WO2024251933A1 - Procédé amélioré pour l'enrichissement de cellules tumorales circulantes à partir de produits de leucophérèse de diagnostic - Google Patents

Procédé amélioré pour l'enrichissement de cellules tumorales circulantes à partir de produits de leucophérèse de diagnostic Download PDF

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WO2024251933A1
WO2024251933A1 PCT/EP2024/065704 EP2024065704W WO2024251933A1 WO 2024251933 A1 WO2024251933 A1 WO 2024251933A1 EP 2024065704 W EP2024065704 W EP 2024065704W WO 2024251933 A1 WO2024251933 A1 WO 2024251933A1
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dla
sample
ctc
ctcs
cellsearch
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Michiel STEVENS
Armagan Kocer
Leonardus Wendelinus Mathias Marie Terstappen
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Twente Universiteit
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57585—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds identifiable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5002—Partitioning blood components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to an improved method and process for rare cell enrichment and detection of circulating tumor cells (CTCs) from Diagnostic LeukApheresis (DLA).
  • CTCs circulating tumor cells
  • DLA Diagnostic LeukApheresis
  • CTCs circulating tumor cells
  • the enumeration of circulating tumor cells (CTCs) from blood can be used for disease prognosis, treatment outcome, and disease relapse prediction.
  • immunomagnetic enrichment is employed to enrich the CTCs from hematopoietic cells.
  • the most prominent example of enrichment methods is the FDA-cleared CellSearch system, which is designed to enrich CTCs from 7.5 mL blood samples.
  • the FDA-cleared CellSearch system which is designed to enrich CTCs from 7.5 mL blood samples.
  • the number of CTCs found in a standard 7.5 mL blood sample is too low for tumor cell characterization, while in patients with non-metastatic disease, the sensitivity and specificity are insufficient to determine the presence of disseminated cancer cells.
  • CTCs Several possibilities exist to increase the number of CTCs, such as capturing both the EpCAM positive as well as the EpCAM negative fraction of CTCs.
  • the capture of EpCAM-negative CTCs is hampered by a lack of markers, while also the prognostic value of these additional CTCs remains unclear.
  • Another option is to draw blood closer to the tumor, for example from tumor-draining veins. Although more CTCs can be recovered this way, it is an invasive procedure normally only possible during surgery, and not necessarily relevant or feasible in the metastatic setting.
  • the third option is to increase the evaluated blood volume, for example by an in-vivo capture system or through Diagnostic LeukApheresis (DLA).
  • DLA Diagnostic LeukApheresis
  • MNCs mononuclear cells
  • CTCs have a similar density as MNCs, these are co-captured in the procedure.
  • diagnostic Leukapheresis is a shortened procedure in which only two to five liters are processed, to minimize the burden on patients.
  • the resulting samples can then be processed using the CellSearch system.
  • aliquots of only 0.2xl0 9 white blood cells (WBC) are processed to ensure the sample can be analyzed. These 0.2xl0 9 aliquots only constitute 2-3% of the collected DLA sample.
  • the inventors of the present invention aimed to process DLA samples obtained from prostate cancer patients using standard CellSearch reagents, but with a reduced enrichment reagent protocol (RER).
  • RER enrichment reagent protocol
  • the inventors compared this approach to the processing of peripheral blood with CellSearch (PB-CS), the previously reported DLA with CellSearch (DLA-CS) and a reduced enrichment reagent protocol that enriches CTCs from l.OxlO 9 WBC (RER+) instead of 0.2xl0 9 WBC.
  • PB-CS peripheral blood with CellSearch
  • DLA-CS DLA with CellSearch
  • RER+ reduced enrichment reagent protocol that enriches CTCs from l.OxlO 9 WBC (RER+) instead of 0.2xl0 9 WBC.
  • the standard CellSearch test was developed to detect CTCs in 7.5 ml of peripheral blood. In the majority of patients, the number of CTCs is insufficient for tumor characterization hence larger blood volumes are needed. DLA offers this opportunity and typically collects CTC from 1-5 liters of blood. The concentration of CTCs per tube of blood does not significantly decrease after DLA suggesting a fast replenishment from the metastatic sites and consequently processing multiple passages of the complete blood volume can lead to a further increase in the number of harvested CTCs.
