Plasma Host-Microbe Proteomics to Predict Complications in High-risk Febrile Neutropenia

A Multicenter Prospective Observational Study on the Plasma Proteomic Profiling of Human and Microbial Proteins for the Early Identification of Biomarker Combinations (Combitypes) Associated With Complications in Oncohematologic Patients With Febrile Neutropenia

Febrile neutropenia (FN) is a common oncologic emergency in patients with hematologic malignancies, associated with high morbidity and mortality. Early identification of patients at higher risk of complications such as sepsis or septic shock is critical to optimize antimicrobial management.

This study aims to characterize the human and microbial plasma proteome using high-resolution mass spectrometry to identify biomarker combinations ("combitypes") capable of predicting complications in oncohematologic patients with FN.

A cohort of 350 adult patients with high-risk FN and initially uncomplicated clinical presentation will be enrolled across three tertiary hospitals. Plasma samples will be collected at fever onset (before antibiotic initiation) and after 48 hours. Proteomic data will be integrated with clinical information using multivariate and machine learning models to develop a predictive model for complications.

Study Overview

• Status: Not yet recruiting
• Conditions: Febrile Neutropenia
• Intervention / Treatment: Biological: Plasma and DNA sample collection for proteomic and genomic analysis

Detailed Description

This multicenter, prospective, observational study will evaluate whether combined proteomic profiles of host and microbial origin can predict complications in patients with hematologic malignancies presenting with high-risk febrile neutropenia (FN).

FN is defined as an oral temperature ≥38.3 °C once or ≥38.0 °C for ≥1 hour in patients with an absolute neutrophil count (ANC) <500 cells/mm³ or expected to decrease below that threshold within 48 hours. Despite empirical broad-spectrum antibiotics, up to 50% of these patients develop sepsis, and 10% progress to septic shock.

Current biomarkers such as C-reactive protein (CRP) or procalcitonin (PCT) have limited specificity in this immunocompromised population. This study proposes a novel integrative proteomic approach based on mass spectrometry to simultaneously quantify host and microbial proteins in plasma, identifying molecular patterns associated with poor outcomes.

Plasma samples (10 mL, EDTA) will be obtained at two time points: the first febrile episode (prior to antibiotic administration) and 48 hours later. Proteins will be processed using PreOmics® ENRICHplus technology and analyzed via LC-MS/MS on an Evosep One-timsTOF Pro2 platform. Differentially expressed proteins will be identified using a data-independent acquisition (DIA-PASEF) workflow and validated in a subset of 200 patients through targeted mass spectrometry.

Clinical, analytical, and microbiological data will be collected via the REDCap platform. Machine learning models (XGBoost, SHAP interpretability) will be used to generate a predictive risk model for complications, integrating proteomic and clinical data.

This study is expected to establish a new decision-support tool for early identification of high-risk FN patients, facilitating personalized antimicrobial strategies and improved prognosis.

• Study Type: Observational
• Enrollment (Estimated): 350

Contacts and Locations

• Study Contact: Name: Jesús Francisco Bermejo Martín, MD PhD; Phone Number: +34923 29 45 41; Email: jfbermejo@usal.es
• Study Contact Backup: Name: Nadia García Mateo, PhD; Phone Number: +34923 29 45 41; Email: nadiagarciamateo@ibsal.es

Study Locations

• Spain
  • Barcelona
    • Barcelona, Barcelona, Spain
      • Hospital Universitari Vall d'Hebron
  • Salamanca
    • Salamanca, Salamanca, Spain, 37007
      • Complejo Asistencial Universitario de Salamanca
      • Contact: Jesús Francisco Bermejo Martín, PhD; Phone Number: +34923 29 45 41; Email: jfbermejo@usal.es
  • Sevilla
    • Seville, Sevilla, Spain
      • Hospital Universitario Virgen Macarena

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Probability Sample

Study Population

Oncohematologic Patients With Febrile Neutropenia

Description

Inclusion Criteria:

• Adults (≥18 years).
• Written informed consent provided by patient or legal representative.
• Diagnosis of hematologic malignancy under induction chemotherapy, post-allogeneic hematopoietic stem cell transplantation, or CAR-T therapy.
• High-risk febrile neutropenia (ANC ≤ 100 cells/mm³, expected duration ≥ 7 days, or significant comorbidities).
• Fever defined as oral temperature ≥38.3 °C once or ≥38.0 °C for ≥1 hour.

• Hospitalized or requiring immediate admission at the time of FN diagnosis.

  ´- Initial uncomplicated clinical presentation, with no previous infection or colonization by multidrug-resistant bacteria.

• Eligible for initial monotherapy with broad-spectrum empirical antibiotic.
• Availability for serial plasma sampling and clinical follow-up.

Exclusion Criteria:

• Age <18 years.
• Low-risk FN according to MASCC/CISNE criteria.
• Initial sample collected after antibiotic administration.
• Decline or inability to provide informed consent.
• Any condition preventing safe participation or reliable sample collection.
• Fever induced by noninfectious causes (considered as adjustment factor, not exclusion).

