FOXM1 drives HPV+ HNSCC sensitivity to WEE1 inhibition.

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.

Detecting middle ear fluid using smartphones.

The presence of middle ear fluid is a key diagnostic marker for two of the most common pediatric ear diseases: acute otitis media and otitis media with effusion. We present an accessible solution that uses speakers and microphones within existing smartphones to detect middle ear fluid by assessing eardrum mobility. We conducted a clinical study on 98 patient ears at a pediatric surgical center. Using leave-one-out cross-validation to estimate performance on unseen data, we obtained an area under the curve (AUC) of 0.898 for the smartphone-based machine learning algorithm. In comparison, commercial acoustic reflectometry, which requires custom hardware, achieved an AUC of 0.776. Furthermore, we achieved 85% sensitivity and 82% specificity, comparable to published performance measures for tympanometry and pneumatic otoscopy. Similar results were obtained when testing across multiple smartphone platforms. Parents of pediatric patients (n = 25 ears) demonstrated similar performance to trained clinicians when using the smartphone-based system. These results demonstrate the potential for a smartphone to be a low-barrier and effective screening tool for detecting the presence of middle ear fluid.

Patterns of cervical node positivity, regional failure rates, and fistula rates for HPV+ oropharyngeal squamous cell carcinoma treated with transoral robotic surgery (TORS).

OBJECTIVES: (1) Report the patterns of cervical node positivity for HPV + oropharyngeal squamous cell carcinoma (OPSCC) treated with transoral robotic surgery (TORS) and a unilateral level II-IV node dissection. (2) Investigate the regional failure rate following this operation. (3) Report the rate of pharyngocutaneous fistula (PCF) formation intraoperatively and postoperatively following TORS/neck dissection. METHODS: Retrospective case series of 88 patients with HPV+ OPSCC treated with TORS and simultaneous neck dissection levels II-IV at the University of Washington from 2010 to 2016. Primary endpoints were PCF, regional recurrence, disease-free survival (DFS), and overall survival (OS). RESULTS: The overall frequency of cervical node positivity was 93%, with 84% in level IIa, 7% in IIb, 23% in III, and 13% in IV. Two patients developed PCF intraoperatively, repaired with a local digastric flap, and no postoperative PCF occurred. Sixteen patients (18%) received surgery alone, 49 patients (56%) received adjuvant radiation, and 23 patients (26%) underwent adjuvant chemoradiation. DFS at 2 years was 95% and OS at 2 years was 100%. No concerning level Ib nodes were identified preoperatively or during surgery, and no regional failures occurred in this location. CONCLUSION: Our data suggests, in TORS for HPV+ OPSCC, neck dissection of levels II-IV accurately stages the neck pathologically and prevents regional recurrences, with adjuvant therapy when indicated, and survival outcomes are excellent. Single-staged operations did not result in any postoperative PCF. Avoiding dissection of level Ib with TORS oropharyngectomy limits morbidity to the marginal mandibular nerve and salivary function, and resulted in no postoperative fistulas with minimal reconstruction interventions.

A Phase I Clinical Trial of AZD1775 in Combination with Neoadjuvant Weekly Docetaxel and Cisplatin before Definitive Therapy in Head and Neck Squamous Cell Carcinoma.

Purpose: The WEE1 tyrosine kinase regulates G2-M transition and maintains genomic stability, particularly in p53-deficient tumors which require DNA repair after genotoxic therapy. Thus, a need arises to exploit the role of WEE1 inhibition in head and neck squamous cell carcinoma (HNSCC) mostly driven by tumor-suppressor loss. This completed phase I clinical trial represents the first published clinical experience using the WEE1 inhibitor, AZD1775, with cisplatin and docetaxel.Patients and Methods: We implemented an open-label phase I clinical trial using a 3+3 dose-escalation design for patients with stage III/IVB HNSCC with borderline-resectable or -unresectable disease, but who were candidates for definitive chemoradiation. Escalating AZD1775 was administered orally twice a day over 2.5 days on the first week, then in combination with fixed cisplatin (25 mg/m2) and docetaxel (35 mg/m2) for 3 additional weeks. The primary outcome measure was adverse events to establish MTD. Secondary measures included response rates, pharmacokinetics (PK), pharmacodynamics, and genomic data.Results: The MTD for AZD1775 was established at 150 mg orally twice per day for 2.5 days. RECISTv1.1 responses were seen in 5 of 10 patients; histologic adjustment revealed three additional responders. The only drug-limiting toxicity was grade 3 diarrhea. The PK C8hr target of 240 nmol/L was achieved on day 4 at all three doses tested. Pharmacodynamic analysis revealed a reduction in pY15-Cdk, and increases in γH2AX, CC3, and RPA32/RPA2 were noted in responders versus nonresponders.Conclusions: The triplet combination of AZD1775, cisplatin, and docetaxel is safe and tolerable. Preliminary results show promising antitumor efficacy in advanced HNSCC, meriting further investigation at the recommended phase II dose. Clin Cancer Res; 24(12); 2740-8. ©2018 AACR.

