Tools and methods for detecting abnormal erythroblasts

Abstract

The present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a pathological state in the bone marrow of a subject, said method comprising at least the step of determining the presence of abnormal erythroblasts in a sample of a body fluid of the subject. The use of abnormal erythroblasts as a marker for diagnosing, detecting, monitoring or prognosticating a pathological state in the bone marrow of a subject is also envisaged, as well as a kit for diagnosing, detecting, monitoring or prognosticating a pathological state in the bone marrow of a subject comprising means for determining the presence in a sample of the subject of (i) CD71 and / or GPA, (ii) CD45, (iii) nucleic acids of rare circulating cells, and (iv) at least one of EpCam or (v) vimentin.

Description

Technical Field

[0001] This invention relates to a method for detecting, diagnosing, monitoring, or predicting pathological conditions in the bone marrow of a subject, the method comprising at least the step of determining the presence of abnormal erythroblasts in a subject's bodily fluid sample. The use of abnormal erythroblasts as markers for diagnosing, detecting, monitoring, or predicting pathological conditions in the bone marrow of a subject is also envisioned, as well as a composition for diagnosing, detecting, monitoring, or predicting pathological conditions in the bone marrow of a subject, comprising tools for determining the presence of at least one of the following in a subject's sample: (i) CD71 and / or GPA, (ii) CD45, (iii) nucleic acids from rare circulating cells, and (iv) epithelial cell adhesion molecule (EpCam) or (v) vimentin. Background Technology

[0002] The driving forces behind human tumorigenesis are multifactorial and vary from individual to individual, often taking several years to manifest systemically. This development manifests as a progression from benign to malignant, from dysplasia to tumor formation, and from local to systemic spread. This complexity hinders a deep or comprehensive understanding of cancer reversibility and tumor growth rates. At the cellular level, the development of malignant tumors can be measured by the number of stable genetic mutations acquired by each clone, as well as the mutations among tumor cells and the resulting phenotypic heterogeneity. It is assumed that a cell must acquire at least six tumor-associated mutations to be malignant. Genetic alterations follow phylogeny, thus representing an evolutionary process, and lead to phenotypic changes, cell dedifferentiation, and loss of function in very specific cellular environments.

[0003] In many cases, a century-old grading system is still in use, which is differentiation-oriented and translates into an assessment of tumor aggressiveness. This assessment of tumor aggressiveness (defined as the tumor's ability to invade other tissue regions and establish distant micrometastases over a given timeframe) translates into prediction and potentially therapeutic implications. At the cellular level, tumor aggressiveness can be directly correlated with clonal doubling time, degree of cell differentiation, and the diversity of genetically distinct tumor cell populations. Therefore, the quality of aggressive tumors increases rapidly, but more importantly, the clonal heterogeneity of pleomorphic tumors increases. This ultimately increases the likelihood that certain tumor cells will thrive in hostile or unnatural distant tissue regions. A specific example of an aggressive cancer type is pancreatic adenocarcinoma.

[0004] The spread of tumor cells and the establishment of stable micrometastases mark the transition from localized disease to systemic disease, thus evolving from a treatable condition into a often fatal one. In particular, bone and bone marrow are hypothesized to constitute a metastatic niche for several cancer types, such as breast, prostate, and lung tumors. Patients with bone metastases are considered “high-risk” because few helpful treatments are available. Most metastatic development often goes undetected or unrecognized until the very end of its growth. It is widely believed that early detection is necessary for most cancer types to prevent early death through various treatment modalities and to detect the disease before it transforms into a systemic manifestation. However, current diagnostic techniques are often limited to identifying early-stage cancers through incidental or organ-specific screenings, such as common breast cancer screening programs.

[0005] In liquid biopsy, the process of tumor cells detaching into the bloodstream is being studied, which is often considered to support or represent the initiation of micrometastases. Factors leading to the timely detachment of tumor cells at the individual and cancer type levels are currently being investigated. The discovery of bone marrow-resident tumor cells (called disseminated tumor cells) in the early stages of breast cancer supports the theory of early tumor cell dissemination and thus demonstrates the universality of cell-based liquid biopsy in detecting early-stage cancer. Further common sense dictates that cell efflux from bone marrow tissue into the circulation increases under any type of stress. Furthermore, non-malignant tumor lesions such as myopathy, adenoma, colon polyps, and pancreatic cysts have been found to cause cells to enter the bloodstream from the affected area.

[0006] Therefore, it is hypothesized that any tissue lesion at any stage can lead to cells entering the circulation, thus forming a rare spectrum of circulating cells. These specific microcellular events, particularly involving malignant, benign, somatic, and stem or progenitor cells in the vast majority of blood cells, are indicative, if not the cause of disease progression, and therefore have significant diagnostic potential.

[0007] Currently, bone marrow is considered a major source of rare cell lines. Under both pathological and physiological conditions, there are driving factors for the repair or maintenance of engraftment of non-hematopoietic and hematopoietic bone marrow-derived cells. Furthermore, the bone marrow itself can be imbalanced, such as inflamed bone marrow tissue, thus triggering uncontrolled cell efflux into the circulation. Therefore, it is necessary to identify bone marrow cell efflux in both intentional processes of cellular immunity and repair systems and in passive, unintentional processes caused by more or less severe damage to the bone marrow. Thus, as discovered by the inventors, systemic cancer evolution is detectable before systemic spread and can be detected at the very beginning of its lesions, proliferative or inflammatory sites.

[0008] Current diagnostic methods for potential cancer patients are quite complex and difficult to master (Schünemann et al., Breast Cancer Screening and Diagnosis: A Synopsis of the European Breast Guidelines, Ann Intern Med, 2020, 172:46-56; Iaccarino and Wiener, Diagnostic Evaluation After Lung Cancer Screening in Real-World Practice: More Questions Than Answers, 2020, Chest 157(2), 247-248). Patients who screen positive must undergo methods confirming malignancy, which are mostly limited to examination of tissue samples at the cellular level by a pathologist and involve exploratory tissue biopsies or cancer surgery. Individual investigations must then be conducted to assess metastasis. Each testing stage typically has limitations and drawbacks. No single diagnostic step can present a complete or accurate picture of the cancer.

[0009] Therefore, there is a need for an effective and accurate diagnostic method that allows for the correct and rapid assessment of tumors and their invasiveness, and the adoption of potential treatment methods. Summary of the Invention

[0010] This invention addresses this need and provides a method for detecting, diagnosing, monitoring, or predicting pathological states in the bone marrow of a subject, the method comprising at least the step of determining the presence of abnormal erythroblasts in a body fluid sample of the subject. The invention is based on the discovery that a population of circulating rare cells of bone marrow origin constitutes a reflection of bone marrow condition. The inventors have surprisingly found that determining the presence of abnormal erythroblasts in a subject's body fluid sample, along with their molecular and phenotypic analysis, can advantageously supplement and thus improve cancer diagnosis. Therefore, the invention is based on the discovery of abnormal native bone marrow cells in the blood circulation of individuals suffering from cancer and other diseases. In the context of known conditions associated with bone marrow damage, the discovery of abnormal erythroblasts of bone marrow origin allows the inventors to achieve the premise that the circulating erythroblast profile reflects, to some extent, the bone marrow state of each individual. Therefore, this observation is translated into an invention of bone marrow analysis without the need for bone marrow aspiration, thereby allowing for highly reliable prediction of bone marrow damage.

[0011] Therefore, in some respects, the present invention is particularly useful for detecting bone metastases. Furthermore, the present invention supports tumor staging based on systemic tumor cell spread by detecting tumor cell invasion in the bone marrow. Thus, the present invention has two diagnostic implications for solid tissue cancers: one is a novel and powerful diagnostic window into tumor invasiveness, and the other is support for characterizing metastases. Therefore, the present invention relates to a potential alternative to bone marrow aspiration, or an equivalent of the diagnostic practice requiring bone marrow aspiration for tissue cancer staging, possessing all its advantages in patient care, sensitivity, and cost. Furthermore, the present invention relates to simplified preclinical and postclinical diagnostic applications for managing acute conditions requiring immediate clinical response or short-term monitoring.

[0012] In a preferred embodiment of the above method, the sample is a venous blood sample or a peripheral blood sample, preferably an inferior vena cava sample or a portal vein sample.

[0013] In another preferred embodiment, the determination includes determining the ploidy of the erythroblast and the potential presence of (i) nuclear budding or lobulation phenotype, (ii) internuclear bridge, (iii) two or more nuclei in a single cell, (iv) megaloblast, (v) macrogonormoblast, (vi) erythroblast with synchronous cytoplasmic division, (vii) erythroblast aggregate containing at least three adherent erythroblasts, and (viii) increased intranuclear ploidy of the erythroblast.

[0014] In another preferred embodiment, the abnormal erythroblasts exhibit increased ploidy, preferably having a binuclear or multinucleate phenotype.

[0015] In another preferred embodiment, the determination further includes determining the presence of at least CD71 and / or GPA in the cells of the subject sample, and optionally one or more of CD44, CD45, VAV1, Kell blood group protein, and nucleic acid.

[0016] In another preferred embodiment of the method according to the invention, abnormal erythroblasts exhibiting increased intranuclear and / or intracellular ploidy, nuclear budding or lobulation phenotype, internuclear bridges, two or more nuclei in a single cell, megaloblastic appearance, or megaloblastic appearance are identified, and CD71 of the erythroblasts in the sample is also identified. + (Positive) and / or GPA + (Positive) and CD45 - (Negative) Biochemical state, indicating bone marrow disorders such as myelodysplastic syndrome / acute myeloid leukemia, lymphoma, essential thrombocytosis, or diabetes.

[0017] Particularly preferably, the determination further includes determining the presence of EpCam and cytokeratin in the cells of the subject sample, and optionally the presence of vimentin.

[0018] In another preferred embodiment, the present invention relates to a method as defined above, wherein the determination further includes determining whether the cells of the subject sample are present in the form of cell clusters.

[0019] In another preferred embodiment, abnormal erythroblasts exhibiting increased intranuclear and intracellular ploidy, nuclear budding or lobulation phenotype, internuclear bridges, two or more nuclei in a single cell, megaloblastic appearance, and megaloblastic appearance were identified, while CD71 of the erythroblasts in the sample was also identified. + (Positive) and / or GPA + (Positive); and CD45 - (Negative) biochemical state, and EpCam identified in the sample. + (Positive) or cytokeratin + (Positive) cells indicate invasive solid tissue cancer.

[0020] In another preferred embodiment, the determination further includes morphological analysis of cells in the sample, preferably performed after May-Grünwald-Giemsa (MGG) staining of the cells.

[0021] Particularly preferably, the cell morphology analysis includes classifying the cells in the sample as:

[0022] -1 class, wherein the sample comprises round or oval cells containing a single nucleus;

[0023] -2, wherein the sample comprises round or oval cells containing at least two nuclei;

[0024] -3 categories, wherein the samples include cell pairs containing constriction; and

[0025] -4, wherein the sample comprises an aggregate of at least three round or oval cells having one or more nuclei.

[0026] In yet another particularly preferred embodiment, class 1 includes further subdividing cells into the following subclasses:

[0027] -1a category, wherein the sample comprises normal erythroblasts with a diameter of about 6.5 μm to about 12.4 μm, having a dense nucleus and a high nucleocytoplasmic ratio;

[0028] -1b category, wherein the sample comprises macrocirculatory erythroblasts with a diameter of about >12.5 μm, having at least one low-density nucleus and low nucleocytoplasmic ratio with a diameter of about 6 to 10 μm;

[0029] -1c class, wherein the samples comprise megaloblasts with asynchronous nucleocytoplasmic and medium to high density chromatin and a high nucleocytoplasmic ratio; and

[0030] -1d class, wherein the sample comprises giant erythroblasts with a diameter of about 4 μm to 6 μm, having a total condensation nuclei without nucleoplasm and a low nucleoplasm ratio.

[0031] In yet another particularly preferred embodiment, the two categories include further subdividing the cells into the following subcategories:

[0032] -2a class, wherein the cells are binucleated;

[0033] -2b class, wherein the cell contains at least 3 nuclei.

[0034] In yet another particularly preferred embodiment, the three categories include further subdividing the cells into the following subcategories:

[0035] Class -3a, wherein the cells do not show nuclear bridging; and

[0036] -3b class, wherein the cells exhibit nuclear bridges, and wherein the nuclei are not equivalent in size, shape, and / or chromatin density.

[0037] In another preferred embodiment, the determination further includes determining the ratio of at least one of the following: (i) class 1a cells to class 1c / 1d cells; (ii) class 1a cells to class 2 cells; and (iii) class 1a cells to class 3 cells, and the ratio of circulating tumor cell (CTC) clusters.

[0038] Particularly preferred, at least one of (i) to (iii) is identified:

[0039] (i) EpCam in the sample + (Positive) and CD45 - (Negative) Circulating tumor cells (CTC) (label 1);

[0040] (ii) CTC cell clusters with a diameter of 6-20 μm are shown in the single CTC cells labeled 1 (label 2);

[0041] (iii) EpCam in the sample - (Negative) and vimentin + (Positive) CTC (marked 3); and additionally

[0042] (iv) The presence of at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells, indicating malignancy of solid tissue carcinoma.

[0043] In another particularly preferred embodiment, it was identified that:

[0044] -Class 1a cells present in the sample at a rate of approximately 10 to 500 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a rate of approximately 1 to 10 cells per milliliter, indicate mild bone marrow damage; or

[0045] -Class 1a cells present in the sample at a level of approximately 1000 to 5000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a level of approximately 10 to 50 cells per milliliter, indicate moderate bone marrow injury; or

[0046] -Class 1a cells present in the sample at a level greater than approximately 10,000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a level greater than approximately 10 cells per milliliter, indicate severe bone marrow injury.

[0047] In another particularly preferred embodiment, the detection of approximately 0.5 to 10 cells of type 2a and type 2b per milliliter of sample indicates moderate bone marrow injury; or the detection of approximately 10 to 50 cells of type 2a and type 2b per milliliter of sample indicates severe bone marrow injury.

[0048] In another particularly preferred embodiment, the identification of about 1 to 10 class 3a cells per milliliter of sample indicates moderate bone marrow injury; or the identification of about 10 to 50 class 3a cells per milliliter of sample and / or the identification of about 1 to 10 class 3b cells per milliliter of sample indicates severe bone marrow injury.

[0049] In another particularly preferred embodiment, the identification of at least one of the four cell types per milliliter of sample indicates severe bone marrow injury.

[0050] In yet another particularly preferred embodiment, mild or moderate bone marrow damage is identified, markers 1, 2 and / or 3 as defined above are identified, and a ratio of 50 or less to 0 as defined above is identified, indicating bone marrow damage, mild residual cancer, or micrometastases associated with invasive solid tissue cancer in the bone marrow that are related to cancer treatment-related non-invasive or dormant cancer.