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
High-risk FN cohort; Adult onco-hematologic inpatients meeting inclusion criteria; two plasma draws at fever onset and 48 h.	Biological: Plasma and DNA sample collection for proteomic and genomic analysis; Collection of 10 mL of peripheral blood in EDTA tubes at fever onset (before antibiotic initiation) and 48 hours later for proteomic and genomic analysis. Samples are processed to obtain plasma and DNA, which will be used for mass spectrometry-based proteomics and potential metagenomic studies.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Identification of plasma host-microbial proteomic signatures (combitypes) associated with major complications in febrile neutropenia.	Evaluation of proteomic profiles (human and microbial) associated with hemodynamic instability, sepsis, septic shock, or death.	Within 7 days from fever onset.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Dynamic changes in plasma proteome over 48 hours	Assessment of longitudinal variations in host and microbial protein levels between baseline and 48 hours.	0-48 hours
Predictive performance of identified combitypes versus conventional biomarkers (CRP, PCT)	Comparison of ROC-AUC for new proteomic models against current inflammatory markers.	Up to 7 days.
Correlation between microbial proteomic profiles and microbiologically documented infections	Association between detected microbial peptides and confirmed pathogens.	During hospitalization (up to 30 days).
Development of a predictive model for complications	A predictive risk model for complications will be developed using machine-learning algorithms (XGBoost) based on the integration of plasma proteomic data and relevant clinical parameters collected at fever onset and during follow-up. The model will be trained and internally validated within the full study cohort using cross-validation techniques to optimize predictive performance and minimize overfitting.	Study duration (36 months).
Validation of selected protein biomarkers by targeted mass spectrometry	Selected protein biomarkers previously identified through discovery-phase proteomic profiling will be validated using targeted LC-MS/MS mass spectrometry in plasma samples from 200 patients within the study cohort. This validation phase will assess the analytical performance (including reproducibility, accuracy, and sensitivity) of the selected biomarkers, as well as their clinical relevance in predicting complications such as hemodynamic instability, sepsis, septic shock, or death. This outcome cannot be divided into sepparate ones, as biomarker expression will be analyzed as a whole, given the fact that until the trial begins, the biomarkers associated with complications in oncohematologic patients with febrile neutropenia will be unkown. This outcome will determine the feasibility of implementing the identified biomarkers as prognostic tools in routine clinical practice for the early risk stratification of patients	By end of study (month 36).

Collaborators and Investigators

• Sponsor: Instituto de Investigación Biomédica de Salamanca
• Investigators: Principal Investigator: Jesús Francisco Bermejo Martín, MD PhD, Centro Asistencial Universitario de Salamanca (CAUSA)

Study record dates

Study Major Dates

• Study Start (Estimated): May 15, 2026
• Primary Completion (Estimated): December 1, 2028
• Study Completion (Estimated): January 1, 2029

Study Registration Dates

• First Submitted: November 18, 2025
• First Submitted That Met QC Criteria: December 9, 2025
• First Posted (Actual): December 11, 2025

Study Record Updates

• Last Update Posted (Actual): April 23, 2026
• Last Update Submitted That Met QC Criteria: April 22, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: septic shock; biomarkers; hematologic malignancy; sepsis; febrile neutropenia; mass spectrometry; proteomics
• Additional Relevant MeSH Terms: Cytopenia; Pathologic Processes; Neoplasms by Site; Neoplasms; Infections; Systemic Inflammatory Response Syndrome; Inflammation; Leukocyte Disorders; Hematologic Diseases; Leukopenia; Shock; Agranulocytosis; Neutropenia; Pathological Conditions, Signs and Symptoms; Hemic and Lymphatic Diseases; Hematologic Neoplasms; Sepsis; Shock, Septic; Febrile Neutropenia
• Other Study ID Numbers: COMBITYPES-FN

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES

IPD Plan Description

Following anonymization, individual participant-level data supporting the publications will be made available. These data include demographic and clinical variables collected in REDCap. A data dictionary, eCRF, and-when possible-analysis scripts (R/Python) and FAIR-compliant metadata will also be provided.

Data will be hosted within the controlled infrastructure of the project (XNAT/Core-lab) and, for controlled dissemination, within the Zenodo community of IBSAL, with DOI assignment and regulated access, in accordance with the GDPR/LOPDGDD and the study protocol as approved by the Research Ethics Committee.

• IPD Sharing Time Frame: Data will be available starting 12 months after publication of the primary results and will remain accessible for at least 5 years thereafter.
• IPD Sharing Access Criteria: Qualified researchers with a methodologically sound proposal may request access. Requests will be reviewed by the study's Data Committee/Steering Committee. Applications should be addressed to the Principal Investigator (Jesús Francisco Bermejo Martín). A Data Use Agreement (DUA) will be required, including commitments to non-reidentification, appropriate data security, and mandatory citation of both the source and dataset DOI. Applicants may also be required to share their analytical code and a publication plan prior to authorization.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ICF; CSR

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No