Personalized Treatment of Recurrent, Metastatic Head and Neck Cancer Guided by Patient-Derived Xenograft Models.

Recurrent or metastatic head and neck squamous cell carcinoma (RMHNSCC) is associated with a poor prognosis and short survival duration. There is an urgent need to identify personalized predictors of drug response to guide the selection of the most effective therapy for each individual recurrence. We tested the feasibility of patient-derived xenografts (PDX) for guiding their RMHNSCC salvage treatment. Fresh tumor samples from eligible, consented patients were implanted into mice. Established tumors were expanded in mouse PDX cohorts to identify responses to candidate salvage drug treatments in parallel testing. Patients alive and suitable for chemotherapy were treated based on responses determined by PDX testing. Nine patient tumors were successfully engrafted in mice with an average time of 89.2±41.7 days. Four patients' PDX models underwent parallel drug testing. Two patients received PDX-guided therapy. In one of these patients, single agents of cetuximab and paclitaxel demonstrated the best responses in the PDX model, and this patient exhibited sequential partial responses to each drug, including a 17-month clinical response to cetuximab. The main limitation of PDX testing for RMHNSCC was the time delay in obtaining testing results. Despite this, parallel PDX testing may be feasible for a subset of patients and appears to correlate with clinical benefit.

HPV-negative head and neck cancers with adverse pathological features carry specific molecular changes that are associated with survival.

BACKGROUND: Adverse pathological features following surgery in head and neck squamous cell carcinoma (HNSCC) are strongly associated with survival and guide adjuvant therapy. We investigated molecular changes associated with these features. METHODS: We downloaded data from the Cancer Genome Atlas and Cancer Proteome Atlas HNSCC cohorts. We compared tumors positive versus negative for perineural invasion (PNI), lymphovascular invasion (LVI), extracapsular spread (ECS), and positive margins (PSM), with multivariable analysis. RESULTS: All pathological features were associated with poor survival, as were the following molecular changes: low cyclin E1 (HR = 1.7) and high PKC-alpha (HR = 1.8) in tumors with PNI; six of 13 protein abundance changes with LVI; greater tumor hypoxia and high Raptor (HR = 2.0) and Rictor (HR = 1.6) with ECS; and low p38 (HR = 2.3), high fibronectin (HR = 1.6), low annexin A1 (HR = 3.1), and high caspase-9 (HR = 1.6) abundances with PSM. CONCLUSIONS: Pathological features in HNSCC carry specific molecular changes that may explain their poor prognostic associations.

A comparison of timing and patterns of treatment failure, and survival outcomes after progression between HPV+ and HPV- patients undergoing chemoradiation for oropharyngeal squamous cell carcinomas.

BACKGROUND: We aimed to analyze and compare the timing and patterns of treatment failure, and survival after progression between HPV-positive (HPV+) and HPV-negative (HPV-) patients undergoing chemoradiation for oropharyngeal squamous cell carcinomas (OPSCC). METHODS: A retrospective review was performed of all patients undergoing primary chemoradiation for OPSCC between 2008 and 2021. Demographic and clinical data were collected. Kaplan-Meier estimates for overall survival (OS), and time to recurrence/metastases (TTR) were compared using the log-rank test, with Cox regression used for multivariable modeling comparing HPV+ and HPV- patients. RESULTS: HPV- patients developed recurrence or metastases at earlier time points than HPV+ patients (8.8 vs. 15.2 months, p < 0.05), due to earlier local/locoregional recurrence and distant metastases, but not isolated regional recurrences. HPV- distant metastases exclusively occurred in a single organ, most commonly the lungs or bone, while HPV+ metastases frequently had multi-organ involvement in a wide variety of locations (p < 0.05). Once progression (recurrence/metastases) was diagnosed, HPV+ patients experienced superior survival to HPV- patients on univariate and multivariate analysis, largely due to improved outcomes after treatment of local/locoregional recurrences (p < 0.05). There were no differences in survival after isolated regional recurrences or distant metastases. CONCLUSION: HPV+ OPSCC patients relapse later compared to HPV- patients in local/locoregional and distant sites. HPV+ patients with local/locoregional recurrence experience superior survival after recurrence, which does not hold true for isolated regional recurrences or distant metastases. These data can be useful to inform prognosis and guide treatment decisions in patients with recurrent OPSCC.

