F4/80+Ly6Chigh Macrophages Lead to Cell Plasticity and Cancer Initiation in Colitis.

BACKGROUND & AIMS: Colorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to colorectal cancer is not well understood. METHODS: To investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate [DSS], 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer. RESULTS: DSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1ß, tumor necrosis factor-α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer. CONCLUSIONS: We have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Practice guidelines for BRCA1/2 tumour testing in ovarian cancer.

The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based BRCA1 and BRCA2 (BRCA1/2) tumour testing in ovarian cancers. This document was drafted by the members of the Canadian College of Medical Geneticists (CCMG) somatic BRCA Ad Hoc Working Group, and representatives from the Canadian Association of Pathologists. The document was circulated to the CCMG members for comment. Following incorporation of feedback, this document has been approved by the CCMG board of directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada; however, they are not inclusive of all information laboratories should consider in the validation and use of NGS for BRCA1/2 tumour testing in ovarian cancers.

Clinical value of next-generation sequencing compared to cytogenetics in patients with suspected myelodysplastic syndrome.

Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%.

Evaluating the impact of lymph node resampling on colorectal cancer nodal stage.

AIMS: Nodal staging in colorectal cancer (CRC) informs prognosis and guides adjuvant treatment decisions. A standard minimum of 12 lymph nodes is widely used, with additional sampling being performed as required. However, there are few data on how lymph node resampling in this context has an impact on nodal stage. The aims of this study were to evaluate the effectiveness of resampling in detecting metastases and tumour deposits, and the impact on stage. METHODS AND RESULTS: A retrospective cohort analysis was performed on CRC resections that underwent resampling because of an initial yield of <12 lymph nodes, from 2008 to 2018. Data relating to patient demographics, specimen, malignancy and prosection were collected. Slides were reviewed to quantify nodal metastases and tumour deposits before and after resampling. Among ≥pN1 cases, logistic regression analysis was performed to evaluate factors that predicted the finding of additional metastases and tumour deposits. The cohort comprised 395 cases: resampling identified nodal metastases and/or tumour deposits in 30 (7.6%) cases; nodal upstaging occurred in 20 (5.1%) cases; and eight (2.0%) cases changed from pN0 to ≥pN1. No factors predicted resampling of positive lymph nodes or tumour deposits, and pN upstaging occurred across a variety of cases. A subgroup analysis was performed to assess the impact of resampling on high-risk features in stage II cases (n = 117). There were 33 (8.5%) patients who no longer had any high-risk features after resampling. CONCLUSIONS: Lymph node resampling has an impact on nodal staging and possible treatment decisions in a considerable proportion of patients, and is recommended in all cases with <12 lymph nodes.

Implementation of an NGS-based sequencing and gene fusion panel for clinical screening of patients with suspected hematologic malignancies.

OBJECTIVES: The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen. METHODS: Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results. RESULTS: The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%. CONCLUSIONS: Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories.

Comprehensive gene expression profiling identifies distinct and overlapping transcriptional profiles in non-specific interstitial pneumonia and idiopathic pulmonary fibrosis.

BACKGROUND: The clinical-radiographic distinction between idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) is challenging. We sought to investigate the gene expression profiles of IPF and NSIP vs. normal controls. METHODS: Gene expression from explanted lungs of patients with IPF (n = 22), NSIP (n = 10) and from normal controls (n = 11) was assessed. Microarray analysis included Significance Analysis of Microarray (SAM), Ingenuity Pathway, Gene-Set Enrichment and unsupervised hierarchical clustering analyses. Immunohistochemistry and serology of proteins of interest were conducted. RESULTS: NSIP cases were significantly enriched for genes related to mechanisms of immune reaction, such as T-cell response and recruitment of leukocytes into the lung compartment. In IPF, in contrast, these involved senescence, epithelial-to-mesenchymal transition, myofibroblast differentiation and collagen deposition. Unlike the IPF group, NSIP cases exhibited a strikingly homogenous gene signature. Clustering analysis identified a subgroup of IPF patients with intermediate and ambiguous expression of SAM-selected genes, with the interesting upregulation of both NSIP-specific and senescence-related genes. Immunohistochemistry for p16, a senescence marker, on fibroblasts differentiated most IPF cases from NSIP. Serial serum levels of periostin, a senescence effector, predicted clinical progression in a cohort of patients with IPF. CONCLUSIONS: Comprehensive gene expression profiling in explanted lungs identifies distinct transcriptional profiles and differentially expressed genes in IPF and NSIP, supporting the notion of NSIP as a standalone condition. Potential gene and protein markers to discriminate IPF from NSIP were identified, with a prominent role of senescence in IPF. The finding of a subgroup of IPF patients with transcriptional features of both NSIP and senescence raises the hypothesis that "senescent" NSIP may represent a risk factor to develop superimposed IPF.