[0051] In another particularly preferred embodiment, moderate bone marrow damage is identified in the absence of type 2b cells per milliliter of sample, identification of markers 1, 2 and / or 3 as defined above, and identification of a ratio of 50 or less to 0 as defined above, indicating the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow.

[0052] In yet another particularly preferred embodiment, the identification of severe bone marrow damage, the identification of markers 1, 2 and / or 3 as defined above, and the identification of a ratio of 50 or less to 0 as defined above, indicate the presence of active micrometastases associated with invasive solid tissue cancer in the bone marrow.

[0053] In yet another particularly preferred embodiment, in the absence of type 2b cells per milliliter of sample, severe bone marrow injury is identified, markers 1, 2 and / or 3 as defined above are identified, and a ratio of 50 or less to 0 as defined above is identified, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0054] In another aspect, the present invention relates to the use of abnormal erythroblasts as markers for diagnosing, detecting, monitoring or predicting pathological conditions in the bone marrow of a subject.

[0055] In a preferred embodiment, the abnormal erythroblasts are present in the amount defined above.

[0056] In another preferred embodiment, the abnormal erythroblasts are used in conjunction with other markers as defined above. Particularly preferred is the pathological condition being malignant solid tissue cancer; more preferably, it is non-invasive solid tissue cancer in the presence of mild bone marrow damage or in the absence of bone marrow damage.

[0057] In another aspect, the present invention relates to a kit for diagnosing, detecting, monitoring or predicting pathological states in the bone marrow of a subject, comprising tools for determining the presence of the following in a subject sample: (i) CD71 and / or GPA, (ii) CD45, (iii) nucleic acids of rare circulating cells, and (iv) at least one of EpCam or (v) vimentin.

[0058] In a preferred embodiment, the tool used to detect EpCam is anti-EpCam antibody MH99 and / or VU-1D9.

[0059] In a further preferred embodiment, the kit also includes tools for detecting the presence of one, two, three, or all of CD44, CD24, CD133, and CD31. Attached Figure Description

[0060] Figure 1 An overview of cells 1 to 4 according to the present invention is provided.

[0061] Figure 2 A diagram illustrating the diagnostic procedure for determining the invasiveness of solid tissue tumors in the bone marrow is shown.

[0062] Figure 3 A test diagram for mild residual disease is shown.

[0063] Figure 4 Mature erythroblasts or normal erythroblasts are shown, i.e., type 1a cells according to the invention.

[0064] Figure 5 Immature erythroblasts, i.e., type 1b cells according to the invention, are shown.

[0065] Figure 6 A typical megaloblast, i.e., a class 1c cell according to the invention, is shown.

[0066] Figure 7 Giant erythroblasts, i.e., class 1d cells according to the present invention, are shown.

[0067] Figure 8 A binucleated erythroblast, i.e., a type 2a cell according to the invention, is shown.

[0068] Figure 9 Synchronous binuclear cell pairs are shown, namely, type 3a cells according to the invention.

[0069] Figure 10 Cell aggregates of different sizes are described, namely the four types of cells according to the present invention. Detailed Implementation

[0070] Although the invention will be described with reference to specific embodiments, this description should not be construed as limiting.

[0071] Before describing in detail exemplary embodiments of the present invention, important definitions for understanding the present invention are given.

[0072] As used in this specification and the appended claims, the singular forms “a” and “an” also include the corresponding plural forms, unless the context clearly indicates otherwise.

[0073] In the context of this invention, the terms "about" and "approximately" refer to precision intervals that, as will be understood by those skilled in the art, still ensure the technical effect of the features in question. These terms typically indicate a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0074] It should be understood that the term "comprising" is not restrictive. For the purposes of this invention, the terms "consisting of" or "substantially consisting of" are considered preferred embodiments of the term "comprising." If a group is defined below as including at least a certain number of embodiments, this means that it also includes groups that preferably consist only of those embodiments.

[0075] Furthermore, the terms "(i)," "(ii)," "(iii)," or "(a)," "(b)," "(c)," "(d)," or "first," "second," "third," etc., in the specification or claims are used to distinguish similar elements and are not necessarily used to describe a sequence or chronological order. It should be understood that the terms used are interchangeable where appropriate, and the embodiments of the invention described herein can operate in a different order than that described or shown herein. If these terms relate to steps of a method or purpose, there is no temporal or time interval continuity between these steps; that is, these steps can be performed simultaneously, or there can be time intervals of seconds, minutes, hours, days, weeks, etc., between these steps, unless otherwise stated.

[0076] It should be understood that the present invention is not limited to the specific methods, schemes, reagents, etc., described herein, and therefore these can be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077] As described above, in one aspect, the present invention relates to a method for detecting, diagnosing, monitoring, or predicting a pathological state in the bone marrow of a subject, the method comprising at least the step of determining the presence of abnormal erythrocytes in a body fluid sample of the subject.

[0078] As used herein, the term "body fluid sample" refers to any suitable type of body fluid sample. Typically, the sample is derived from the body fluids of a subject, including one or more cells or cell derivatives. Body fluids contemplated by this invention may include, for example, whole blood, fluids of the lymphatic system, saliva, nasal discharge, sputum, ear discharge, genital fluid, milk, colostrum, placental fluid, amniotic fluid, sweat, synovial fluid, ascites, cerebrospinal fluid, bile, gastric juice, aqueous humor, vitreous humor, gastrointestinal fluid, secretions, exudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, liquid feces, fluids collected from sites of immune response, fluids collected from collection sites, bronchoalveolar lavage fluid, or urine. In further embodiments, materials such as biopsy materials, for example, materials from all suitable organs, such as lymph node materials or bone marrow materials, may also be used. For ease of extraction, biopsy materials are typically homogenized and / or resuspended in a suitable buffer solution as described above. Preferably, the sample is a venous blood sample or a peripheral blood sample. Particularly preferred is the sample from the inferior vena cava, pulmonary artery, pulmonary vein, or portal vein. In a specific embodiment, the sample is not a blood smear.

[0079] Blood samples, including those from the vena cava, pulmonary artery, pulmonary vein, and portal vein (excluding peripheral blood), can be collected in any suitable form or manner. Typically, sample collection is invasive and is best performed during or shortly after surgery. Peripheral blood can preferably be collected from the antecubital vein.

[0080] Preferably, all types of bodily fluid samples, such as blood samples, are collected in volumes suitable for subsequent processing, analysis, transport, or storage. In particularly preferred embodiments, volumes of 3 mL to 30 mL are used, more preferably 5 mL to 10 mL or 3 mL to 10 mL. In specific embodiments, bodily fluid samples are retained in volumes of 5 μL to 250 μL, for example, after an enrichment step, or without enrichment. Accordingly, cells may be retained in volumes of 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 50 μL, 75 μL, 100 μL, 125 μL, 150 μL, 175 μL, 200 μL, 250 μL, or any other volume between the foregoing values. The term “enrichment” as used herein refers to any suitable form of cell enrichment procedure, preferably an automated enrichment procedure. The enriched sample can be concentrated, for example, in a suitable solution of 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 50 μL, 75 μL, 100 μL, 125 μL, 150 μL, 175 μL, 200 μL, 250 μL, or any other volume between these values, such as a cell-friendly solution, like phosphate-buffered saline supplemented with 2%–10% fetal bovine serum or 0.5% bovine serum albumin. Further details are available from the examples, such as Example 1 below. More preferably, the sample can be stored in a commonly used container tube. Such containers may contain additional components necessary for sample stabilization as pretreatment components to prevent degradation or disintegration. In some embodiments, particularly in the case of blood samples, the container tube contains EDTA, such as EDTA-K2, EDTA-K3, or EDTA-2Na. More preferably, sodium heparin (Na-Heparin) is used. The components and the sample can be stored at any suitable temperature, such as room temperature. Storage can be performed at any suitable time. Storage is preferably no longer than 24 hours. Furthermore, storage is preferably performed in a place free from light or heat. Particularly preferred is storage in the dark. The storage procedure can maintain cell viability for most cell types for a short period. In cases where storage is extended to several days, such as 4, 5, 6, 7 days or longer, preferably 7 days, body fluid samples, such as blood samples, require cell fixation or the addition of cell membrane stabilizing agents at the time of collection, as is well known to those skilled in the art. A preferred approach is to use a product commercially available called CellSave vacutainers. The commercially available method. In another embodiment, Cell-Free DNA BCT can be used. TM One method for maintaining long-term storage is to use blood collection equipment with formaldehyde-free stabilizing reagents, which can preserve cell-free DNA in blood samples for up to 14 days at room temperature.

[0081] Preferably, the sample contains low or very low levels of abnormal erythroblasts, such as rare cells in an environment with high levels of normal white blood cells. For example, the level of abnormal erythroblasts in the sample is less than 3 × 10⁻⁶ of the total white blood cells. -6 %, 3×10 -5 %, 3×10 -4 0.01%. This very low level of abnormal erythroblasts reflects the etiology of a bone marrow-related pathological condition that is not caused by mutations or genetic defects, which typically affect most or all erythroblasts. Instead, it is due to the indirect influence of interacting components such as pathogens or tumors on erythroblasts.

[0082] As used herein, the terms “subject” or “patient” refer to mammals, specifically humans. In specific embodiments, a subject is a person of post-pubertal age, such as an adult of any age, or a person undergoing puberty, such as a person of any age. In very specific embodiments, a subject is neither a child or adolescent in puberty nor a person who has not yet entered puberty. In further embodiments, a subject may be asymptomatic but troubled by a systemic pathology. Thus, a subject may, for example, exhibit normal erythropoiesis during an attack of the disease, which is negatively affected during the appropriate course of the disease. In specific embodiments, the systemic component of the systemic pathology has a negative impact on erythropoiesis. For example, it can be a pathogen or tumor cell that interacts (e.g., directly interacts) with erythroid lineage cells in the red bone marrow. More information is available from appropriate literature sources, such as Front Immunol., 2016; 7,364. In another specific embodiment, the systemic component of the systemic pathology may have a negative impact on erythropoiesis. For example, it may be a pathogen or tumor cell recognized by erythroid cells. In another specific embodiment, systemic components of a systemic pathology can negatively impact erythropoiesis, such as tumor cells or pathogens that produce inflammatory mediators in the red bone marrow. In another specific embodiment, systemic components of a systemic pathology can negatively impact erythropoiesis, such as tumor cells, bone marrow cells, or pathogens that alter the bone marrow microenvironment, for example, through pH changes, cell crowding, or reactive oxygen species deposition. In another embodiment, systemic components of a systemic pathology can negatively impact erythropoiesis, including blood chemical imbalances that lead to tissue damage within the bone marrow.

[0083] The term "erythroblast" as used in this article refers to the precursors of erythrocytes. These cells typically undergo a cellular maturation process that begins as proerythroblasts, a process expected to occur only in the bone marrow under physiological conditions. Cells at the final stage of erythroblast maturation in the bone marrow are generally called normoblasts and are present at very low levels in the circulation of healthy donors. Immature erythroblasts are less common (see Schreier et al., 2018, Annals of translational medicine, 6, 20). Normal or healthy erythroblast maturation typically involves three morphological aspects: cell size, nuclear chromatin density, and nucleus / cell ratio. Normal, non-abnormal erythroblasts are usually round cells containing a round nucleus at the center throughout the maturation process, which shrinks as they begin maturation from relatively large cells. These large cells can be larger than 20 μm in diameter. During maturation, the diameter typically decreases to about 7 μm or less. As the cell shrinks, the nucleus typically shrinks along with the densification of the chromatin. This shrinkage usually occurs at a different ratio compared to the cytoplasm. Therefore, the nitrogen-to-carbon (N / C) ratio is at its maximum during atrophy. Consequently, normal or healthy erythroblasts may appear in different sizes and nuclear densities, but all exhibit characteristic high cellular and nuclear roundness.

[0084] The term "abnormal erythroblasts" as used in this article refers to one or more phenotypic or molecular features that are significantly different from those of normal or healthy erythroblasts described above. Typical erythroblast abnormalities include mitotic processes found in the blood that are contrary to the erythroblast maturation expected under physiological conditions. Furthermore, erythroblast abnormalities often involve synchronous and asynchronous cell division. This asynchrony in cell division leads to aneuploidy (polyploidy).

[0085] As used in this article, “circulating erythroblasts” refers to erythroblasts present in body fluids, particularly blood or lymph. For example, circulating erythroblasts (cEBs) can be normal or non-abnormal circulating erythroblasts. Therefore, they are designated as class 1a or class 1b erythroblasts. Definitions of these classes are provided below. Alternatively, circulating erythroblasts can take the form of abnormal erythroblasts as defined above. Circulating erythroblasts are typically of bone marrow origin.

[0086] In this article, the term "cellular abnormality" used in the context of erythroblasts or erythroblast development refers to any asymptomatic or symptomatic, latent or overt, chronic or acute condition in the bone marrow where intrinsic or extrinsic cells are affected, which can be detected by circulating erythroblast quantification. Abnormality quantification typically requires assessing the count of each type of circulating erythroblast above a threshold. Therefore, cellular abnormalities are understood as pathological conditions of the bone marrow that can be detected by circulating erythroblast quantification.

[0087] According to the present invention, the presence of non-abnormal circulating erythroblasts in body fluids (e.g., blood or lymph) at concentrations exceeding a specific (particularly as defined herein) threshold, i.e., class 1a or 1b erythroblasts as defined herein, indicates cellular abnormality. The presence of only abnormal circulating erythroblasts in body fluids (e.g., blood or lymph) indicates cellular abnormality. Therefore, when they are found in body fluids (particularly blood), their presence in circulation provides valuable diagnostic information about bone marrow status.

[0088] Cellular abnormalities in bone marrow are known in the prior art, for example, Goasguen et al., 2018, British Journal of Haematology 182.4, 526-533. However, specific abnormalities associated with bodily fluids (e.g., blood) in patients with solid tissue cancer have not been known until now. Contrary to what is expected by those skilled in the art, the inventors have discovered abnormal erythroblasts in the bodily fluids, particularly blood, of patients with solid tissue cancer. This finding strongly suggests a similar situation in the bone marrow. According to the invention, the detection of erythroblast abnormalities in bodily fluids such as blood or lymph can be used diagnostically to detect leukemia, anemia, diabetes, or general bone marrow disorders. The detection of erythroblast abnormalities typically involves determining the cell frequency per milliliter of whole sample volume, such as whole blood, as well as additional phenotypic and genotypic abnormalities of these cells, such as polyploidy.