HPV16 Intratypic Variants in Head and Neck Cancers: A North American Perspective.

Human papillomavirus (HPV) is the major causative agent for cervical and many head and neck cancers (HNCs). HPVs randomly acquire single nucleotide polymorphisms (SNPs) that may become established via positive selection. Within an HPV type, viral isolates differing by <2% in the L1 region are termed "variants" and classified based on combinations of SNPs. Studies in cervical cancer demonstrate clear differences between HPV16 intratypic variants in terms of persistence of infection, tumor histology, cancer risk, and death. Much less is known about the frequency of HPV16 variants in HNC, and their effects on clinical outcomes. We combined HPV16 positive (HPV16+) HNC samples from a local Southwestern Ontario, Canada cohort with those from the Cancer Genome Atlas to create a larger North American cohort of 149 cases with clinical data and determined the distribution of intratypic variants and their impact on clinical outcomes. Most isolates were lineage A, sublineage A1, or A2, with roughly half exhibiting the T350G polymorphism in E6. Univariable analysis identified significant differences between 350T and 350G intratypic variants in clinical T, N, and O staging, as well as disease-free survival. Multivariable analysis failed to identify any clinical factor as a statistically significant covariate for disease-free survival differences between 350T and 350G. Significant differences in several measures of B-cell mediated immune response were also observed between 350T and 350G intratypic variants. We suggest that HPV genetic variation may be associated with HNC clinical characteristics and may have prognostic value.

Loss of LRP1B expression drives acquired chemo and radio-resistance in HPV-positive head and neck cancer.

OBJECTIVES: Although human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15-20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity. MATERIALS AND METHODS: Whole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo. RESULTS: Through integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo. CONCLUSION: Taken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases.

In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging.

BACKGROUND: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. METHODS: Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans. RESULTS: No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC. CONCLUSION: We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy. RELEVANCE STATEMENT: Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness. KEY POINTS: • Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

The HPV Induced Cancer Resource (THInCR): a Suite of Tools for Investigating HPV-Dependent Human Carcinogenesis.

Human papillomaviruses (HPVs) are highly infectious and cause the most common sexually transmitted viral infections. They induce hyperproliferation of squamous epithelial tissue, often forming warts. Virally encoded proteins reprogram gene expression and cell growth to create an optimal environment for viral replication. In addition to their normal roles in infection, functional alterations induced by viral proteins establish conditions that frequently contribute to human carcinogenesis. In fact, ~5% of human cancers are caused by HPVs, with virtually all cervical squamous cell carcinomas (CESC) and an increasing number of head and neck squamous cell carcinomas (HNSC) attributed to HPV infection. The Cancer Genome Atlas (TCGA) molecularly characterized thousands of primary human cancer samples in many cancer types, including CESC and HNSC, and created a comprehensive atlas of genomic, epigenomic, and transcriptomic data. This publicly available genome-wide information provides an unprecedented opportunity to expand the knowledge of the role that HPV plays in human carcinogenesis. While many tools exist to mine these data, few, if any, focus on the comparison of HPV-positive cancers with their HPV-negative counterparts or adjacent normal control tissue. We have constructed a suite of web-based tools, The HPV Induced Cancer Resource (THInCR), to utilize TCGA data for research related to HPV-induced CESC and HNSC. These tools allow investigators to gain greater biological and medical insights by exploring the impacts of HPV on cellular gene expression (mRNA and microRNA), altered gene methylation, and associations with patient survival and immune landscape features. These tools are accessible at https://thincr.ca/. IMPORTANCE The suite of analytical tools of THInCR provides the opportunity to investigate the roles that candidate target genes identified in cell lines or other model systems contribute to in actual HPV-dependent human cancers and is based on large-scale TCGA data sets. Expression of target genes, including both mRNA and microRNA, can be correlated with HPV gene expression, epigenetic changes in DNA methylation, patient survival, and numerous immune features, like leukocyte infiltration, interferon gamma response, T cell response, etc. Data from these analyses may immediately provide evidence to validate in vitro observations, reveal insights into mechanisms of virus-mediated alterations in cell growth, behavior, gene expression, and innate and adaptive immunity and may help hypothesis generation for further investigations.

