Prospective manipulation of the gut microbiome with microbial ecosystem therapeutic 4 (MET4) in HPV-related locoregionally-advanced oropharyngeal cancer squamous cell carcinoma (LA-OPSCC) undergoing primary chemoradiation: ROMA2 study.

BACKGROUND: Gut microbiome modulation to boost antitumor immune responses is under investigation. METHODS: ROMA-2 evaluated the microbial ecosystem therapeutic (MET)-4 oral consortia, a mixture of cultured human stool-derived immune-responsiveness associated bacteria, given with chemoradiation (CRT) in HPV-related oropharyngeal cancer patients. Co-primary endpoints were safety and changes in stool cumulative MET-4 taxa relative abundance (RA) by 16SRNA sequencing. Stools and plasma were collected pre/post-MET-4 intervention for microbiome and metabolome analysis. RESULTS: Twenty-nine patients received ≥1 dose of MET-4 and were evaluable for safety: drug-related adverse events (AEs) occurred in 13/29 patients: all grade 1-2 except one grade 3 (diarrhea). MET-4 was discontinued early in 7/29 patients due to CRT-induced toxicity, and in 1/29 due to MET-4 AEs. Twenty patients were evaluable for ecological endpoints: there was no increase in stool MET-4 RA post-intervention but trended to increase in stage III patients (p = 0.06). MET-4 RA was higher in stage III vs I-II patients at week 4 (p = 0.03) and 2-month follow-up (p = 0.01), which correlated with changes in plasma and stool targeted metabolomics. CONCLUSIONS: ROMA-2 did not meet its primary ecologic endpoint, as no engraftment was observed in the overall cohort. Exploratory findings of engraftment in stage III patients warrants further investigation of microbiome interventions in this subgroup.

Chromatin- and nucleosome-associated features in liquid biopsy: implications for cancer biomarker discovery.

Cell-free DNA (cfDNA) from the bloodstream has been studied for cancer biomarker discovery, and chromatin-derived epigenetic features have come into the spotlight for their potential to expand clinical applications. Methylation, fragmentation, and nucleosome positioning patterns of cfDNA have previously been shown to reveal epigenomic and inferred transcriptomic information. More recently, histone modifications have emerged as a tool to further identify tumor-specific chromatin variants in plasma. A number of sequencing methods have been developed to analyze these epigenetic markers, offering new insights into tumor biology. Features within cfDNA allow for cancer detection, subtype and tissue of origin classification, and inference of gene expression. These methods provide a window into the complexity of cancer and the dynamic nature of its progression. In this review, we highlight the array of epigenetic features in cfDNA that can be extracted from chromatin- and nucleosome-associated organization and outline potential use cases in cancer management.

MEDIPIPE: an automated and comprehensive pipeline for cfMeDIP-seq data quality control and analysis.

SUMMARY: Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) has emerged as a promising liquid biopsy technology to detect cancers and monitor treatments. While several bioinformatics tools for DNA methylation analysis have been adapted for cfMeDIP-seq data, an end-to-end pipeline and quality control framework specifically for this data type is still lacking. Here, we present the MEDIPIPE, which provides a one-stop solution for cfMeDIP-seq data quality control, methylation quantification, and sample aggregation. The major advantages of MEDIPIPE are: (i) ease of implementation and reproducibility with Snakemake containerized execution environments that will be automatically deployed via Conda; (ii) flexibility to handle different experimental settings with a single configuration file; and (iii) computationally efficiency for large-scale cfMeDIP-seq profiling data analysis and aggregation. AVAILABILITY AND IMPLEMENTATION: This pipeline is an open-source software under the MIT license and it is freely available at https://github.com/pughlab/MEDIPIPE.

Sensitive tumour detection and classification using plasma cell-free DNA methylomes.

The use of liquid biopsies for cancer detection and management is rapidly gaining prominence1. Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations2-5. By contrast, large-scale epigenetic alterations-which are tissue- and cancer-type specific-are not similarly constrained6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns.

Prediction of acute myeloid leukaemia risk in healthy individuals.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure1. The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion2,3. However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)4-8. Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

Treatment outcomes and survival following definitive (chemo)radiotherapy in HPV-positive oropharynx cancer: Large-scale comparison of DAHANCA vs PMH cohorts.

