Flow Cytometric Analysis of Peripheral Blood Neutrophil Myeloperoxidase Expression and Myelodysplastic Syndromes

Myelodysplastic Syndromes Clinical Trial

— MPO-MDS-PILOT  

Official title:

Flow Cytometric Analysis of Peripheral Blood Neutrophil Myeloperoxidase Expression for Ruling Out Myelodysplastic Syndromes: A Pilot Diagnostic Accuracy Study

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT03363399
• Other study ID #: 38RC17.249
• Secondary ID: 2017-A02361-52
• Status: Completed
• Start date: February 22, 2018
• Est. completion date: September 6, 2018

Study information

• Verified date: September 2018
• Source: University Hospital, Grenoble
• Contact: n/a
• Is FDA regulated: No
• Study type: Observational

Clinical Trial Summary

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal bone marrow neoplasms that predominate in the elderly, with a median age at diagnosis of 70 years. MDS are characterized by peripheral blood cytopenia and morphologic dysplasia for one or more hematopoietic cell lineage, reflecting ineffective hematopoiesis.

The diagnostic work-up of MDS includes a bone marrow aspirate and biopsy, which is an invasive procedure, for cytomorphologic and cytogenetic evaluations. Because the prevalence of disease is lower than 20% in subjects referred for suspected MDS, many patients are exposed to unnecessary bone marrow aspiration-related discomfort and harms.

An objective assay is highly desirable for accurately ruling out MDS based on peripheral blood samples, which may obviate the need for invasive bone marrow aspiration and biopsy in patients with negative results.

Few studies have investigated the value of peripheral blood flow cytometric analysis for the diagnosis of MDS and/or chronic myelomonocytic leukemia (CMML). Although promising, these studies lacked replication of their results, used a case-control design, which was prone to spectrum bias, or yielded imprecise diagnostic accuracy estimates due to relatively limited sample sizes.

Anecdotal evidence supports the potential of flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression for the diagnosis of MDS and CMML. Myeloperoxidase is an enzyme synthetized during myeloid differentiation that constitutes the major component of neutrophil azurophilic granules. Myeloperoxidase expression may reflect neutrophil hypogranulation, which is a classical although subjective dysplastic feature of MDS. Flow cytometric analysis of myeloperoxidase expression in bone marrow neutrophil granulocytes has been used for discriminating low versus high grade MDS. Yet a study reporting on the accuracy of flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression for the diagnosis of MDS is still lacking, to our knowledge.

In this study, the investigators hypothesize that flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood may accurately rule out MDS and obviate the need for bone marrow aspiration and biopsy, with sensitivity approaching 100%, in routine practice.

In this observational diagnostic accuracy study, burden will be null for recruited patients. No specific intervention is assigned to participants. All diagnostic testing, procedures, and medication ordering are performed at the discretion of attending physicians. Flow cytometry analysis of peripheral blood neutrophil myeloperoxidase expression will not require additional blood sample. A test result will have no impact on patient management. No follow-up visits are planned in this cross-sectional study.

Description:

The primary objective of MPO-MDS-pilot study is to estimate the accuracy of flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of myelodysplastic syndromes (MDS) and/or chronic myelomonocytic leukemia (CMML).

The secondary objectives are:

1. to provide accuracy estimates for prespecified thresholds of neutrophil myeloperoxidase expression in peripheral blood in ruling out MDS and/or CMML.

2. to estimate the prevalence of alternate diagnosis established by bone marrow cytomorphology.

The MPO-MDS-pilot project is a multicenter, phase II, cross-sectional, diagnostic accuracy study of consecutive unselected patients referred for suspected MDS or CMML.

Screening: All consecutive patients referred to the immuno-hematology lab at the study sites for suspicion of MDS will be screened for eligibility. A lab physician will review inclusion and exclusion criteria, using computerized medical and laboratory records.

Recruitment: Participants will be included in the study once all the screening activities have been conducted and only if the patient meets all inclusion and none exclusion criteria. The consent for flow cytometry analysis of peripheral blood sample and data collection through chart review will be sought under a regime of "non-opposition" (opt-out): after appropriate written information is delivered, cross-sectional data will be collected except in case of opposition from the patient. All patients included in the study will be assigned a unique patient identification number. This number will be used to identify the patient throughout the study.

Index test: Flow cytometry analysis of neutrophil myeloperoxidase expression in peripheral blood will be performed within 24 h of MDS diagnostic evaluation and blinded to the reference standard.