[0089] Hoping to be free from theoretical constraints, circulating erythroblasts are currently considered a common finding in healthy donors (Schreier et al., 2018, Annals of translational medicine, 6, 20; Fachin et al., 2017, Scientific reports 7, 1, 1-11). Circulating erythroblasts can be found at different stages of maturation, suggesting that this rare cell population of bone marrow origin, particularly circulating erythroblasts, can reflect bone marrow events. The physiological environment of cell efflux is currently considered either accidental or due to intentional processes. Accidental efflux of mature erythroblasts can be explained by their proximity to their circulation in the perivascular niche. In this paper, the clonal or mitotic status of erythroblasts residing in the bone marrow may also lead to local competition for space within erythroblast islands, ultimately resulting in cell efflux at the blood barrier in the bone marrow. However, this does not explain the presence of immature erythroblasts, which are often part of the endosteal niche, suggesting a more active role, such as an intentional process of cell efflux. It is hypothesized that circulating erythroblast efflux is enhanced under pathological conditions. Hoping to be free from theoretical constraints, it is expected that pathological conditions within the bone marrow are also reflected through the status of circulation. Therefore, according to the present invention, for example, erythroblast signals found in the blood may indicate a condition or imbalance in the bone marrow.

[0090] For example, a specific type of imbalance may be caused by disseminated tumor cells (DTCs) that have settled in perivascular niches or deeper, causing uncontrolled or unexplained disturbances in the bone marrow microenvironment, such as localized inflammation. Nesting of disseminated tumor cells in bone and bone marrow may be associated with the specific expression of adhesion membrane molecules that bind them to bone marrow stromal cells and bone matrix, thus making bone marrow an important "breeding ground" for disseminated tumor cells. Furthermore, bone marrow is considered a "training ground" for metastatic circulating tumor cells (CTCs). Therefore, rather than being a site of metastasis itself, it constitutes an important diagnostic target for characterizing the metastatic process. Consequences of nesting may include, for example, impaired peripheral bone marrow blood barrier function and bone marrow cell proliferation. Both of these events can lead to the unintentional outflow of normal and abnormal bone marrow cells, as well as tumor cells, which can then be detected as circulating rare cells in the peripheral blood circulation.

[0091] In the context of this invention, it is assumed that bone marrow-derived circulating erythroblasts play a crucial role in characterizing metastasis, thus allowing the identification of early developmental stages or processes occurring prior to detectable secondary tumor growth. A typical scenario envisioned in this invention is the presence of micrometastases, which, when overlooked, lead to incorrect staging and significantly increase the risk of ineffective treatment. Micrometastases are anticipated to be a result, consequence, or coincidence of bone marrow infiltration.

[0092] Given the research on genetic instability, it is believed that the characterization process can be traced not only to micrometastases or precancerous lesions, but also to precancerous lesions. Based on the teachings of this invention, tumor identification and characterization should follow different analytical pathways, preferably prior to treatment, including genetic analysis of certain driver mutations that best indicate the likelihood of invasion and growth at distant sites in the body. In contrast to methods predicting invasiveness, the currently envisioned metastatic characterization diagnostic attempt is part of the characterization of individual tumor evolution and relies on the assessment of the invasive status at certain time points and locations. The most critical location is the bone marrow.

[0093] Tumor cell dissemination is considered an early event, most likely occurring as early as the pre-malignant stage. Therefore, the tumor must release surviving tumor cells into the circulation at the earliest possible time of circulating tumor cell (CTC) production. Importantly, detecting CTCs may not allow for a diagnostic statement of tumor invasiveness, but only of the presence of tumor lesions. On the other hand, semi-disseminated tumor cells are clear evidence of invasion and colonization found in sites outside the primary tumor. As taught in this invention, identifying disseminated tumor cells through cell-based liquid biopsy of the peripheral blood system requires indirect evidence of bone marrow infiltration. This can therefore be achieved by identifying distinct classes of circulating rare cells definitively identified as originating from the bone marrow. According to the teachings of this invention, these cells, namely circulating tumor cells, indicate bone marrow imbalance.

[0094] As used herein, the term "pathological state in the bone marrow of a subject" refers to a disease state or pre-disease state in the bone marrow tissue of a subject. For example, such a disease state or pre-disease state can be a bone marrow-related condition leading to leukemia and anemia, or it can be bone marrow injury or induced by bone marrow injury, which can be, for example, mild, moderate, or severe bone marrow injury. A disease state or pre-disease state can also be associated with an existing or developing tumor or vegetation or its precursor form. In a very specific embodiment, the pathological state in the bone marrow of a subject is not related to the diseases described for non-human animals, particularly mice. In a further very specific embodiment, the pathological state can be a disease state or pre-disease state in the bone marrow tissue of a subject other than congenital erythropoietic anemia in a subject of pre-pubertal or adolescent age. In a further very specific embodiment, the pathological state can be a disease state or pre-disease state in the bone marrow tissue of a subject other than mutations in the genes KLF1, CDAN1, SEC23B, or GATA-1 (e.g., the E325K mutation in the KLF1 gene and / or mutations caused by said mutations). Further information is available from appropriate literature sources, such as Haematologica, 2012; 97(12):1786–1794. In a specific embodiment, the pathological condition of the subject's bone marrow may be malignant solid tissue cancer. Examples of such malignant solid tissue cancers include lung cancer, breast cancer, and colon cancer. Alternatively, it may be a tumor or a tumor precursor in the form of metastatic or tumor-derived cell aggregates. In a further embodiment, the disease may be myelodysplastic syndrome, acute or chronic myeloid leukemia, acute or chronic lymphocytic leukemia, lymphoma involving the bone marrow, essential thrombocythemia, or diabetes, or non-neoplastic acquired anemia, including anemia caused by iron deficiency, arsenic poisoning, and vitamin B12 deficiency.

[0095] In a specific embodiment, the pathological state of the subject's bone marrow is bone marrow damage caused by metastasis or bone marrow damage caused by invasive tumors.

[0096] Particularly preferably, the pathological condition of the subject's bone marrow is not due to genetic abnormalities of red blood cells or erythroblasts, such as due to gene defects or mutations in red blood cells, erythroblasts, hematopoietic-related cells, or corresponding stem cells, for example, one of the aforementioned gene defects. More preferably, the pathological condition of the subject's bone marrow is associated with systemic pathology, particularly systemic components of systemic pathology (such as pathogens or tumor cells) that negatively affect erythropoiesis.

[0097] An important aspect of a subject's bone marrow pathology is bone marrow injury. Typically, bone marrow injury is caused by bone marrow disorders such as leukemia or anemia. Additionally, bone marrow injury can occur during systemic inflammation, which can include diseases such as diabetes (see Fadini et al., 2014, 3, 8, 949-957), cancer and lymphoma, or diseases related to radiation-induced or cytotoxic drug-induced bone marrow injury acquired through cancer treatment.

[0098] The term "mild bone marrow injury" as used herein refers to a clinical condition in which the subject feels asymptomatic and healthy (subjective perception) and may or may not exhibit abnormal blood results common in the art. This category can include both static conditions and dynamic developments in terms of lifestyle or manifested pathology. In terms of disease, mild impairment typically indicates the onset or early stage of disease. For example, mild bone marrow injury may be associated with solid tissue cancer, where mild impairment may represent dormant tumor cells or slight residual disease present in the bone marrow in either developmental mode; the impairment may reverse or progress. In terms of lifestyle, mild impairment is often irreversible with lifestyle changes. Mild bone marrow injury may result in a physiological outflow of erythroblasts, which may resemble the outflow of other cells derived from stem cells or progenitor cells that are bone marrow-based. Even a significant outflow of erythroblasts is not necessarily associated with physical damage to the vascular niche and therefore suggests normal physiology. Therefore, in accordance with the teachings of this application, cellular abnormalities associated with mild bone marrow injury are associated with dysregulated cell maturation.

[0099] The term "moderate bone marrow injury" as used in this article refers to an existing pathological condition, such as in the early stages of disease, that indicates or predicts an increase in severity, i.e., leading to severe bone marrow injury, or predicts a continued disease evolution, i.e., from local to systemic disease. This is equivalent to moderate bone marrow injury. It can also predict the occurrence of secondary complications such as diabetic neuropathy. Furthermore, dysregulation of erythroblasts, cellular processes that indicate chemical damage such as inflammatory processes, may be associated with moderate bone marrow injury. As described below, these processes may or may not occur simultaneously with physical destruction of the bone marrow and bone matrix. The grading of "moderate bone marrow injury" typically depends on the number of abnormal erythroblasts identified.

[0100] The term "severe bone marrow injury" as used herein refers to an individual's pathological state, typically at an advanced stage of disease. In the case of cancer, this category supports the possibility of bone metastasis. Based on the teachings of this application, it is assumed that bone tumors are typically induced by one or more disseminated tumor cells in the bone marrow, particularly hematopoietic niche-resident tumor cells. These cells can cause dysregulation of osteoblast and osteoclast maturation, leading to osteosclerosis and osteolysis, which interact in bone matrix destruction and tumor cell growth, respectively. Physical damage to the microvessels within the bone marrow can correspondingly result in the presence of specific large aggregates of circulating erythroblasts, i.e., the four classes of erythroblasts defined herein. Furthermore, the cytokines involved are shown to influence erythroblast maturation, resulting in mitotic cells found in circulation (in body fluids, particularly blood), i.e., identified as classes 2 and 3 of erythroblasts as defined herein. Therefore, the presence of class 4 erythroblasts in body fluids (such as blood) is considered a result of physical destruction of the bone marrow, thereby indicating severe bone marrow damage.

[0101] As described in this article, bone marrow damage leads to the presence of cellular abnormalities and / or can be detected by the presence of cellular abnormalities, particularly erythroblast abnormalities, especially in the context of leukemia and anemia.

[0102] Another example of erythroblast abnormalities being associated with disease is diabetes and the presence of solid tissue cancer. Hoping not to be bound by theory, it is hypothesized that several genetic or epigenetic mutations may be the cause of a typical phenotype.

[0103] Another example is myelodysplastic syndrome with erythroid dysplasia, which typically includes nuclear alterations such as abnormal chromatin aggregation, binucleation / multinucleation, or nuclear bridging. In this syndrome, significant defects in histone release from EB may be found due to deficiencies in the caspase 3 process, which are thought to inhibit chromatin condensation and terminal differentiation required for EB maturation, leading to the pathogenesis of megaloblastic alterations in erythroblastogenesis disorders (see Baobing et al., 2019, Cancer Medicine, 8.3, 1169-117). In another example, a genetic factor for CDAII has been identified: a specific mutation in the SEC23B gene, attributed to causing multinucleation (Pellegrin et al., 2019 British Journal of Haematology, 184.5, 876).

[0104] The term “detection of pathological state in the bone marrow of a subject” as used herein refers to the ability to determine the presence of a pathological state in the bone marrow of a subject as defined above, preferably a bone marrow-related condition associated with leukemia or anemia, or bone marrow damage, or a developing or existing tumor or growth, such as malignant solid tissue cancer, or to identify such a disease or condition in the subject.

[0105] The determination or identification of the pathological state in the bone marrow of a subject as defined above can be achieved by comparing the phenotypic or genotypic markers related to the pathological state described in this invention with normal or healthy controls.

[0106] A pathological condition in the bone marrow of a subject, as defined above, can be detected when at least one, preferably more than one, phenotypic or genotypic marker is present or significantly increased compared to a normal / healthy control or level as defined herein. In a preferred embodiment, such a control or level as defined herein is obtained from or is representative of a healthy subject who has never been troubled by a pathological condition in their bone marrow as defined herein.

[0107] In another preferred embodiment of the invention, if the level of a phenotypic or genotypic marker is similar to a control or level as defined herein, such as a control or level derived from the bone marrow of a subject who has been independently diagnosed with a pathological condition in their bone marrow, then the pathological condition in the subject's bone marrow can be detected.

[0108] In a preferred embodiment, the detection of pathological conditions in the subject's bone marrow involves detecting abnormal erythroblasts in the subject's body fluids (preferably blood samples).

[0109] As used herein, the term "diagnostic pathological state in the bone marrow of a subject" refers to a condition or disease that a subject may believe leads to the pathological state as defined herein. Such conditions may include bone marrow-related conditions associated with leukemia or anemia, or tumors or growths, such as malignant solid tissue cancer.

[0110] The diagnosis of a pathological condition in the bone marrow of a subject, as defined above, can be achieved by comparing phenotypic or genotypic markers associated with the pathological condition described in this invention with normal or healthy controls. In specific embodiments, the diagnosis may include additional steps, such as independent histological, biochemical, or genetic examinations. Further envisioned is an AI-based comparison method based on available training data from healthy and independently diagnosed pathological subjects.

[0111] A pathological condition in the bone marrow of a subject, as defined herein, can be diagnosed when at least one, preferably more than one, phenotypic or genotypic marker is present or significantly increased compared to a normal / healthy control or level as defined herein. In a preferred embodiment, such a control or level as defined herein is obtained from or represents a healthy subject who is not considered to have a condition or disease that would lead to the pathological condition as defined herein.

[0112] The term "diagnosis" also refers to the conclusions drawn through the above-mentioned comparative process, particularly the specific process described below.

[0113] As used herein, the term "monitoring pathological status in the bone marrow of a subject" refers to a pathological status accompanying a diagnosis or test in the bone marrow of a subject, such as during treatment or over a specific time period, typically 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other time period. The term "accompanying" refers to a disease state as defined herein, and in particular, changes in these disease states can be detected by comparing a sample with a phenotypic or genotypic marker associated with the pathological state as described herein with a normal or healthy control as defined herein. In another embodiment, an established (e.g., independently established) pathological bone marrow or a cell line derived therefrom may be used as a positive control. This accompaniment can occur in any type of periodic time period, such as weekly, every 2 weeks, monthly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, every 1.5 years, every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years, during any time period, such as in 2 weeks, 3 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months; 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years.

[0114] As used herein, the term "predicting the pathological state in a subject's bone marrow" refers to the prediction of the course or outcome of a diagnosed or detected bone marrow-related condition, as defined herein, associated with leukemia or anemia, bone marrow damage, or developing or existing tumors or growths (e.g., malignant solid tissue cancer), over a given period, during or after treatment. The term also refers to determining the chance of survival or recovery from the disease, and predicting the subject's expected survival time. Specifically, prognosis may involve determining the subject's likelihood of survival over a future period of time (e.g., 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other timeframe).

[0115] Determining the presence of abnormal erythroblasts in a subject's bodily fluids (e.g., blood) may include any appropriate activity or procedure known to those skilled in the art or available from suitable literature sources, such as Fachin et al., 2017, Scientific Reports 7, 1, 1-11; and Schreier et al., 2018, Annals of Translational Medicine, 6, 20. In a preferred embodiment, the determination includes at least determining the ploidy of the erythroblasts. As known to those skilled in the art, the term "ploidy" refers to the number of complete sets of chromosomes in a cell. Mammalian cells typically have a diploid level, i.e., having 2 sets of chromosomes. The ploidy level is also diploid for normal or healthy erythroblasts. In abnormal erythroblasts, the ploidy level may differ from 2, for example, >2, such as 4, 6, or more. The ploidy level may be reflected in a single cell nucleus (with more chromosomes), or more than one nucleus per cell, or both, i.e., increased ploidy within the cell nucleus and more than one nucleus per cell.