  • Adaptation of the CellSearch peripheral blood test to DLA resulted in a DLA-CS protocol that uses DLA aliquots of 0.2xl0 9 leukocytes meaning that on average, the DLA product of 112 mL of blood could be processed per CellSearch test.
  • the inventors introduced the RER protocol, which is comparable to or betterthan the DLA-CS protocol, while using 10% of the enrichment reagents of a CellSearch test.
  • the RER+ protocol uses 50% of the enrichment reagents to process a five times larger sample input, thereby overcoming the cost and time restrictions of the DLA-CS procedure.
  • CellSearch is semi-automated and RER is a manual procedure, causing a higher operator dependence and likely more variability.
  • the magnetic incubation in the CellSearch system is eight times three minutes, and in RER three times 10 minutes magnetic incubation is used.
  • staining is done using the same reagents at the same concentrations.
  • RER uses a smaller staining volume.
  • the system removes a portion of the unbound magnetic particles before the sample is transferred to the cartridge. The reason for this is that these hinder the imaging and identification of CTCs. In the RER protocol, this step is not necessary because there are much fewer ferrofluid particles present.
  • the enrichment of CTCs is followed by the identification of the captured cells and in many cases the isolation of single CTCs for further analysis.
  • the RER protocol demonstrates the possibility of enriching CTCs from aliquots of 0.2xl0 9 WBC using only 10% of the CellSearch enrichment reagents while obtaining a comparable number of CTCs.
  • the lower number of total cells in the enriched samples when using the RER protocol is likely due to the lower amount of ferrofluid used. Surprisingly, there is no correlation seen in the total number of cells captured in DLA-CS and RER. A possible reason for this could be a threshold effect.
  • the reduced number of magnetic particles causes many of these cells to bind insufficient particles to be retained during separation. The specific antibody-antigen binding of the particles would in these cases outcompete the non-specific interaction, resulting in a retained binding efficiency for CTCs. If this is the case, an even lower reagent volume may further reduce non-specific capture without a loss of capture efficiency. As a too-high sample concentration or too-low magnetic particle concentration will inevitably lead to a reduction in CTC recovery, further optimization is needed.
  • the invention provides a method for circulating tumor cell enrichment and enumeration workflow analysis in a subject, comprising: (a) obtaining (or providing) an apheresis sample containing CTCs (obtained) from a subject; (b) processing an apheresis aliquot in a system comprising: (i) concentrating the apheresis aliquot, preferably so as to reach a concentration of 0.05 xl0 9 -0.5xl0 9 , or 0.1 xl0 9 -0.3xl0 9 , more preferably 0.2xl0 9 white blood cells (WBC) per mL (e.g.
  • WBC white blood cells
  • the method for circulating tumor cell enrichment and enumeration workflow analysis in a subject can also be termed as method for obtaining a composition enriched in circulating tumor cells (CTCs).
  • Enrichment or enriched means that the obtained composition (bound portion) has a higher concentration of CTCs relative to the starting sample/aliquot.
  • the method may or may not comprise enumeration workflow analysis, which refers to enumerating the number of CTSs and/or their characteristics.
  • the method is ex vivo.
  • the concentrating in i. is performed by centrifuging.
  • step i) starts with pre-treating the apheresis aliquot (or sample) with DNase.
  • DNase may be used in a concentration of 5-55, preferably 10-30 pg/ml, preferably in the presence of an activator such as MgSO4, preferably in a concentration of 5-50, preferably 10-30 pM (e.g. for a 2xl0 9 WBC aliquot of leukapheresis sample).
  • an activator such as MgSO4
  • incubation time is 1-100, preferably 2-30, more preferably 5-15 min, preferably at 10-30 degrees Celsius, more preferably 15-25 degrees Celsius.
  • the fluorescent antibodies/peptides preferably bind CTC or CTC-antigen.