Lymph Node Yield as a Predictor of Survival in Pathologically Node Negative Oral Cavity Carcinoma.

OBJECTIVE: Even after a pathologically node-negative (pN0) neck dissection for oral cavity squamous cell carcinoma (SCC), patients may develop regional recurrence. In this study, we (1) hypothesize that an increased number of lymph nodes removed (lymph node yield) in patients with pN0 oral SCC predicts improved survival and (2) explore predictors of survival in these patients using a multivariable model. STUDY DESIGN: Case series with chart review. SETTING: Administrative database analysis. SUBJECTS AND METHODS: The SEER database was queried for patients diagnosed with all-stage oral cavity SCC between 1988 and 2009 who were determined to be pN0 after elective lymph node dissection. Demographic and treatment variables were extracted. The association of lymph node yield with 5-year all-cause survival was studied with multivariable survival analyses. RESULTS: A total of 4341 patients with pN0 oral SCC were included in this study. The 2 highest lymph node yield quartiles (representing >22 nodes removed) were found to be significant predictors of overall survival (22-35 nodes: hazard ratio [HR] = 0.854, P = .031; 36-98 nodes: HR = 0.827, P = .010). Each additional lymph node removed during neck dissection was associated with increased survival (HR = 0.995, P = .022). CONCLUSION: These data suggest that patients with oral SCC undergoing elective neck dissection may experience an overall survival benefit associated with greater lymph node yield. Mechanisms behind the demonstrated survival advantage are unknown. Larger nodal dissections may remove a greater burden of microscopic metastatic disease, diminishing the likelihood of recurrence. Lymph node yield may serve as an objective measure of the adequacy of lymphadenectomy.

Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity in head and neck carcinoma.

OBJECTIVE: Patients with head and neck squamous cell carcinoma (HNSCC) containing TP53 mutation and 3p deletion ("double-hit") have poorer prognosis compared to patients with either event alone ("single-hit"). The etiology for worse clinical outcomes in patients with "double-hit" cancers is unclear. We compared radiosensitivity of cell lines containing both TP53 mutations and deletion of Fragile Histidine Triad (FHIT, the gene most commonly associated with 3p deletion) to "single-hit" lines with only TP53 mutation. We compared radiosensitivity in a "single-hit" cell line with TP53 mutation converted to "double-hit" using RNA interference targeting FHIT. Finally, we compared matrixmetalloproteinase-2/9 (MMP-2/9) activity, a previously-established biomarker for tumor aggressiveness, in xenograft tumors derived from these cell lines. MATERIALS/METHODS: TP53 mutation and FHIT deletion profiles of HNSCC lines were established using Cancer Cell Line Encyclopedia (CCLE). We used RNA-interference to convert a "single-hit" cell line (SCC4) to "double-hit". Cultured cells were examined for radiosensitivity and cisplatin sensitivity. MMP-2/9 activity was evaluated in "double-hit" versus "single-hit" tumors using ratiometric activatable cell-penetrating peptide (RACPP) in tongue (n=17) and flank xenografts (n=4). RESULTS: Radiotherapy caused greater double-stranded DNA breaks in "single-hit" vs naturally occurring and engineered "double-hit" cells. In-vivo, "double-hit" xenografts demonstrated higher MMP-2/9 activity compared to "single-hit" xenografts (p<0.01). There was no difference in cisplatin sensitivity between the cell lines. CONCLUSIONS: TP53 mutation combined with FHIT deletion correlates with decreased radiosensitivity in HNC cell lines. Xenograft from "double-hit" cells exhibit increased MMP-2/9 activity. These findings may in part account for the worse clinical outcome seen in patients with HNSCC "double-hit" tumors.

Fluorescently labeled peptide increases identification of degenerated facial nerve branches during surgery and improves functional outcome.

Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL), complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41) can improve intraoperative visualization of chronically (up to 9 months) denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair.

Treatment of Merkel cell carcinoma of the head and neck: a systematic review.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare and aggressive neurocutaneous malignancy that frequently arises in sun-exposed areas of the head and neck. Standard therapy focuses on wide local excision (WLE) with adjuvant locoregional radiotherapy. However, treatment is often complicated by concerns for cosmesis and for preservation of the head and neck neurovasculature. OBJECTIVE: To explore treatment-related outcomes of the head and neck MCC. METHODS: A MEDLINE and Google Scholar search was performed for studies focusing on the head and neck MCC treatment. RESULTS: The search terms produced 100 articles. Seventeen studies met eligibility/screening criteria, yielding 868 patients. Three of the 6 relevant studies found a significant difference in disease-free survival (DFS) between surgery and surgery plus adjuvant radiation. Two studies found no difference in DFS or overall survival (OS) in patients receiving chemotherapy. Two studies found no difference in DFS between radiotherapy and surgery with adjuvant radiation. No difference in OS was found between WLE and Mohs surgery. CONCLUSION: In an uncomplicated head and neck MCC, treatment with surgery and adjuvant radiotherapy is effective in increasing survival and reducing recurrence. Radiotherapy alone may be appropriate for inoperable regions. Primary chemotherapy seems to have a limited role; however, few studies explored this treatment modality.