[0116] In a very specific embodiment, the determination of the presence of abnormal erythroblasts is performed solely in the subject's bodily fluids. Therefore, in this very specific embodiment, the determination of the presence of abnormal erythroblasts in other tissues, such as bone marrow, is excluded. In another very specific embodiment, the combination of analytical steps disclosed in Jaffray et al., 2013, Blood Cells, Molecules and Diseases, 51, 71-75 is excluded.

[0117] Methods for determining erythroblast ploidy are known to those skilled in the art or are available from suitable literature sources, such as Zhang et al., 2020, Cancer Letters, 469, 355-366. Fluorescence detection is preferred, and fluorescence in situ hybridization (FISH) is more commonly used to determine erythroblast ploidy in body fluid samples. Further details are available from suitable literature sources, such as Ehtisham et al., 2016, International Research Journal of Clinical Medicine, 1, 4, 23-29.

[0118] Determining the ploidy of erythroblasts in body fluids may additionally include determining the presence of one or more phenotypic states of erythroblasts. These phenotypes can be detected or observed when erythroblasts are analyzed following nuclear staining and subsequent microscopic examination. Preferred staining methods use dyes such as NucSpot, RedDot, DMAO, SYBR Green, thiazolium green, thiazolium orange, DAPI, acridine orange, p-phenylenediamine, DRAQ5, ethidium homodimer I or III, or Hoechst 33342. Microscopic examination is preferably performed using a UV microscope or a UV-VIS microscope. Further preferred is the use of an automated imaging system to determine the ploidy of erythroblasts, which allows for automated image analysis, such as as defined herein.

[0119] Among these phenotypic states are nuclear budding or lobulation phenotypes. In this phenotype, erythroblasts display protrusions or vegetations within the nucleus. The nucleus is usually present in a condensed state.

[0120] Another example of an abnormal erythrocytic phenotype is the presence of internuclear bridges. In this phenotype, the cell contains at least two nuclei connected by DNA containing structures in the form of bridges, also known as chromatin bridges. These are preferably determined at 400x magnification or higher, or at a resolution of approximately 6.7 μm per pixel or lower. Further details are available from appropriate literature sources, such as Bethlenfalvay et al., 1986, American Journal of Hematology, 21.3, 315-322.

[0121] Another example of an abnormal erythrocyte phenotype is the presence of two or more nuclei in a single cell. For example, a single cell may contain two, three, four, five, or more nuclei. These nuclei may be completely separated, or they may appear in anaphase and telophase of divisions, similar to mitosis. As mentioned above, the two or more nuclei may be connected by internuclear bridges.

[0122] Another example of an abnormal erythroblastic phenotype is the presence of megaloblasts. The term "megaloblast" as used herein refers to erythroblasts with a large nucleus relative to their size. Megaloblasts are typically 50–150% larger or 1.5–2.5 times larger than normal erythrocytes. The occurrence of megaloblasts is thought to be due to impaired DNA replication, delaying nuclear maturation and cell division. Megaloblasts often exhibit dissociation between nuclear and cytoplasmic maturation.

[0123] According to the invention, another example of an abnormal erythroblastic phenotypic state is the presence of giant erythroblasts. As used herein, the term "giant erythroblast" refers to an abnormally large erythroblast that can be as large as or larger than a megaloblast. It typically contains a very small nucleus, exhibiting total chromatin condensation. Furthermore, the cell maturation of this cell type is asynchronous and dissociative. Compared to megaloblasts, giant erythroblasts do not exhibit the dissociation between nuclear and cytoplasmic maturation.

[0124] According to the invention, another example of an abnormal erythroblast phenotypic state is erythroblasts undergoing synchronous cytokinesis. These erythroblasts are groups of three erythroblasts that are at the same stage of cytokinesis. Erythroblasts undergoing synchronous cytokinesis are typically the three cell types defined herein, which may or may not be bridged. They consist of two pairs of cells that are equal or unequal in size in terms of cell diameter and nuclear area. These pairs may be clearly separated or appear as a single entity, suggesting different states of cytokinesis. The nuclei are always locally separated, indicating the end of telophase.

[0125] Examples of abnormal erythroblast phenotypes were also envisioned, in which erythroblast aggregates appeared. These aggregates typically contained at least three, such as three, four, five, six, seven, nine, ten or more, fused together.

[0126] Another example of an abnormal erythroblast phenotype is increased ploidy within the erythroblast nucleus. Therefore, the nucleus of an erythroblast may contain 4, 6, 8, or more sets of chromosomes, i.e., a ploidy level of 4, 6, 8, or more. In specific embodiments, such as those associated with leukemia, erythroblasts may also contain 3, 5, 7, 9, or more sets of chromosomes, i.e., a ploidy level of 3, 5, 7, 9, or more. The nucleus of such erythroblasts may have an increased size and / or a higher density.

[0127] In a preferred embodiment of the invention, the aberrant erythroblasts exhibit increased ploidy, for example, ploidy levels of 4, 6, 8, or more. In a particularly preferred embodiment, the aberrant erythroblasts exhibit a binuclear or multinucleated phenotype. As used herein, a “binuclear phenotype” refers to the presence of two nuclei per cell. As described above, these nuclei can be isolated or connected by internuclear bridges. A “multinucleated phenotype” refers to the presence of two or more nuclei per cell, for example, 3, 4, 5, 6, 7, 8, or more. As described above, these nuclei can be isolated, or (at least some) can be connected by internuclear bridges.

[0128] In another embodiment, an acidified serum test is performed using bodily fluid samples, particularly blood samples, to detect, diagnose, monitor, or predict pathological conditions in the subject's bone marrow. This test, also known as the Ham test, involves placing red blood cells in a weak acid. This test primarily examines whether red blood cells become more fragile when placed in a weak acid. The test is typically based on visual observation and can distinguish between positive and negative results.

[0129] If the acidified serum test as described above shows erythrocyte lysis in the blood sample, and if abnormal erythroblasts exhibiting ploidy as described above are identified in the sample, it can be inferred that the subject from whom the sample originated is troubled by a pathological condition in their bone marrow. Specifically, the subject is believed to be troubled by a bone marrow-related disorder of hereditary erythroblastic polynucleosis (congenital erythropoiesis-type II).

[0130] In another set of embodiments, the invention envisions identifying one or more erythroblast-related biochemical or molecular markers. Thus, the biochemical state of one or more of these markers can be determined. As used herein, the term "biochemical state" refers to the presence or absence of a marker (e.g., a protein or a derivative thereof), and / or the amount or concentration of said protein. For a "positive" biochemical state, the protein needs to be detected above a threshold level known to those skilled in the art using suitable, preferably standardized methods, such as those available from suitable literature or internet sources. More preferably, the relevant information, along with calibration tests, etc., can be provided by the manufacturer of the kit for detecting the corresponding biomarker. Those skilled in the art can adjust conclusions about the presence of biomarkers to the specific parameters of the test employed.

[0131] Similarly, for a "negative" biochemical state, the protein must be undetectable using suitable, preferably standardized methods known to those skilled in the art, or the detected protein must be below a threshold level known to those skilled in the art, for example, obtainable from suitable literature or internet sources. More preferably, the relevant information, along with calibration tests, can be provided by the manufacturer of the kit used to detect the corresponding biomarker. For example, a so-called isotype control assay can be performed. This assay is typically necessary in fluorescence flow cytometry and can detect the non-specific binding level of the antibody isotype. This level is typically interpreted as the background noise level. Those skilled in the art can adjust the conclusion regarding the absence of the biomarker to specific parameters of the test used, or to the background noise level as defined above.

[0132] In some embodiments, it is envisioned that, in order to detect, diagnose, monitor, or predict pathological conditions in a subject's bone marrow, the presence of the marker CD71 in cells, particularly erythroblasts, is determined.

[0133] CD71 (also known as transferrin receptor protein 1 (TfR1)) is considered essential for the transfer of iron from transferrin into cells via endocytosis. This protein is a transmembrane glycoprotein composed of two monomers linked by disulfide bonds. It is considered a precursor marker for erythroid cells. Hoping not to be bound by theory, it is assumed that CD71 is selectively and ubiquitously expressed at high levels in erythroid precursors at all stages of maturation, including in normal and dysplastic bone marrow (see Dong et al., 2011. Am JSurg Path, 35(5), 723-732). Further information can be found in Schreier et al., 2018. Annals of Translational Medicine, 6, 20. For the detection of CD71, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include OKT9 and H68.4.

[0134] In some embodiments, it is envisioned that the presence of GPA markers on cells, particularly erythroblasts, be determined in order to detect, diagnose, monitor, or predict pathological conditions in the bone marrow of a subject. GPA may be measured together with CD71, or in other embodiments, in lieu of CD71 measurement.

[0135] GPA (also known as glycoprotein A or CD235a1) is a sialic acid glycoprotein of 151 amino acids, expressed only on the cell membrane of erythroid cells at approximately 500,000 copies per cell from the early stages of erythroid cell formation until the formation of mature erythrocytes. The glycoprotein gene is located on chromosome 4 and has two allele forms: M and N, differing by two amino acids. The M allele has Ser1 and Gly5, while the N allele has Leu1 and Glu5. This marker does not distinguish between primitive and mature cells in erythroid cell lines and is therefore used as an auxiliary marker to confirm the nature of the positively identified cells under discussion (see also Grant et al., 1974, Proceedings of the National Academy of Sciences, 71.12, 4653-4657). CD235a is typically detected by monoclonal antibody ligand. Detecting antibodies can show both the M and N alleles simultaneously. In the tested applications, the relevant clones (e.g., 10F7MN) were pre-titrated and tested using flow cytometry analysis of normal human blood cells. The dose used per test was 5 μL (0.25 μg). The test was defined as the amount of antibody (μg) stained in a final volume of 100 μL of cell sample. The cell number should be determined empirically, but can be up to 10. 5 Up to 10 8 Within the range of cells / test. In this method, flow cytometry is not suitable for detecting rare events, therefore analysis by microscopy is required.

[0136] In further embodiments, the presence of additional biochemical or molecular markers on cells, particularly erythroblasts, is envisioned. These additional markers include CD44, CD45, CD24, CD133, CD31, VAV1, and Kell blood group protein. These markers can be measured individually along with CD71 and / or GPA, for example, combinations of CD71 and / or GPA plus CD44, CD71 and / or GPA plus CD45, CD71 and / or GPA plus VAV1, etc., or they can be measured as different groups combined with CD71 and / or GPA, such as CD71 and / or GPA plus CD44 and CD45 and VAV1; or CD71 and / or GPA plus CD45 and VAV1 and Kell blood group protein; or CD71 and / or GPA plus CD44 and CD45 and VAV1; or CD71 and / or GPA plus CD44 and CD45 and VAV1 and Kell blood group protein, etc. The group may include any 2, 3 or 4 items from the marker groups CD44, CD45, CD24, CD133, CD31, VAV1, and Kell blood group protein.

[0137] CD44 is a receptor for hyaluronic acid and can interact with various ligands such as osteopontin, collagen, or MMPs. It is a cell surface glycoprotein involved in intercellular interactions, cell adhesion, and migration. Due to splicing variants, this protein exists in several isoforms. It can also contain different sugar residues. One variant is the sialylated form, called the HCELL glycoform, which is considered a ligand for lectins. Preferably, phagocytic glycoprotein type 1 of CD44 is used as a biochemical marker, for example, by a suitable monoclonal antibody. CD44 expression is considered to be maturity-dependent, indicating high expression in early stages and low expression in later stages. More information is available from appropriate literature sources, such as Chen et al., 2009, Proceedings of the National Academy of Sciences 106.41, 17413-17418. For the detection of CD44, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include IM7 and Hermes-1.

[0138] CD45 is a receptor-type protein tyrosine phosphatase, a transmembrane protein present on all differentiated hematopoietic cells except erythrocytes and plasma cells, and has several isoforms. CD45 comprises an extracellular domain, a transmembrane segment, and two tandemly linked intracytoplasmic catalytic domains, thus belonging to the receptor-type PTP family. It is a regulator of T-cell and B-cell antigen receptor signaling. This invention envisions recognizing at least five isoforms of the currently known CD45 antigen: RA, RO, RB, RAB, RBC, and RABC. Monoclonal antibodies reactive to all isoforms are preferred. Preferred examples of such antibodies are anti-human MEM-28 or HI30. These antibodies can be mixed and then react with CD45 bound to the cell membrane of leukocytes. Therefore, CD45 detection using antibodies as described above, i.e., CD45 staining, can be used as a counterstain with a positive identification marker such as CD71 to exclude leukocyte characteristics of the target cells. More information can be obtained from appropriate sources, such as Schreier et al., 2017, Journal of Translational Medicine 15.1(7),6.

[0139] CD24 is a double-chain glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed at multiple stages of B cell development, from the pre-B cell compartment of the bone marrow to mature surface Ig-positive B cells. Plasma cell expression is usually low or negative. CD24 is also expressed in most B-cell acute lymphoblastic leukemias, B-cell CCL, and B-cell non-Hodgkin lymphoma. CD24 may play a role in regulating B cell proliferation, maturation, and controlling autoimmunity. The expression pattern of human CD24 is similar to that of its homologous protein, the mouse heat-stable antigen (HAS). HAS is a mouse glycoprotein composed of 31 amino acids, which are attached to the cell membrane by immobilized glycosylphosphatidylinositol (GPI). Of these amino acids, 16 are Ser, Thr, and ASn residues, which can be O-glycosylated and N-glycosylated (see Kay al., 1991, J Immunol, 147:1412-1416). Potential O-glycosylation sites are primarily located in the N- and C-terminal regions of CD24. Therefore, CD24 is expected to have a dumbbell-shaped shape. CD24 glycosylation is thought to be cell type dependent. Glycosylated CD24 has a wide molecular weight range of 35 kDa–70 kDa (see Kristiansen et al., 2004, J Mol Histology 35:255–262). Unlike mouse HAS, human CD24 is typically not expressed in erythrocytes and thymocytes and is found only in early B lymphocytes (Kay et al., 1991, J Immunol, 147:1412–1416). Human CD24 has been used as a marker for early B cells. CD24 has also been used as a marker for epidermal cells of the kidneys and brain during developmental stages. CD24 knockout mice show no functional defects other than B lymphocyte development (see Nielsen et al., 1997, Blood 89:1245-1258; Shirasawa et al., 1993, Dev Dyn 198:1-13), indicating that CD24 is involved in the proliferation and maturation of pre-B lymphocytes. For the detection of CD24, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include clones ML5 and M1 / 69.

