EP4217746A1 - Krebsdiagnose mittels bildgebungsdurchflusszytometrie - Google Patents

Krebsdiagnose mittels bildgebungsdurchflusszytometrie

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Publication number
EP4217746A1
EP4217746A1 EP21794957.7A EP21794957A EP4217746A1 EP 4217746 A1 EP4217746 A1 EP 4217746A1 EP 21794957 A EP21794957 A EP 21794957A EP 4217746 A1 EP4217746 A1 EP 4217746A1
Authority
EP
European Patent Office
Prior art keywords
cells
sample
cancer
mcm
cell sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21794957.7A
Other languages
English (en)
French (fr)
Inventor
Ida FISCHER
Raif YUECEL
Peter Perenyi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytosystems Ltd
Original Assignee
Cytosystems Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2015122.1A external-priority patent/GB202015122D0/en
Priority claimed from GBGB2015211.2A external-priority patent/GB202015211D0/en
Application filed by Cytosystems Ltd filed Critical Cytosystems Ltd
Publication of EP4217746A1 publication Critical patent/EP4217746A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology

Definitions

  • the present invention relates to diagnostic methods and systems and particularly to diagnostic methods and systems for analysing abnormal cells and more particularly to analysing abnormal cells for the diagnosis of cancer.
  • Bladder cancer is described as a growth of abnormal tissue, known as a tumour, develops in the bladder lining.
  • Bladder cancer is responsible for over 3% of new cancer diagnoses and is the 10 th most prevalent malignancy worldwide. Over 90% of diagnosed patients are older than 55 years, with a mean age at diagnosis of 73 years. It must be noted that men are four times more likely than women to develop the disease. Also, BC affects whites about two times more often than blacks or Hispanics however, exact genetic link has not been entirely revealed yet. As the incidence rate of BC has mitigated, the disease-related mortality has decreased for women but remains unchanged for men.
  • BC has the highest lifetime treatment costs per patient of all cancers and is only exacerbated by significant spend related to the caused bacterial and fungal infections due to the applied gold standard diagnostic methodologies. Its incidence ranks first among malignant cancers of the urinary system and second only to prostate cancer in Western countries.
  • Urine cytology has been the mainstay of BC screening for decades. The process involves a urine specimen being collected and the bladder cells recovered, prepared, and viewed on a brightfield microscopy slide manually by a cytopathologist.
  • Urine cytology is a fairly cheap, non- invasive, and clinically widely accepted methodology, that has gained popularity among clinicians worldwide in the past due to its excellent positive predictive value.
  • the methodology in question has high specificity (96.9%) but poor sensitivity (29.1%), which properties make it a potent diagnostic tool to detect high grade cancer, but incompetent for early detection of low-grade cancer.
  • the poor sensitivity of cytology infers the decreased survival rate and the mitigated efficiency of the available treatment options.
  • Cytopathologists are highly trained clinicians, who can analyse, and interpret, clinically processed urine samples regarding to chromosomal and cell morphological abnormalities. Although, the characteristics of the aforementioned abnormalities are accurately described and well-known, human diagnosis-making process is not as objective as desired according to published studies.
  • Cystoscopy is named as the gold standard methodology to diagnose BC.
  • the process is an invasive and expensive medical intervention, which causes high level of anxiety and discomfort for the patients in every cases.
  • the process performed by a urologist, or in some countries a specialist nurse, in clinical environment therefore, well-trained staff and special equipment are essential to carry out the procedure.
  • Cystoscopy has high specificity (97.3%) and sensitivity (96.0%), which properties come with outstanding positive and negative predictive values.
  • the procedure can miss up to 40% of cancer according to published data, and it can be responsible for procedure acquired urinary tract infection (UTI) of between 2.7%-35.0% of which 10.1%-16.3% patients can progress to septicaemia.
  • UTI procedure acquired urinary tract infection