  • the method may also be applied on other types of samples, such as blood sample, cerebral spinal fluid sample, bone marrow fluid sample, ascites sample, urine sample, or pleural effusion sample.
  • the immunomagnetic particles are CellSearch Ferrofluid.
  • the incubation with the immunomagnetic particles may be performed in the presence of a magnetic field. In some cases, controlled clustering of the immunomagnetic particles is performed during incubation.
  • the staining reagents may, for example, be CellSearch staining reagents.
  • the imaging microscope system may conveniently be a CellTracks Analyzer II.
  • the obtained apheresis sample may be stored in CellSave vacutainers.
  • the CellTracks Analyzer II system may include an analysis cartridge.
  • the subject is a patient with metastatic hormone sensitive prostate cancer.
  • the apheresis may be Diagnostic LeukApheresis (DLA) and may, for example, be obtained in a Spectra Optia® system.
  • DLA Diagnostic LeukApheresis
  • the processed blood volume is 2 to 5 liters.
  • the DLA aliquot may, for example, consist of 0.2xl0 9 WBC or lxlO 9 WBC or any value in the range 0.2xl0 9 WBC to lxlO 9 WBC or even to 10xl0 9 WBC.
  • the solution used for fluorescent antibody staining may be a solution containing a permeabilization reagent, nuclear stain, staining reagent, and Cell Buffer for incubating the sample at 37°C for 20 minutes.
  • the solution may be manually transferred to a CellSearch sample cartridge for placement into a CellSearch Magnest holder.
  • the CellSearch sample cartridge may be scanned in a CellTracks Analyzer II.
  • the CellTracks Analyzer II files may be processed using StarDist segmentation followed by a deep learning approach.
  • enumerating a sufficient number of CTCs provides a testing platform for an integrated use of clinical workflow.
  • the invention provides a diagnostic test kit for identification and/or characterization of one or more CTCs in an apheresis sample consisting of a workflow analysis comprising: (a) optionally obtaining an aliquot of apheresis material; (b) a capture reagent; and (c) instructions for how to prepare the sample for analysis with a method as described above, where the kit characterizes CTCs used in disease prognosis, treatment outcome and disease relapse prediction in the subject.
  • the instructions may further include how to interrogate a sample, for example using a CellTracks Analyzer II system.
  • the invention provides a method for circulating tumor cell enrichment and enumeration workflow analysis in a subject, comprising: (a) obtaining (or providing) an apheresis sample containing CTCs (obtained) from a subject; (b) pretreating the sample using a nuclease, preferably DNase. This may reduce clumps and/or remove free DNA in the sample; (c) enriching the tumor cells using immunomagnetic particles (comprising CTC- antigen binding antibody); (d) optionally staining the (enriched) sample for cell identification using fluorescent antibodies/peptides; and (e) identification of CTC e.g.
  • this method may further comprise any of the optional method steps described above in relation to the first aspect of the invention.
  • the method for circulating tumor cell enrichment and enumeration workflow analysis in a subject can also be termed as method for obtaining a composition enriched in circulating tumor cells (CTCs).
  • Enrichment or enriched means that the obtained composition (bound portion) has a higher concentration of CTCs relative to the starting sample/aliquot.
  • the method may or may not comprise enumeration workflow analysis, which refers to enumerating the number of CTSs and/or their characteristics.
  • the method is ex vivo.
  • step b) pretreats the sample/aliquot with DNase.
  • DNase may be used in a concentration of 5-55, preferably 10-30 pg/ml, preferably in the presence of MgSCU preferably in a concentration of 5-50, preferably 10-30 pg/ml (e.g. for a 2xl0 9 WBC aliquot of leukapheresis sample).
  • incubation time is 1-100, preferably 2-30, more preferably 5-15 min, preferably at 10-30 degrees Celsius, more preferably 15-25 degrees Celsius.
  • step b. further includes concentrating the apheresis aliquot/sample so as to reach a concentration of 0.05 xl0 9 -0.5xl0 9 , or 0.1 xl0 9 -0.3xl0 9 , more preferably 0.2xl0 9 white blood cells (WBC) per mL (e.g. by removal of plasma).