[0140] CD133, also known as Prominin-1, is a glycoprotein known to be expressed by immature hematopoietic stem cells rather than mature blood cells. CD133 is particularly expressed in hematopoietic stem cells, endothelial progenitor cells, and neural stem cells. CD133 has been found present or enriched in cell populations of several human solid tumors, such as colon cancer, melanoma, and brain tumors (e.g., glioblastoma). CD133 is a probable marker of cancer stem cells, meaning cancer cells possess stem cell-like characteristics, such as the ability to differentiate into multiple cell types and generate new tumors. Furthermore, CD133 protein has been found to be located at membrane protrusions and is typically expressed on adult stem cells. One proposed function of CD133 is to maintain "stemness" by inhibiting differentiation. The presence or absence of CD133 can often distinguish between endothelial progenitor cells and mature endothelial cells. For the detection of CD133, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include 13A4 and ab19898.

[0141] CD31, or PECAM-1 (platelet endothelial cell adhesion molecule-1), is an inhibitory co-receptor that regulates T-cell and B-cell signaling through a tyrosine-based dual immunoreceptor inhibitory motif (ITIM). ITIM, upon phosphorylation mediated by related kinases, provides docking sites for protein tyrosine phosphatases. CD31 is widely expressed within the vascular lumen, primarily located at junctions between adjacent cells. CD31 is a multifunctional molecule playing various roles in regulating integrin-mediated cell adhesion, transendothelial migration, angiogenesis, apoptosis, negative regulation of immune receptor signaling, autoimmunity, macrophage phagocytosis, IgE-mediated allergic reactions, and thrombosis. It is one of the key regulatory molecules in the vascular system. CD31 has also been found on endothelial cells and neutrophils and has been shown to participate in leukocyte migration across the endothelium. CD31 consists of a single-chain molecule containing six Ig-like extracellular domains, a short transmembrane fragment, and a cytoplasmic tail containing two ITIMs. The structure of CD31 is specifically and constitutively expressed on cells at the vascular interface. CD31 is also associated with inflammatory processes, and anti-CD31 monoclonal antibodies have been reported to block neutrophil recruitment in vivo (see Nakada et al., 2000, J. Immunol., 164:452-462). In some embodiments, it is envisioned that the presence of CD31 markers on cells be determined to detect, diagnose, monitor, or predict a subject's pathological condition. In specific embodiments, its presence is detected, diagnosed, monitored, or predicted on endothelial cells to provide evidence of vascular injury. Furthermore, the polyploidy and nuclear heterogeneity of such cells are associated with tumor growth (more information can be found, e.g., Hida et al., 2004, Cancer research, 64, 22:8249-8255). For the detection of CD31, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include WM59 and HEC7.

[0142] VAV1 is a proto-oncogene of the Dbl family of guanine nucleotide exchange factors, belonging to the Rho family of GTP-binding proteins. VAV1 is believed to have hematopoietic functions and may play a role in the development and activation of B cells and T cells. Further details are available from appropriate literature sources such as Bustelo et al., 1993, Cell Growth & Differentiation, 4(4), 297–308, or Fray et al., 2020, Journal of Cell Science. For the detection of VAV1, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include 2E11 and ZV003.

[0143] Kell blood group protein, also known as CD238, is a 93 kDa transmembrane zinc-dependent endopeptidase believed to be involved in the cleavage of Enthelin-3. This protein is a type II transmembrane glycoprotein. The encoding gene contains several alleles, resulting in a highly polymorphic Kell blood group antigen set (antigens are defined as peptides contained in Kell blood group protein). Kell blood group protein can be detected using antibodies, preferably conjugated antibodies such as A-10, which can be conjugated to phycoerythrin or FITC, Alexa Fluor 680, or 790. Further details are available from appropriate literature sources, such as Chen et al., 2009, Proceedings of the National Academy of Sciences, 106.41(9):17413-17418. For the detection of CD238, any suitable antibody or other binding entity can be used. Monoclonal antibodies are preferred. Examples of such antibodies include OTI8E1, ETI5E6, and BRIC203.

[0144] In a further embodiment, the presence of nucleic acids in the cells of the subject sample is preferably determined by methods as defined above, such as staining cells with a suitable DNA dye as described above. In a specific embodiment, the presence of nucleic acids in the cells of the subject sample is determined in addition to CD71 and / or GPA, or optionally in addition to one or more of CD44, CD45, CD24, CD133, CD31, VAV1, and Kell blood group proteins.

[0145] In specific embodiments, biochemical markers, such as erythroblasts or circulating tumor cells (CTCs), can be found on cells, and their combinations and / or amounts can be obtained from Table 1 below. Therefore, in a particularly preferred embodiment, detection of markers and marker combinations associated with the indicated amounts can clearly identify the cell types described in the left column. Further details can be obtained from the embodiments, which describe the tests and experiments contemplated by the present invention as general methods.

[0146] Table 1: The presence of specific cell markers according to the present invention

[0147]

[0148]

[0149] *dim: Slightly higher than the background signal

[0150] **Low:** Detectable, but still at a very low level.

[0151] ***High: Significantly higher than the average level

[0152] ****EMT: Transformation of epithelial cells into mesenchymal cells

[0153] In a preferred embodiment, the presence of one or more phenotypic markers as defined above, combined with one or more biochemical markers as defined above (e.g., in Table 1), can advantageously be used to identify cells in a subject's sample and thereby identify pathological conditions in the subject's bone marrow that lead to a specific bone marrow-related condition. For example, identifying abnormal erythroblasts with increased nuclear and intracellular ploidy, while simultaneously identifying CD71 of the erythroblasts in the sample. + (Positive) and / or GPA + (Positive); and CD45 - A (negative) biochemical status can be considered an indication of bone marrow disorders, such as myelodysplastic syndrome / acute myeloid leukemia, lymphoma, essential thrombocythemia, or diabetes. In a further preferred embodiment, abnormal erythroblasts exhibiting increased nuclear and intracellular ploidy may additionally display nuclear budding and / or lobulation phenotypes and / or internuclear bridges and / or two or more nuclei in a single cell and / or (i) megaloblastic appearance or (ii) megaloblastic appearance. In a further specific embodiment, in addition to CD71+ (positive) and / or GPA+ (positive); and CD45 - In addition to (negative) biochemical status, abnormal erythroblasts may also show CD44. + (Positive), VAV1 + (Positive), Kell blood type protein + (Positive) Biochemical status. For the diagnostic relevance of certain cell types, some biochemical markers are redundant or may have overlapping diagnostic relevance. In such cases (as can be deduced from the general description herein), they may or may not be used for the detection of erythroblasts or CTCs. For example, the use of the biomarkers Kell blood group protein or VAV1 is considered optional and can be used as an overlapping diagnostic tool. The decision to use such a marker can be made based on the diagnostic setup, availability of the testing reagents, the specific instrument used, the choice of staining method, etc. The corresponding parameters and thresholds are known to a technician.

[0154] In further embodiments, it is envisioned that an additional set of biochemical or molecular markers may be present. These additional markers include EpCam and cytokeratin. These markers may be determined individually with CD71 and / or GPA as described above, or with any of the CD44, CD45, CD24, CD133, CD31, VAV1, and Kell blood group proteins, or they may be determined as different sets of combinations with CD71 and / or GPA, CD44, CD45, CD24, CD133, CD31, VAV1, and Kell blood group proteins, such as EpCam plus CD71 and / or GPA, or cytokeratin plus CD71 and / or GPA; or EpCam plus CD71 and / or GPA plus the marker group CD44. 4. Any 1, 2, 3, or 4 of the following blood group proteins: CD45, VAV1, and Kell; or any 1, 2, 3, or 4 of the following blood group proteins: cytokeratin plus CD71 and / or GPA plus a labeling group: CD44, CD45, CD24, CD133, CD31, VAV1, and Kell; or EpCam plus cytokeratin plus CD71 and / or GPA; or EpCam plus cytokeratin plus CD71 and / or GPA plus a labeling group: CD44, CD45, CD24, CD133, CD31, VAV1, and Kell. A particularly preferred combination is CD71 and / or GPA plus EpCam plus CD45.

[0155] In some embodiments, the use of reagents capable of detecting the markers, such as specific antibodies, as a reagent mixture for all cells in the sample is also envisioned. Therefore, it is possible to perform differential detection of erythroblasts and / or CTCs using such a multiplex approach. To increase specificity, it is preferable to use diagnostically redundant or overlapping markers in these multiplex approaches, i.e., to detect them. In some embodiments, this can be achieved by using different staining agents or dyes that are optically distinguishable. Modification of the mixture is also envisioned as needed and for the cells to be detected, for example, according to the information provided in Table 1.

[0156] EpCam, also known as epithelial cell adhesion molecule or CD326, mediates intraepithelial calcium (Ca) adhesion in epithelial cells. 2+EpCam is a transmembrane glycoprotein that does not rely on isotype-dependent intercellular adhesion. It also participates in cell signaling, migration, proliferation, and differentiation. EpCam expression can be used to characterize cancer cells, particularly colon cancer. EpCam cell surface antigens can be detected by conjugated antibody binding, preferably with fluorescently labeled antibodies. The detection of EpCam expression in circulating tumor cells is considered to indicate that the cells are epithelial progenitor-like cells, or that EpCam expression was acquired during a circulating or distant homing event. The detection of EpCam-positive blast-like cells in healthy donors implicitly indicates circulating epithelial progenitor cells, and in the case of mature cells, implicitly indicates circulating epithelial cells, such as squamous or columnar cells. More information on EpCam detection is available from appropriate literature sources, such as Allard et al., 2004, Clinical Cancer Research, 10.20, 6897-6904.

[0157] Cytokeratin, also known as CK keratin, is a type of keratin typically found in the cytoskeleton of epithelial tissue cytoplasm. Cytokeratin is described as squamous keratin, belonging to the HMWCK family, or simple keratin, belonging to the LMWCK family. They are further subdivided into basic CK and acidic CK. There are at least 20 CK types. In a preferred embodiment, cytokeratin is detected using an antibody, such as monoclonal IgG or any other suitable antibody form. More preferably, the antibody detects more than one member of the cytokeratin family. Therefore, a pan-cytokeratin antibody, such as monoclonal antibody C-11, can be used. In alternative embodiments, a mixture or combination of antibodies binding to different cytokeratin types can be used. For example, the combination may include antibodies that bind to the following cytokeratins: cytokeratin 1 (e.g., LHK1) and / or cytokeratin 3 (e.g., AE5) and / or cytokeratin 4 (e.g., SN74-03) and / or cytokeratin 5 (e.g., EP1601Y) and / or cytokeratin 6 (e.g., SN71-07) and / or cytokeratin 7 (e.g., RCK105 or OV-TL12 / 30) and / or cytokeratin 8 (e.g., M20) and / or cytokeratin 10 (e.g., DE-K10) and / or cytokeratin 15 (e.g., LHK15) and / or cytokeratin 16 (e.g., LL025) and / or cytokeratin 17 (e.g., E3) and / or cytokeratin 18 (e.g., DC10) and / or cytokeratin 19 (e.g., 4E8) and / or cytokeratin 20 (e.g., Ks20.8), and / or monoclonal or polyclonal antibodies that bind to any other cytokeratin.

[0158] In specific embodiments, antibodies are conjugated with suitable markers, such as HRP, FITC, Alexa Fluor, Pycroerythrin, etc. Cytokeratin detection typically requires cell fixation and permeabilization because filament proteins are expressed within the cells. Different cytokeratin types may be used depending on the site or cancer type. In further embodiments, particularly if the cancer type is unknown, a pan-cytokeratin antibody mixture may be used. Circulating CD45 in the presence of the nucleus... - In (negative) cells, cytokeratins are commonly used as single epithelial markers. However, compared to EpCam... + / CD45 - Compared to the phenotype, the specificity is reduced, suggesting that other rare cells express cytokeratin. The commonly used standardized assay uses the cytokeratin / CD45 / DAPI phenotype based on the CellSearch system, as disclosed in Allard et al., 2004, Clinical Cancer Research, 10.20, 6897-6904.

[0159] In further embodiments, it is envisioned that an additional set of biochemical or molecular markers exists. Its use is optional. In some embodiments, it is advantageous to also test these markers. This additional set of markers includes vimentin. This set of markers can be measured alone with CD71 and / or GPA as described above, or with any one of the CD44, CD45, CD24, CD133, CD31, VAV1, Kell blood group protein, EpCam, and cytokeratin groups, or it can be measured as a different set of combinations with CD71 and / or GPA, CD44, CD45, CD24, CD133, CD31, VAV1, Kell blood group protein, EpCam, and cytokeratin, for example, vimentin plus CD71 and / or GPA; or vimentin plus CD71 and / or GPA plus any 1, 2, 3, 4, 5, or 6 of the marker group CD44, CD45, CD24, CD133, CD31, VAV1, Kell blood group protein, EpCam, and cytokeratin.

[0160] Vimentin is a type III intermediate filament (IF) protein commonly expressed in mesenchymal cells. Vimentin is therefore an intracellular filament protein similar to cytokeratin. It contains a central α-helix capped at each end by non-helical amino and carboxyl domains. Vimentin plays a crucial role in supporting and anchoring organelles in the cytosol, particularly in maintaining cell shape, cytoplasmic integrity, and stabilizing cytoskeletal interactions. Vimentin is commonly used as a marker for sarcoma tumors. Vimentin is preferably detected by fluorescent dye-conjugated antibodies. + / CD45 -The presence of this phenotype typically indicates that tumor cells are transitioning from an epithelial cell type to a mesenchymal cell type, hence the term epithelial-to-mesenchymal transition (EMT-CTC). A negative result allows for some prognosis. Vimentin is thought to be expressed in many normal non-hematopoietic cell types and plays a distinct role in tumor cell attack. Further details can be found in appropriate literature sources, such as Polioudaki et al., 2015, BMC cancer, 15.1, 399.

[0161] In a further embodiment, determining the presence of abnormal erythroblasts in a subject's bodily fluid sample further includes determining whether the cells in the subject's sample are present in the form of cell clusters. As used herein, the term "cell cluster" refers to two or more groups of erythroblasts in the analyzed liquid sample. These cell groups originate from cell division and therefore constitute an even-numbered cell population. The number of clustered cells can be 2, 4, 6, 8, 10, 12, 14, 16, 18 to 20, preferably 2, 4, 6, 8, or 10. In another embodiment, determining the presence of abnormal erythroblasts in a subject's bodily fluid sample further includes determining whether the cells in the subject's sample are present in the form of cell aggregates. As used herein, the term "cell aggregate" refers to three or more groups of erythroblasts in the analyzed liquid sample. These cell groups originate from cell aggregation events and therefore constitute an even- or odd-numbered cell population. The number of aggregated cells can be 3, 4, 5, 6, 7, 8, 9, etc. Preferably 3 cells.

