CMMC-ANIMA-02

University Hospital Würzburg

Dr. Delphine Chabut

Global Medical Affairs Head

Menarini Silicon Biosystems S.p.A.

Via Giuseppe di Vittorio 21/B3, I-40013 Castel Maggiore (BO) Italia

E-mail:dchabut@menarini.ch

Dr. Francesco Picardo

Medical Affairs Manager

Menarini Silicon Biosystems S.p.A.

Via Giuseppe di Vittorio 21/B3, I-40013 Castel Maggiore (BO) Italia

E-mail: fpicardo@siliconbiosystems.com

The emergence of several new effective drugs over the recent past dramatically improved patient outcomes in Multiple Myeloma (MM), prolonging the median survival by 4 years and more [1, 2]. Complete response (CR) rates have increased in parallel, as well as the need to develop more sensitive methods to define deeper responses and monitor minimal residual disease (MRD). Minimal Residual Disease (MRD) refers to the small number of cancer cells that remain in the body after treatment. In multiple myeloma, MRD detection is crucial for assessing the depth of response to treatment and predicting patient outcomes. Furthermore, MRD assessment is of paramount importance for future prognosis, treatment monitoring, and MRD-driven treatment strategies. Measurement of MRD in bone marrow by both next-generation sequencing (NGS) of variable diversity joining (V(D)J) rearrangements or next-generation flow cytometry (NGF) is highly predictive of survival in MM [3,4] and may therefore represent a biomarker to adapt treatment strategies [5,6]. However, serial assessments of MRD involve repeated sampling, which implies the invasiveness and the risks of repeated BM aspirations. A different way of capturing tumor heterogeneity and potentially decide upon treatment over the course of time in a minimally invasive method in MM patients may be the use of liquid biopsies from peripheral blood. Menarini Silicon Biosystems developed an automated assay to enumerate and characterize Circulating Multiple Myeloma Cells (CMMCs) from peripheral blood of patients with plasma cell disorders. Briefly, a sample of 10 ml peripheral blood is collected in the CellRescue, a dedicated tube that preserves CMMC stabilizing CD138 expression at room temperature for 120 hours. A 4 ml aliquot is placed in the Celltracks Autoprep for automated sample preparation which includes the addition of anti-CD138 ferrofluid conjugated (capture antibody) and anti-CD38 (detection antibody), DAPI and anti-CD45/CD19 (exclusion antibody). Then, the sample is transferred to Celltracks Analyzer II where a proprietary software will provide to the operator a gallery of images of "events" to be reviewed. An event is then classified as CMMC if is CD38+, DAPI+, CD45/CD19-. The results will be provided as "CMMC/4ml". [7] So far, no peripheral blood assay to evaluate MRD has been validated. Both NGS and Euroflow methods in blood have shown low sensitivity with respectively, 44% and 40% of false negative in respect of bone marrow assessment. [8,9] CMMC assay potentially can detect down to 1 cell/4 ml, equivalent approximately to a sensitivity of 1 x ^10-7 cell, higher than NGS (^10-6) and NGF (^10-6). Thus, our aim is to compare the CMMC assay performance in blood towards Euroflow NGF (mandatory) and Adaptive Clonoseq (optional) methods in bone marrow which are considered the reference standard for MRD assessment.

Primary Objective:

To contribute to set up a pooled dataset, with multiple sources, aimed to:

• Correlation of the CMMC assay with the NGF-MRD data from the bone marrow using multiparametric flow cytometry (EuroFlow) to determine the concordance between the two methods.

Secondary Objectives:

• Correlation of quantitative CMMCs results with clinical, laboratory and survival data.

  • Correlation of quantitative CMMC data with extramedullary disease.

Exploratory Objectives:

• Phenotypic analysis of circulating tumor plasma cells (CTCs) by NGF-Euroflow method.

  • Additional MRD assessment to detect paraprotein by mass spectrometry as well as circulating tumor multiple myeloma DNA (ctDNA/cmmDNA) in peripheral blood to further compare these results with BM-NGF and blood-based CMMC results.

    • Characterization of phenotypical and genome integrity on CMMCs.

The PB sample for CMMC enumeration is obtained within 3 days of BM collection for MRD assessment, if possible before BM aspiration to avoid the potential effects of the bone marrow procedure on the peripheral blood samples. EuroFlow data (mandatory), ClonoSEQ data (optional), mass spectrometry data (optional), ctDNA/cmmDNA data (optional) and CTCs (optional) will be collected at baseline and at first MRD assessment. Intermediate and final reports on data analyses and other specific analyses conducted at Fraunhofer ITEM and BZKF sites will be shared between parties.

EuroFlow will be performed at Würzburg University Hospital when MRD and CTCs will be assessed.

Mass spectrometry will be performed at Würzburg University Hospital when MRD will be assessed.

ctDNA/cmmDNA analysis will be performed at Augsburg University Hospital when MRD will be assessed.

CMMC enumeration using CellSearch system will be performed at Systemic Cancer Progression Laboratory (SCPL) of University of Regensburg at the specified points in time (see fig.1).

CMMC isolation and single cell processing or archiving of the samples in glycerol will be performed at Fraunhofer ITEM Regensburg.