  • the concentrating is performed by centrifuging.
  • the fluorescent antibodies/peptides preferably bind CTC or CTC-antigen.
  • the method may also be applied on other types of samples, such as blood sample, cerebral spinal fluid sample, bone marrow fluid sample, ascites sample, urine sample, or pleural effusion sample.
  • the invention provides a diagnostic test kit for identification and/or characterization of one or more CTCs in an apheresis sample consisting of a workflow analysis comprising: (a) an aliquot of apheresis material; (b) a capture reagent; and (c) instructions for how to prepare the sample for analysis with a method as described in the preceding paragraph, where the kit characterizes CTCs used in disease prognosis, treatment outcome and disease relapse prediction in the subject.
  • Fig 1 A schematic representation of the RER procedure to process DLA aliquots. The bottom row indicates the time for each step. The red/blue rectangle represents the BD iMag (cell separation magnet). FF: ferrofluid, CE: capture enhancement reagent.
  • Fig 2 Panel A Average number of cells and platelets per mL
  • Panel B Percentage of the volume consisting of cells and platelets in blood and DLA as collected as well as during magnetic particle incubation in PB-CS, DLA-CS, and RER procedures.
  • Fig 3 (A) Comparison of CTC recovery between DLA-CS and RER procedures using 0.2xl0 9 WBC from 30 DLA samples of metastatic prostate cancer patients. (B) Number of CTC detected in 30 peripheral blood and regular DLA samples using CellSearch, RER, and RER+ (6 samples) procedures.
  • Fig 4 Comparison of the total number of cells after processing of DLA samples with CS- DLA and RER protocols for 30 leukapheresis samples of prostate cancer patients. (B) The total number of cells after processing whole blood and DLA samples using CellSearch, RER, and RER+ (6 samples) protocols.
  • Fig 5 (A) Final sample purity and (B) relative CTC recovery compared to blood. Results represent final samples as found after enrichment of 7.5 mL of blood, 0.2xl0 9 or l.OxlO 9 WBC from DLA and processed using CellSearch, RER, or RER+.
  • Fig 6Snippets of the DAPI channel showing nucleated cells in the enriched samples containing 100,000, 200,000, 300,000, and 400,000 cells.
  • Fig 7 (A) Coupled number of CTC and the total number of cells with both methods for all 30 samples and (B) relative number of CTC and the total number of cells for all 30 samples.
  • Fig 8 Graph showing recovery of CTC from 19 DLA samples of prostate cancer patients with or without DNase pre-treatment.
  • Fig 9 Graph showing a comparison of the total number of cells after processing of 19 DLA samples with or without DNase pre-treatment.
  • Fig 10 Representation of images having the nuclear stain DAPI from samples after enrichment with and without DNase pre-treatment. Rows contain images from the same patient sample.
  • CTCs circulating tumor cells
  • PB-CS CellSearch
  • DLA Diagnostic LeukApheresis
  • DLA samples were obtained from 28 metastatic Hormone Sensitive Prostate Cancer patients (mHSPC) before initiation of treatment and with greater than 2 CTCs in a 7.5 mL sample of blood.
  • mHSPC metastatic Hormone Sensitive Prostate Cancer patients
  • patients underwent a second leukapheresis procedure after becoming castration-resistant resulting in a total of 30 samples.
  • Leukapheresis was performed per the optimized procedure described by Mout et al. on a Spectra Optia (Terumo, Lakewood, Co, USA). Samples were collected in accordance with the Declaration of Helsinki as part of a study approved by the medical ethical committee of the Erasmus Medical Center.
  • CTCexp CTCexp
  • the MNC population is targeted for extraction.
  • the sample however contains impurities; some erythrocytes, granulocytes, and platelets are co-captured during the procedure.
  • the DLA samples are concentrated by the removal of plasma to reach a concentration of 0.2xl0 9 WBC/mL.