Proteome and phosphoproteome signatures of recurrence for HPV+ head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and the human papillomavirus (HPV+)-driven subtype is the fastest rising cancer in North America. Although most cases of HPV+ HNSCC respond favorably to the treatment via surgery followed by radiochemotherapy, up to 20% recur with a poor prognosis. The molecular and cellular mechanisms of recurrence are not fully understood. Methods: To gain insights into the mechanisms of recurrence and to inform patient stratification and personalized treatment, we compared the proteome and phosphoproteome of recurrent and non-recurrent tumors by quantitative mass spectrometry. Results: We observe significant differences between the recurrent and non-recurrent tumors in cellular composition, function, and signaling. The recurrent tumors are characterized by a pro-fibrotic and immunosuppressive tumor microenvironment (TME) featuring markedly more abundant cancer-associated fibroblasts, extracellular matrix (ECM), neutrophils, and suppressive myeloid cells. Defective T cell function and increased epithelial-mesenchymal transition potential are also associated with recurrence. These cellular changes in the TME are accompanied by reprogramming of the kinome and the signaling networks that regulate the ECM, cytoskeletal reorganization, cell adhesion, neutrophil function, and coagulation. Conclusions: In addition to providing systems-level insights into the molecular basis of recurrence, our work identifies numerous mechanism-based, candidate biomarkers and therapeutic targets that may aid future endeavors to develop prognostic biomarkers and precision-targeted treatment for recurrent HPV+ HNSCC.

Head and neck cancer can be caused by the human papillomavirus. While this type of cancer responds well to chemotherapy given simultaneously with radiation, a significant proportion of cases recur within a few years, leading to illness and sometimes death in these patients. It is therefore important to understand the mechanisms of recurrence in order to develop better treatments. By comparing the levels of proteins and protein phosphorylation­a type of modification that affects how proteins work­between tumors from patients with or without recurrence, we found that the cells surrounding recurrent tumors show signs of fibrosis­the development of fibrous connective tissue­and suppression of the body's immune responses. This suggests that therapies directed towards the regulators of fibrosis and immune suppression may help to overcome recurrent head and neck cancer.

The Impact of Surgical Resectability on Outcomes for Patients Undergoing Primary Radiation Treatment for Human Papillomavirus-Related Oropharyngeal Cancer.

PURPOSE: Primary radiation therapy with or without chemotherapy (RT/CRT) is the most common treatment for oropharyngeal squamous cell carcinomas (OPSCC), but there has been an increase in transoral surgery (TOS) for T1-2 tumors. Because only a subset of T1-2 tumors are TOS-favorable, nonrandomized comparisons between RT/CRT and TOS could be confounded by indication. We aimed to compare outcomes of potential TOS-candidates versus non-TOS candidates, among patients who underwent RT/CRT for early T-stage OPSCC. METHODS AND MATERIALS: For patients treated with RT/CRT for early-stage human papilloma virus positive OPSCC between 2014 and 2018, pretreatment imaging was reviewed by 3 head and neck surgeons, blinded to outcomes, to assess primary-site appropriateness for TOS, and extracapsular extension (ECE) was scored by a head and neck neuroradiologist. We compared outcomes based on surgical favorability pertaining to (1) the primary site tumor alone and (2) the primary site and an absence of ECE. Kaplan-Meier estimates for overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS) were compared using the log-rank test, with Cox regression used for multivariable modeling. RESULTS: One hundred and forty-three patients were evaluated, of which 121 were male (84.6%), the median age was 59.4 years, and all of them were p16 positive (100%). The primary site was TOS-favorable in 115 of 143 (80.4%). Patients with TOS-favorable primary site experienced superior 5-year OS (89.8% vs 71.2%, P = .017), DSS (90.4% vs 63.4%, P = .022), and RFS (83% vs 49.4%, P = .04) compared with TOS-unfavorable patients. Similarly, patients with a TOS-favorable primary site and no ECE on imaging 101 of 143 (70.6%), had improved OS, DSS, and RFS (P < .05) compared with TOS-unfavorable patients. CONCLUSIONS: In this first study to assess surgical favorability as a prognostic factor among patients with T1/2 p16+ OPSCC, patients with TOS-favorable early-stage OPSCC have better outcomes than TOS-unfavorable patients. This provides valuable prognostic information for patients, and also suggests the risk of confounding by indication in nonrandomized comparisons of treatment modalities.

Tumor molecular differences associated with outcome disparities of Black patients with head and neck cancer.