We compare outcomes in two large-scale contemporaneously treated HPV-positive (HPV+) oropharynx cancer (OPC) cohorts treated with definitive radiotherapy/chemoradiotherapy (RT/CRT). p16-confirmed HPV+ OPC treated between 2007 and 2015 at PMH and DAHANCA were identified. Locoregional failure (LRF), distant metastasis (DM), and overall survival (OS) were compared. Multivariable analysis (MVA) calculated adjusted-hazard-ratio (aHR) with 95% confidence interval (95% CI), adjusting for cohort, age, gender, performance status, smoking pack-years, T-category and N-category and chemotherapy. Compared to PMH (n = 701), DAHANCA (n = 1174) contained lower TNM-8T-categories (T1-T2: 77% vs 56%), N-categories (N0-N1: 77% vs 67%) and stages (stage I: 63% vs 44% (all P < .001). PMH used standard-fractionation CRT in 69% (481) while 31% (220) received hypofractionated or moderately accelerated RT-alone. All DAHANCA patients were treated with moderately accelerated RT; 96% (1129) received nimorazole (NIM) and 73% (856) concurrent weekly cisplatin. DAHANCA had shorter overall-treatment-time (P < .001), lower gross tumor (66-68 vs 70 Gy) and elective neck (50 vs 56 Gy) doses. Median follow-up was 4.8 years. DAHANCA had higher 5-year LRF (13% vs 7%, aHR = 0.47 [0.34-0.67]), comparable DM (7% vs 12%, aHR = 1.32 [0.95-1.82]), but better OS (85% vs 80%, aHR = 1.30 [1.01-1.68]). CRT patients had a lower risk of LRF (aHR 0.56 [0.39-0.82]), DM (aHR 0.70 [0.50-1.00]) and death (aHR 0.39 [0.29-0.52]) vs RT-alone. We observed exemplary outcomes for two large-scale trans-Atlantic HPV+ OPC cohorts treated in a similar manner. Concurrent chemotherapy was a strong, independent prognostic factor for all endpoints. Our findings underscore the need for a very careful approach to de-intensification of treatment for this disease.

Longitudinal health utility and symptom-toxicity trajectories in patients with head and neck cancers.

BACKGROUND: This study examined long-term health utility and symptom-toxicity trajectories among patients with head and neck cancer (HNC). METHODS: For patients diagnosed with HNC (2014-2019), Health Utility Index 3 (HUI-3), Edmonton Symptom Assessment Scale (ESAS), and MD Anderson Symptom Inventory (MDASI) surveys (including both the core and head and neck cancer modules) were prospectively collected at multiple time points (at the baseline, after surgery, during radiotherapy, and 3, 6, 12, and 24 months after treatment). Locally estimated scatterplot smoothing plots were generated to describe HUI-3, ESAS, and MDASI trajectories over time by clinicodemographic factors, treatment modality, and tumor subsite. Contributions of clinical factors were assessed with univariable and multivariable analyses. RESULTS: In 800 patients, the treatment modality and the tumor subsite produced unique HUI-3, ESAS, and MDASI trajectories. Patients treated with surgery alone experienced rapid improvements in HUI-3, ESAS, and MDASI scores postoperatively. Among patients treated with chemoradiotherapy, patients with nasopharyngeal carcinoma had greater declines in HUI-3 during treatment in comparison with patients with oropharyngeal carcinoma, but they had similar ESAS/MDASI scores. Among patients treated with radiotherapy, patients with laryngeal carcinoma had better HUI-3/ESAS/MDASI scores than those with oropharyngeal carcinoma during treatment, but they slowly converged after treatment. Female sex, an age > 75 years, a household income < $40,000, a Charlson comorbidity score > 1, an Eastern Cooperative Oncology Group performance status > 0 (at the baseline), and current smoking were independently associated with worse HUI-3 trajectories. HUI-3 had mild to moderate correlations (ρ = 0.2-0.5) with individual symptom-toxicity trajectories. CONCLUSIONS: Long-term HUI-3 trajectories are associated with tumor subsite, clinicodemographic, and treatment factors, and this may be partly explained by relationships with symptoms/toxicities. Separate evaluations by subsite and treatment should occur in health utility and symptom-toxicity studies of HNC. LAY SUMMARY: This study indicates that the long-term health utility and symptoms/toxicities of patients with the most common head and neck cancers (ie, squamous cell carcinomas and nasopharyngeal carcinomas) differ over time with a variety of factors, including the tumor anatomic site, treatment volume, clinicodemographic characteristics (eg, age, human papillomavirus status, tumor stage, gender, smoking status, alcohol status, education, and comorbidities), and treatment modalities. Generalizations across all head and neck cancers should be strongly discouraged. Future studies should evaluate health utility, symptoms and toxicities, and patient need assessments separately for each anatomic site and treatment modality.

Clinical presentation and outcome of human papillomavirus-positive nasopharyngeal carcinoma in a North American cohort.