  • Differential blood counts of DLA and whole blood samples were taken on the same day and determined on a DxH 500 hematology analyzer (Beckman Coulter, Utrecht, The Netherlands). The number of blood cells as well as their total volume per mL during magnetic particle incubation was then calculated for the blood and DLA product during PB-CS, DLA-CS, and RER processing.
  • the sample was supplemented with 2ml 'Cell buffer' (phosphate buffered saline (Merck, Darmstadt, Germany) supplemented with bovine serum albumin (Merck), EDTA (Merck), casein (Merck) and mouse serum (Invitrogen, Carlsbad, USA) and placed in the magnet for 20 minutes after which the unbound fraction was aspirated using a glass Pasteur pipet and syringe pump set to 1 mL/min. The bound fraction was resuspended in ImL of cell buffer before performing a second separation of 10 minutes in the magnet.
  • 2ml 'Cell buffer' phosphate buffered saline (Merck, Darmstadt, Germany) supplemented with bovine serum albumin (Merck), EDTA (Merck), casein (Merck) and mouse serum (Invitrogen, Carlsbad, USA) and placed in the magnet for 20 minutes after which the unbound fraction was aspirated using a glass Pasteur pipet and syringe pump set
  • samples containing l.OxlO 9 WBC were first incubated with 20 pg/mL DNase I (Roche, Basel, Switzerland) together with 20 pM MgSC to prevent aggregation.
  • the enrichment was performed analogously to the RER procedure.
  • all volumes up to the separation step in figure 1 were multiplied by 5, and largerconsumables were used where needed.
  • aspirating supernatant to 5 mL in a 12 mL round bottom tube (Greiner bio) and using 75 pL CellSearch ferrofluid and 75 pL CellSearch capture enhancement.
  • RER+ samples were transferred to 12x75mm centrifuge tubes after the initial separation and processed further using the standard RER protocol. Staining of RER and RER+ samples was performed identically.
  • DNase as an effective prevention of leukocyte aggregates in the immunomagnetic processing of leukapheresis material: Next to the sheer number of WBC that is non-specially co-captured in the immune-magnetic enrichment of CTC, also the presence of cell aggregates limits the tolerable cell density during CTC identification, and with that the processable DLA volume. Additionally, these aggregates prevent efficient single-cell analysis and the identification of pre-existing tumor-associated clusters. Close examination of these aggregates has revealed that the reason for their aggregation is the presence of free DNA in the resulting samples.
  • the inventors of the present invention introduced the application of a DNase pre-treatment for DLA material prior to sample processing.
  • this pre-treatment the free DNA material in the DLA material is removed, while the DNA material in the CTC and WBC is preserved.
  • the improved sample quality of the resulting samples will allow the processing of more samples as automated analysis can be better applied to the clearly distinguishable cells in the DNase pre-treated samples.
  • Fig 10 a gallery of single images from 4 different prostate cancer DLA samples enriched with or without DNase pre-treatment is shown, demonstrating the improved sample quality and absence of cell aggregates due to the application of DNase in this setting.
  • the number of cells and platelets per mL during magnetic particle incubation in CS-DLA is on average only 7% (median 7%, range 4% to 9%) of that in PB-CS.
  • the number of cells and platelets per mL is about equal to that in PB-CS (average 98%, median 96%, range 59% to 131%).
  • volume fraction The percentage of volume taken up by cells and platelets (volume fraction) in PB-CS was on average 26% (median 26%, range 20% to 32%), and for the DLA-CS procedure on average 1.5% (median 1.4%, range 1.1% to 2.2%). In the RER procedure, the volume fraction is 16% (median 14%, range 11% to 23%), which is much closer to, but still below the volume fraction in PB-CS.
  • the number of cells and platelets per mL in the RER procedure is similar to that in CS-PB, while the cell volume concentration is below that of PB-CS.
  • concentration of capture reagents in the assay is the same for all three procedures, the capture reagent per cell is approximately the same for PB-CS and RER, and approximately 12-fold higher in PB-CS.