BACKGROUND: Numerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored. METHODS: We downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n = 43) and White (n = 354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used. RESULTS: Black patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed. CONCLUSIONS: There are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes.

Mindfulness-Oriented Recovery Enhancement vs Supportive Group Therapy for Co-occurring Opioid Misuse and Chronic Pain in Primary Care: A Randomized Clinical Trial.

IMPORTANCE: Successful treatment of opioid misuse among people with chronic pain has proven elusive. Guidelines recommend nonopioid therapies, but the efficacy of mindfulness-based interventions for opioid misuse is uncertain. OBJECTIVE: To evaluate the efficacy of Mindfulness-Oriented Recovery Enhancement (MORE) for the reduction of opioid misuse and chronic pain. DESIGN, SETTING, AND PARTICIPANTS: This interviewer-blinded randomized clinical trial enrolled patients from primary care clinics in Utah between January 4, 2016, and January 16, 2020. The study included 250 adults with chronic pain receiving long-term opioid therapy who were misusing opioid medications. INTERVENTIONS: Treatment with MORE (comprising training in mindfulness, reappraisal, and savoring positive experiences) or supportive group psychotherapy (control condition) across 8 weekly 2-hour group sessions. MAIN OUTCOMES AND MEASURES: Primary outcomes were (1) opioid misuse assessed by the Drug Misuse Index (self-report, interview, and urine screen) and (2) pain severity and pain-related functional interference, assessed by subscale scores on the Brief Pain Inventory through 9 months of follow-up. Secondary outcomes were opioid dose, emotional distress, and ecological momentary assessments of opioid craving. The minimum intervention dose was defined as 4 or more completed sessions of MORE or supportive group psychotherapy. RESULTS: Among 250 participants (159 women [63.6%]; mean [SD] age, 51.8 [11.9] years), 129 were randomized to the MORE group and 121 to the supportive psychotherapy group. Overall, 17 participants (6.8%) were Hispanic or Latino, 218 (87.2%) were White, and 15 (6.0%) were of other races and/or ethnicities (2 American Indian, 3 Asian, 1 Black, 2 Pacific Islander, and 7 did not specify). At baseline, the mean duration of pain was 14.7 years (range, 1-60 years), and the mean (SD) morphine-equivalent opioid dose was 101.0 (266.3) mg (IQR, 16.0-90.0 mg). A total of 203 participants (81.2%) received the minimum intervention dose (mean [SD], 5.7 [2.2] sessions); at 9 months, 92 of 250 participants (36.8%) discontinued the study. The overall odds ratio for reduction in opioid misuse through the 9-month follow-up period in the MORE group compared with the supportive psychotherapy group was 2.06 (95% CI, 1.17-3.61; P = .01). At 9 months, 36 of 80 participants (45.0%) in the MORE group were no longer misusing opioids compared with 19 of 78 participants (24.4%) in the supportive psychotherapy group. Mixed models demonstrated that MORE was superior to supportive psychotherapy through 9 months of follow-up for pain severity (between-group effect: 0.49; 95% CI, 0.17-0.81; P = .003) and pain-related functional interference (between-group effect: 1.07; 95% CI, 0.64-1.50; P < .001). Participants in the MORE group reduced their opioid dose to a greater extent than those in the supportive psychotherapy group. The MORE group also had lower emotional distress and opioid craving. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, among adult participants in a primary care setting, the MORE intervention led to sustained improvements in opioid misuse and chronic pain symptoms and reductions in opioid dosing, emotional distress, and opioid craving compared with supportive group psychotherapy. Despite attrition caused by the COVID-19 pandemic and the vulnerability of the sample, MORE appeared to be efficacious for reducing opioid misuse among adults with chronic pain. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02602535.

Introduction and expression of PIK3CAE545K in a papillary thyroid cancer BRAFV600E cell line leads to a dedifferentiated aggressive phenotype.

Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC.

3p Arm Loss and Survival in Head and Neck Cancer: An Analysis of TCGA Dataset.

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort; patients either had <1% or >97% of the arm deleted. 3p arm loss, defined as >97% deletion in HPV-positive patients and >50% in HPV-negative patients, had no impact on survival (p > 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p < 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR < 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR < 0.1), and pathway analysis revealed low lymphoid-non-lymphoid cell interactions and cytokine signaling (FDR < 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR < 0.1) and elevated hypoxia (FDR < 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients.