  • UroVysion Bladder Cancer Kit is an FDA approved molecular test designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization in urine specimens from persons with haematuria suspected of having bladder cancer.
  • UroVysion is a non-invasive urine-based test although, it is not categorized as a stand-alone diagnostic tool due to its inadequate specificity (89.7%) and sensitivity (61.9%). UroVysion test detects common chromosome abnormalities in bladder cancer nevertheless, the presence of the chromosome abnormality does not mean that the patient has bladder cancer, it can even indicate genetic disposition, which can lead to unnecessary testing of patients with the risks of developing UTI due cystoscopy. Of course, other promising candidates have been already on the market with various molecular scientific protocols, mainly genetic based, but their acceptance by the healthcare professionals are not as ascendant as the UroVysion molecular test. Therefore, what is needed are technologies that overcome the above-mentioned disadvantages.
  • a method of diagnosis comprising: providing a cell sample; contacting said cell sample with an antibody capable of binding a minichromosome maintenance (MCM) polypeptide(s); introducing the cell sample into an imaging flow cytometer; analysing one or more cells within the imaging flow cytometer; measuring visual properties of the cells in the sample; making a diagnosis based on the measured visual properties.
  • MCM minichromosome maintenance
  • the step of analysing the one or more cells comprises capturing an image of the cells within the flow cytometer.
  • a diagnostic or prognostic assay for the determination of cancer in a subject comprising the method of the invention wherein cells comprising the characteristics, for example at least two, three of four of those listed in Table 2 are an indicator of the presence of cancer.
  • a method for the treatment of cancer in a subject comprising:
  • a method of diagnosis comprising: providing a cell sample; introducing the cell sample into an imaging flow cytometer; analysing one or more cells within the imaging flow cytometer; measuring visual properties of the cells in the sample; making a diagnosis based on the measured visual properties.
  • the present invention may provide a method of cancer diagnosis comprising:
  • the visual properties/characteristics may include at least two, for example 2 to 20, or 2 to 10, such as 3 to 8 (3, 4, 5, 6, 7 or 8), properties.
  • the properties may be selected from the properties shown in Table 2.
  • the visual properties, or characteristics, of the cells may include two or more of cell size, cell shape characteristics, cell texture characteristics, cell contrast/intensity characteristics, cell density characteristics and cell location characteristics.
  • the term "cell” may be used interchangeably with “nucleus” to include visual properties such as features of nucleus size, nucleus shape characteristics, nucleus texture characteristics, nucleus contrast/intensity characteristics, nucleus density characteristics and nucleus characteristics.
  • Specific visual properties may include nuclear staining/intensity, nuclear shape, nuclear compactness, Cell or nuclear circularity, nuclear to cytoplasm ratio, nuclear to cytoplasm circularity, nuclear to cytoplasm perimeter.
  • the method advantageously further comprises summing the number of individual cells which have determined characteristics which correlate with the predetermined characteristics.
  • the MCM is selected from the group consisting of MCM 2, 3, 4, 5, 6 and 7.
  • the MCM may be a combination of two or more different MCMs, for example, two different MCMs selected from the group consisting of MCM 2, 3, 4, 5, 6 and 7.
  • the MCM may include MCM2 and one other MCM selected from MCM 3, 4, 5, 6 and 7.
  • the MCM may include MCM5 and one other MCM selected from MCM 2, 3, 4, 6 and 7.
  • the MCM is selected from the group consisting of MCM 2, 5 and 7.
  • the MCM is MCM 2.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes (e.
  • Antibodies may be polyclonal or monoclonal.
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic.
  • Antibodies which are specific for a MCM may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit) with the protein or a fragment thereof or a cell or virus which expresses the protein or fragment. "Specific” is generally used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s), e. g., has less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with any other molecule.
  • the antibody may be a monoclonal antibody having an antigen binding domain specific for MCM.