  • the capture reagent per cell volume is compared to PB-CS approximately 2-fold higher in the RER protocol, and approximately 10-fold higher in DLA-CS.
  • the DLA-CS of an 0.2xl0 9 WBC aliquot leads to a median 5.6-fold (mean 5.6, range 1.4 to 14.6-fold) increase in CTC compared to the 7.5 mL whole blood PB-CS.
  • RER leads to a similar increase (median 4.4, mean 5.5, range 2 to 18.6-fold).
  • another 3.8-fold median increase in CTC was observed compared to DLA-CS and 3.4-fold compared to RER, or a total median increase of 19.3-fold (mean 22.9, range 4.3 - 50.1 to fold) in CTC compared to PB-CS.
  • the number of CTC found in all samples with the different enrichment methods is shown in figure 3B.
  • the standard CellSearch test was developed to detect CTCs in 7.5 ml of peripheral blood. In the majority of patients, the number of CTCs is insufficient for tumor characterization hence larger blood volumes are needed 6 . DLA offers this opportunity and typically collects CTC from 1-5 liters of blood. The concentration of CTCs per tube of blood does not significantly decrease after DLA suggesting a fast replenishment from the metastatic sites and consequently processing multiple passages of the complete blood volume can lead to a further increase in the number of harvested CTCs.
  • Adaptation of the CellSearch peripheral blood test to DLA resulted in a DLA-CS protocol that uses DLA aliquots of 0.2xl0 9 leukocytes meaning that on average, the DLA product of 112 mL of blood could be processed per CellSearch test.
  • DLA-CS tests one could perform greater than 30 DLA-CS tests to process the complete DLA product from five liters of blood, it would be cost and time prohibitive and makes subsequent interrogation of the tumor cells difficult.
  • the major limitation to processing larger volumes was the large number of leukocytes remaining after enrichment.
  • the RER protocol which is comparable to or better than the DLA- CS protocol, while using 10% of the enrichment reagents of a CellSearch test.
  • the RER+ protocol uses 50% of the enrichment reagents to process a five times larger sample input, thereby overcoming the cost and time restrictions of the DLA-CS procedure.
  • CellSearch is semi-automated and RER is a manual procedure, causing a higher operator dependence and likely more variability.
  • the magnetic incubation in the CellSearch system is eight times three minutes, and in RER three times 10 minutes magnetic incubation is used.
  • staining is done using the same reagents at the same concentrations.
  • RER uses a smaller staining volume and staining is performed at 37°C.
  • the system removes a portion of the unbound magnetic particles before the sample is transferred to the cartridge. The reason for this is that these hinder the imaging and identification of CTCs. In the RER protocol, this step is not necessary because there are much fewer ferrofluid particles present.
  • the enrichment of CTCs is followed by the identification of the captured cells and in many cases the isolation of single CTCs for further analysis.
  • the RER protocol demonstrates the possibility of enriching CTCs from aliquots of 0.2xl0 9 WBC using only 10% of the CellSearch enrichment reagents while obtaining a comparable number of CTCs.

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Abstract

L'invention concerne un procédé d'analyse de produits DLA incorporant des aliquotes de leucocytes pour un traitement utilisant CellSearch (DLA-CS). Le protocole de réactif d'enrichissement réduit (RER) est utilisé pour traiter des aliquotes de leucocytes de 0,2x109 avec 10 fois moins de réactifs d'enrichissement que DLA-CS. En utilisant 1,0x10 9 aliquotes de leucocytes, une augmentation de 4 fois dans les cellules tumorales par rapport à DLA-CS et une augmentation de 24 fois par rapport à PB-CS ont été obtenues. En utilisant 10 fois moins de réactif de capture CellSearch, nous avons traité des aliquotes de leucophérèse standards sans perte dans la récupération de cellules tumorales, tout en atteignant une pureté plus élevée. Le procédé permet de traiter 26 % de l'échantillon de leucophérèse totale à l'aide de réactifs CellSearch, ce qui permet un nombre suffisant de CTC pour une caractérisation de cellules tumorales chez la plupart des patients atteints d'un cancer de la prostate métastatique. L'utilisation de la DNAse permet d'empêcher l'agglutination normalement observée des cellules pendant la procédure d'enrichissement magnétique, ce qui permet une meilleure identification et un traitement ultérieur des CTC enrichies.
