Implementation of an ISO 15189 accredited next generation sequencing service for cell-free total nucleic acid (cfTNA) analysis to facilitate driver mutation reporting in blood: the experience of a clinical diagnostic laboratory.

AIMS: Next generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging clinical tool that has rapidly expanded clinical utility. Gene mutations in cell-free total nucleic acids (cfTNA) circulating in the blood are representative of whole tumour biology and can reveal different mutations from different tumour sites, thus addressing tumour heterogeneity challenges. METHODS: The novel Ion Torrent Genexus NGS system with automated sample preparation, onboard library preparation, templating, sequencing, data analysis and Oncomine Reporter software was used. cfTNA extracted from plasma was verified with the targeted pan-cancer (~50 genes) Oncomine Precision Assay (OPA). Assessment criteria included analytical sensitivity, specificity, limits of detection (LOD), accuracy, repeatability, reproducibility and the establishment of performance metrics. RESULTS: An ISO 15189 accredited, minimally invasive cfTNA NGS diagnostic service has been implemented. High sensitivity (>83%) and specificity between plasma and tissue were observed. A sequencing LOD of 1.2% was achieved when the depth of coverage was >22 000×. A reduction (>68%) in turnaround time (TAT) of liquid biopsy results was achieved: 5 days TAT for in-house analysis from sample receipt to a final report issued to oncologists as compared with >15 days from reference laboratories. CONCLUSION: Tumour-derived somatic variants can now be reliably assessed from plasma to provide minimally invasive tumour profiling. Successful implementation of this accredited service resulted in:Appropriate molecular profiling of patients where tumour tissue is unavailable or inaccessible.Rapid TAT of plasma NGS results.

Implementation of an ISO15189 accredited next-generation sequencing service with the fully automated Ion Torrent Genexus: the experience of a clinical diagnostic laboratory.

AIMS: Next-generation sequencing (NGS) is integral to the delivery of personalised medicine for targeted cancer therapy. Average turnaround times (TAT) from reference laboratories with advanced expertise in sequencing are typically 2-3 weeks. Prolonged TAT for biomarker analysis can adversely affect patient outcomes. The project aim was to establish an accredited NGS service integrated within a routine clinical diagnostic laboratory, in a designated tertiary cancer centre with no previous experience in NGS or bioinformatics. METHODS: Platform selected was the novel Ion Torrent Genexus Sequencer with automated onboard library preparation, templating, sequencing and data analysis, with subsequent reporting using Oncomine Reporter software.Entire workflow validation was performed with a targeted panel, the Oncomine Precision Assay, on formalin-fixed paraffin embedded clinical tumour samples. Oncomine Reporter software was used to report on variants including mutations, copy number variations and fusions across 50 key genes.Samples included surgical resections, biopsies, cytology and commercial reference material. Assessment of criteria included analytical sensitivity, specificity, limit of detection, accuracy, repeatability and reproducibility, with the establishment of performance metrics and quality parameters. RESULTS: High sensitivity, specificity and reproducibility were achieved. DNA/RNA input requirements optimised to >10 ng, and sequencing performance established with a limit of detection of 5% when depth of coverage of 2500X was reached. This NGS service attained ISO15189 accreditation with no non-conformances and >56% reduction in TAT. CONCLUSION: Successful implementation, clinical validation and accreditation of a novel NGS technology was achieved in this institution, with a significantly improved TAT of results to oncologists.

Cytokeratin 7 and 19 expression in oropharyngeal and oral squamous cell carcinoma.

PURPOSE: The precise etiopathogenesis of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC), and reasons for predilection for crypt epithelium, remain uncertain. The purpose of this study is to investigate the interaction between HPV and specific cytokeratins 7 (CK7) and 19 (CK19) in crypt epithelium. METHODS: This is a retrospective cohort study of patients presenting between 1999 and 2015 at a tertiary referral center. CK7 and CK19 positivity and H Scores were determined by immunohistochemistry. Disease-specific and overall survival rates were analyzed. RESULTS: There were 253 patients presenting with OPSCC (134), squamous cell carcinoma (SCC) of unknown primary site (22), and oral tongue SCC (97). Primary tumor CK7 and CK19 positivity and H Scores were significantly higher in HPV-positive OPSCC than HPV-negative OPSCC and oral tongue SCC. Higher CK19 Scores, but not CK7 Scores, were also seen in regional metastases from HPV-positive OPSCC than other sites. No impact on disease-specific or overall survival was identified on multivariate analysis. CONCLUSION: The increased expression of CK7 and CK19 in HPV-positive OPSCC compared to HPV-negative disease supports the theory for a role for these cytokeratins in the etiopathogenesis of HPV-related OPSCC.

Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma.

BACKGROUND: The density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma. METHODS: Fifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression. RESULTS: CD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival. CONCLUSIONS: Thus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

The role of tumour morphology in assigning HPV status in oropharyngeal squamous cell carcinoma.

OBJECTIVES: There is no consensus on the optimal approach to human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC). Our objective was to classify OPSCC as HPV positive or negative based on (1) morphology alone, (2) p16 status alone, (3) combined morphology and p16 status with additional HPV testing in discordant cases in keeping with 2012 College of American Pathologists (CAP) guidelines (combined approach), and to evaluate and compare survival outcomes. MATERIALS AND METHODS: Retrospective review of 168 patients, 146 with OPSCC and 22 with cervical SCC of unknown primary site (SCCUP). Morphology was classified as keratinizing or non-keratinizing, p16 immunohistochemistry (IHC) performed and additional HPV DNA PCR testing undertaken in cases in which morphology and p16 status were discordant. Survival statistics were evaluated and compared for the 3 different approaches to classification. RESULTS: On univariate analysis all 3 classification methods significantly predicted for overall survival (OS). Both p16 status and the combined approach also predicted for disease specific survival (DSS), whereas morphology fell just outside significance (p = 0.06). On multivariate analysis only the combined approach retained significance for both OS and DSS, whilst morphology was also significant for DSS. CONCLUSIONS: Our findings confirm that tumour morphology significantly predicts for survival in OPSCC. However, we found combined tumour morphology and p16 IHC, with additional testing for discordant cases to be superior to either morphology or p16 IHC alone. Further study is required to establish the optimal testing method for HPV in OPSCC particularly in low prevalence populations.

