When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate high-resolution taxonomic and functional information at once. However, the technique is limited by missing information about host-to-microbe ratios observed in different body compartments. This limitation makes it difficult to plan shotgun sequencing assays, especially in the context of high sample multiplexing and limited sequencing output and is of particular importance for studies employing the recently described shallow shotgun sequencing technique. In this study, we evaluated the use of a quantitative PCR (qPCR)-based assay to predict host-to-microbe ratio prior to sequencing. Combining a two-target assay involving the bacterial 16S rRNA gene and the human beta-actin gene, we derived a model to predict human-to-microbe ratios from two sample types, including stool samples and oropharyngeal swabs. We then validated it on two independently collected sample types, including rectal swabs and vaginal secretion samples. This assay enabled accurate prediction in the validation set in a range of sample compositions between 4% and 98% nonhuman reads and observed proportions varied between -18.8% and +19.2% from the expected values. We hope that this easy-to-use assay will help researchers to plan their shotgun sequencing experiments in a more efficient way. IMPORTANCE When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate large amounts of data. However, in sample compositions with low or variable microbial density, shallowing sequencing can negatively affect microbial community metrics. Here, we show that variable sequencing depth decreases measured alpha diversity at differing rates based on community composition. We then derived a model that can determine sample composition prior to sequencing using quantitative PCR (qPCR) data and validated the model using a separate sample set. We have included a tool that uses this model to be available for researchers to use when gauging shallow sequencing viability of samples.
Abstract Introduction: Pembrolizumab monotherapy and pembrolizumab plus platinum-based chemotherapy are the standard of care for first-line treatment of recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Pembrolizumab plus lenvatinib showed promising antitumor activity and manageable safety in metastatic HNSCC in the phase 1b/2 Study 111/KEYNOTE-146 trial. The randomized, open-label, phase 2 LEAP-009 (NCT04428151) study is designed to evaluate the safety and efficacy of lenvatinib plus pembrolizumab or lenvatinib monotherapy versus chemotherapy in R/M HNSCC upon progression after treatment with platinum-based therapy and a PD-1/L1 inhibitor. Materials and Methods: Eligible patients have locally incurable, histologically confirmed R/M HNSCC (of the oral cavity, oropharynx, hypopharynx, and/or larynx), ECOG PS of 0 or 1, no ulceration and/or fungation of disease on skin, and disease progression on or after platinum-based therapy (with/without cetuximab) and PD-1/L1 inhibitor (progression must be within 12 weeks from last dose and patients must have received ≥2 doses). Patients will be randomly assigned 3:3:2 to receive oral lenvatinib 20 mg every day plus pembrolizumab 200 mg IV every 3 weeks (≤35 pembrolizumab cycles), standard-of-care chemotherapy (investigator’s choice of docetaxel, paclitaxel, cetuximab, or capecitabine), or oral lenvatinib monotherapy 24 mg every day. Treatment will continue until centrally verified disease progression, unacceptable toxicity, or withdrawal. Patients who experience centrally verified disease progression in chemotherapy or lenvatinib monotherapy arms may cross over to lenvatinib plus pembrolizumab at time of disease progression with sponsor consultation. Randomization will be stratified by PD-L1 tumor proportion score (<50% vs ≥50%) and ECOG PS (0 vs 1). Imaging will be performed every 6 weeks through year 1 and every 9 weeks thereafter. Response will be assessed per RECIST v1.1 by blinded independent central review. Safety will be monitored throughout the study and for 30 days following treatment end (90 days for serious AEs) and graded per NCI CTCAE v5.0. The primary end point is objective response rate and secondary end points are progression-free survival, duration of response, overall survival, and safety. Results: Planned enrollment is approximately 400, and recruitment is currently underway in Asia, Australia, Europe, and North America. Conclusion: Results of LEAP-009 will provide clarity on the efficacy and safety of lenvatinib with or without pembrolizumab versus chemotherapy for patients with R/M HNSCC upon progression after platinum and immunotherapy. Citation Format: Kevin J. Harrington, Hye Ryun Kim, Sebastien Salas, Marc Oliva, Robert Metcalf, Mogens Bernsdorf, Ji-Won Kim, Ezra E. W. Cohen, Lillian Siu, Danny Rischin, Lisa Licitra, Jan Vermorken, Quynh Le, Makoto Tahara, Jean-Pascal Machiels, Karen O'Hara, Kumudu Pathiraja, Burak Gumuscu, Behzad Bidadi, Barbara Burtness. Lenvatinib with or without pembrolizumab versus chemotherapy for treatment of recurrent or metastatic head and neck squamous cell carcinoma that progressed on platinum therapy and immunotherapy: Phase 2 LEAP-009 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-003.
Ann Oncol 2018; 29: 1727–1740 (doi: 10.1093/annonc/mdy225) The funding statement has been corrected to read “This research has been funded by the Comprehensive Program of Cancer Immunotherapy & Immunology (CAIMI) supported by the Banco Bilbao Vizcaya Argentaria Foundation (FBBVA) (grant 89/2017).” Early-drug development in the era of immuno-oncology: are we ready to face the challenges?Annals of OncologyVol. 29Issue 8PreviewThe classical development of drugs has progressively faded away, and we are currently in an era of seamless drug-development, where first-in-human trials include unusually big expansion cohorts in the search for early signs of activity and rapid regulatory approval. The fierce competition between different pharmaceutical companies and the hype for immune combinations obliges us to question the current way in which we are evaluating these drugs. In this review, we discuss critical issues and caveats in immunotherapy development. Full-Text PDF Open Archive