Acquired resistance to immunotherapy and chemoradiation in MYC amplified head and neck cancer.

The proto-oncogene MYC encodes a nuclear transcription factor that has an important role in a variety of cellular processes, such as cell cycle progression, proliferation, metabolism, adhesion, apoptosis, and therapeutic resistance. MYC amplification is consistently observed in aggressive forms of several solid malignancies and correlates with poor prognosis and distant metastases. While the tumorigenic effects of MYC in patients with head and neck squamous cell carcinoma (HNSCC) are well known, the molecular mechanisms by which the amplification of this gene may confer treatment resistance, especially to immune checkpoint inhibitors, remains under-investigated. Here we present a unique case of a patient with recurrent/metastatic (R/M) HNSCC who, despite initial response to nivolumab-based treatment, developed rapidly progressive metastatic disease after the acquisition of MYC amplification. We conducted comparative transcriptomic analysis of this patient's tumor at baseline and upon progression to interrogate potential molecular processes through which MYC may confer resistance to immunotherapy and/or chemoradiation and used TCGA-HNSC dataset and an institutional cohort to further explore clinicopathologic features and key molecular networks associated with MYC amplification in HNSCC. This study highlights MYC amplification as a potential mechanism of immune checkpoint inhibitor resistance and suggest its use as a predictive biomarker and potential therapeutic target in R/M HNSCC.

A nanoengineered topical transmucosal cisplatin delivery system induces anti-tumor response in animal models and patients with oral cancer.

Despite therapeutic advancements, oral cavity squamous cell carcinoma (OCSCC) remains a difficult disease to treat. Systemic platinum-based chemotherapy often leads to dose-limiting toxicity (DLT), affecting quality of life. PRV111 is a nanotechnology-based system for local delivery of cisplatin loaded chitosan particles, that penetrate tumor tissue and lymphatic channels while avoiding systemic circulation and toxicity. Here we evaluate PRV111 using animal models of oral cancer, followed by a clinical trial in patients with OCSCC. In vivo, PRV111 results in elevated cisplatin retention in tumors and negligible systemic levels, compared to the intravenous, intraperitoneal or intratumoral delivery. Furthermore, PRV111 produces robust anti-tumor responses in subcutaneous and orthotopic cancer models and results in complete regression of carcinogen-induced premalignant lesions. In a phase 1/2, open-label, single-arm trial (NCT03502148), primary endpoints of efficacy (≥30% tumor volume reduction) and safety (incidence of DLTs) of neoadjuvant PRV111 were reached, with 69% tumor reduction in ~7 days and over 87% response rate. Secondary endpoints (cisplatin biodistribution, loco-regional control, and technical success) were achieved. No DLTs or drug-related serious adverse events were reported. No locoregional recurrences were evident in 6 months. Integration of PRV111 with current standard of care may improve health outcomes and survival of patients with OCSCC.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Molecular drivers of oral cavity squamous cell carcinoma in non-smoking and non-drinking patients: what do we know so far?

Oral cavity squamous cell carcinoma (OCSCC) is one of the most common head and neck cancers worldwide. It is well known that risk factors for OCSCC include tobacco and excess alcohol consumption. However, in recent years, OCSCC incidence has been increasing in patients without these traditional risk factors. The cause of this increase is unclear and various genetic, environmental, and infectious factors have been hypothesized to play a role. Additionally, there are expert opinions that oral cancer in non-smoking, non-drinking (NSND) patients have a distinct phenotype resulting in more aggressive disease presentation and poorer prognosis. In this review, we summarize the current state of knowledge for oral cavity cancer in patients without traditional risk factors.

Application of liquid biopsy as multi-functional biomarkers in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a molecularly heterogeneous disease, with a 5-year survival rate that still hovers at ~60% despite recent advancements. The advanced stage upon diagnosis, limited success with effective targeted therapy and lack of reliable biomarkers are among the key factors underlying the marginally improved survival rates over the decades. Prevention, early detection and biomarker-driven treatment adaptation are crucial for timely interventions and improved clinical outcomes. Liquid biopsy, analysis of tumour-specific biomarkers circulating in bodily fluids, is a rapidly evolving field that may play a striking role in optimising patient care. In recent years, significant progress has been made towards advancing liquid biopsies for non-invasive early cancer detection, prognosis, treatment adaptation, monitoring of residual disease and surveillance of recurrence. While these emerging technologies have immense potential to improve patient survival, numerous methodological and biological limitations must be overcome before their implementation into clinical practice. This review outlines the current state of knowledge on various types of liquid biopsies in HNSCC, and their potential applications for diagnosis, prognosis, grading treatment response and post-treatment surveillance. It also discusses challenges associated with the clinical applicability of liquid biopsies and prospects of the optimised approaches in the management of HNSCC.

A robust and interpretable gene signature for predicting the lymph node status of primary T1/T2 oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) affects more than 30 000 individuals in the United States annually, with smoking and alcohol consumption being the main risk factors. Management of early-stage tumors usually includes surgical resection followed by postoperative radiotherapy in certain cases. The cervical lymph nodes (LNs) are the most common site for local metastasis, and elective neck dissection is usually performed if the primary tumor thickness is greater than 3.5 mm. However, postoperative histological examination often reveals that many patients with early-stage disease are negative for neck nodal metastasis, posing a pressing need for improved risk stratification to either avoid overtreatment or prevent the disease progression. To this end, we aimed to identify a primary tumor gene signature that can accurately predict cervical LN metastasis in patients with early-stage OSCC. Using gene expression profiles from 189 samples, we trained K-top scoring pairs models and identified six gene pairs that can distinguish primary tumors with nodal metastasis from those without metastasis. The signature was further validated on an independent cohort of 35 patients using real-time polymerase chain reaction (PCR) in which it achieved an area under the receiver operating characteristic (ROC) curve and accuracy of 90% and 91%, respectively. These results indicate that such signature holds promise as a quick and cost effective method for detecting patients at high risk of developing cervical LN metastasis, and may be potentially used to guide the neck treatment regimen in early-stage OSCC.

