Assessment of targeted therapy opportunities in sinonasal cancers using patient-derived functional tumor models.

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer. METHODS: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations. RESULTS: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies. CONCLUSION: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine.

Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma: Association with Metabolic Tumor Burden Determined with FDG-PET/CT.

BACKGROUND: The detection of circulating tumor DNA (ctDNA) with next-generation sequencing (NGS) in venous blood is a promising tool for the genomic profiling of head and neck squamous cell carcinoma (HNSCC). The association between ctDNA findings and metabolic tumor burden detected with FDG-PET/CT imaging is of particular interest for developing prognostic and predictive algorithms in HNSCC. METHODS: Twenty-six prospectively enrolled HNSCC patients were eligible for further analysis. All patients underwent tumor tissue and venous liquid biopsy sampling and FDG-PET/CT before definitive oncologic treatment. An NGS-based commercial panel was used for a genomic analysis of the samples. RESULTS: Maximum variant allele frequency (VAF) in blood correlated positively with whole-body (WB) metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (r = 0.510, p = 0.008 and r = 0.584, p = 0.002, respectively). A positive liquid biopsy was associated with high WB-TLG using VAF ≥ 1.00% or ≥5.00% as a cut-off value (p = 0.006 or p = 0.003, respectively). Additionally, ctDNA detection was associated with WB-TLG when only concordant variants detected in both ctDNA and tissue samples were considered. CONCLUSIONS: A high metabolic tumor burden based on FDG imaging is associated with a positive liquid biopsy and high maximum VAF. Our findings suggest a complementary role of metabolic and genomic signatures in the pre-treatment evaluation of HNSCC.

The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients' cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells' response to anti-cancer treatments. MATERIALS/METHODS: Fresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed. RESULTS: Cancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions. CONCLUSIONS: Our study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

Programmed death-ligand 1 and tumor-infiltrating lymphocytes (TILs) - low TIL density may predict poorer long-term prognosis in T1 laryngeal cancer.

We evaluated the prognostic role of programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in T1 glottic laryngeal squamous cell carcinoma (LSCC). T1 glottic LSCC patients (n = 174) treated at five Finnish university hospitals between 2003 and 2013 were included. Tissue microarray (TMA) blocks were used for PD-L1 immunohistochemistry. TILs were scored from intratumoral and stromal regions in whole tissue sections. Of 174 patients, 92 (53%) had negative, 66 (38%) intermediate, and 16 (9%) high PD-L1 levels. Of 80 patients whose TILs were analyzed, 50 (63%) had low and 30 (38%) high stromal TIL density. Patients with a local recurrence or a new primary tumor of the larynx had lower TIL density than had other patients (p = 0.047). High PD-L1 expression with low stromal TIL density was associated with inferior 5-year disease-specific survival (85% vs. 100%, p = 0.02). In conclusion, in patients treated for T1 glottic LSCC, low stromal TIL density was associated with local recurrences and new primary tumors of the larynx. High PD-L1 expression with low stromal TIL density may be associated with worse survival in T1 glottic LSCC.

Pictorial Review of MRI Findings in Acute Neck Infections in Children.

Pediatric neck infections and their complications, such as abscesses extending to deep neck compartments, are potentially life-threatening acute conditions. Medical imaging aims to verify abscesses and their extensions and exclude other complications. Magnetic resonance imaging (MRI) has proven to be a useful and highly accurate imaging method in acute neck infections in children. Children and adults differ in terms of the types of acute infections and the anatomy and function of the neck. This pictorial review summarizes typical findings in pediatric patients with neck infections and discusses some difficulties related to image interpretation.

Patient and health care delays in large (class T3-T4) oral, oropharyngeal, and laryngeal carcinomas.

BACKGROUND: Head and neck cancers (HNCs) are often diagnosed at an advanced stage. We investigated the lengths and factors associated with patient, primary health care (PHC), and specialist care (SC) delays in T3-T4 oral, oropharyngeal, and laryngeal cancer. METHODS: A nationwide prospective questionnaire-based study (n = 203) with the 3-year long data collection period. RESULTS: The median patient, PHC and SC delays were 58, 13, and 43 days, respectively. Lower level of education, heavy alcohol use, hoarseness, difficulties breathing, and eventual palliative treatment associated with a longer patient delay. A lump on the neck or facial swelling associated with a shorter PHC delay. Conversely, if symptoms were treated as an infection, PHC delay was longer. The treatment modality and tumor site affected SC delay. CONCLUSIONS: Patient delay stands as the most notable factor contributing to delays before treatment. HNC symptom awareness thus remains especially important among HNC risk groups.

Timing and frequency of oropharyngeal squamous cell carcinoma recurrences after treatment with curative intent.

