Novel Flow-cytometry Approaches to Improve the Detection of Tumor Cells in CTCL

Design, Development, and Validation of Novel "Next Generation Flow" Approaches for Rapid, Specific, Sensitive, and Reproducible Detection of Tumor Cells in Cutaneous T-cell Lymphoma.

Identification and quantitation of circulating tumor cells in patients with cutaneous T-cell lymphoma -mycosis fungoides (MF)/Sézary syndrome (SS)- are required for diagnosis and precising the actual staging and response to treatment. The current flow cytometry techniques used in clinical laboratories do not correctly allow to compare results in a clinical setting. Furthermore, now we know that the phenotype of tumor cells partially overlaps with that of normal TCD4+ cells, and it is rather heterogeneous. The GENERAL OBJECTIVE of this project is to apply flow-cytometry standardized strategies for rapid, specific, sensitive, and reproducible detection and quantitation of tumor cells in patients with MF/SS. For this purpose, in the first phase of the project we will design an optimal combination of markers to detect tumor cells by spectral flow-cytometry, and then the specificity and analytical sensitivity of the new combination/procedure will be assessed in blood samples -to be later applied to skin samples-, and finally reference databases will be created for the automatic analysis of cytometry data. In a second phase of the project, the developed method will be validated in a multicenter manner, through the demonstration of its practical applicability and clinical utility (speed and precision) in blood samples (and skin, where appropriate) for diagnosis, staging, and treatment monitoring. In parallel, the tumor microenvironment (residual normal immune system) will be explored -by applying the panel designed in the first phase together with additional immune-monitoring panels by flow cytometry-, and its relationship with clinical-biological heterogeneity of the tumor will be analyzed. In the two phases of the project, cytometry data will be compared with the gold standard approach to identify tumor T cells (through the identification of clonal rearrangement by PCR and/or NGS, performed on cell populations previously sorted by flow cytometry).

Study Overview

• Status: Recruiting
• Conditions: Cutaneous T-Cell Lymphoma
• Intervention / Treatment: Diagnostic test: Detection and quantitation of (blood) tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS).

Detailed Description

Primary cutaneous lymphomas are a group of tumors derived from lymphocytes (mostly T lymphocytes) that primarily affect the skin (such as mycosis fungoides, MF), although they can spread beyond the skin in more advanced stages - for example, to the blood - or may already be disseminated initially, as is the case with Sézary syndrome (SS), a tumor with very aggressive behavior from the beginning, which by definition already has blood dissemination at diagnosis. They are very rare tumors and difficult to diagnose because often the skin lesions are indistinguishable from benign processes (the latter much more common than cutaneous lymphomas). For this reason, it is not uncommon for the diagnosis to be delayed, with the consequent negative impact on the patient's prognosis if it is a tumor. Currently, the diagnosis of blood dissemination (the amount of which is also essential because it impacts the prognosis, the worse the higher tumor burden in the blood) is performed by flow cytometry, which has replaced conventional morphology. However, the flow cytometry techniques currently used in most clinical laboratories are not standardized among different centers, and therefore the comparability of the results (even over time, in the same center) and their use in the clinical settings is difficult.

In this project, the investigators HYPOTHESIZE that the application of novel "next-generation flow" strategies proposed by the international collaborative group EuroFlow (to which the applicant of this proposal belongs) would improve the detection of circulating tumor cells in patients with cutaneous T-cell lymphoma, which would allow improving the clinical management of the patients. Therefore, in this project, the aim as a GENERAL OBJECTIVE to apply standardized strategies of next-generation flow cytometry developed by the EuroFlow Consortium for the rapid, specific, sensitive, and reproducible detection and quantification of tumor cells in patients with MF/SS, through: i) the application of standardized sample staining procedures with antibody panels designed after previous rounds of testing, which would allow distinguishing tumor T lymphocytes from normal T cells (specificity); ii) adapting the technique to analyze a large number of cells, in order to achieve a high sensitivity of detection, also crucial for a more precise monitoring of residual disease in blood after treatment with the new therapies available in cutaneous T-cell lymphomas; and iii) development of automatic analysis strategies of cytometry data, so that the results do not depend on the personal experience of the cytometrist.