Evaluation of a teaching strategy based on integration of clinical subjects, virtual autopsy, pathology museum, and digital microscopy for medical students.

BACKGROUND: Learning pathology is fundamental for a successful medical practice. In recent years, medical education has undergone a profound transformation toward the development of an integrated curriculum incorporating both basic science and clinical material. Simultaneously, there has been a shift from a magisterial teaching approach to one centered around problem-based learning. Now-a-days, informatics tools are expected to help better implement these strategies. AIM: We applied and evaluated a new teaching method based on an active combination of clinical problems, gross pathology, histopathology, and autopsy pathology, all given through informatics tools, to teach a group of medical students at the Universidad de Santander, Colombia. DESIGN: Ninety-four medical students were followed in two consecutive semesters. Students were randomized to receive teaching either through traditional methodology or through the new integrated approach. RESULTS: There was no significant difference between the intervention group and the control group at baseline. At the end of the study, the scores in the intervention group were significantly higher compared to the control group (3.91/5.0 vs. 3.33/5.0, P = 0.0008). Students and tutors endorsed the benefits of the integrated approach. Participants were very satisfied with this training approach and rated the program an 8.7 out of 10, on average. CONCLUSION: This study confirms that an integrated curriculum utilizing informatics systems provides an excellent opportunity to associate pathology with clinical medicine early in training of medical students. This can be possible with the use of virtual microscopy and digital imaging.

Primary diffuse large B-cell lymphoma associated with chronic osteomyelitis of the knee.

Diffuse large B-cell lymphoma (DLBCL) associated with chronic inflammation is a recently adopted category of DLBCL, which describes an aggressive B-cell lymphoma raised in the setting of non-immune chronic inflammation. Primary presentation of this subtype of DLBCL in bone is extremely rare. Here, we present the case of a 27 year old woman with DLBCL of the right distal femur, identified after a three-year history of chronic osteomyelitis. In this report, we describe the clinical and histopathologic features of this unusual presentation of DLBCL and discuss aspects relevant to diagnosis and treatment of this entity.

Matrix-metalloproteinases in head and neck carcinoma-cancer genome atlas analysis and fluorescence imaging in mice.

OBJECTIVE: (1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). STUDY DESIGN: Retrospective human cohort study; prospective animal study. SETTING: Translational research laboratory. SUBJECTS AND METHODS: Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. RESULTS: Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). CONCLUSIONS: Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

Neuroradiology and histopathology in two cases of adult medulloblastoma.

Medulloblastoma (MB) is the most common central nervous system neoplasm in children and only rarely presents in the adult population. Recent molecular biology findings have characterized MB as a heterogeneous neoplasm distinguished by well-defined tumour subsets each with specific histologic and molecular features. Available immunohistochemical stains can now be used to differentiate the distinct molecular types of MB. This report analyzed the histopathologic and neuroradiologic features of two new cases of adult MB. Imaging studies in these patients revealed the morphological appearance of high-grade, well-circumscribed heterogeneous tumours with necrosis, located laterally within the posterior cranial fossa. Histopathology of resected samples demonstrated high-grade tumours (WHO grade IV) containing sheets of undifferentiated neural cells with high mitotic activity and evidence of necrosis. The histopathologic and molecular characteristics of these cases of MB are reviewed for potential applications in new molecular methods of imaging.

Maternal dietary tryptophan deficiency alters cardiorespiratory control in rat pups.

Malnutrition during pregnancy adversely affects postnatal forebrain development; its effect upon brain stem development is less certain. To evaluate the role of tryptophan [critical for serotonin (5-HT) synthesis] on brain stem 5-HT and the development of cardiorespiratory function, we fed dams a diet ∼45% deficient in tryptophan during gestation and early postnatal life and studied cardiorespiratory variables in the developing pups. Deficient pups were of normal weight at postnatal day (P)5 but weighed less than control pups at P15 and P25 (P < 0.001) and had lower body temperatures at P15 (P < 0.001) and P25 (P < 0.05; females only). Oxygen consumption (Vo(2)) was unaffected. At P15, deficient pups had an altered breathing pattern and slower heart rates. At P25, they had significantly lower ventilation (Ve) and Ve-to-Vo(2) ratios in both air and 7% CO(2). The ventilatory response to CO(2) (% increase in Ve/Vo(2)) was significantly increased at P5 (males) and reduced at P15 and P25 (males and females). Deficient pups had 41-56% less medullary 5-HT (P < 0.01) compared with control pups, without a difference in 5-HT neuronal number. These data indicate important interactions between nutrition, brain stem physiology, and age that are potentially relevant to understanding 5-HT deficiency in the sudden infant death syndrome.