[0162] In a further preferred embodiment, determining the presence of abnormal erythroblasts in a subject's bodily fluid sample includes morphological analysis of the cells in the sample. As used herein, the term "morphological analysis" refers to observing cells using suitable optical or electronic methods to determine the cell's morphology, such as geometry, geometric irregularity, etc.; shape; diameter; size, such as cell area; the form, size, and location of subcellular entities, such as the nucleus, such as its area, perimeter, or form; the ratio of cells or subcellular entities, such as the nucleus / cytoplasm ratio; behavior with respect to certain signals or environmental conditions; adhesion behavior of adjacent cells; and other microbiological or histological parameters known to those skilled in the art. For example, the term geometry includes additional geometric descriptive parameters such as perimeter, roundness, eccentricity, elongation, roundness, convexity, or nuclear eccentricity. Therefore, in some embodiments, the term "morphological analysis" may also include the nuclear morphology of the cells. Typically, morphological analysis is performed with the aid of a microscope or microscope system (preferably a digital microscope system). Alternatively, morphological analysis can be performed using an automated analyzer. Contemplated analysis systems include the Cellavision DM96, Diffmaster Octavia, and Cobas M511 analyzers. Further details are known to those skilled in the art or may be obtained from suitable sources, such as Merino et al., 2018, Intl. Journal of Laboratory Hematology, 40(1), 54-61. In a preferred embodiment, cell analysis is performed using an automated microscopy system, such as the Perkin Elmer Operetta high-content screening system or the Merck Amnis image streaming technology.

[0163] Further, the use of suitable software programs, particularly image analysis programs, is envisioned to assist morphological analysis. Such programs can be further integrated with artificial intelligence systems or neural networks, such as DCNN. Suitable examples of such programs include image analysis packages from Imaris, CellProfiler, Kaleido, Columbus, and Zeiss (e.g., ZEN or Olympus), which are typically used in conjunction with their respective microscopy systems. Independent software solutions based on public open-source elements are particularly preferred.

[0164] In some embodiments, according to the invention, cell morphology analysis is also accompanied by a cell staining procedure. This cell staining may result in the staining of one or more cell compartments or cell components. For example, staining may be nucleic acid staining as described herein, such as based on the use of DAPI, which allows the detection of cellular regions with high nucleic acid concentrations. An exemplary method is Giemsa staining, which uses a mixture of methylene blue, eosin, and Azure B.

[0165] A preferred staining method is May-Grünwald-Giemsa (MGG) staining, commonly used for nuclear staining. This method uses the dyes methylene blue and eosin Y or eosin b. Eosin dyes are typically used to stain basic regions within cells, such as DNA-binding proteins. A further proposed combination of May-Grünwald staining and Giemsa staining is termed Pappenheim staining.

[0166] Further staining methods can be used to detect other cellular compartments, such as the cell membrane. Examples of suitable staining agents include cell membrane staining agents, foveolar lectin A, wheat germ lectin (WGA) conjugates, 1,6-diphenyl-1,3,5-hexatriene, biotin-DHPE, biotin-X-DHPE, DiA (4-(4-(hexadecylaminostyryl)-N-methylpyridinium iodide)), DiB, DiD, or 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindocyanine).

[0167] In a central embodiment of the invention, morphological analysis of cells includes or allows for the classification of cells, particularly erythroblasts, in a sample into different categories. Corresponding parameter definitions can be used in automated systems or software programs to distinguish the cell categories defined below, or can be provided to experts to perform classification according to the definitions. It is also envisioned that the corresponding training data be used for machine learning or artificial intelligence (AI) methods, where neural networks are trained to detect and classify cells according to the provided definitions. Thus, corresponding cell images can be obtained and classified for use as training data for artificial intelligence methods, which are also envisioned as part of the invention; or they can be stored in a database, such as on a cloud-based server, for future or parallel use.

[0168] This classification includes distinguishing erythroblasts derived from bodily fluid samples (such as blood samples) as follows:

[0169] -1 category: In this category, body fluid samples include round or oval cells containing a single nucleus;

[0170] -2 category: In this category, body fluid samples include round or oval cells containing at least two nuclei;

[0171] -Class 3: In this category, body fluid samples include those containing contracted cell pairs; and

[0172] -Class 4: In this category, body fluid samples include aggregates of at least three round or oval cells with one or more nuclei.

[0173] The aforementioned geometric parameters may be suitable for encoding in an automated system. They can be set, for example, based on an exemplary image and a definition of divergence or deviation from the exemplary form, such as a deviation of 10%, 20%, 30%, or 35%.

[0174] In a further preferred embodiment, the aforementioned cell categories may be further divided into one or more subcategories based on the presence of certain cell types, nuclear diameter values, or cell diameter. The term "diameter" as used herein refers to the distance between the two furthest points on the cell periphery, particularly obtained from cell images obtained through microscopy or other cell imaging methods described herein.

[0175] For class 1, it can be further subdivided into the following subclasses:

[0176] -1a category, wherein the sample contains normal erythroblasts with a diameter of about 6.5 μm to 12.4 μm, having a dense nucleus and a high nucleocytoplasmic ratio;

[0177] -1b class, wherein the sample contains macrocirculatory erythroblasts with a diameter >12.5 μm, having at least one low-density nucleus and low nucleocytoplasmic ratio with a diameter of 6-10 μm;

[0178] -1c class, wherein the samples comprise megaloblasts with asynchronous nucleocytoplasmic and medium to high density chromatin and a high nucleocytoplasmic ratio; and

[0179] -1d class, wherein the sample comprises giant erythroblasts with a diameter of 4 μm to 6 μm, having a total condensed nucleus without nucleoplasmic asynchrony and a low nucleoplasmic ratio.

[0180] In addition, determining whether cells in a subject sample are present in the form of cell clusters may be accompanied by (preferably, optionally) determining the presence of vimentin or EpCam as described above.

[0181] Type 1a cells are morphologically or phenotypically normal erythroblasts. They typically range in diameter from about 6.5 μm to 12.4 μm. The cell nuclei are dense, with a high nucleocytoplasmic ratio. The aberrant behavior of type 1a cells may be attributed to their number, particularly their quantity in a defined volume of bodily fluid sample (e.g., blood). In some embodiments, the number of type 1a cells may be 1 × 10⁻⁶ per milliliter of sample (e.g., whole blood). 4 Individual cells. According to the preferred embodiments of the invention as defined herein, when class 1a cells are used as biomarkers for bone marrow injury, they possess clinical interpretability by allowing the grading of bone marrow injury into mild, moderate, and severe injury, as explained herein.

[0182] Class 1b cells include macrocirculating erythroblasts. These macroerythroblasts have a diameter >12.5 μm. They typically have a low-density nucleus with a diameter of 6 to 10 μm and a low nucleocytic ratio. These macroerythroblasts are considered normal early erythroblasts, hence their larger size ranging from approximately 12.5 to 25 μm. Compared to class 1a cells, they have lower nuclear chromatin density and a lower nucleocytic ratio (N / C) compared to class 1a cells and megaloblasts, but a higher N / C ratio compared to macroerythroblasts.

[0183] Class 1c cells include megaloblastoid or megaloblast-like cells, i.e., cells morphologically similar to megaloblasts. The presence of these cells in samples (e.g., blood samples) is generally considered an indicative factor or cellular marker of myelodysplastic leukemia. According to the invention, class 1c cells are morphologically similar to megaloblasts but are abnormal erythroblasts. Class 1c cells typically contain large nuclei with heterogeneous chromatin density, preferably exceeding 7.5 μm in diameter. Furthermore, these cells have a high N / C ratio. This characteristic distinguishes class 1c cells from other megaloblasts, which typically exhibit a lower N / C ratio and lower chromatin density. According to the invention, the presence of class 1c cells in bodily fluid samples such as blood is considered indicative of bone marrow tumors. In some embodiments, they can also be detected in cancer patient samples.

[0184] According to the present invention, 1d-type cells comprise giant erythroblasts with a diameter of approximately 4 μm to 6 μm, exhibiting morphological characteristics of giant erythroblasts. 1d-type cells typically exhibit a very small, high-density nucleus, resembling the most mature stage, but with a larger cytoplasm. 1d-type cells are considered abnormal for erythropoiesis, particularly when certain damage is involved in erythroblast maturation. According to the present invention, the presence of 1d-type cells in bodily fluid samples such as blood is considered indicative of bone marrow tumors. In some embodiments, they can also be detected in samples from cancer patients.

[0185] For category 2, it can be further subdivided into the following subcategories:

[0186] -2a class, wherein the cells are binucleated;

[0187] -2b class, wherein the cell contains at least 3 nuclei;

[0188] In type 2 cells, the nuclei of two or three cells are clearly distinguishable from each other, for example, by being located in different positions within the cell and / or not connected by any junctional structures or appearing to be undergoing nuclear division. The density of the nuclei or chromatin is preferably the same or similar. The severity of the abnormality can be indicated by the unequal size of the nuclei and the chromatin density in each cell. Such cells may have the cell morphology of any of the cells described in type 1.

[0189] Due to presumed pathological differences, cells are generally classified into binuclear (type 2a) and multinucleated (type 2c) cells. Binuclear cells (type 2a) represent sufficient morphological characteristics and arise from damage during cytokinesis (potentially including a lack of intracellular flow or cellular stability). However, these cells may differ in cell size and shape, nuclear size distribution, distance between the two nuclei, and nuclear density. The presence of nucleic acids in type 2 cells is independent of mitosis; that is, the cell may exhibit this phenotype in dividing or completely separating cells. Type 2b cells generally share the same morphological characteristics as type 2a cells, except that they contain at least three nuclei. In type 2b cells, unintentional intranuclear mitosis is thought to have occurred, thus constituting a pathological condition resulting in the production of multiple sets of chromosomes in a single cell.

[0190] The three categories can be further subdivided into the following subcategories:

[0191] Class -3a, in which cells do not show nuclear bridging; and

[0192] -3b class, wherein the cells exhibit nuclear bridges, and wherein the nuclei are not equivalent in size, shape, and / or chromatin density.

[0193] This type of cell may exhibit a contractile morphological characteristic. The term "contraction" as used in this article refers to a morphology that causes the cell to present an asymmetrical shape and contract at a certain point. The contractile phenotype is thought to be associated with mitotic events in the cell.

[0194] In the context of the three cell types, the term "nuclear bridge" refers to a nuclear morphology in which the nucleus enlarges and partially divides into two still connected parts. The connection between these two parts takes the form of a bridge or arch. In some embodiments, nuclear bridges can be found in contractile regions within the cell, i.e., the regions where nuclear separation occurs during mitosis.

[0195] Therefore, class 3 cells are morphologically distinguishable from other cell categories defined in this paper because they contain specific populations of cells undergoing mitosis; that is, they represent the morphology of cell pairs during cytokinesis. This interpretation stems from observations of paired cells exhibiting cell membrane contraction, which rules out the possibility of two attached cells aggregating. Class 3a cells typically exhibit a fairly normal morphology, similar to class 1a cells, but in a dividing state. Unlike class 1a cells, class 3a cells are present in bodily fluid samples, such as blood.

[0196] Unlike class 3a cells, class 3b cells typically represent widespread abnormalities, indicating more severe damage caused by cancer. This cell subclass displays paired cells that appear to be in a dividing state, with two nuclei of different sizes. The cells also show nuclear bridging and chromatin density changes associated with morphological features of impaired division.

[0197] Type 4 cells appear as at least three round or oval cell clusters, each cell possessing one or more nuclei. Therefore, Type 4 cells exhibit the morphological characteristics of cell clusters as defined herein. These cells appear to be in a dividing state or aggregated into erythrocytes, separated from erythroid islands within the bone marrow. The presence of this cell type typically reflects physical damage to the bone marrow. Metastatic growth may be present within the bone marrow for Type 4 cells. In some embodiments, Type 4 cells may also include anomalous conditions. Aside from clustering or aggregation, there is no difference between Type 4 and Type 3b cells.

[0198] In a particularly preferred embodiment, determining the presence of abnormal erythroblasts in a subject's bodily fluid sample further includes determining the ratio of at least one of the following: (i) class 1a cells to class 1c / 1d cells; (ii) class 1a cells to class 2 cells; and (iii) class 1a cells to class 3 cells, as well as the ratio of circulating tumor cell (CTC) clusters. The cell ratios according to certain classes as defined herein are determined by counting cells within a specific volume of bodily fluid sample (preferably a specific volume of blood) as described herein. The volume itself may be suitably chosen, depending on the sampling or sample preparation procedure. Since the invention is based on the determination of ratios, any suitable sample volume or size is contemplated. For statistical reasons, a larger sample volume is preferred. It is also contemplated that ratio determination be performed on more than one sample from a patient. Cell counting can be performed in a simple counting chamber or with the aid of a cell imaging system or procedure, such as those described herein.

[0199] The term circulating tumor cells (CTCs) used in this article refers to cells that detach from a primary tumor, enter blood vessels or lymphatic vessels, and are carried throughout the body via the bloodstream. CTCs can serve as seeds for the subsequent growth of other tumors, i.e., metastasize to distant organs. CTCs originating from tumors (including carcinomas, sarcomas, and melanomas) can be classified based on the expression of epithelial markers or their size and apoptotic status. One class (Group 1) of CTCs exhibits an intact, viable nucleus; expression of EpCam and cytokeratin, indicating epithelial origin; absence of CD45, indicating that the cell is not hematopoietic; and their larger size, irregular shape, or subcellular morphology. Another class (Group 2) of CTCs is cytokeratin-negative, characterized by the absence of EpCam or cytokeratin, which may indicate the presence of an undifferentiated phenotype (circulating cancer stem cells) or an acquired mesenchymal phenotype (EMT). Another group (Group 3) of CTCs are apoptotic CTCs, otherwise belonging to Group 1, i.e., typical CTCs undergoing apoptosis. Group 4 cells were small CTCs, positive for cytokeratin, and negative for CD45, but similar in size and shape to leukocytes. Group 5 cells were small or large cells, and were Epcam + (Positive), CK and CD45 were negative.

[0200] Circulating tumor cell clusters (CTC clusters) comprise two or more independent CTCs linked together. A CTC cluster may contain cells from any of the groups 1 through 5 of CTCs as defined herein. These clusters typically possess cancer-specific biomarkers that allow them to be identified as CTCs. Identification of CTC clusters is considered an indicator of advanced tumor evolution.

[0201] In a preferred embodiment, at least one of (i) to (iv) is identified in the subject sample: (i) EpCam in the sample + (Positive) and CD45 - (ii) Negative circulating tumor cells (CTCs) (label 1); (iii) CTC clusters in the sample showing single CTC cells of approximately 6-20 μm in diameter (label 2); (iv) EpCam in the sample - (Negative) and vimentin + (Positive) CTCs (label 3), for example, CTCs that are positive for vimentin expression and nuclear staining, but negative for CD45 expression. These CTCs may be positive for EpCam expression. CTCs with the above phenotype are called epithelial-to-mesenchymal transition CTCs, abbreviated as EMTCTC; and additionally (iv) the presence of at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells, indicating malignancy of solid tissue carcinoma.

[0202] In some embodiments, a ratio of 50 or less to 0 as described above can include ratios of about 50 to 0, about 45 to 0, about 40 to 0, about 35 to 0, about 30 to 0, about 25 to 0, about 20 to 0, about 15 to 0, about 10 to 0, about 5 to 0, about 3 to 0, about 2 to 0, or any other value (integer) between or below said values. Specifically, this ratio applies only to cases where erythroid abnormalities are present and indicates the presence of the diseases or conditions described above and below, particularly invasive cancers.