BACKGROUND: The objective of this study was to describe the clinical presentation and outcomes of human papillomavirus (HPV)-positive nasopharyngeal cancer (NPC) versus Epstein-Barr virus (EBV)-positive NPC and HPV-positive oropharyngeal cancer (OPC). METHODS: Clinical characteristics and presenting signs/symptoms were compared between patients who had viral-related NPC versus viral-related OPC treated with intensity-modulated radiotherapy from 2005 to 2020 and who were matched 1:1 (by tumor and lymph node categories, smoking, age, sex, histology, and year of diagnosis). Locoregional control (LRC), distant control (DC), and overall survival (OS) were compared using the 2005-2018 cohort to maintain 2 years of minimum follow-up. Multivariable analysis was used to evaluate the cohort effect. RESULTS: Similar to HPV-positive OPC (n = 1531), HPV-positive NPC (n = 29) occurred mostly in White patients compared with EBV-positive NPC (n = 422; 86% vs. 15%; p < .001). Primary tumor volumes were larger in HPV-positive NPC versus EBV-positive NPC (median volume, 51 vs. 23 cm3 ; p = .002), with marginally more Level IB nodal involvement. More patients with HPV-positive NPC complained of local pain (38% vs. 3%; p = .002). The median follow-up for the 2005-2018 cohort was 5.3 years. Patients who had HPV-positive NPC (n = 20) had rates of 3-year LRC (95% vs. 90%; p = .360), DC (75% vs. 87%; p = .188), and OS (84% vs. 89%; p = .311) similar to the rates in those who had EBV-positive NPC (n = 374). Patients who had HPV-positive NPC also had rates of LRC (95% vs. 94%; p = .709) and OS (84% vs. 87%; p = .440) similar to the rates in those who had HPV-positive OPC (n = 1287). The DC rate was lower in patients who had HPV-positive disease (75% vs. 90%; p = .046), but the difference became nonsignificant (p = .220) when the analysis was adjusted for tumor and lymph node categories, smoking, and chemotherapy. CONCLUSIONS: HPV-positive NPC and EBV-positive NPC seem to be mutually exclusive diseases. Patients who have HPV-positive NPC have greater local symptom burden and larger primary tumors but have similar outcomes compared with patients who have EBV-positive NPC or HPV-positive OPC.

Transitions in oral and gut microbiome of HPV+ oropharyngeal squamous cell carcinoma following definitive chemoradiotherapy (ROMA LA-OPSCC study).

BACKGROUND: Oral and gut microbiomes have emerged as potential biomarkers in cancer. We characterised the oral and gut microbiomes in a prospective observational cohort of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients and evaluated the impact of chemoradiotherapy (CRT). METHODS: Saliva, oropharyngeal swabs over the tumour site and stool were collected at baseline and post-CRT. 16S RNA and shotgun metagenomic sequencing were used to generate taxonomic profiles, including relative abundance (RA), bacterial density, α-diversity and ß-diversity. RESULTS: A total of 132 samples from 22 patients were analysed. Baseline saliva and swabs had similar taxonomic composition (R2 = 0.006; p = 0.827). Oropharyngeal swabs and stool taxonomic composition varied significantly by stage, with increased oral RA of Fusobacterium nucleatum observed in stage III disease (p < 0.05). CRT significantly reduced the species richness and increased the RA of gut-associated taxa in oropharyngeal swabs (p < 0.05), while it had no effect in stool samples. These findings remained significant when adjusted by stage, smoking status and antibiotic use. CONCLUSIONS: Baseline oral and gut microbiomes differ by stage in this HPV+ cohort. CRT caused a shift towards a gut-like microbiome composition in oropharyngeal swabs. Stage-specific features and the transitions in oral microbiome might have prognostic and therapeutic implications.

Definitive radiotherapy for localized follicular lymphoma staged by 18F-FDG PET-CT: a collaborative study by ILROG.

Radiotherapy (RT) can be curative in patients with localized follicular lymphoma (FL), with historical series showing a 10-year disease-free survival of 40 to 50%. As 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography with computerized tomography (PET-CT) upstages 10 to 60% of patients compared to CT, we sought to evaluate outcomes in patients staged by PET-CT, to determine if more accurate staging leads to better patient selection and results. We conducted a multicenter retrospective study under the direction of the International Lymphoma Radiation Oncology Group (ILROG). Inclusion criteria were: RT alone for untreated stage I to II FL (grade 1-3A) with dose equivalent ≥24 Gy, staged by PET-CT, age ≥18 years, and follow-up ≥3 months. End points were freedom from progression (FFP), local control, and overall survival (OS). A total of 512 patients treated between 2000 and 2017 at 16 centers were eligible for analysis; median age was 58 years (range, 20-90); 410 patients (80.1%) had stage I disease; median RT dose was 30 Gy (24-52); and median follow-up was 52 months (3.2-174.6). Five-year FFP and OS were 68.9% and 95.7%. For stage I, FFP was 74.1% vs 49.1% for stage II (P < .0001). Eight patients relapsed in-field (1.6%). Four had marginal recurrences (0.8%) resulting in local control rate of 97.6%. On multivariable analysis, stage II (hazard ratio [HR], 2.11; 95% confidence interval [CI], 1.44-3.10) and BCL2 expression (HR, 1.62; 95% CI, 1.07-2.47) were significantly associated with less favorable FFP. Outcome after RT in PET-CT staged patients appears to be better than in earlier series, particularly in stage I disease, suggesting that the curative potential of RT for truly localized FL has been underestimated.