Lack of matrix metalloproteinase 3 in mouse models of lung injury ameliorates the pulmonary inflammatory response in female but not in male mice.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a complex pulmonary disorder in which the local release of cytokines and chemokines appears central to the pathophysiology. OBJECTIVE: Based on the known role of matrix metalloproteinase-3 (MMP3) in inflammatory processes, the objective was to examine the role of MMP3 in the pathogenesis of ARDS through the modulation of pulmonary inflammation. MATERIALS AND METHODS: Female and male, wild type (MMP3+/+) and knock out (MMP3-/-) mice were exposed to two, clinically relevant models of ARDS including (i) lipopolysaccharide (LPS)-induced lung injury, and (ii) hydrochloric acid-induced lung injury. Parameters of lung injury and inflammation were assessed through measurements in lung lavage including total protein content, inflammatory cell influx, and concentrations of mediators such as TNF-α, IL-6, G-CSF, CXCL1, CXCL2, and CCL2. Lung histology and compliance were also evaluated in the LPS model of injury. RESULTS: Following intra-tracheal LPS instillation, all mice developed lung injury, as measured by an increase in lavage neutrophils, and decrease in lung compliance, with no overall effect of genotype observed. Increased concentrations of lavage inflammatory cytokines and chemokines were also observed following LPS injury, however, LPS-instilled female MMP3-/- mice had lower levels of inflammatory mediators compared to LPS-instilled female MMP3+/+ mice. This effect of the genotype was not observed in male mice. Similar findings, including the MMP3-related sex differences, were also observed after acid-induced lung injury. CONCLUSION: MMP3 contributes to the pathogenesis of ARDS, by affecting the pulmonary inflammatory response in female mice in relevant models of lung injury.

Evaluation of HER2 immunohistochemistry expression in non-standard solid tumors from a Single-Institution Prospective Cohort.

Aim: Human epidermal growth factor receptor-2 (HER2) is a well-established prognostic and predictive biomarker. It is an FDA-approved therapeutic target for HER2 positive breast, gastroesophageal, and more recently, lung and colon cancers. It is an emerging biomarker in biliary tract, bladder, cervical, endometrial, ovarian, and pancreatic cancers. The emergence of new indications warrants further characterization of HER2 expression in diverse cancer populations. This study investigated HER2 expression in solid tumour samples and the feasibility of obtaining these results. Methods: Prospective consent was obtained at a Canadian tertiary academic cancer center from adult oncology patients who were referred for molecular genetic testing of malignant tissue samples. Standard HER2-targeted malignancies were considered breast and gastroesophageal, and were excluded from this study. Between July 2020 and November 2023, 499 samples of solid tumors underwent immunohistochemistry (IHC) HER2 staining. A median turnaround time (TAT) of 14 days would be considered feasible for clinical decision making. Results: The mean age (± SD) of participants was 67 ± 12.5 years, with 270 (54%) male and 229 (46%) female. HER2 protein expression was measured in 42 unique cancer types. IHC levels of 0, 1+, 2+, and 3+ were reported and were 43%, 12%, 35%, and 10% of all analyzable samples respectively (tissue inadequate in 3% of samples). The median TAT for HER2 expression results from time of request to result in release was 18 (interquartile range, 11 to 30) days. Conclusions: HER2 protein expression varies widely between different cancer types. TAT for HER2 IHC results was a median of 18 days, which is close to our feasibility cut-off.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

The effect of HLA-DRB1*04:01 on a mouse model of atherosclerosis.