Tumour Cell Anaplasia and Multinucleation as Prognosticators in Oropharyngeal Squamous Cell Carcinoma.

Human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCC) tend to have good outcomes, however a subset does not share this favourable prognosis. The aim of this paper is to investigate the utility of tumour cell anaplasia and multinucleation as prognostic markers in oropharyngeal squamous cell carcinoma. Retrospective review of 104 patients with OPSCC or squamous cell carcinoma of unknown primary site (SCCUP) who underwent primary resection and/or lymph node dissection. Slides of both primary and nodal metastatic disease were assessed for the presence of anaplasia and multinucleation. 53 patients were HPV-positive. Anaplasia was more frequent in males (p = 0.005), smokers (p = 0.003), and HPV-negative disease (p = 0.04). HPV status and > 10 pack-year smoking history were independent predictors of recurrence-free survival (RFS) and disease-specific survival (DSS). Neither anaplasia, nor multinucleation, at the primary site or in cervical metastases, had any significant impact on RFS or DSS. We did not find either anaplasia or multinucleation to have any significant prognostic impact in OPSCC. However, given the small number of adverse events in the HPV-positive cohort, we may have lacked sufficient power to detect significance in what was the subgroup of primary interest. Our study highlights the challenge of identifying markers of poor prognosis in HPV-positive OPSCC.

High concordance of BRAF mutational status in matched primary and metastatic melanoma.

BACKGROUND: Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. METHODS: Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. RESULTS: Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. CONCLUSIONS: Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.

Impact of positive margins on outcomes of oropharyngeal squamous cell carcinoma according to p16 status.

BACKGROUND: Currently, positive surgical margins in head and neck cancer are considered to be an indicator for postoperative chemoradiotherapy (CRT) over radiotherapy (RT) alone. However, there are less data regarding the impact of margin status on human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC). METHODS: We performed a retrospective review of 55 patients with oropharyngeal SCC undergoing primary surgical treatment. The impact of margin status on disease-specific survival (DSS) was studied according to p16 status. RESULTS: Twenty-one patients had positive margins. Adjuvant treatment in these cases was CRT (n = 6), RT alone (n = 14), and none (n = 1). Among p16-negative patients, positive margins and dysplasia at margins predicted significantly worse DSS. Among patients with p16-positive disease, margin status had no impact on DSS. CONCLUSION: Patients with p16-positive oropharyngeal SCC and positive margins after excision maintain a low risk of recurrence despite most receiving RT alone as adjuvant treatment. These findings raise questions regarding the additional benefit of postoperative CRT in this group.

Association of Extracapsular Spread With Survival According to Human Papillomavirus Status in Oropharynx Squamous Cell Carcinoma and Carcinoma of Unknown Primary Site.

IMPORTANCE: The presence of extracapsular spread (ECS) of metastatic nodes is considered a poor prognosticator in head and neck cancer, with postoperative chemoradiation therapy often recommended over radiation therapy alone in such cases. However, there is less clarity regarding the effect of ECS on human papillomavirus-associated oropharynx squamous cell carcinoma (OPSCC) or carcinoma of unknown primary site (CUP). OBJECTIVE: To investigate the association of ECS according to human papillomavirus status in OPSCC and CUP with survival. DESIGN, SETTING, AND PARTICIPANTS: This investigation was a retrospective cohort study performed between August 1998 and March 2015 at an academic teaching hospital. Participants were 83 patients with OPSCC (n = 62) or CUP (n = 21) undergoing neck dissection as part of initial treatment. MAIN OUTCOME AND MEASURES: Human papillomavirus status was determined by p16 immunohistochemistry. The presence of ECS was extrapolated from pathology reports, and the extent of ECS was determined by rereview of original pathology slides. Disease-specific survival (DSS) and recurrence-free survival (RFS) were assessed. RESULTS: Among 83 patients (71 male), there were 45 p16-positive and 38 p16-negative tumors. Fifty-one patients had ECS, which was graded as extensive in 43 cases. The median follow-up was 31 months for all patients and 50 months for surviving patients. Among the entire cohort, adverse predictors of RFS were p16-negative status (hazard ratio [HR], 9.4; 95% CI, 3.3-27.2) and ECS (HR, 6.5; 95% CI, 2.0-21.6). Adverse predictors of DSS were p16-negative status (HR, 16.8; 95% CI, 3.9-71.2) and ECS (HR, 8.3; 95% CI, 2.0-35.3). Among p16-negative patients, ECS was significantly associated with worse RFS (HR, 9.7; 95% CI, 1.3-72.3) and DSS (HR, 8.7; 95% CI, 1.1-62.7). In contrast, among p16-positive patients, ECS had no effect on RFS (HR, 1.1; 95% CI, 0.2-7.8) or DSS (HR, 1.2; 95% CI, 0.1-18.7). CONCLUSIONS AND RELEVANCE: The presence of ECS appears to be associated with survival in OPSCC and CUP according to p16 status. Our findings raise questions regarding the benefits of postoperative chemoradiation therapy in p16-positive patients with ECS.