 1 dose-escalation study will be a 3 + 3 design with staggered enrollment. A total of 21 to 30 subjects (9 to 18 in phase 1 study, 12 in expansion cohort) will be enrolled. The primary endpoint is determining the recommended phase 2 dose (RP2D) of IFNγ-stimulated BM-MSCs to enable further studies on the efficacy of BM-MSCs. Patients' bone marrow will be aspirated, and BM-MSCs will be expanded, stimulated with IFNγ, and injected into the submandibular gland. The RP2D will be determined by dose-limiting toxicities occurring within 1 month of BM-MSC injection. Secondary outcomes of saliva amounts and composition, ultrasound of salivary glands, and quality of life surveys will be taken at 3-, 6-, 12-, and 24-month visits. DISCUSSION: Autotransplantation of IFNγ-stimulated BM-MSCs in salivary glands after radiation therapy or chemoradiation therapy may provide an innovative remedy to treat xerostomia and restore quality of life. This is the first therapy for radiation-induced xerostomia that may be curative. TRIAL REGISTRATION: World Health Organization International Clinical Trials Registry Platform: NCT04489732.

Advances in Organ Preservation for Laryngeal Cancer.

OPINION STATEMENT: At the University of Wisconsin, all treatment of head and neck cancer patients begins with discussion at our multi-disciplinary tumor board. Most patients with T4 disease, with existing laryngeal dysfunction, considered unlikely to complete definitive CRT or who have a high risk of persistent aspiration after non-operative management undergo total laryngectomy. A laryngeal sparing approach is attempted on most other patients. Radiotherapy is delivered over 6.5 weeks, preferably with concurrent weekly cisplatin. If the patient is hesitant of chemotherapy or has contraindications to cisplatin, concurrent cetuximab may be offered. Patients treated with RT alone are often treated to the same dose, but via an accelerated schedule by adding a 6th fraction per week. The 6th fraction is given by delivering two treatments at least 6 h apart on a weekday of the patient's choosing. We consider the following to be major risk factors for clinically significant weight loss during treatment: a 10% or greater loss of weight in the 6 months prior to starting treatment, delivery of concurrent cisplatin, and treatment of the bilateral neck with radiation. Patients who have 2-3 of these characteristics are often given gastrostomy tubes prophylactically. Patients are seen 2 weeks after completion of therapy, and then every 3 months after completion for 2 years. A CT neck and PET-CT are performed at the first 3-month visit. They are seen twice in year three, and then yearly until years 5-7. At each of these visits, we have a low threshold to present the patient at our multidisciplinary tumor board for consideration of salvage laryngectomy if there are signs of progression.

Using 4D dose accumulation to calculate organ-at-risk dose deviations from motion-synchronized liver and lung tomotherapy treatments.

Tracking systems such as Radixact Synchrony change the planned delivery of radiation during treatment to follow the target. This is typically achieved without considering the location changes of organs at risk (OARs). The goal of this work was to develop a novel 4D dose accumulation framework to quantify OAR dose deviations due to the motion and tracked treatment. The framework obtains deformation information and the target motion pattern from a four-dimensional computed tomography dataset. The helical tomotherapy treatment plan is split into 10 plans and motion correction is applied separately to the jaw pattern and multi-leaf collimator (MLC) sinogram for each phase based on the location of the target in each phase. Deformable image registration (DIR) is calculated from each phase to the references phase using a commercial algorithm, and doses are accumulated according to the DIR. The effect of motion synchronization on OAR dose was analyzed for five lung and five liver subjects by comparing planned versus synchrony-accumulated dose. The motion was compensated by an average of 1.6 cm of jaw sway and by an average of 5.7% of leaf openings modified, indicating that most of the motion compensation was from jaw sway and not MLC changes. OAR dose deviations as large as 19 Gy were observed, and for all 10 cases, dose deviations greater than 7 Gy were observed. Target dose remained relatively constant (D95% within 3 Gy), confirming that motion-synchronization achieved the goal of maintaining target dose. Dose deviations provided by the framework can be leveraged during the treatment planning process by identifying cases where OAR doses may change significantly from their planned values with respect to the critical constraints. The framework is specific to synchronized helical tomotherapy treatments, but the OAR dose deviations apply to any real-time tracking technique that does not consider location changes of OARs.

Impact of immediate cryopreservation on the establishment of patient derived xenografts from head and neck cancer patients.

BACKGROUND: Patient-derived xenografts established from human cancers are important tools for investigating novel anti-cancer therapies. Establishing PDXs requires a significant investment and many PDXs may be used infrequently due to their similarity to existing models, their growth rate, or the lack of relevant mutations. We performed this study to determine whether we could efficiently establish PDXs after cryopreservation to allow molecular profiling to be completed prior to implanting the human cancer. METHODS: Fresh tumor was split with half used to establish a PDX immediately and half cryopreserved for later implantation. Resulting tumors were assessed histologically and tumors established from fresh or cryopreserved tissues compared as to the growth rate, extent of tumor necrosis, mitotic activity, keratinization, and grade. All PDXs were subjected to short tandem repeat testing to confirm identity and assess similarity between methods. RESULTS: Tumor growth was seen in 70% of implanted cases. No growth in either condition was seen in 30% of tumors. One developed a SCC from the immediate implant but a lymphoproliferative mass without SCC from the cryopreserved specimen. No difference in growth rate was seen. No difference between histologic parameters was seen between the two approaches. CONCLUSIONS: Fresh human cancer tissue can be immediately cryopreserved and later thawed and implanted to establish PDXs. This resource saving approach allows for tumor profiling prior to implantation into animals thus maximizing the probability that the tumor will be utilized for future research.

Immunoavidity-Based Capture of Tumor Exosomes Using Poly(amidoamine) Dendrimer Surfaces.

Tumor-derived blood-circulating exosomes have potential as a biomarker to greatly improve cancer treatment. However, effective isolation of exosomes remains a tremendous technical challenge. This study presents a novel nanostructured polymer surface for highly effective capture of exosomes through strong avidity. Various surface configurations, consisting of multivalent dendrimers, PEG, and tumor-targeting antibodies, were tested using exosomes isolated from tumor cell lines. We found that a dual layer dendrimer configuration exhibited the highest efficiency in capturing cultured exosomes spiked into human serum. Importantly, the optimized surface captured a > 4-fold greater amount of tumor exosomes from head and neck cancer patient plasma samples than that from healthy donors. Nanomechanical analysis using atomic force microscopy also revealed that the enhancement was attributed to multivalent binding (avidity) and augmented short-range adhesion mediated by dendrimers. Our results support that the dendrimer surface detects tumor exosomes at high sensitivity and specificity, demonstrating its potential as a new cancer liquid biopsy platform.