PCT/EP2024/065704 2023-06-08 2024-06-07 Procédé amélioré pour l'enrichissement de cellules tumorales circulantes à partir de produits de leucophérèse de diagnostic Pending WO2024251933A1 (fr)

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Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
A. NANOUN.H. STOECKLEIND. DOERRC. DRIEMELL.W.M.M. TERSTAPPENF. A. W. C.: "Training an automated circulating tumor cell classifier when the true classification is uncertain", SUBMITTED, 2023
ALLARD, W. J. ET AL.: "Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases", CLIN. CANCER RES., vol. 10, 2004, XP002489234, DOI: 10.1158/1078-0432.CCR-04-0378
ANDREE, K. C. ET AL.: "Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap", INT. J. CANCER, vol. 143, 2018, pages 2584 - 2591, XP055788804, DOI: 10.1002/ijc.31752
BESSMAN, J. D.JOHNSON, R. K.: "Erythrocyte volume distribution in normal and abnormal subjects", BLOOD, vol. 46, 1975, pages 369 - 379
BULFONI, M. ET AL.: "In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis", BREAST CANCER RES., vol. 18, 2016, pages 1 - 15
CHAPMAN, E. H.KUREC, A. S.DAVEY, F. R.: "Cell volumes of normal and malignant mononuclear cells", J. CLIN. PATHOL., vol. 34, 1981, pages 1083 - 1090
CHEMI, F. ET AL.: "Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse", NOT. MED., vol. 25, 2019, pages 1534 - 1539, XP037154998, DOI: 10.1038/s41591-019-0593-1
COUMANS, F. A. W.LIGTHART, S. T.UHR, J. W.TERSTAPPEN, L: "W. M. M. Challenges in the Enumeration and Phenotyping of CTC", CLIN. CANCER RES., vol. 18, 2012, pages 5711 - 8
COUMANS, F.TERSTAPPEN, L.: "Methods and Protocols", 2015, SPRINGER, article "Detection and Characterization of Circulating Tumor Cells by the CellSearch Approach BT - Whole Genome Amplification", pages: 263 - 278
COUMANS, FTERSTAPPEN, L: "Detection and characterization of circulating tumor cells by the CellSearch approach", WHOLE GENOME AMPLIFICATION: METHODS AND PROTOCOLS, 2015
CRISTOFANILLI, M. ET AL.: "Circulating tumor cells: A novel prognostic factor for newly diagnosed metastatic breast cancer", J. CLIN. ONCOL., vol. 23, 2005, XP002570579
DE BONO, J. S. ET AL.: "Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer", CLIN. CANCER RES., vol. 14, 2008, pages 6302 - 6309, XP055129201, DOI: 10.1158/1078-0432.CCR-08-0872
DE WIT, S. ET AL.: "The detection of EpCAM+and EpCAM-circulating tumor cells", SCI. REP., vol. 5, 2015, XP055758468, DOI: 10.1038/srep12270
FAUGEROUX V ET AL.: "Genetic characterization of a Unique Neuroendocrine Transdifferentiation Prostate Circulating Tumor Cell - Derived eXplant (CDX) Model", NATURE COMMUNICATIONS, vol. 11, 2020, pages 1884, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-020-15426>
FEHM, T. N.: "Diagnostic leukapheresis for CTC analysis in breast cancer patients:CTC frequency, clinical experiences and recommendations for standardized reporting.", CYTOM. PART A, vol. 93, 2018, pages 1213 - 1219, XP072330560, DOI: 10.1002/cyto.a.23669
FISCHER, J. C. ET AL.: "Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients", PROC. NATL. ACAD. SCI. U. S. A., vol. 110, 2013, pages 16580 - 16585, XP093116615, DOI: 10.1073/pnas.1313594110
