Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: •Use phosphate buffer (PB) as the blocking solution solvent.•Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

Background: The COVID-19 pandemic placed considerable strain on the healthcare system, leading to the re-allocation of resources and implementation of new practice guidelines. The objective of this study is to assess the impact of COVID-19 guideline modifications on head and neck cancer (HNC) care at two tertiary care centers in Canada. Methods: A retrospective cohort study was conducted. HNC patients seen at two tertiary care centers before and after the onset of the COVID-19 pandemic (pre-pandemic: July 1st, 2019, to February 29th, 2020; pandemic: March 1st, 2020, to October 31st, 2020) were included. The pre-pandemic and pandemic cohorts were compared according to patient and tumor characteristics, duration of HNC workup, and treatment type and duration. Mean differences in cancer care wait times, including time to diagnosis, tumor board, and treatment as well as total treatment package time and postoperative hospital stay were compared between cohorts. Univariate and multivariate analyses were used to compare characteristics and outcomes between cohorts. Results: Pre-pandemic (n = 132) and pandemic (n = 133) patients did not differ significantly in sex, age, habits, or tumor characteristics. The percentage of patients who received surgery only, chemo/radiotherapy (CXRT) only, and surgery plus adjuvant CXRT did not differ significantly between cohorts. Pandemic patients experienced a significant time reduction compared to pre-pandemic patients with regards to the date first seen by a HNC service until start of treatment ([ ] [ ] = 48.7 and 76.6 days respectively; p = .0001), the date first seen by a HNC service until first presentation at tumor board ([ ] [ ] = 25.1 and 38 days respectively; p = .001), mean total package time for patients who received surgery only ([ ] [ ] = 3.7 and 9.0 days respectively; p = .017), and mean total package time for patients who received surgery plus adjuvant CXRT ([ ] [ ] = 80.2 and 112.7 days respectively; p = .035). Conclusion: The time to treatment was significantly reduced during the COVID-19 pandemic as compared to pre-pandemic. This transparent model of patient-centered operative-room prioritization can serve as a model for improving resource allocation and efficiency of HNC care during emergency and non-emergency scenarios.

ThyroSeq V3 (TsV3) tests for various genetic alterations, including gene expression alterations (GEAs), to improve diagnostic accuracy and clinical decision-making for indeterminate thyroid nodules. This study aimed to clarify the clinico-pathological features and outcomes of GEA-positive thyroid nodules, which have not yet been well-described in the literature. A retrospective chart review was performed whereby patients were included if they underwent thyroid surgery between January 2018 and May 2022 at two McGill University teaching hospitals and their surgery was preceded by pre-operative molecular TsV3 testing. In total, 75 of the 328 patients with thyroid nodules (22.9%) who underwent molecular testing and surgery were GEA-positive. On surgical pathology, GEA-positive nodules showed a significantly higher malignancy rate compared to their GEA-negative counterparts (90.7% vs. 77.7%, respectively, p = 0.011). Among those that were malignant, 48.5% had at least one aggressive pathological feature, including histological subtype, extra-thyroidal extension, or lymph node metastasis. BRAF V600E mutation had a significantly greater association with aggressive malignant GEA-positive nodules compared to non-aggressive ones (p < 0.001). This study demonstrates that GEA may be an effective diagnostic and prognostic tool for thyroid nodule management. However, further investigation is needed to characterize the clinico-pathological features of GEA in isolation and in association with other gene alterations.