Pharmacological characterisation of GSK3335103, an oral αvß6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-ß (TGFß) via the alpha-V beta-6 (αvß6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvß6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvß6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvß6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvß6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvß6 integrin and inhibit the activation of TGFß in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvß6 engagement, TGFß signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvß6 inhibitor that attenuates TGFß signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Detection of Circulating Tumor DNA in Patients with Thyroid Nodules.

OBJECTIVE: Detection of circulating tumor DNA (ctDNA) in cancer patients can potentially serve as a noninvasive, sensitive test of disease status. The purpose of this study was to determine the ability to detect BRAF (V600E) mutations in the plasma of patients with thyroid nodules, with the goal of distinguishing between benign and malignant nodules. METHODS: Consecutive patients with thyroid nodules who consented for surgery were recruited. Plasma samples were obtained preoperatively and one month postoperatively. Quantitative PCR was used to determine the levels of the BRAF (V600E) mutation preoperatively and postoperatively. RESULTS: A total of 109 patients were recruited. On final pathology, 38 (32.8%) patients had benign thyroid nodules, 45 (38.8%) had classical papillary thyroid cancer (PTC), 23 (19.8%) had nonclassical PTC, and 3 (2.6%) had follicular thyroid cancer. 15/109 patients had detectable BRAF (V600E) ctDNA in their preoperative samples-all of them having classical PTC. Higher T-stage and extrathyroidal extension in PTC were associated with positive BRAF (V600E) ctDNA (p < 0.05). Eighty-eight pairs of preoperative and postoperative plasma samples were collected and analyzed. Of these eighty-eight paired samples, a total of 13/88 (14.8%) patients had detectable BRAF (V600E) ctDNA in their preoperative samples-all of them having classical PTC. 12 of these 13 patients had no detectable BRAF (V600E) postoperatively, while one remaining patient had a significant decline in his levels (p < 0.05). CONCLUSION: BRAF (V600E) circulating thyroid tumor DNA can be detected in plasma and is correlated with a final diagnosis of the classical variant of PTC. Given that a postoperative drop in BRAF (V600E) ctDNA levels was observed in all cases suggests its utility as a tumor marker.

Does HPV Subtype Predict Outcomes in Head and Neck Cancers?

BACKGROUND: Recently, reanalysis of The Cancer Genome Atlas study demonstrated that human papillomavirus (HPV) genotypes in head and neck cancers other than HPV-16 have inferior survival to HPV-16-positive tumors. We aimed to examine the association of HPV subtypes and survival in a large cohort of patient samples from our institution. METHODS: Fresh frozen primary site biopsy samples were collected either in clinic or at the time of surgery. Patient demographic, staging, and survival data were also collected. Tumors were tested for HPV subtypes by quantitative polymerase chain reaction (qPCR). Univariable and multivariable analyses were performed using Cox proportional hazards regression. RESULTS: 280 patient biopsy samples were collected between 2011 and 2017. Mean ± standard deviation (SD) age was 61.9 ± 11.1 years and most patients (78%) were male. The majority of cancers were of the oral cavity (60%) or oropharynx (25%) and 30% had HPV-positive disease. Median follow-up was 3.76 years and 96/280 patients (34%) developed recurrences. Patients with p16-positive versus negative disease had significantly improved 5-year overall survival (OS, 77.6% vs. 53.3%; p=0.009) and progression-free survival (PFS, 67.3% vs. 41.0%, p=0.006). Similarly improved 5-year OS and PFS were observed for patients with HPV-positive versus negative disease (65.0% vs. 55.0%, p=0.084; 53.3% vs. 43.2%, p=0.072, resp.). Patients with HPV-16 compared to other HPV diseases had worse 5-year OS and PFS (62.1% vs. 88.9%, p=0.273; 49.0% vs. 88.9%, p=0.081, resp.). CONCLUSIONS: In contrast to the data derived from The Cancer Genome Atlas, patients with HPV-16 tumors trended towards decreased PFS and OS compared with tumors driven by other HPV genotypes. Further larger multi-institutional studies are necessary to understand the relationship between other HPV genotypes and survival in head and neck squamous cell carcinomas.