[0203] The term "solid tissue cancer" as used in this article refers to different types of localized solid tumors, including sarcomas, melanomas, and carcinomas. Examples of hypothetical cancers include colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head cancer, neck cancer, neuroblastoma, nephroblastoma, or retinoblastoma.

[0204] The term "malignant tumor" as used in this article refers to malignant tumors in the context of oncology. Its typical characteristics include anaplasia, invasiveness, and metastasis. Malignant tumors often also exhibit genomic instability, thus typically containing 10,000 to 100,000 mutations throughout their genome, as assessed by whole-genome sequencing. Malignant tumors often exhibit tumor heterogeneity, containing multiple subclones. The expression of DNA repair enzymes is also typically reduced due to epigenetic methylation of DNA repair genes or microRNA changes controlling DNA repair gene expression.

[0205] In another preferred embodiment, the presence of class 1a cells at an amount of about 10 to 500 cells per milliliter of sample and class 1b / 1c / 1d cells at an amount of about 1 to 10 cells per milliliter of sample indicate mild bone marrow injury as defined above.

[0206] When values ​​or numbers are involved, the term “1b / 1c / 1d” or any other combination of certain cell classes with a “ / ” as used herein refers to a combination or sum of cells of classes such as 1b, 1c and 1d in a sample, or a combination or sum of cell classes described before and after the “ / ” symbol.

[0207] In another embodiment, the presence of class 1a cells at an amount of approximately 1,000 to 5,000 cells per milliliter of sample and class 1b / 1c / 1d cells at an amount of approximately 10 to 50 cells per milliliter of sample indicated moderate bone marrow injury.

[0208] In another embodiment, the presence of class 1a cells at a level greater than approximately 10,000 cells per milliliter of sample and class 1b / 1c / 1d cells at a level greater than approximately 10 cells per milliliter of sample were identified as indicators of severe bone marrow injury.

[0209] As used herein, the term "amount of 1 to 10 cells per milliliter" means the presence of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cells per milliliter of a bodily fluid sample (e.g., a blood sample) as defined herein. The term "amount of 10 to 50 cells per milliliter" as used herein means the presence of approximately 10, 20, 30, 40, or 50 cells, or any value between those values, per milliliter of a bodily fluid sample (e.g., a blood sample) as defined herein. The term "amount of 10 to 500 cells per milliliter" as used herein means the presence of approximately 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 cells, or any value between those values, per milliliter of a bodily fluid sample (e.g., a blood sample) as defined herein. As used herein, the term "amount of 1,000 to 5,000 cells per milliliter" means approximately 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 cells, or any value between these, found in one milliliter of a bodily fluid sample (e.g., a blood sample) as defined herein. The term "amount of 0.5 to 10 cells per milliliter" as used herein means approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cells found in one milliliter of a bodily fluid sample (e.g., a blood sample) as defined herein. Furthermore, non-integer values ​​such as 1.5, 2.5, etc., may be present. The term "0.5 cells found per milliliter of bodily fluid sample" as used herein means 1 cell found in 2 milliliters of bodily fluid, or 2 cells found in 4 milliliters of bodily fluid, etc. Therefore, "1.5 cells per milliliter" means 3 cells found in 2 mL, etc.

[0210] In a particularly preferred embodiment, the method described herein includes identifying mild or moderate bone marrow damage as described above, identifying markers 1, 2 and / or 3 as described above, and identifying bone marrow damage, mild residual cancer disease, or micrometastases associated with invasive solid tissue cancer in the bone marrow, with a ratio of 50 or less to 0 as described above, indicating cancer treatment-related non-invasive or dormant cancer disease.

[0211] Therefore, in specific embodiments, the present invention envisions a combination of features: the identification of class 1a cells at an amount of approximately 10 to 500 cells per milliliter of sample and class 1b / 1c / 1d cells at an amount of approximately 1 to 10 cells per milliliter of sample, and further, the discovery of EpCam in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating mild bone marrow injury; and / or clusters of CTCs measuring approximately 6 to 20 μm in size were found in the sample, showing EpCam. + (Positive) and CD45 -(Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells; indicating micrometastases associated with dormant cancer, minimal residual cancer, or invasive solid tissue cancer in the bone marrow.

[0212] In another specific embodiment, the invention envisions a combination of features: the presence of class 1a cells at an amount of approximately 1000 to 5000 cells per milliliter of sample and class 1b / 1c / 1d cells at an amount of approximately 10 to 50 cells per milliliter of sample, and further, the discovery of EpCam in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating moderate bone marrow injury; and / or clusters of CTCs measuring approximately 6 to 20 μm in size were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) biochemical marker combination; and / or EpCam found in the sample. - (Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells; indicating micrometastases associated with dormant cancer, minimal residual disease, or invasive minimal residual disease or progressively invasive solid tissue cancer in the bone marrow.

[0213] In another specific embodiment, the invention envisions a combination of features: the identification of class 1a cells in amounts greater than about 10,000 cells per milliliter of sample and class 1b / 1c / 1d cells in amounts greater than about 10 cells per milliliter of sample, and further, the discovery of EpCam in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating severe bone marrow injury; and / or clusters of CTCs with cells ranging in size from approximately 6 to 20 μm were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin +(Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells; indicating micrometastases associated with active development of invasive solid tissue cancer in the bone marrow.

[0214] According to the present invention, class 1a quantification is associated with bone marrow injury. Current methods for abnormal erythroblasts in circulation are either not sensitive enough or lack the ability to detect even the subtlest morphological differences. According to the present invention, the erythroblast concentration level associated with a severe stage of disease is 1 × 10⁻⁶ cells per milliliter of body fluid sample (e.g., blood sample). 4 Up to 2×10 5 Within the range of normal red blood cells. In a specific embodiment, the amount of type 1a cells can be correlated with bone marrow damage according to Table 2 below.

[0215] Table 2:

[0216]

[0217] Additionally, the number of 1b / 1c / 1d cells may have additional value in determining bone marrow injury. For example, in cases of mild bone marrow injury, the number of 1b / 1c / 1d cells is quite low, ranging from approximately 1 to 10 cells per milliliter of sample.

[0218] In addition, in cases of moderate bone marrow injury, the number of 1b / 1c / 1d cells increases slightly to approximately 10 to 50 cells per milliliter of sample.

[0219] Furthermore, in cases of severe bone marrow injury, the number of 1b / 1c / 1d cells is similar to that in cases of moderate bone marrow injury, i.e., greater than 10 cells per milliliter of sample. However, the number may also exceed 50 cells per milliliter. Typically, the number will be in the range of approximately 10 to 50 cells, more preferably in the range of approximately 10 to 20 cells.

[0220] For class 1c cells, a cutoff value indicating mild bone marrow injury has been identified as 2.1 cells per milliliter of sample (e.g., blood), for example, as shown and described in Example 1. It is contemplated that this cutoff value can vary depending on the system and device used, for example, due to changes in the detection mechanism, different resolution, etc. Those skilled in the art will be able to adapt the currently mentioned cutoff values ​​to further (e.g., future) systems by performing comparison and / or calibration procedures. In some embodiments, moderate bone marrow injury in a subject (e.g., a cancer patient) is associated with a cell count range of approximately 10 to 50 cells per milliliter of sample (e.g., blood). In specific embodiments, more than 20 cells per milliliter of sample (e.g., blood) indicates severe bone marrow injury. In further embodiments, severe bone marrow injury is considered to be associated with the likelihood or presence of bone metastasis.

[0221] In another preferred embodiment, the amount of class 2a and class 2b cells identified per milliliter of sample was approximately 0.5 to 10 cells, indicating moderate bone marrow injury.

[0222] In another preferred embodiment, the amount of class 2a and class 2b cells identified per milliliter of sample was approximately 10 to 50 cells, indicating severe bone marrow injury.

[0223] The two cell types defined above are considered specific to severe conditions such as leukemia, cancer, and anemia. Therefore, the detection of one or more cells in a sample suggests bone marrow damage. In some specific embodiments, a threshold of approximately 0.3 to 0.6 cells per milliliter of sample is considered to indicate bone marrow damage. This translates to a minimum LOD of 0.5 to 1 cell per milliliter. Further details are available in Example 3, which also explains possible calculation methods for determining the threshold. The presence of two cell types exceeding the threshold indicates at least moderate damage. In specific embodiments, the distinction between multinucleated (type 2b) and binucleated (type 2a) cells also allows for differentiation between metastatic leukemia and congenital erythropoiesis-type II (CDAII). According to the invention, in both cases, only type 2a cells and CTCs above the threshold are present, thus the absence of type 2b cells indicates externally induced bone marrow damage. The formation of type 2a and type 2b cells is subject to different pathologies. Hoping to avoid being bound by theory, it is assumed that the dinuclearization represented by class 2b cells is due to cellular intrinsic genetic factors resulting from bone marrow primary pathology, as is the case in leukemia. Strict binucleation is generally associated with chemical disturbances in erythroid mitotic processes (especially erythroblast cytokinesis), induced, for example, by externally induced activity of intramedullary inflammatory tumor cells. Conversely, the presence of both class 2a and class 2b cells suggests intrinsic bone marrow damage, implying bone marrow disorders such as anemia and leukemia. In a further embodiment, the presence of class 2a and class 2b cells without CTCs rules out potential bone metastases. In a further embodiment, the presence of class 2a and class 2b cells, along with the presence of EpCam+ cells, involves both external and internal bone marrow damage.

[0224] Therefore, the identification of moderate bone marrow lesions as defined in the context of type 2 cells and the absence of type 2b cells per milliliter of sample, the identification of markers 1, 2, and / or 3 as defined above, and the identification of a ratio of 50 or less as defined above, indicate the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow. The term "progressive micrometastases" as used herein refers to systemic spread from the bone marrow via micrometastases; however, micrometastases may not develop into bone metastases.

[0225] In another embodiment, severe bone marrow injury as defined in the context of type 2 cells is identified in the absence of type 2b cells per milliliter of sample, identification of markers 1, 2 and / or 3 as defined above, and identification of a ratio of 50 or less to 0 as defined above, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0226] Therefore, in specific embodiments, the present invention envisions a combination of features: since the amount of class 2a and class 2b cells per milliliter of sample is identified as approximately 0.5 to 10 cells, and no additional class 2b cells are present per milliliter of sample, and EpCam is found in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating moderate bone marrow injury; and / or clusters of CTCs measuring approximately 6 to 20 μm in size were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin + (Positive) CTCs, and identifying at least one of the following ratios of 50 or less to 0: (i) class 1a cells to class 1c / 1d cells; (ii) class 1a cells to class 2 cells; and (iii) class 1a cells to class 3 cells; indicating the presence of active micrometastases associated with invasive solid tissue cancer in the bone marrow. The terms “active micrometastases” or “active developing micrometastases” as used herein refer to a cancerous state that is developing into metastasis.

[0227] Therefore, in another specific embodiment, the present invention envisions a combination of features: since the amount of type 2a and type 2b cells identified per milliliter of sample is approximately 10 to 50 cells, and no additional type 2b cells are present per milliliter of sample, and EpCam is found in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating severe bone marrow injury; and / or clusters of CTCs with cells ranging in size from approximately 6 to 20 μm were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) Class 1a cells with Class 1c / 1d cells; (ii) Class 1a cells with Class 2 cells; and (iii) Class 1a cells with Class 3 cells; indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0228] In another preferred embodiment, the amount of class 3a cells identified per milliliter of sample was approximately 1 to 10 cells, indicating moderate bone marrow injury.

[0229] In another preferred embodiment, the identification of about 10 to 50 class 3a cells per milliliter of sample and / or about 1 to 10 class 3b cells per milliliter of sample indicates severe bone marrow injury.

[0230] According to certain embodiments of the invention, type 3a cells may be present in individuals with bone marrow damage unrelated to leukemia or cancer at very low concentrations or amounts. Further details can be obtained, for example, from Example 6. The presence of these cells is generally considered a phenomenon of bone marrow damage caused by various etiologies.

[0231] Compared to class 3a cells, class 3b cells represent a wide range of genetic abnormalities, indicating more severe damage that may be caused by cancer. Therefore, class 3b cells indicate severe bone marrow damage, while class 3a cells indicate milder damage.

[0232] Therefore, the identification of moderate bone marrow damage as defined in the context of 3 cell types and the absence of 2b cells per milliliter of sample, the identification of markers 1, 2 and / or 3 as defined above, and the identification of a ratio of 50 or less to 0 as defined above, indicate the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow.

[0233] In another embodiment, severe bone marrow injury as defined in the context of class 3 cells is identified in the absence of class 2b cells per milliliter of sample, identification of markers 1, 2 and / or 3 as defined above, and identification of a ratio of 50 or less to 0 as defined above, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0234] Therefore, in specific embodiments, the present invention envisions a combination of features: since the amount of type 3a cells identified per milliliter of sample is approximately 1 to 10 cells, and no additional type 2b cells are present per milliliter of sample, and EpCam is found in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating moderate bone marrow injury; and / or clusters of CTCs measuring approximately 6 to 20 μm in size were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) class 1a cells with class 1c / 1d cells; (ii) class 1a cells with class 2 cells; and (iii) class 1a cells with class 3 cells; indicating the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow.

[0235] Therefore, in another specific embodiment, the invention envisions a combination of features: approximately 10 to 50 cells of class 3a cells per milliliter of sample and / or approximately 1 to 10 cells of class 3b cells per milliliter of sample, and the absence of additional class 2b cells per milliliter of sample, and the presence of EpCam in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating severe bone marrow injury; and / or clusters of CTCs with cells ranging in size from approximately 6 to 20 μm were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. - (Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) Class 1a cells with Class 1c / 1d cells; (ii) Class 1a cells with Class 2 cells; and (iii) Class 1a cells with Class 3 cells; indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0236] In another preferred embodiment, the identification of at least one of the four cell types per milliliter of sample indicates severe bone marrow injury.

[0237] The presence of type 4 cells typically reflects severe bone marrow damage above a critical threshold. In a further embodiment, the presence of type 4 cells in a body fluid sample can diagnose or detect metastatic growth within the bone marrow.