• Documented multiple myeloma according to IMWG criteria

• Hematopoietic stem cell transplantation eligible (NDTEMM) or ineligible (NDTIMM) newly diagnosed MM patients (NDMM) who undergo MRD assessment as for clinical standard practice and/or for treatment based on local protocols or relapsed/refractory MM (RRMM) patients who start a new line of treatment and undergo MRD assessment as for clinical standard practice and/or for treatment based on local protocols.

• Male or female subjects, 18 years of age or older

• Voluntary written informed consent must be given before performance of any study-related procedure not part of normal medical care, with the understanding that the subject may withdraw consent at any time without prejudice to future medical care.

• Life expectancy of ≥ 6 months

200 patients including NDMM and RRMM patients will be enrolled into the CMMC-ANIMA-02 protocol. Patients assessed for MRD with BM aspirate analyzed with NGF EuroFlow (mandatory) and clonoSEQ (optional) assay will undergo additional PB sampling of 10 ml in CellRescue tubes and 27 ml in EDTA tubes within +3days from BM aspiration, preferably before BM aspiration and will be included in CMMC-ANIMA analysis as per primary objective of CMMC-ANIMA-02.

PB samples for CMMCs analyses will be analyzed in the SCPL, University of Regensburg, with CMMCs assay in conjunction with CellSearch System. CellSearch image galleries will be provided to MSB for blinded central analysis, further research analysis and cut off definition. In case of discrepancy, an arbiter will decide the effective value of the CMMC enumeration. After completion of the CellSearch™ CMMC test, the enriched cells will be recovered and stored in glycerol at -20°C or isolated for single cell whole genome amplification.

As detailed in the contract with Fraunhofer ITEM-Regensburg, the following selection criteria for subsequent cell isolation and single cell whole genome amplification (WGA) of CMMCs will be applied:

• All CellSearch samples with more than 50 CMMC will be stored in glycerol using an SOP provided by MSB.

• Samples with 5-50 CMMC will be subjected to immediate CMMC isolation, single cell WGA and genome integrity assessment.

Samples with less than 5 cells will be considered unsuited for cell isolation and will not be processed further.

Cells will be stored for no longer than 5 years from study completion.

Detection of paraprotein by mass spectrometry in PB and NGF CTC assays, will be performed at Würzburg University Hospital according to standardized protocols when MRD will be assessed.

Detection of ctDNA/cmmDNA in PB will be performed by CAPP-Seq at Augsburg University Hospital according to standardized methods when MRD will be assessed.

• EuroFlow, ClonoSEQ, mass spectrometry, ctDNA/cmmDNA and CTC results, as well as clinical and laboratory data, will be collected for subsequent analysis.

Data collected from this CMMC-ANIMA-02 will be pooled with other sources to determine the following endpoints:

• Primary endpoint

  • Correlation/concordance between CMMC and NGF-MRD assays.

• Secondary endpoint

  • Correlation of CMMCs assessment with:

    • % BM malignant plasma cell infiltration

    • M spike levels

    • K/L ratio

    • 12 months PFS rate correlation

    • Quantitative correlation between CMMC number and PFS rate

    • Single cell genomic molecular characterization

    • Presence of extramedullary disease

• Exploratory Endpoints

  • Assessment of MRD in peripheral blood through mass spectrometry, ctDNA/cmmDNA and CTC NGF methods and comparison with BM-NGF and CMMC MRD results.

  • Distribution of CMMCs DNA Genome Integrity Index measured on Single cells, stratified for patient (e.g. clinical parameters or assay parameters such as CMMC number) and process parameters (e.g. time from blood draw to enrichment, time from glycerol storage to cell isolation and WGA).

  • CMMC image analysis by machine learning algorithms.

  • Correlation of available functional imaging studies (including CT scans, MRI scans and FDG/Pentixafor PET-CT scans) with MRD bone marrow and peripheral blood methods and clinical data.

• Planned cohort size: 200 patients (150 NDMM, 50 RRMM) for CMMC-ANIMA pooled analysis

Partner Sites Bavarian Center for Cancer Research (BZKF):

University Hospital Würzburg

University Hospital, TU Munich

University Hospital Augsburg

University Hospital Regensburg

University Hospital Erlangen

University Hospital, LMU Munich

University of Regensburg

Fraunhofer ITEM Regensburg

Data results of overall and BZKF/Fraunhofer specific analysis will be shared between parties. The comparison between CMMC assay and BM-MRD will be assessed as follows:

• Concordance of reported positive MRD calls;
• Concordance of reported MRD negative calls;

• Concordance of reported MRD positive and negative calls.

Furthermore, correlation among CMMC number with BM% plasma cell infiltration, M spike level and K/L ratio measured within 3 days before or after PB sampling will also be evaluated.

Statistical analyses for exploratory endpoints including mass spectrometry, ctDNA/cmmDNA and NGF-CTCs will be performed based on individual requirements primarily interrogating

• Correlation of mass spectrometry, ctDNA/cmmDNA and NGF-CTC based MRD assessment from PB with BM-based MRD

• Concordance of PB based MRD assays

• Assessment of clonal evolution from ctDNA/cmmDNA data