All HPV-negative head and neck cancers are not the same: Analysis of the TCGA dataset reveals that anatomical sites have distinct mutation, transcriptome, hypoxia, and tumor microenvironment profiles.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) affects various anatomical sites, which often dictates whether the cancer is managed with primary surgery or radiation. This study aimed to assess differences in single nucleotide variation (SNV), copy number, mRNA abundance, methylation, and tumor microenvironment (TME) between HPV-negative oral cavity (OC), oropharyngeal (OPC), hypopharyngeal (HPC), and laryngeal (LC) cancers within The Cancer Genome Atlas (TCGA). METHODS: We downloaded the clinical information and molecular data for the TCGA HNSCC cohort from the data portal and published literature. The TME was estimated using mRNA abundance data. We conducted our analyses within the Bioconductor statistical framework in the R environment. CNA and mRNA abundance results were correlated and grouped with SNV results for downstream pathway analysis. RESULTS: LC had a higher mutational burden than OC and OPC (p <10-4). LC tumors were enriched in CSMD3, NSD1, DCHS2 and ANK2 SNVs, while OC tumors were enriched in CASP8 SNVs (FDR < 0.1). LCs were enriched for neuronal and glycosylation pathways, while OCs were enriched for extracellular matrix pathways. B cells and endothelial cells were more abundant in LC while monocytes were more abundant in OC (FDR < 0.1). OPC was the most hypoxic, followed by OC then LC (FDR < 0.05). OC had greater methylation of Hox genes than LC. Subsite analysis revealed that oral tongue cancers had fewer CASP8 and FBN2 mutations and higher dendritic cell abundance than other oral cavity cancers. CONCLUSIONS: We identified significant genomic, transcriptional, and microenvironmental differences between HPV-negative HNSCC. Further study is warranted to determine if these findings portend differential response to specific treatment modalities.

Low expression of NSD1, NSD2, and NSD3 define a subset of human papillomavirus-positive oral squamous carcinomas with unfavorable prognosis.

BACKGROUND: Frequent mutations in the nuclear receptor binding SET domain protein 1 (NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear. METHODS: Using data from over 500 HNSCC patients from The Cancer Genome Atlas (TCGA), we compared the relative level of mRNA expression of NSD1, NSD2, and NSD3 in HPV+ and HPV- HNSCC. Correlation analyses were performed between T-cell infiltration and the relative level of expression of NSD1, NSD2, and NSD3 mRNA in HPV+ and HPV- HNSCC. In addition, overall survival outcomes were compared for both the HPV+ and HPV- subsets of patients based on stratification by NSD1, NSD2, and NSD3 expression levels. RESULTS: Expression levels of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC. CONCLUSION: These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.

Modern treatment outcomes for early T-stage oropharyngeal cancer treated with intensity-modulated radiation therapy at a tertiary care institution.

BACKGROUND: Transoral surgery (TOS), particularly transoral robotic surgery (TORS) has become the preferred modality in the United States for the treatment of early stage oropharyngeal cancer, largely due to assumptions of fewer toxicities and improved quality of life compared to primary radiotherapy (RT). However, these assumptions are based on retrospective analysis, a subset of which utilize primary RT groups not limited to T1-2 stage tumors for which transoral robotic surgery is FDA approved. Thus, there is potential for underestimating survival and overestimating toxicity, including treatment related mortality, in primary RT. METHODS: Consecutive cases of early T-stage (T1-T2) oropharyngeal cancer presenting to the London Health Sciences Centre between 2014 and 2018 treated with RT or chemoradiation (CRT) were reviewed. Patient demographics, treatment details, survival outcomes and toxicity were collected. Toxicities were retrospectively graded using the Common Terminology Criteria for Adverse Events criteria. RESULTS: A total of 198 patients were identified, of which 82% were male and 73% were HPV-positive. Sixty-eight percent of patients experienced a grade 2 toxicity, 48% a grade 3 and 4% a grade 4. The most frequent toxicities were dysphagia, neutropenia and ototoxicity. The rates of gastrostomy tube dependence at 1 and 2 years were 2.5% and 1% respectively. There were no grade 5 (fatal) toxicities. HPV-positive patients experienced improved 5-year overall survival (86% vs 64%, p = 0.0026). CONCLUSIONS: Primary RT or CRT provides outstanding survival for early T-stage disease, with low rates of severe toxicity and feeding tube dependence. This study provides a reference for comparison for patients treated with primary transoral surgery.