Objectives: HLA-DRB1 is associated with an increased risk of cardiovascular disease in patients with rheumatoid arthritis (RA). This study aimed to determine the effect of HLA-DRB1 on atherosclerotic cardiovascular disease (ASCVD) using a novel mouse model. Methods: Mice transgenic for HLA-DRB1*04:01 (DR4tg) were crossed with low density lipoprotein receptor knock-out (Ldlr-/-) mice that develop atherosclerosis when fed a high fat, high cholesterol (HFHC) diet. Male and female DR4tgLdlr-/- (n = 48), Ldlr-/- (n = 24), DR4tg (n = 24), and C57Bl/6 (B6) background (n = 24) mice were fed HFHC or regular diet (RD) for 12 weeks. Blood samples were analyzed for serum lipoproteins using a colorimetric assay. C-reactive protein (CRP) and oxidized LDL (OxLDL) were measured using ELISA. Atherosclerosis in the aortas was assessed using the lipid stain, Sudan IV. The presence of citrulline in atherosclerotic plaque was determined by immunohistochemistry. Results: Sera low-density lipoprotein cholesterol (LDL-C) levels were higher in HFHC-fed Ldlr-/- versus DR4tgLdlr-/--; p = 0.0056, but the aortic plaque burden and degree of citrullination in the plaque were similar for these two strains. The ratio of pro-atherogenic OxLDL to LDL levels was higher in DR4tgLdlr-/- than Ldlr-/-mice; p = 0.0017. All mice had an increase in CRP when fed a HFHC diet, most pronounced for DR4tgLdlr-/-; p = 0.0009. There were no significant sex differences for DR4tgLdlr-/- mice; however, male Ldlr-/- mice had worse atherosclerosis. B6 and DR4tg mice did not have significant elevations in serum cholesterol levels and did not develop atherosclerosis. Conclusions: Expression of HLA-DRB1 resulted in an elevation of OxLDL and a reduction in the male bias for atherosclerosis, mimicking what is observed in RA.

Molecular profiling of solid tumors by next-generation sequencing: an experience from a clinical laboratory.

Background: Personalized targeted therapies have transformed management of several solid tumors. Timely and accurate detection of clinically relevant genetic variants in tumor is central to the implementation of molecular targeted therapies. To facilitate precise molecular testing in solid tumors, targeted next-generation sequencing (NGS) assays have emerged as a valuable tool. In this study, we provide an overview of the technical validation, diagnostic yields, and spectrum of variants observed in 3,164 solid tumor samples that were tested as part of the standard clinical diagnostic assessment in an academic healthcare institution over a period of 2 years. Methods: The Ion Ampliseq™ Cancer Hotspot Panel v2 assay (ThermoFisher) that targets ~2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes was validated, and a total of 3,164 tumor DNA samples were tested in 2 years. A total of 500 tumor samples were tested by the comprehensive panel containing all the 50 genes. Other samples, including 1,375 lung cancer, 692 colon cancer, 462 melanoma, and 135 brain cancer, were tested by tumor-specific targeted subpanels including a few clinically actionable genes. Results: Of 3,164 patient samples, 2,016 (63.7%) tested positive for at least one clinically relevant variant. Of 500 samples tested by a comprehensive panel, 290 had a clinically relevant variant with TP53, KRAS, and PIK3CA being the most frequently mutated genes. The diagnostic yields in major tumor types were as follows: breast (58.4%), colorectal (77.6%), lung (60.4%), pancreatic (84.6%), endometrial (72.4%), ovary (57.1%), and thyroid (73.9%). Tumor-specific targeted subpanels also demonstrated high diagnostic yields: lung (69%), colon (61.2%), melanoma (69.7%), and brain (20.7%). Co-occurrence of mutations in more than one gene was frequently observed. Conclusions: The findings of our study demonstrate the feasibility of integrating an NGS-based gene panel screen as part of a standard diagnostic protocol for solid tumor assessment. High diagnostic rates enable significant clinical impact including improved diagnosis, prognosis, and clinical management in patients with solid tumors.

