Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2

Does the Incorporation of Chemotherapy to Adjuvant Radiation Skew the Influence of Treatment Package Time in High-risk Oral Cavity Carcinoma? A Multi-institutional Collaborative Study

Background: Treatment package time (TPT) defined as days elapsing from surgery to the end of adjuvant radiation therapy (RT) is known to impact outcomes in head and neck cancer patients receiving adjuvant RT alone. Objectives: We sought to explore the influence of adding concomitant chemotherapy to adjuvant RT (CRT) on the effect of TPT in high risk oral cavity squamous cell carcinoma (OCSCC). Methods: We queried our multi-institutional oral cavity collaborative database with 1282 cases to identify OCSCC diagnosed between 2005-2015 that received adjuvant CRT after surgery. All included cases had at least a high-risk feature (extracapsular nodal extension (ECE) and/or positive final surgical margin (PM)) and were treated within 180 days beyond surgery. TPT was calculated in days between surgery date and the last RT fraction and was stratified in 10 days increments (10D-INC). Kaplan-Meier curves, log-rank p-values and uni/multi-variate analyses (MVA) were used to investigate the interaction between TPT in 10D-INC and Overall (OS), locoregional failure free (LRFS) and distant metastases free (DMFS) survival. Results: We identified 187 cases treated with CRT who met out inclusion criteria after excluding cases with inadequate RT and those with unknown treatment dates. Median age was 58 years (24-87), males were 66% and ever smokers were 69% with median smoking pack years of 30. ECE and PM were detected in 32% and 85% respectively; and oral tongue then floor of mouth constituted 49% and 18% of the study cohort. Median RT dose delivered was 66 Gy and median cisplatin dose received was 200 mg/m2 per patient. For the entire cohort median TPT was 98 days (63-162) divided between time to start RT of 51 days (29-109) and median total RT duration of 45 days (33-97). Two- and 5-years OS were significantly better for TPT≤90 days (28%) than TPT\u3e90 days of 71% vs. 65% (2-years) and 62% vs. 45% (5-years); p=0.05 respectively. However, there was no difference for LRFS or DMFS (p\u3e0.05). Clinico-pathological features, smoking index as well as RT and cisplatin doses were non-different between TPT≤90 vs. \u3e90 days; nevertheless, more extensive lymph node (LN) dissection (p=0.039) and unplanned reoperation (p=0.037) were associated with TPT\u3e90 days. On MVA, TPT in 10D-INC was independently detrimental for OS (HR:1.14; CI [1-1.28]; p=0.043) in addition to perineural invasion, age and positive LN (p\u3c0.05 for all). Conclusions: TPT was associated with worsened OS in one of the largest multi-institutional cohorts treated with modern modalities with no influence on LRFS or DMFS for high-risk OCSCC managed with adjuvant CRT. The addition of concurrent chemotherapy to adjuvant RT is suggested to negate the established impact of TPT on oncologic outcomes. Worse OS with prolonged TPT seemed to be driven by peri-operative complications and poor performance status

Outcomes of post-operative treatment with concurrent systemic therapy and radiotherapy (RT) in intermediate (INT) risk resected oral cavity squamous cell carcinoma (OCSCC): A multi-institutional collaboration

Background: Patients (pts) with adverse pathologic factors in resected OCSCC excluding positive surgical margins or extranodal extension represent a group of INT risk disease. Though not standard of care, adjuvant CRT is often used in INT pts. We conducted a multi-institutional study to evaluate factors associated with improved outcomes in INT pts treated with or without chemotherapy. Methods: An IRB-approved collaborative database of patients with primary OCSCC (Stage I-IVB AJCC 7th edition) treated with primary surgical resection between 1/1/2005 and 1/1/2015 with or without adjuvant therapy was established from 6 academic institutions. Pts were categorized by pathologic features and adjuvant therapy. Kaplan Meier curves, log-rank p-values and multivariate analysis (MVA) were used to describe outcomes by treatment including locoregional control (LRC) and disease free survival (DFS). Results: From a total sample size of 1270 patients, 455 INT risk pts were treated with primary surgical resection and adjuvant therapy; 95 received CRT, 274 received RT alone, and 86 received RT without recorded chemotherapy. 49% of pts had perineural invasion (PNI), 24.8% lymphovascular space invasion, 21.5% poorly differentiated histology, 47.3% with pT3/4 disease, and 27.9% with \u3e 2 lymph node positive (LN+). 55.8% of CRT pts were treated with cisplatin. \u3e 2 LN+ was the only significant predictor of LRC (HR 1.49, p= 0.049). PNI and \u3e 2 LN+ were significant predictors of DFS (HR 1.52, p= 0.003 and HR 1.76, p\u3c 0.001). On MVA, after adjusting for \u3e 2 LN+, treatment with cisplatin-RT was borderline significant for LRC (HR 0.52, p= 0.08). 3 year LRC in pts with \u3e 2 LN+ was 84.4% in pts treated with cisplatin-RT compared with 64.9% for RT alone. Conclusions: The addition of cisplatin-based CRT to INT risk pts is controversial but among pts with \u3e 2 LN+ there was a trend toward benefit. This study is limited by small numbers of pts treated with CRT, though these results highlight the need for further investigation in this population to identify INT pts who would benefit from adjuvant therapy intensification