BACKGROUND: The increasing number of patients under surveillance after treatment of human papillomavirus-related oropharyngeal squamous cell carcinoma (OPSCC) places a great burden on healthcare providers. AIMS/OBJECTIVES: The aim of this study was to explore OPSCC recurrences in a long follow-up period: their site, frequency and timepoint after primary treatment, treatment and outcome. The secondary aim was to investigate if the recurrences are diagnosed on routine follow-up visits, and if the p16 status will have an effect on the pattern of recurrences. MATERIAL AND METHODS: We analyzed recurrences within a 10-year follow-up period after completed curatively intended treatment among OPSCC patients in Finland treated between 2000 and 2009. Patient-, tumor-, treatment- and follow-up -related parameters were investigated. RESULTS: Out of 495 patients with no residual tumor during the first six months, 71 (14%) were diagnosed with a recurrence, of which 47 were locoregional and 28 were treated with curative intent. Of the recurrences, 86% were diagnosed during the first 36 months after primary treatment. Only ten recurrences appeared after 36 months. The median OS after recurrence was 10.9 months. CONCLUSIONS AND SIGNIFICANCE: Routine follow-up longer than three years after treatment seems not to be effective in terms of detecting OPSCC recurrences.

MRI of acute neck infections: evidence summary and pictorial review.

Infection of the deep neck spaces is a life-threatening acute illness that requires prompt diagnosis and treatment. Magnetic resonance imaging (MRI) offers unsurpassed soft tissue discrimination and is therefore well suited for imaging neck infections. Recently, the feasibility, diagnostic accuracy, and clinical significance of this method have been documented in patients with acute neck infections. This review article summarizes the scientific evidence, provides a practical guide to image acquisition and interpretation, reviews the most common imaging findings, and discusses some difficult diagnoses and pitfalls in acute neck infections, to help both radiologists and clinicians in managing these critically ill patients.

MRI of odontogenic maxillofacial infections: diagnostic accuracy and reliability.

OBJECTIVES: To determine the diagnostic accuracy of emergency magnetic resonance imaging (MRI) in odontogenic maxillofacial infections, the clinical and surgical significance of MRI findings, and whether MRI can identify the tooth responsible for the infection. METHODS: A retrospective cohort study reviewed 106 emergency neck MRI scans of patients with neck infections of odontogenic origin. The diagnostic accuracy of MRI in identifying abscesses was studied relative to surgical findings. Correlations were analyzed between various MRI findings and clinical results and outcomes, such as the surgical approach (intraoral vs. extraoral). The ability of MRI findings to predict the causative tooth was assessed in a blinded multi-reader setting. RESULTS: Of the 106 patients with odontogenic infections, 77 (73%) had one or more abscesses. Imaging showed a sensitivity, specificity, and accuracy of 0.95, 0.84, and 0.92, respectively, for MRI diagnosis of an odontogenic abscess. Among the imaging findings, mediastinal edema was the strongest predictor of extraoral surgery. MRI showed bone marrow edema in the majority of patients, and multi-reader assessment showed good reliability. MRI was also able to predict the causative tooth accurately. CONCLUSIONS: Emergency neck MRI can accurately detect odontogenic abscesses and reliably point to the causative tooth. These results can increase the utility and reliance on emergency MRI in clinical decision-making.

TSPO is a potential independent prognostic factor associated with cellular respiration and p16 in head and neck squamous cell carcinoma.

Background: Treatment resistance and relapse are common problems in head and neck squamous cell carcinoma (HNSCC). Except for p16, no clinically accepted prognostic biomarkers are available for HNSCC. New biomarkers predictive of recurrence and survival are crucial for optimal treatment planning and patient outcome. High translocator protein (TSPO) levels have been associated with poor survival in cancer, but the role of TSPO has not been extensively evaluated in HNSCC. Materials and methods: TSPO expression was determined in a large population-based tissue microarray cohort including 611 patients with HNSCC and evaluated for survival in several clinicopathological subgroups. A TCGA HNSCC cohort was used to further analyze the role of TSPO in HNSCC. Results: TSPO expression was downregulated in more aggressive tumors. Low TSPO expression associated with worse 5-year survival and was an independent prognostic factor for disease-specific survival. Subgroup analyses showed that low TSPO expression associated with worse survival particularly in p16-positive oropharyngeal cancer. In silico analyses supported the prognostic role of TSPO. Cellular respiration had the highest significance in pathway analyses for genes expressed positively with TSPO. Conclusion: Decreased TSPO expression associates with poor prognosis in HNSCC. TSPO is a prognostic biomarker in HNSCC to potentially guide treatment stratification especially in p16-positive oropharyngeal cancer.

Epidemiological Study of p16 Incidence in Head and Neck Squamous Cell Carcinoma 2005-2015 in a Representative Northern European Population.

The incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) has increased globally. Our research goal was to study HNSCC incidence in a representative Northern European population and evaluate the utility of the HPV surrogate marker p16 in clinical decision-making. All new HNSCC patients diagnosed and treated in Southwest Finland from 2005-2015 (n = 1033) were identified and analyzed. During the follow-up period, the incidence of oropharyngeal (OPSCC) and oral cavity squamous cell carcinoma (OSCC) increased, while the incidence of laryngeal squamous cell carcinoma (LSCC) decreased. This clinical cohort was used to generate a population-validated tissue microarray (PV-TMA) archive for p16 analyses. The incidence of p16 positivity in HNSCC and OPSCC increased in southwest Finland between 2005 and 2015. p16 positivity was mainly found in the oropharynx and was a significant factor for improved survival. p16-positive OPSCC patients had a better prognosis, regardless of treatment modality. All HNSCC patients benefited from a combination of chemotherapy and radiotherapy, regardless of p16 expression. Our study reaffirms that p16 expression offers a prognostic biomarker in OPSCC and could potentially be used in cancer treatment stratification. Focusing on p16 testing for only OPSCC might be the most cost-effective approach in clinical practice.

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma.

OBJECTIVES: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. MATERIALS AND METHODS: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further cross-validated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. RESULTS: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome thisradioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. CONCLUSION: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.

Magnetic resonance imaging findings in pediatric neck infections-a comparison with adult patients.

BACKGROUND: Differences in the functioning of the immune system and the anatomical proportions of the neck between children and adults lead to different manifestations of deep neck infections. Magnetic resonance imaging (MRI) may serve as an alternative to computed tomography (CT) as the primary imaging modality. OBJECTIVE: To study characteristic MRI findings and the diagnostic accuracy of MRI in pediatric deep neck infections. MATERIALS AND METHODS: We retrospectively studied a cohort of pediatric patients who underwent a neck 3-tesla MRI study over a five-year period. Inclusion criteria were: 1) emergency MRI findings indicating an infection, 2) infection as the final clinical diagnosis, 3) diagnostic image quality verified by the radiologist reading the study and 4) age under 18 years. Patient record data, including surgery reports, were compared with the MRI findings. RESULTS: Data of 45 children were included and analysed. Compared to adults, children had a higher incidence of retropharyngeal infection and lymphadenitis, and a lower incidence of peritonsillar/parapharyngeal infection. MRI showed evidence of an abscess in 34 children. Of these 34 patients, 24 underwent surgery, which confirmed an abscess in 21 but no abscess in three patients. In addition, three patients underwent surgery without MRI evidence of abscess, and an abscess was found in one of these cases. The measures of diagnostic accuracy among the children were sensitivity 0.96, specificity 0.77, positive predictive value 0.89, negative predictive value 0.91 and accuracy 0.89. Compared with adults, children had lower C-reactive protein, but a similar proportion of them had an abscess, and abscess size and rate of surgery were similar. CONCLUSION: Despite the differences in the infection foci, emergency MRI in children had equal diagnostic accuracy to that in adults.

Dysphagia, hypothyroidism, and osteoradionecrosis after radiation therapy for head and neck cancer.

OBJECTIVES: To analyze the long-term side effects of radiation therapy (RT) for head and neck cancer (HNC). METHODS: Retrospective chart analysis of all 688 HNC patients treated during 2010-2015 at Turku University Hospital, Finland. All patients who survived for more than a year after RT/chemoRT were included (n = 233). Intensity modulated RT (IMRT) with standard fractionation was applied in each case. RESULTS: One hundred and six patients (45%) reported persisting dysphagia, for which neck RT increased risk. Definitive neck RT to high-risk volume did not increase late toxicity risks compared to elective neck RT. Radiation-induced hypothyroidism (29%, n = 67) was more common among younger patients and females. Osteoradionecrosis (12%, n = 29) was more common in the oral cavity cancer group (20.7%, n = 92) compared to all other subsites. CONCLUSIONS: Late toxicities of RT for HNC are common. Age, gender, tumor subsite, and neck RT affect susceptibility to long-term side effects. LEVEL OF EVIDENCE: 4.

Survival and Larynx Preservation in Early Glottic Cancer: A Randomized Trial Comparing Laser Surgery and Radiation Therapy.

PURPOSE: The prognosis of glottic T1a laryngeal squamous cell carcinoma (LSCC) is excellent with both transoral laser surgery (TLS) and radiation therapy (RT). Our aim was to compare TLS and RT treatment results in a randomized study. METHODS AND MATERIALS: Of 56 male patients with glottic T1a LSCC, 31 were randomized for TLS and 25 for RT. Survival and larynx preservation data were collected from medical records. RESULTS: Five-year overall survival (OS) was 87%, disease-specific survival (DSS) was 97%, and recurrence-free survival (RFS) was 81% in patients treated with TLS. Five-year OS was 92%, DSS was 100%, and RFS was 88% in patients treated with RT. The primary treatment method was not associated with OS, RFS, or DSS in a log-rank test. The larynx preservation rate was similar in both groups (TLS, 97%; RT, 92%; P = .575). CONCLUSIONS: In a prospective randomized setting oncological outcomes of both treatment modalities (TLS or RT) for T1a LSCC were similar.