Dysregulated transforming growth factor-beta mediates early bone marrow dysfunction in diabetes.

Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.

T-cell clonality testing for the diagnosis of T-cell large granular lymphocytic leukemia: Are we identifying pathology or incidental clones?

INTRODUCTION: T-cell clonality testing by T-cell receptor (TCR) gene rearrangement is key to the diagnosis of T-cell lymphoproliferative disorders such as T-cell large granular lymphocytic (T-LGL) leukemia. Benign clonal T-cell expansions, however, are commonly found in patients without identifiable disease, a condition referred to as T-cell clones of uncertain significance (T-CUS). In practice, T-cell clonality testing is performed for a range of reasons and results are often challenging to interpret given the overlap between benign and malignant clonal T-cell proliferations and uncertainties in the management of T-CUS. METHODS: We conducted a 5-year retrospective cohort study of 211 consecutive patients who underwent PCR-based T-cell clonality testing for suspected T-LGL leukemia at our institution to characterize the use of T-cell clonality testing and its impact on patient management. RESULTS: Overall, 46.4% (n = 98) of individuals tested had a clonal T-cell population identified. Patients with a monoclonal T-cell population were more likely to be older, have rheumatoid arthritis and have higher lymphocyte counts compared to patients with polyclonal populations. The majority of patients eventually diagnosed and treated for T-LGL leukemia had rheumatoid arthritis and lower neutrophil counts compared to untreated patients with monoclonal T-cell populations. A diagnosis of T-LGL leukemia was made in only a minority of patients (n = 48, 22.7%), and only a small proportion were treated (n = 17, 8.1%). CONCLUSION: Our study suggests that T-cell clonality testing most commonly identifies incidental T-cell clones with only a minority of patients receiving a diagnosis of T-LGL leukemia and fewer requiring active treatment. These finding indicate an opportunity to improve utilization of T-cell clonality testing in clinical practice to better target patients where the results of testing would impact clinical management.

CAP-ACP Workload Model for Advanced Diagnostics in Precision Medicine.

OBJECTIVES: In precision medicine, where oncologic management is tailored to the individual's clinical and genetic profiles, advanced diagnostic testing provides prognostic information and guides management in a growing number of malignancies. There is a need to capture the work pathologists perform to meet this demand by providing medically relevant, timely, and accurate testing results. This work includes not only direct patient consults (interpretation of results and issuing reports) but the administrative and medical oversight as well as the research needed to provide the necessary quality assurance, quality control, direction, and framework for the laboratory. METHODS: An expert panel of Canadian pathologists involved in advanced diagnostics was convened to establish and beta test a model for workload assessment in advanced diagnostics. RESULTS: All aspects of the advanced diagnostics workload were detailed and applied to models based on members' experience, including medical oversight, administration, and the introduction of new testing and platforms. Models for biomarker testing were developed for simple and complex or multiplexed assays, and a detailed model was developed to assess the workload for next-generation sequencing-based assays. CONCLUSIONS: This paper provides the first detailed proposal for capturing an advanced diagnostic workload to enable appropriate pathologist allotment for performing all the steps required to run an advanced diagnostic service.

Reducing cytogenetic testing in the era of next generation sequencing: Are we choosing wisely?

INTRODUCTION: In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. METHODS: Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. RESULTS: Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. CONCLUSIONS: Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

Detection of Circulating Tumor DNA in Patients with Thyroid Nodules.