  • Monoclonal antibodies specific for MCM are known in the art, for example, anti-MCM2 antibody may be obtained from the Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ and as described in WO2016/207632.
  • the specific antibody is typically labelled with a detectable label, for example a radioactive label, labels detectable by spectroscopy techniques such as NMR as well as optically detectable labels.
  • a detectable label for example a radioactive label, labels detectable by spectroscopy techniques such as NMR as well as optically detectable labels.
  • the label may be attached to the antibody using conventional chemistry known in the art of antibody imaging. Such labelling allows those cells that are bound to the antibody to be detected/visualised.
  • the invention may include the step of coupling the antibody to an optically detectable label.
  • the optically detectable label may be detectable by emission spectroscopy, such as by fluorescence spectroscopy.
  • the optically detectable label is a fluorophore such as DyLight 405, 488, 550, 650, 680, 800, or may be selected from the fluorophores listed in Table 1.
  • the anti-MCM antibody is conjugated to an immunofluorescent dye, such as DY680.
  • the reactivities of an antibody on normal and test samples may be determined by any appropriate means.
  • Other labels include fluorochromes, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.
  • Other labels include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example.
  • They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Alkaline phosphatase or horseradish peroxidase are generally employed.
  • the method of the invention further comprises the step of providing a biological sample from a patient and obtaining an isolated cell sample from said biological sample.
  • the biological sample may be any type of tissue sample or fluid sample.
  • said biological sample is a sample of isolated bodily fluid, for example urine, semen, faeces, vaginal fluid or seminal fluid.
  • the biological sample is urine.
  • the cells may be isolated from urine by filtration or centrifugation.
  • the methods of the invention are typically for the diagnosis, prognosis and or treatment of cancer.
  • the cancer may be selected from the group consisting of bladder cancer, prostate cancer, kidney cancer, colon cancer, testicular, uterine, scrotum, endometrial, peritoneal cancer (for example of the peritoneal fluid), oesophageal or liver cancer.
  • the method of the invention is particularly useful in the diagnosis of bladder cancer and prostate cancer, preferably bladder cancer.
  • the methods of the invention may allow for the diagnosis of high grade cancers and/or low grade cancers.
  • the term "subject" is typically a mammal, preferably human.
  • the method of the invention includes one or more washing steps before the cell sample is loaded into the flow cytometer, for example, to separate the cells conjugated to the labelled antibody from other components in sample, including excess assay reagent, stains, tissue fragments cellular debris.
  • the cells may be washed using any suitable wash buffer, for example Perm buffer (Becton Dickinson) as manufacturer's instruction + 1% Foetal Calf Serum (FCS).
  • the present invention utilises flow cytometry, typically intranuclear IFC (inIFC), to detect MCM2 in cell samples obtained from urine.
  • Imaging flow cytometry IFC is used to produce images of the labelled cells.
  • IFC combines the high-throughput, multiparameter capabilities of conventional flow cytometry with morphological and spatial information, all at single-cell resolution. Multichannel digital images of hundreds of thousands of individual cells can be captured within minutes and include several fluorescence channels as well as bright field (transmitted light) and dark field (scattered light).
  • the throughput of IFC means that it is especially well suited to the analysis of rare cell types such as circulating tumour cells (which are cancer cells that escaped from a primary tumour and circulate in the bloodstream) and transition states, such as cell cycle phases (mitosis).
  • IFC can quantify multiple properties of constituents of interest (including proteins, nucleic acids, glycolipids etc.) in multiple subcellular compartments (nucleus, mitochondria, etc.).