Structure-Activity Relationship of 3-Methylcytidine-5'-α,ß-methylenediphosphates as CD73 Inhibitors.

We recently reported N4-substituted 3-methylcytidine-5'-α,ß-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ∼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Clinical and prognostic significance of emergency MRI findings in neck infections.

OBJECTIVES: Due to its superior soft-tissue contrast and ability to delineate abscesses, MRI has high diagnostic accuracy in neck infections. Whether MRI findings can predict the clinical course in these patients is unknown. The purpose of this study was to determine the clinical and prognostic significance of various MRI findings in emergency patients with acute neck infections. MATERIALS AND METHODS: We retrospectively reviewed the 3-T MRI findings of 371 patients with acute neck infections from a 5-year period in a single tertiary emergency radiology department. We correlated various MRI findings, including retropharyngeal (RPE) and mediastinal edema (ME) and abscess diameter, to clinical findings and outcomes, such as the need for intensive care unit (ICU) treatment and length of hospital stay (LOS). RESULTS: A total of 201 out of 371 patients (54%) with neck infections showed evidence of RPE, and 81 out of 314 patients (26%) had ME. Both RPE (OR = 9.5, p < 0.001) and ME (OR = 5.3, p < 0.001) were more prevalent among the patients who required ICU treatment than among those who did not. In a multivariate analysis, C-reactive protein (CRP) levels, RPE, and maximal abscess diameter were independent predictors of the need for ICU treatment, and CRP, ME, and maximal abscess diameter were independent predictors of LOS. CONCLUSION: In patients with an acute neck infection that requires emergency imaging, RPE, ME, and abscess diameter, as shown by MRI, are significant predictors of a more severe illness. KEY POINTS: • Two hundred one out of 371 patients (54%) with neck infection showed evidence of retropharyngeal edema (RPE), and 81 out of 314 patients (26%) had mediastinal edema (ME). • Maximal abscess diameter, RPE, and C-reactive protein (CRP) were independent predictors of the need for intensive care unit (ICU) treatment, and maximal abscess diameter, ME, and CRP were independent predictors of length of hospital stay. • Prognostic significance of MRI findings was evident also while controlling for CRP values.

T1 glottic laryngeal cancer: the role of routine follow-up visits in detecting local recurrence.

PURPOSE: We assessed the treatment outcome and the benefits of routine follow-up visits in T1 glottic laryngeal squamous cell carcinoma (LSCC). METHODS: Medical records of patients diagnosed with stage T1 glottic LSCC (N = 303) in five Finnish university hospitals between 2003 and 2015 were reviewed. Moreover, data from the Finnish Cancer Registry and the Population Register Center were collected. RESULTS: Of all 38 recurrences, 26 (68%) were detected during a routine follow-up visit, and over half (21 of 38, 55%) presented without new symptoms. Primary treatment method (surgery vs. radiotherapy) was not connected with 5-year disease-specific survival (DSS) or laryngeal preservation rate. CONCLUSION: The majority of recurrences were detected on a routine follow-up visit, and local recurrences often presented without new symptoms. Routine post-treatment follow-up of T1 glottic LSCC seems beneficial. TRIAL REGISTRATION: Trial registration number and date of registration HUS/356/2017 11.12.2017.

Practical aspects of sentinel node biopsy in oral cavity cancer: all nodes that emit a signal are important.

BACKGROUND AND OBJECTIVES: Sentinel node biopsy (SNB) is a safe and effective way to examine an N0 neck in early-stage oral cavity cancer (OCC). In this study, we evaluated the variables of SNB detection, surgery, and outcome. MATERIAL AND METHODS: Ninety-two patients with N0 OCC examined with SNB were included. Number and brightness of nodes detected on preoperative imaging and during surgery were analyzed and compared with histological findings. Patients with recurrent disease were evaluated separately and the effect of smoking and alcohol consumption was analyzed. RESULTS: Eighteen patients had at least malignant cells in the sentinel lymph node (SLN); 18 patients had recurrent disease and nine patients died from the cancer. The negative predictive value of SNB was 95%. Six patients did not have metastases in the node with the strongest signal, but metastases were found in an SLN with a weaker signal. Smoking and alcohol consumption did not affect disease-specific or overall survival. CONCLUSION AND SIGNIFICANCE: SNB has been confirmed to be safe and effective in early stage N0 OCC. However, it is important to carefully harvest up to four hottest SLNs that emit a signal. Treatment of patients with only isolated tumor cells (ITC) in the SLN appears to be necessary.