Reference standard: The diagnosis of MDS will be established according to the World Health Organization (WHO) classification, based on clinical data, peripheral blood cytopenia, cytomorphology of peripheral blood and bone marrow aspirate, and cytogenetic analysis. The criteria for MDS diagnosis are 1) the presence of ≥10% dysplastic cells in any hematopoietic lineage, 2) the exclusion of acute myeloid leukemia (defined by the presence of ≥20% peripheral blood or bone marrow blasts), and 3) the exclusion of reactive etiologies of dysplasia. Cytopenia is defined by hemoglobin concentration <10 g/dL, platelet count <100x109/L, and/or absolute neutrophil count <1.8 x109/L. Yet a diagnosis of MDS could be made with milder levels of cytopenia. Idiopathic cytopenia of uncertain significance (ICUS) is defined by unexplained mild persistent cytopenia for at least 6 months and the failure to establish the diagnosis to MDS according to published guidelines. Consistent with WHO classification, MDS subcategorization will rely on the degree of dysplasia (unilineage versus multilineage), blast percentages, presence of ring sideroblasts, and cytogenetic analysis (del(5q)). The criteria for CMML diagnosis are 1) the presence of persistent peripheral blood monocytosis ≥1 x109/L, and 2) monocyte accounting for more than 10% of the white blood cell differential count. Evaluation of bone marrow cytomorphology will be performed prospectively by experienced hematopathologists who are blinded to the index test results.

Patients with confirmed suspicion of MDS: Participants for whom the diagnosis of MDS (or CMML) is confirmed by the reference standard will be categorized as patients with confirmed suspicion of MDS.

Patients with unconfirmed suspicion of MDS: Participants for whom the diagnosis of MDS (or CMML) is ruled out by the reference standard will be categorized as patients with unconfirmed suspicion of MDS. This latter subgroup will include patients with ICUS, as defined in accordance with published guidelines

Follow-up: No follow-up visit is planned in this cross-sectional diagnostic accuracy study.

Recruitment information / eligibility

• Status: Completed
• Enrollment: 60
• Est. completion date: September 6, 2018
• Est. primary completion date: September 6, 2018
• Accepts healthy volunteers: No
• Gender: All
• Age group: 50 Years and older

Eligibility

Inclusion Criteria:

• Age =50 years;

• Clinical suspicion of myelodysplastic syndromes (MDS) and / or chronic myelomonocytic leukemia (CMML);

• Peripheral blood cytopenia defined by hemoglobin concentration <10 g/dL, platelet count <100 x109/L, and/or absolute neutrophil count <1.8 x109/L.

Exclusion Criteria:

• History of or active documented MDS or CMML;

• Admission to the intensive care unit;

• Suspicion of hematological disease other than MDS requiring bone marrow aspirate and/or biopsy (i.e., acute leukemia,…);

• Absolute neutrophil count <0.5 x109/L;

• Not affiliated with a social security system;

• Homelessness;

• Incarcerated;

• Inability to understand research information and/or to provide non-opposition, because of language restriction, dementia, or altered mental status;

• Refusal to participate;

• Previous enrollment in the study.

Related Conditions & MeSH terms

• Chronic Myelomonocytic Leukemia
• Leukemia, Myelomonocytic, Chronic
• Leukemia, Myelomonocytic, Juvenile
• Myelodysplastic Syndromes
• Preleukemia
• Syndrome

Intervention

Diagnostic Test:
• Flow cytometry analysis of neutrophil myeloperoxidase expression
Flow cytometry analysis of neutrophil myeloperoxidase expression in peripheral blood samples will be performed within 24 h of MDS diagnostic evaluation and blinded to the reference standard. Peripheral blood samples will be collected in 5 ml (EDTA) anticoagulant plastic tubes and processed within 24 h maximum of collection. The samples will be stored at 4°C overnight. Blood samples containing at least 105 neutrophils will be incubated with a panel of antibodies conjugated to fluorochromes, according to the manufacturers' recommendations. At least 10,000 cell events will be acquired on a 3-laser, 8-color flow cytometer and analyzed using Becton Dickinson (BD) Fluorescence-activated cell sorting (FACS) Diva Software version 6. Each marker will be expressed as median, geometric and arithmetic mean, regular and robust coefficient of variation.

Locations

Country	Name	City	State
France	Centre Hospitalier Universitaire Estaing	Clermont-Ferrand
France	Centre Hospitalier Universitaire Grenoble Alpes	Grenoble

Sponsors (1)

Lead Sponsor	Collaborator
University Hospital, Grenoble

Country where clinical trial is conducted

France, 

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Area under the Receiver Operating Characteristic (ROC) curve of neutrophil myeloperoxidase expression in peripheral blood	The primary outcome is the discrimination of the index test (i.e., flow cytometry analysis of neutrophil myeloperoxidase expression in peripheral blood) for the diagnosis of myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMLL) quantified by the area under the ROC curve point estimate along with its 95% confidence interval.	Baseline
Secondary	Sensitivity point estimates (along with 95% confidence interval) of neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of MDS or CMML		Baseline
Secondary	Specificity point estimates (along with 95% confidence interval) of neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of MDS or CMML		Baseline
Secondary	Negative predictive value point estimates (along with 95% confidence interval) of neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of MDS or CMML		Baseline
Secondary	Positive predictive value point estimates (along with 95% confidence interval) of neutrophil myeloperoxidase expression in peripheral blood for the diagnosis of MDS or CMML		Baseline
Secondary	Prevalence point estimate (along with 95% confidence interval) for alternate diagnosis established by bone marrow cytomorphology.		Baseline