  • the rich information makes IFC ideal for high-content analysis, as well as machine learning (ML) and artificial intelligence (Al) functions, raising the possibility to profile complex cell phenotypes, identify rare cells and transition states, and, importantly, discover further useful targets for BC diagnosis. Equipped with 20x, 40x, or 60x objectives and up to two charge-coupled device cameras, IFC allows thousands of morphological and spatial properties to be measured for each individual cell. These include bright field, dark field, and up to ten fluorescent channels.
  • IFC is well-suited to image nonadherent or dissociated cells, key for many clinical applications such as analyses of bodily fluids like urine or blood, whose structures can be distorted or damaged by placement onto a slide.
  • the instrument can capture thousands of individual cellular morphological features, what dataset is then processed by machine learning (ML) and Al components to provide the exceptional specificity, sensitivity, NPV and PPV of the product.
  • ML machine learning
  • aspects of the present disclosure include processing and analysing a cell sample produced by contacting a biological sample with a labelled anti-MCM antibody.
  • the IFC device is configured as a flow-through system for sequentially processing and immediately analysing the processed cell samples.
  • Figure 1 Detection of anti-MCM2-Dy680 in (1A) HEK293, (IB) RT112, (1C) DU145, and (ID) HT1197 cells. SurePath fixed, permeabilized with P3, and labelled with the 1:100 diluted antibody. Signal detection in cells can be seen below.
  • FIG. 2 (2A) Detection of anti-MCM2-Dy680 in RT112 cells. Effect of additional fixation was tested on SurePath stored cells. Cells were treated with (brightest grey) or without (bright grey) Cel I Fix after SurePath, then permeabilized with P3 and labelled with the 1:100 diluted antibody. (2B) Detection of anti-MCM2-Dy680 in HEK cells. SurePath fixed, permeabilized with P3, and labelled with the 1:100 diluted antibody.
  • CW BD cell wash (Becton Dickinson) as manufacturer's instruction + 1% Foetal Calf Serum (FCS) d.
  • Conjugated MCM2 antibody of Cytosystems antibody conjugated with direct immunofluorescent dye, e.g. Dy680.
  • the MCM2 antibody (WO2016/207632) is prepared from the hybridoma cell line stored and grown by Bio-Rad Laboratories and thereafter conjugated to dye (Dy680).
  • the assay is performed with cells, concentrated by centrifugation or filtration from biological samples taken from a patient.
  • the samples are normally urine collected from the patient as voided urine (non- invasive).
  • Both the RT112 and the HT1197 are bladder cancer cell lines
  • the DU 145 (ATCC® HTB-81TM) is a prostate cancer cell line
  • the HEK293 is an is embryonic kidney immortalized cell line, which represents the epithelial morphology and possibility to form tumours in nude mice.
  • HEK 293 cells are used for their ease of growth and transfection, making them a common cell culture in cancer research.
  • HEK 293 is employed as a highly malignant tumour model as its tumorigenicity increases significantly in high-passage.
  • Control cells provided from specific cell lines of RT112 (Sigma Aldrich, 85061106) and/or HT1197 (ATCC®, CRL-1473TM) were cell cultured in appropriate media (EMEM + 10% FBS + 1% NEAA + 1% Penicillin/Streptomycin) under appropriate conditions (37°C and 5%CC>2).
  • Cells were harvested at concentration of 2-3xl0 6 cells/ml by centrifuging them at 300g for 5 minutes at room temperature (20-24°C) followed by washing of cells twice with 2ml BD cell wash (Becton Dickinson, according to manufacturer's protocol) plus 1% foetal calf serum (FCS). Washed cells were centrifuged at 300g for 5 minutes at room temperature and fixed in 1 ml BD SurePath preservative buffer solution for 30 minutes (up to 2 weeks) at room temperature (20-24°C).
  • CSCs Clinical sample cells
  • the filtered CSCs were collected by processing 30-50 ml urine through the DCCD (according to manufacturer's protocol). The vial containing the filtered CSCs stored until further processing according to manufacturer's instruction.
  • centrifugation collected CSCs were collected by centrifuging 30-50ml urine at 800g for 10 minutes. After discarding the supernatant, the cell pellet was resuspended in BD SurePath preservative buffer and stored in fridge (2-8°C) or at room temperature (18-25°C) until further processing (for a maximum of 14 days). Both control and CSCs were fixed in BD SurePath preservative buffer solution.
  • the cell pellet was resuspended in 200 pl BD cell wash (w/o FCS), from which 100 pl was transferred into unstained tube and the other 100 pl were transferred into stained tube (these steps, when samples had been in higher quantity, were transferred into separate and labelled wells of sterile 96-wells plate).