Influence of Treatment Package Time on outcomes in High-Risk Oral Cavity Carcinoma in patients receiving Adjuvant Radiation and Concurrent Systemic Therapy: A Multi-Institutional Oral Cavity Collaborative study

OBJECTIVES: To explore the influence of treatment package time(TPT) in high-risk oral cavity squamous cell carcinoma(OCSCC) receiving adjuvant radiotherapy with concurrent chemotherapy(CRT). MATERIALS AND METHODS: We queried our multi-institutional OCSCC collaborative database for cases diagnosed between 2005 and 2015 who underwent surgery followed by adjuvant CRT. All patients had high-risk features: extranodal extension(ENE) and/or positive surgical margin(PM). TPT was days between surgery to last radiotherapy fraction. Kaplan-Meier curves, log-rank p-values and multivariate analysis(MVA) were used to investigate the impact of TPT on overall(OS), disease-free(DFS), locoregional failure-free(LRFS) and distant metastases-free(DMFS) survival. RESULTS: We identified 187 cases: median age 58 (range, 24-87 years), males 66%, and ever smokers 69%. ENE and PM were detected in 85% and 32%, and oral tongue and floor of the mouth constituted 49% and 18%, respectively. Median radiotherapy and cisplatin doses received were 66 Gy and 200 mg/m2. Overall, median TPT was 98 (range, 63-162 days). OS was worse for TPT \u3e 90-days (n = 134) than TPT ≤ 90 (n = 53) at two-(65% vs. 71%) and five-years (45% vs. 62%); p = 0.05, with similar results for DFS. No influence on LRFS or DMFS was noted. More lymph nodes(LN) dissected(P = 0.039), T3-4 disease(P = 0.017), and unplanned reoperations(P = 0.037) occurred with TPT \u3e 90-days. On MVA, TPT in 10-day increments was independently detrimental for OS (Hazard Ratio: 1.14; 95 %Confidence Interval [1-1.28]; P = 0.043), perineural invasion, age and positive LN (p \u3c 0.05 for all). CONCLUSION: In one of the largest multi-institutional cohorts, TPT \u3e 90-days predicted worse OS for high-risk OCSCC receiving adjuvant CRT. All efforts are needed to optimize perioperative care and baseline conditions for favorable outcomes

Outcomes of Post-Operative Treatment with Concurrent Chemoradiotherapy (CRT) in High-Risk Resected Oral Cavity Squamous Cell Carcinoma (OCSCC): A Multi-Institutional Collaboration

Adjuvant chemoradiation (CRT), with high-dose cisplatin remains standard treatment for oral cavity squamous cell carcinoma (OCSCC) with high-risk pathologic features. We evaluated outcomes associated with different cisplatin dosing and schedules, concurrent with radiation (RT), and the effect of cumulative dosing of cisplatin. An IRB-approved collaborative database of patients (pts) with primary OCSCC (Stage I–IVB AJCC 7th edition) treated with primary surgical resection between January 2005 and January 2015, with or without adjuvant therapy, was established from six academic institutions. Patients were categorized by cisplatin dose and schedule, and resultant groups compared for demographic data, pathologic features, and outcomes by statistical analysis to determine disease free survival (DFS) and freedom from metastatic disease (DM). From a total sample size of 1282 pts, 196 pts were identified with high-risk features who were treated with adjuvant CRT. Administration schedule of cisplatin was not significantly associated with DFS. On multivariate (MVA), DFS was significantly better in patients without perineural invasion (PNI) and in those receiving ≥200 mg/m2 cisplatin dose (p < 0.001 and 0.007). Median DFS, by cisplatin dose, was 10.5 (<200 mg/m2) vs. 20.8 months (≥200 mg/m2). Our analysis demonstrated cumulative cisplatin dose ≥200 mg/m2 was associated with improved DFS in high-risk resected OCSCC pts

Evaluating compliance with process-related quality metrics and survival in oral cavity squamous cell carcinoma: Multi-institutional oral cavity collaboration study

Background: Process-related measures have been proposed as quality metrics in head and neck cancer care. A recent single-institution study identified four key metrics associated with increased survival. This study sought to validate the association of these quality metrics with survival in a multi-institutional cohort. Methods: Multicenter retrospective study of patients with oral cavity squamous cell (1/2005-1/2015). Baseline patient and disease characteristics and compliance with quality metrics was evaluated. Association between compliance with quality metrics with overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) was evaluated using Cox proportional hazards models. Results: Failure to comply with two or more of the quality metrics was associated with worse OS, DFS, and DSS. Adherence to all or all but one of the quality metrics was found to be associated with improved survival. Conclusions: Process-related quality metrics are associated with increased survival in patients with oral cavity squamous cell carcinoma in a multi-institutional cohort