IMPACT. The availability of a new standardized flow cytometry strategy for sensitive and specific detection (and quantification) of circulating tumor cells in blood (and skin) of patients with cutaneous T-cell lymphoma will provide an essential tool to support dermo-hematological diagnosis, with the possibility of translating to routine diagnosis in the very short term after the completion of the project, for its application in hospitals that have a Cytometry/Immunopathology Laboratory, with the aim of: i) Making an early and accurate (differential) diagnosis between lymphoma - benign lesion in patients with skin lesions; ii) Detecting tumor cells in blood with greater sensitivity than conventional methods, to more precisely establish blood dissemination of a primary cutaneous T-cell lymphoma, and to assess the response to current treatments in a more reliable and sensitive way

• Study Type: Observational
• Enrollment (Estimated): 100

Contacts and Locations

• Study Contact: Name: Julia M Almeida Parra, Prof.; Phone Number: 5813 +34 923 29 45 00; Email: jalmeida@usal.es
• Study Contact Backup: Name: Ricardo López Pérez, PhD; Phone Number: 55144 +34 923 29 12 00; Email: uicec.gestion@ibsal.es

Study Locations

• Spain
  • Salamanca, Spain, 37007
    • Recruiting
    • Instituto de Investigacion Biomedica de Salamanca (IBSAL)
    • Contact: Ricardo López Pérez, PhD; Phone Number: 55144 +34 923 29 12 00; Email: uicec.gestion@ibsal.es
    • Contact: Esperanza López Franco, PhD; Phone Number: 55779 +34 923 29 12 00; Email: uicec.coordinacion@ibsal.es

Participation Criteria

Eligibility Criteria

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

• Sampling Method: Non-Probability Sample

Study Population

Cases of cutaneous lymphoma/reactive erythroderma will be recruited from the routine clinical practice of the Cutaneous T-Cell Lymphoma Monographic Clinic at the University Hospital of Salamanca, which is conducted in a multidisciplinary manner under the responsibility of the Dermatology and Hematology services, involving two members of the research team (ECA and MLP). Diagnosis will be made according to the current criteria of the EORTC-OMS; patients will be included both at diagnosis and at any other stage of the tumor during follow-up

Description

Inclusion Criteria:

• Patients with cutaneous T-cell lymphoma
• Over 18 years old
• Sign the informed consent

Exclusion Criteria:

• Under 18 years old
• Do not sign the informed consent

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Cutaneous T-cell lymphoma; Patients with cutaneous T-cell lymphoma	Diagnostic test: Detection and quantitation of (blood) tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS).; Specific, sensitive, and reproducible (blood) detection and quantitation of tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS)
Control group 1; patients with benign/reactive erythroderma	Diagnostic test: Detection and quantitation of (blood) tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS).; Specific, sensitive, and reproducible (blood) detection and quantitation of tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS)
Control group 2; patients with systemic inflammatory processes regardless of whether they have cutaneous involvement	Diagnostic test: Detection and quantitation of (blood) tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS).; Specific, sensitive, and reproducible (blood) detection and quantitation of tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS)
Control group 3; healthy adult subjects, age- and sex-matched with patients	Diagnostic test: Detection and quantitation of (blood) tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS).; Specific, sensitive, and reproducible (blood) detection and quantitation of tumor cells in patients with mycosis fungoides (MF) / Sézary syndrome (SS)

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Optimizing Marker Combination for Specific Identification and Quantification of Sézary Cells using Spectral Flow Cytometry	Designing an optimal marker combination for the specific identification and quantification of Sézary cells (particularly in blood) using spectral flow cytometry, enabling their discrimination from normal/reactive TCD4+ cells, and including markers for T-cell identification, T-cell maturation, aberrant markers, and markers to assess T-cell clonality	18 months

Collaborators and Investigators

• Sponsor: Instituto de Investigación Biomédica de Salamanca
• Collaborators: Carlos III Health Institute
• Investigators: Principal Investigator: Julia M Almeida Parra, Prof., Instituto de Investigación Biomédica de Salamanca

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2024
• Primary Completion (Estimated): September 30, 2026
• Study Completion (Estimated): December 31, 2026

Study Registration Dates

• First Submitted: April 20, 2024
• First Submitted That Met QC Criteria: April 20, 2024
• First Posted (Actual): April 24, 2024

Study Record Updates

• Last Update Posted (Actual): May 1, 2025
• Last Update Submitted That Met QC Criteria: April 29, 2025
• Last Verified: April 1, 2025

More Information

Terms related to this study

• Keywords: Lymphoma; Mycosis fungoides; T Cell; Flow cytometry; Cutaneous; Sézary syndrome
• Additional Relevant MeSH Terms: Neoplasms; Immune System Diseases; Neoplasms by Histologic Type; Lymphatic Diseases; Lymphoproliferative Disorders; Immunoproliferative Disorders; Lymphoma, Non-Hodgkin; Lymphoma; Lymphoma, T-Cell; Lymphoma, T-Cell, Peripheral; Lymphoma, T-Cell, Cutaneous
• Other Study ID Numbers: PI23/00486; PI2023 08 1416 (Registry Identifier: Ethics Committee for Drug Research of the Salamanca Health Area)

Plan for Individual participant data (IPD)

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

Drug and device information, study documents

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