  • 100 pl diluted MCM2-Dy680 (1:50 dilution of 0.5mg/ml stock in BD cell wash (w/o FCS)) and 100 pl BD cell wash (w/o FCS) were transferred into the stained tube and the unstained tube, respectively.
  • the tubes were incubated for 60 minutes (lhour) at room temperature (20-24°C).
  • EDF Extended Depth of Field
  • the 96-wells plate containing samples was loaded into the lmageStream®X Mk II. Instrument was turn on lhour prior measurements were taken. After 1 hour, fluidics start-up was run and ASSIST quality was control checked according to manufacturer's instructions.
  • Pre-set measurement template was loaded with necessary instrument settings, including 40x objective, low flow speed setting, lasers: 405nm laser lOmW, 642nm laser 150mW, 785nm laser 1.48mW, enable channels (Ch07 for NB, Ch09 for brightfield, Chll for DyLight680, Chl2 for side scatter), dot plots and histograms creation for recording (Area M09 vs Aspect Ratio M09 gating single cell population, Gradient RMS M09 gating on focused events).
  • the software pre-created regions for all the individual samples that were merged to represent each sample on a plot.
  • basic analysis regions were created on all events, then the regions were crosschecked using the image panel to region of contents, such as Area M09 vs Aspect Ratio M09 gating single cell population, and Gradient RMS M09 gating on focused events.
  • the stained and unstained sample were plotted for comparison on the intensity plot for Chll (MCM2 staining intensity).
  • MCM2 staining intensity The median fluorescence intensity values and all other features for each population from the statistics table were collected.
  • the individual cell profiles and cell population profiles were analysed.
  • the features that describes cancer cells based on cancer lines RT112, DU145, HT-1197, and HEK293 compared to normal epithelial bladder cells were that cancer cells expressed MCM2-Dy680 in their nucleus at high intensity and in high quantity.
  • Significant change in nuclear shape index, compactness, and circularity in cells expressing the MCM2-Dy680 were associated with cancer.
  • the area of nuclei to cytoplasm ratio in cancerous cells exceeded 0.5 while there was a significant decrease in both their nuclear and cytoplasmic circularity ( ⁇ 0.5) compared to healthy or only replicating cells (>0.5), where 1.0 is the perfect circle.
  • the nuclear and cytoplasmic perimeter ratio of cancerous cells were greater than 0.7.
  • the extent of nuclear MCM2 presence in samples were 0-15 ⁇ 3% and 15 ⁇ 3% were associated with low- and high-grade cancers, respectively.
  • Table 1 Fluorochrome Chart lmageStream®X Mkll Table 2. The features included in the creation of individual cell profiles according to categories.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
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  • Hematology (AREA)
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  • Hospice & Palliative Care (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
EP21794957.7A 2020-09-24 2021-09-23 Krebsdiagnose mittels bildgebungsdurchflusszytometrie Withdrawn EP4217746A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB2015122.1A GB202015122D0 (en) 2020-09-24 2020-09-24 Assay
GBGB2015211.2A GB202015211D0 (en) 2020-09-25 2020-09-25 Assay
PCT/IB2021/058693 WO2022064422A1 (en) 2020-09-24 2021-09-23 Diagnosis of cancer by imaging flow cytometry

Publications (1)

Publication Number Publication Date
EP4217746A1 true EP4217746A1 (de) 2023-08-02

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Application Number Title Priority Date Filing Date
EP21794957.7A Withdrawn EP4217746A1 (de) 2020-09-24 2021-09-23 Krebsdiagnose mittels bildgebungsdurchflusszytometrie

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EP (1) EP4217746A1 (de)
WO (1) WO2022064422A1 (de)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1422526A1 (de) * 2002-10-28 2004-05-26 MTM Laboratories AG Verfahren zur verbesserten Diagnose von Dysplasien
WO2006052822A2 (en) * 2004-11-05 2006-05-18 Cytolution, Inc. Methods and devices for screening cervical cancer
US11054426B2 (en) * 2011-01-07 2021-07-06 Cytosystems Ltd Diagnostic method
GB201100223D0 (en) * 2011-01-07 2011-02-23 Cytosystems Ltd Diagnostic method
GB201511196D0 (en) 2015-06-25 2015-08-12 Cytosystems Ltd Monoclonal antibodies

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