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Loss of GSK-3ß mediated phosphorylation in HtrA2 contributes to uncontrolled cell death with Parkinsonian phenotype.

HtrA2, a proapoptotic mitochondrial serine protease, promotes cellular protection against oxidative damage. Literature reports show positive correlation between loss of HtrA2 protease activity and Parkinson's Disease (PD) susceptibility. Homozygous loss-of-function mutations in murine-HtrA2, and when they rarely occur in humans result in severe neurodegeneration and infantile death. Here, we report a novel heterozygous pathogenic HTRA2 variant, c.725C > T (p.T242M) in Indian PD patients. Although, this mutation exhibits no significant conformational changes compared to the wild-type, functional studies with HtrA2-T242M transfected neurons reveal common features of PD pathogenesis such as dysfunction, altered morphology and mitochondrial membrane depolarization. Despite exhibiting two-fold decrease in enzyme activity, observation of excessive cell-death due to over-expression of the mutant has been correlated with it being constitutively active. This interesting behavioral anomaly has been attributed to the loss of phosphorylation-mediated regulatory checkpoint at the T242M mutation site that is otherwise controlled by glycogen synthase kinase-3ß (GSK-3ß). This study, with seamless amalgamation of biophysical and biomedical research unravels a mechanistic pathway of HtrA2 regulation and delineates its biological role in PD. Therefore, this investigation will not only prove beneficial toward devising therapeutic strategies against HtrA2-associated diseases mediated by GSK-3ß but also suggest new avenues for treatment of Parkinsonian phenotype.

Modulatory Role of Single Nucleotide Polymorphisms of Distinct Genetic Pathways on Clinical Behavior of Medullary Thyroid Carcinoma.

BACKGROUND: Role of RET proto-oncogene as predisposing gene for Medullary Thyroid Carcinoma is well established which provides the basis for clinical management of patients. However clinical behavior of MTC varies considerably among patients. Several studies have investigated whether SNPs in low penetrance genes could modulate the clinical behavior of MTC but with conflicting or inconclusive results. The present study aimed to investigate the modifier effect of 13 SNPs of three distinct genetic pathways -Detoxification, Cell cycle regulation and RET on the clinico-pathological features of hereditary and sporadic MTC. METHODS: SNPs were genotyped using RFLP or TaqMan method. The genotypes were correlated with various clinico-pathological parameters (age and calcitonin levels at MTC diagnosis, tumor volume, nodal and distant metastasis). RESULTS: Nodal metastasis was the only clinico-pathological parameter showing significant association with any SNP. In the hereditary MTC group (n=77), incidence of nodal metastases was significantly higher in wild type allele for Cyp1A1m1, CDKN2A and CDKN2C (p=0.01 for all three). In sporadic MTC group (n=361) CDKN2C wild type allele had higher nodal metastasis (p=0.03). CONCLUSION: In this largest MTC cohort with comprehensive analysis of modulatory role of 13 most frequently studied SNPs with MTC clinical outcome, we observed a statistically significant association of few SNPs with nodal metastasis. However as these SNPs did not show association with any other clinico-pathological parameters like tumor volume or Calcitonin, they may not be true modifier of MTC. Additional large cohort studies with clinico-pathological details and long-term follow-up are needed to identify genetic modifiers of MTC behavior.

Genetic risk association of CDKN1A and RET gene SNPs with medullary thyroid carcinoma: Results from the largest MTC cohort and meta-analysis.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare subtype of thyroid cancer. Other than gain-of-function RET mutations, no other genetic, lifestyle or environmental risk associations have been established for MTC. Several case-control studies and meta-analysis have examined the risk association of different SNPs with MTC in different populations but with contradictory or inconclusive results. METHODS: In a large cohort of 438 Indian MTC cases and 489 gender and ethnicity matched healthy controls from 1000 genome project, a comprehensive risk association of 13 SNPs of three pathways-detoxification, cell cycle regulation and RET was performed along with meta-analysis of RET SNPs. RESULTS: Multivariate logistic regression analysis identified a protective risk association of CDKN1ASer31Arg SNP with both hereditary (OR 0.26; 95% confidence interval [CI] 0.13-0.55; P < .001) and sporadic MTC (OR 0.53; 95% CI 0.36-0.78; P = .001). An increased risk association was identified for NAT2Y94Y SNP (OR 1.62, 95% CI 1.17-2.25, P = .004) and CDKN2A3'UTR SNP (OR 1.89, 95% CI 1.19-2.98, P = .006) with sporadic MTC and RET S904S with hereditary MTC (OR 2.82, 95% CI 1.64-4.86, P < .001). Meta-analysis of RET SNPs including our cohort identified increased risk association of all four RET SNPs with MTC. CONCLUSION: In this largest SNP risk association study for MTC and the only risk association study of the 13 most commonly studied MTC associated SNPs in a single cohort of this rare cancer, a significant protective risk association of CDKN1ASer31Arg SNP with MTC was shown for the first time. Meta-analysis identified significant risk association of all four RET SNPs, not observed in previous meta-analysis.