OBJECTIVE: Detection of circulating tumor DNA (ctDNA) in cancer patients can potentially serve as a noninvasive, sensitive test of disease status. The purpose of this study was to determine the ability to detect BRAF (V600E) mutations in the plasma of patients with thyroid nodules, with the goal of distinguishing between benign and malignant nodules. METHODS: Consecutive patients with thyroid nodules who consented for surgery were recruited. Plasma samples were obtained preoperatively and one month postoperatively. Quantitative PCR was used to determine the levels of the BRAF (V600E) mutation preoperatively and postoperatively. RESULTS: A total of 109 patients were recruited. On final pathology, 38 (32.8%) patients had benign thyroid nodules, 45 (38.8%) had classical papillary thyroid cancer (PTC), 23 (19.8%) had nonclassical PTC, and 3 (2.6%) had follicular thyroid cancer. 15/109 patients had detectable BRAF (V600E) ctDNA in their preoperative samples-all of them having classical PTC. Higher T-stage and extrathyroidal extension in PTC were associated with positive BRAF (V600E) ctDNA (p < 0.05). Eighty-eight pairs of preoperative and postoperative plasma samples were collected and analyzed. Of these eighty-eight paired samples, a total of 13/88 (14.8%) patients had detectable BRAF (V600E) ctDNA in their preoperative samples-all of them having classical PTC. 12 of these 13 patients had no detectable BRAF (V600E) postoperatively, while one remaining patient had a significant decline in his levels (p < 0.05). CONCLUSION: BRAF (V600E) circulating thyroid tumor DNA can be detected in plasma and is correlated with a final diagnosis of the classical variant of PTC. Given that a postoperative drop in BRAF (V600E) ctDNA levels was observed in all cases suggests its utility as a tumor marker.

Low expression of NSD1, NSD2, and NSD3 define a subset of human papillomavirus-positive oral squamous carcinomas with unfavorable prognosis.

BACKGROUND: Frequent mutations in the nuclear receptor binding SET domain protein 1 (NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear. METHODS: Using data from over 500 HNSCC patients from The Cancer Genome Atlas (TCGA), we compared the relative level of mRNA expression of NSD1, NSD2, and NSD3 in HPV+ and HPV- HNSCC. Correlation analyses were performed between T-cell infiltration and the relative level of expression of NSD1, NSD2, and NSD3 mRNA in HPV+ and HPV- HNSCC. In addition, overall survival outcomes were compared for both the HPV+ and HPV- subsets of patients based on stratification by NSD1, NSD2, and NSD3 expression levels. RESULTS: Expression levels of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC. CONCLUSION: These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.

Does HPV Subtype Predict Outcomes in Head and Neck Cancers?

BACKGROUND: Recently, reanalysis of The Cancer Genome Atlas study demonstrated that human papillomavirus (HPV) genotypes in head and neck cancers other than HPV-16 have inferior survival to HPV-16-positive tumors. We aimed to examine the association of HPV subtypes and survival in a large cohort of patient samples from our institution. METHODS: Fresh frozen primary site biopsy samples were collected either in clinic or at the time of surgery. Patient demographic, staging, and survival data were also collected. Tumors were tested for HPV subtypes by quantitative polymerase chain reaction (qPCR). Univariable and multivariable analyses were performed using Cox proportional hazards regression. RESULTS: 280 patient biopsy samples were collected between 2011 and 2017. Mean ± standard deviation (SD) age was 61.9 ± 11.1 years and most patients (78%) were male. The majority of cancers were of the oral cavity (60%) or oropharynx (25%) and 30% had HPV-positive disease. Median follow-up was 3.76 years and 96/280 patients (34%) developed recurrences. Patients with p16-positive versus negative disease had significantly improved 5-year overall survival (OS, 77.6% vs. 53.3%; p=0.009) and progression-free survival (PFS, 67.3% vs. 41.0%, p=0.006). Similarly improved 5-year OS and PFS were observed for patients with HPV-positive versus negative disease (65.0% vs. 55.0%, p=0.084; 53.3% vs. 43.2%, p=0.072, resp.). Patients with HPV-16 compared to other HPV diseases had worse 5-year OS and PFS (62.1% vs. 88.9%, p=0.273; 49.0% vs. 88.9%, p=0.081, resp.). CONCLUSIONS: In contrast to the data derived from The Cancer Genome Atlas, patients with HPV-16 tumors trended towards decreased PFS and OS compared with tumors driven by other HPV genotypes. Further larger multi-institutional studies are necessary to understand the relationship between other HPV genotypes and survival in head and neck squamous cell carcinomas.