HEJAZIAN, M.LI, W.NGUYEN, N. T.: "Lab on a chip for continuous-flow magnetic cell separation", LAB CHIP, vol. 15, 2015, pages 959 - 970, XP055741445, DOI: 10.1039/C4LC01422G
KIM, T. H. ET AL.: "A temporary indwelling intravascular aphaeretic system for in vivo enrichment of circulating tumor cells", NOT. COMMUN, vol. 10, 2019
LAMBROS MARYOU B. ET AL: "Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis", vol. 24, no. 22, 15 November 2018 (2018-11-15), US, pages 5635 - 5644, XP093089263, ISSN: 1078-0432, Retrieved from the Internet <URL:https://aacrjournals.org/clincancerres/article-pdf/24/22/5635/2047630/5635.pdf> DOI: 10.1158/1078-0432.CCR-18-0862 *
LAMBROS MB: "Single Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis.", CLINCANRES, vol. 24, no. 22, 2018, pages 5635 - 5644, XP093089263, DOI: 10.1158/1078-0432.CCR-18-0862
MICHIEL STEVENS ET AL: "Improved enrichment of circulating tumor cells from diagnostic leukapheresis product", CYTOMETRY A, WILEY-LISS, HOBOKEN, USA, vol. 103, no. 11, 2 August 2023 (2023-08-02), pages 881 - 888, XP072529807, ISSN: 1552-4922, DOI: 10.1002/CYTO.A.24779 *
MOUT, L. ET AL.: "Generating human prostate cancer organoids from leukapheresis enriched circulating tumour cells", EUR. J. CANCER, vol. 150, 2021, pages 179 - 189
RADFAR PAYAR ET AL: "Single-cell analysis of circulating tumour cells: enabling technologies and clinical applications", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 40, no. 9, 17 March 2022 (2022-03-17), pages 1041 - 1060, XP087145971, ISSN: 0167-7799, [retrieved on 20220317], DOI: 10.1016/J.TIBTECH.2022.02.004 *
SLAVKA, G. ET AL.: "Mean platelet volume may represent a predictive parameter for overall vascular mortality and ischemic heart disease", ARTERIOSCLER. THROMB. VASC. BIOL, vol. 31, 2011, pages 1215 - 1218
STEVENS MICHIEL ET AL: "StarDist Image Segmentation Improves Circulating Tumor Cell Detection", CANCERS, vol. 14, no. 12, 13 June 2022 (2022-06-13), pages 1 - 13, XP093089293, DOI: 10.3390/cancers14122916 *
STEVENS, M.: "StarDist Image Segmentation Improves Circulating Tumor Cell Detection", CANCERS, 2022, pages 14
STOECKLEIN, N. H.FISCHER, J. C.NIEDERACHER, D.TERSTAPPEN, L. W. M. M.: "Challenges for CTC-based liquid biopsies: Low CTC frequency and diagnostic leukapheresis as a potential solution", EXPERT REV. MOL. DIAGN., vol. 16, 2016, pages 147 - 164
TAMMINGA MENNO ET AL: "Leukapheresis increases circulating tumour cell yield in non-small cell lung cancer, counts related to tumour response and survival", BRITISH JOURNAL OF CANCER, NATURE PUBLISHING GROUP UK, LONDON, vol. 126, no. 3, 30 November 2021 (2021-11-30), pages 409 - 418, XP037682341, ISSN: 0007-0920, [retrieved on 20211130], DOI: 10.1038/S41416-021-01634-0 *
TING-BEALL, H.NEEDHAM, D.HOCHMUTH, R: "Volume and osmotic properties of human neutrophils", BLOOD, vol. 81, 1993, pages 2774 - 2780
WU JUNHAO ET AL: "Magnetic-Based Enrichment of Rare Cells from High Concentrated Blood Samples", CANCERS, vol. 12, no. 4, 10 April 2020 (2020-04-10), pages 933, XP055811083, DOI: 10.3390/cancers12040933 *
YU, M ET AL.: "Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition", SCIENCE, vol. 339, 2013, pages 580 - 584

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