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Clinical Studies

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



Gut microbiome modulation to boost antitumor immune responses is under investigation.


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.


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.


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.

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Fig. 1: Ecological primary endpoints.
Fig. 2: Exploratory stage subgroup analysis.
Fig. 3: Plasma and stool short chain fatty acids exploratory analysis in stage subgroups.

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Data availability

The datasets generated and analyzed during the current study will be made available in public repository. OTU tables are provided in the Supplementary Material.


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We would like to thank all patients and their families for their participation. We acknowledge and are grateful for the support of the Head and Neck Discovery Program, the Tomcyzk AI and Microbiome Working Group, the Bartley-Smith/Wharton, the Gordon Tozer, the Wharton Head and Neck Translational, Dr. Mariano Elia, Petersen-Turofsky Funds and the ‘The Joe & Cara Finley Center for Head & Neck Cancer Research’ at the Princess Margaret Cancer Foundation. MO would like to acknowledge the SEOM Foundation and Cris Contra el Cancer Foundation for their financial-grant support of his fellowship at Princess Margaret Cancer Center; and the Conquer Cancer Foundation 2019 Young Investigator Award, Instituto de Salud Carlos III (ICIII) Rio Hortega Pre-Doctoral Program and M-AES research support grants and Dr. Spreafico Research Funding to the ROMA Project.


This study was partly supported by MO ASCO Conquer Cancer Foundation Young Investigator Award (YIA) 2019, by the Princess Margaret Hospital DMOH 2019 Grant and by AS Division of Medical Oncology, Department of Medicine, University of Toronto Strategic Planning Innovation Grant.

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Authors and Affiliations



MO, LLS, BC and AS developed the concept and design of the ROMA LA-OPSCC-002 study. Patient recruitment and sample collection was performed by MO, GW, AS, LLS, RT and AH. Clinical data collection, analysis and curation was performed by MO, GW and AS. KC performed 16 S qPCR quantification, sequencing library preparation and taxonomic profiling (with CAGEF). MO, AH and AR conducted microbiome statistical analyses and figure generation under supervision of BC.

Corresponding authors

Correspondence to Bryan Coburn or Anna Spreafico.

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Competing interests

MO has consulting/advisory arrangements with Merck, MSD and Transgene. Research support from Merck and Roche. The institution receives clinical trial support from Abbvie, Ayala Pharmaceutical, MSD, ALX Oncology, Debiopharm International, Merck, ISA Pharmaceuticals, Roche Pharmaceuticals, Boehringer Ingelheim, Seagen, Gilead. Travel accommodations expenses: BMS, MSD, Merck. AS has consulting/advisory arrangements with Merck, Bristol-Myers Squibb, Novartis, Oncorus, Janssen, Medison & Immunocore. The institution receives clinical trial support from: Novartis, Bristol-Myers Squibb, Symphogen AstraZeneca/Medimmune, Merck, Bayer, Surface Oncology, Northern Biologics, Janssen Oncology/Johnson & Johnson, Roche, Regeneron, Alkermes, Array Biopharma/Pfizer, GSK, Treadwell, ALX Oncology, Amgen, Servier. RMN has consulting/advisory arrangements with Merck, Bristol-Myers Squibb, Roche, Seattle Genetics, Astra Zeneca, Pfizer, Boehringer Ingelheim. Speaker Bureau honoraria from Merck, MSD and Boehringer Ingelheim. GW has consulting/advisory arrangements with Gilead, Novartis, Pfizer, AstraZeneca, Boehringer-Ingelheim. Research support from Pfizer. Travel grants from BMS, Abbvie.

Ethics approval and consent to participate

ROMA LA-OPSCC-002 was a prospective interventional trial that involved MET-4 drug administration and the collection and analysis of oropharyngeal swabs, stool and plasma samples. The study did not determine the eligibility to receive standard-of-care treatment with definitive chemoradiotherapy. The study was approved by the Princess Margaret Cancer Center Institutional Research Ethics Board (Study ID 19-5079.10) and was conducted in accordance with the Declaration of Helsinki. All patients provided written, signed, informed consent to participate.

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Oliva, M., Heirali, A., Watson, G. et al. 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. Br J Cancer 130, 1936–1942 (2024).

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