[0238] Therefore, in another embodiment, severe bone marrow injury as defined in the context of 4 cell types is identified in the absence of 2b cells per milliliter of sample, identification of markers 1, 2 and / or 3 as defined above, and identification of a ratio of 50 or less to 0 as defined above, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0239] Therefore, in a further specific embodiment, the present invention envisions a combination of features: since at least one cell of the four cell types was identified per milliliter of sample, and no additional 2b cell types were present per milliliter of sample, and EpCam was found in the sample. + (Positive) and CD45 - (Negative) circulating tumor cells (CTCs), thus indicating severe bone marrow injury; and / or clusters of CTCs with cells ranging in size from approximately 6 to 20 μm were found in the sample, showing EpCam. + (Positive) and CD45 - (Negative) combination of biochemical markers; and / or the presence of EpCam in the sample. -(Negative) and vimentin + (Positive) CTCs, and identified at least one of the following ratios of 50 or less to 0: (i) Class 1a cells with Class 1c / 1d cells; (ii) Class 1a cells with Class 2 cells; and (iii) Class 1a cells with Class 3 cells; indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

[0240] In another aspect, the present invention relates to the use of abnormal erythroblasts as markers for diagnosing, detecting, monitoring, or predicting pathological states in the bone marrow of a subject. Abnormal erythroblasts are erythroblasts as defined above. In a preferred embodiment, abnormal erythroblasts are cells of class 1c, 1d, 2a, 2b, 3a, 3b, or 4 as defined herein. Pathological states are generally associated with diseases or conditions as defined herein.

[0241] In a preferred embodiment, the abnormal erythroblasts are identified as described above. In a specific embodiment, erythroblasts are observed, counted per milliliter of sample, and classified according to the classification schemes disclosed herein (classes 1a to 4). During this classification and counting, detecting certain quantities and proportions of erythroblasts can be used to infer the pathological state in the subject's bone marrow. For example, observation and classification may produce the following quantities and scenarios:

[0242] In the sample, class 1a cells were present at a concentration of approximately 10 to 500 cells per milliliter, while class 1b / 1c / 1d cells were present at a concentration of approximately 1 to 10 cells per milliliter. Under these conditions, the detected abnormal erythroblasts could be used to infer and identify mild bone marrow damage.

[0243] In the sample, class 1a cells were present at a concentration of approximately 1,000 to 5,000 cells per milliliter, while class 1b / 1c / 1d cells were present at a concentration of approximately 10 to 50 cells per milliliter. Under these conditions, the detection of abnormal erythroblasts could be used to infer and identify moderate bone marrow damage.

[0244] In the sample, class 1a cells were present at a concentration of more than approximately 10,000 cells per milliliter, and class 1b / 1c / 1d cells were present at a concentration of more than approximately 10 cells per milliliter. Under these conditions, the detected abnormal erythroblasts could be used to infer and identify severe bone marrow damage.

[0245] Type 2a and 2b cells were present in quantities of approximately 0.5 to 10 cells per milliliter of sample. In this case, the detected abnormal erythroblasts could be used to infer identification of moderate bone marrow damage.

[0246] Type 2a and 2b cells were present in quantities of approximately 10 to 50 cells per milliliter of sample. In this case, the detected abnormal erythroblasts could be used to infer identification of severe bone marrow damage.

[0247] Type 3a cells were present in quantities of approximately 1 to 10 cells per milliliter of sample. In this case, the detected abnormal erythroblasts could be used to infer identification of moderate bone marrow damage.

[0248] The presence of type 3a cells at a rate of approximately 10 to 50 cells per milliliter of sample, and / or the presence of type 3b cells at a rate of approximately 1 to 10 cells per milliliter of sample, in these cases, the detection of abnormal erythroblasts can be used to infer identification of moderate bone marrow damage.

[0249] In each milliliter of sample, four cell types are present at least one cell. In this case, the detected abnormal erythroblasts can be used to infer identification of severe bone marrow damage.

[0250] The detection of these scenarios may be mutually exclusive or at least partially overlapping. Therefore, according to a specific embodiment of the invention, class 1a cells can be present in all cases, i.e., class 1a cells can be present in combination with class 1b, 1c, 1d, 2, 3, or 4 cells. Thus, the presence of class 1a cells indicates a healthy or diseased state, depending on the presence of other cell classes, etc., as described herein. Class 1b cells can be present in combination with class 1a cells, or in combination with class 1a, 2, 3, or 4 cells. If class 1a and 1b cells are found without other classes, the subject is considered healthy unless the quantity is abnormal. Conversely, if class 1b cells are present with class 1a and 2 cells; or with class 1a, 3, and 4 cells, the subject is considered diseased. Furthermore, class 1a and 1b cells may be present with class 2 cells, which also indicates a disease state. Additionally, the presence of class 1a and 3 cells may also indicate a disease state. Furthermore, the presence of type 4 cells alongside types 1a, 2, and / or 3 cells may indicate a disease state. Additionally, the presence of type 1c and / or type 1d cells alongside type 2 cells indicates a disease state. Furthermore, the presence of type 3 and / or type 4 cells alongside type 1c and / or type 1d cells also indicates a disease state.

[0251] In some embodiments, the aberrant erythroblasts are used in combination with additional markers as defined above. For example, aberrant erythroblasts are used in combination with markers for circulating tumor cells (CTCs). In a preferred embodiment, the invention envisions the identification of aberrant erythroblasts as defined above, as well as the identification of CTCs displaying certain biochemical markers. For example, a CTC might be EpCam. + (Positive) and CD45 - (Negative). Alternatively, CTCs may have clusters of CTC cells, with individual cells ranging from 6 to 20 μm in diameter, and are EpCam. + (Positive) and CD45 - (Negative). In another option, CTC might be EpCam.- (Negative) and vimentin + (Positive) CTC.

[0252] Based on the described use, it can be inferred that the pathological condition is malignant solid tissue cancer, such as non-invasive solid tissue cancer in the presence of mild or no bone marrow damage. Further details are provided above in the context of the method of the invention. These details are also included as additional embodiments in the use claims.

[0253] In another aspect, the present invention relates to a kit for diagnosing, detecting, monitoring, or predicting pathological states in the bone marrow of a subject, comprising tools for determining the presence of (i) CD71 and / or GPA, (ii) CD45, (iii) nucleic acids from rare circulating cells, and (iv) at least one of EpCam or (v) vimentin in a subject sample. In a further embodiment, the kit may also include tools for detecting the presence of one, two, three, or all of CD44, CD24, CD133, and CD31.

[0254] As used herein, the term "instrument for determination" refers to the instruments defined above in the context of CD71, GPA, CD45, EpCam, vimentin, CD44, CD24, CD133, and CD31, as well as nucleic acids, preferably antibodies binding to CD71, such as those defined above, and / or antibodies binding to GPA, such as those defined above, and / or antibodies binding to CD45, such as those defined above, and / or antibodies binding to EpCam, such as those defined above, and / or antibodies binding to vimentin, such as those defined below, and / or antibodies binding to CD44, such as those defined above, and / or antibodies binding to CD24, such as those defined above, and / or antibodies binding to CD133, such as those defined above, and / or antibodies binding to CD31, such as those defined above, as well as staining dyes for nucleic acids, such as DAPI or Hoechst 33342, etc. Instruments for detecting EpCam and vimentin have been defined above, preferably antibodies binding to EpCam and antibodies binding to vimentin. In a specific embodiment, the kit includes tools for detecting EpCam, anti-EpCam antibody MH99, and / or VU-1D9. In a further specific embodiment, the kit includes tools for detecting vimentin, such as anti-vimentin antibodies V9, J144, RV202, SP20, and / or VI-01.

[0255] As used herein, the term "tool for identifying nucleic acids in rare circulating cells" refers to dyes used to stain the nucleic acids of rare circulating cells. This term can be further extended to tools for obtaining said nucleic acids, such as tools for enriching said cells or for specifically binding nucleic acids to these cells. In some embodiments, tools for identifying nucleic acids in rare circulating cells may further or alternatively include tools for determining chromosomal abnormalities such as polyploidy and aneuploidy. Such tools include components for FISH, etc., or components for LCFM, such as Hoechst 33342, etc. Relevant information is well known to those skilled in the art or is available from suitable sources, such as Godek, 2018, Methods CellBiol.; 144:15–32 and Gomes et al., 2018, Cell Div, 13:6. The kit of the present invention is preferably a diagnostic kit. It can be formulated according to conventional procedures known to those skilled in the art. For example, the diagnostic kit may include a sterile isotonic buffered aqueous solution. If necessary, the kit may also include a solubilizer.

[0256] The kit may also include additional ingredients such as chloroquine, proton polar compounds such as propylene glycol, polyethylene glycol, glycerin, EtOH, 1-methyl-L-2-pyrrolidone or derivatives thereof, or non-proton polar compounds such as dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof. It may also include surfactants, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents. The above compounds are added under conditions limited by pH.

[0257] The diagnostic kits of the present invention may also include preservatives. In some kits according to the invention, preservatives include, but are not limited to: butyl paraben; ethyl paraben; imidazolidinyl urea; methyl paraben; o-phenylphenol; propyl paraben; quaternary ammonium-14; quaternary ammonium-15; sodium dehydroacetate; zinc pyrithione, etc. The amount of preservative used should be sufficient to effectively prevent or delay the growth of microorganisms. Typically, the amount of preservative used is from about 0.1% to about 1% of the total kit weight, preferably from about 0.1% to about 0.8%, and most preferably from about 0.1% to about 0.5%.

[0258] In a further embodiment, the present invention relates to a kit for diagnosing, detecting, monitoring, or predicting pathological states in the bone marrow of a subject. The kit may include one or more tools for determining the presence of the following in a subject sample: (i) CD71 and / or GPA, (ii) CD45, (iii) nucleic acids from rare circulating cells, and (iv) at least one of EpCam or (v) vimentin, as defined above. Preferably, the kit may include an antibody binding to CD71, and / or an antibody binding to GPA, and / or an antibody binding to CD45, and / or a dye for nucleic acid staining, such as DAPI. In another set of preferred embodiments, the kit may additionally include tools for detecting the presence of one, two, three, or all of CD44, CD24, CD133, and CD31, as described above.

[0259] Preferably, the kit can be formulated as a diagnostic kit, such as those described above, and may include suitable carriers, etc. According to the invention, components or ingredients of the diagnostic kit may be contained in one or more containers or separate entities. The properties of the reagents are determined by the detection method to which the kit is applicable.

[0260] Optionally, the kit may include a packaging insert or an instruction leaflet. The terms "packaging insert" or "instruction leaflet" refer to the instruction manual typically included in the commercial packaging of diagnostic products, which contains information such as usage, calibration, and / or warnings. The instruction leaflet may be part of the kit.

[0261] The following embodiments and accompanying drawings are provided for illustrative purposes. Therefore, it should be understood that the embodiments and drawings should not be construed as limiting. Those skilled in the art will readily envision further modifications to the principles set forth herein.

[0262] Example

[0263] Example 1

[0264] Isolation, staining and analysis of circulating rare cells

[0265] The procedure for preparing whole blood samples for circulating rare cell analysis is described, represented as a cell-based liquid biopsy, used here to support rapid fluorescence microscopy identification of erythroblasts, epithelial cells, and cells undergoing epithelial-to-mesenchymal transition.

[0266] Material:

[0267] -10 mL of whole blood from a healthy adult donor, stored in a heparin sodium blood storage container for up to 24 hours.

[0268] -Incubation buffer: Isotonic phosphate buffer supplemented with 3% fetal bovine serum.

[0269] Washing solution: isotonic phosphate buffer

[0270] - Red blood cell lysis buffer: 154mM NH4Cl, 10mM NaHCO3, 2mM EDTA

[0271] - Hematology centrifuge, equipped with a swing bucket rotor.

[0272] -Neubauer blood cell counter

[0273] -Antibody mixture;

[0274] - Permeabilization solution 2 for cell fixation (BD Bioscience)

[0275] - Immunomagnetic beads for reacting with unwanted cells (SanoLibio GmbH, Munich).

[0276] -Automatic concentration unit (Walderbach series, SanoLibio GmbH, Munich)

[0277] -Perkin Elmer, Operetta High Content Imaging System.

[0278] Experimental procedure:

[0279] As known to those skilled in the art, red blood cells were lysed using a suitable lysis buffer (RBC lysis buffer). The purified leukocytes from the initial 5 mL of whole blood were concentrated in the separation buffer by centrifugation in a hematologic centrifuge and allowed to stand for 15 minutes. The stock concentration of purified leukocytes was assessed using a standard hematologic counter (as required for optimal enrichment results), followed by an automated enrichment procedure. The enriched sample was concentrated in 30 μL of a cell-friendly solution containing rare circulating cells, leukocytes ranging from 1000 to 3000 cells, and cell debris. After enrichment, the cell suspension was fixed and perforated using permeabilization solution 2 according to the manufacturer's instructions, and analyzed under fluorescence microscopy using staining with anti-CD45PE (ebioscience), anti-CD71FITC, anti-EpCamSB650 (ebioscience), α-VimentinFITC (ebioscience), and anti-pan-CKPerCPCy5 (ebioscience), each using 1 μL of undiluted dye solution, and incubated in the freeze-dark for 25 minutes. Following nuclear staining, 0.5 μL of Hoechst 33342 DNA dye (ThermoFischer) was used. The suspension was washed in 1.5 mL of PBS and then concentrated by centrifugation at 300 × g for 5 min at 4 °C. The precipitate was resuspended in 70 μL of cell-friendly solution and loaded into one well of a dedicated 384-well plate suitable for high-resolution imaging at 40x magnification using the Operetta system (PerkinElmar), which records bright-field channels as well as UV, green, yellow, orange, and red fluorescence emission channels. Columbus analysis software was used as the screening and image analysis tool. Positive cells, including those labeled with EpCam, CD71, cytokeratin, and vimentin, were identified by the formation of cell-like circles when membrane staining was consistent with bright-field morphology; in the absence of typical ring formation, positive Hoechst staining was identified by positive CD45PE staining across the entire emission spectrum from 520 nm to 650 nm.

[0280] Example 2

[0281] Classification of biomarker types based on morphology

[0282] Material:

[0283] As described in Example 1.

[0284] Experimental procedure:

[0285] Cellular material for morphological analysis was produced using the experimental procedure described in Example 1.

[0286] Cell description:

[0287] Table 3:

[0288]

[0289]

[0290] Example 3

[0291] Healthy donor trials

[0292] Healthy donor trials were conducted to assess the threshold for abnormalities for the given cell types listed in Table 4.

[0293] Materials and general procedures:

[0294] As described in Example 1.

[0295] Research Procedure:

[0296] The anomaly (=specificity) threshold is determined by considering the blank limit (LoB) and the detection limit (LoB). LoB is the highest expected concentration of the apparent analyte to be detected when testing parallel blank samples containing no analyte, calculated as follows:

[0297] LoB = Mean blank value + 1.645 (blank SD);

[0298] LoD is the lowest analyte concentration that can be reliably distinguished from LoB and is feasible for detection. It is calculated using the measured LoB and detections on parallel samples known to contain low concentrations of the analyte, as follows:

[0299] LoD = LoB + 1.645 (SD for low-concentration samples). The critical value is equal to the detection limit.

[0300] Cutoff values ​​were derived from measurements of 15 healthy donors, who were selected based on subjective well-being, absence of disease in the past two months, and normal levels of routine blood tests (including C-reactive protein). LoB and LoD were given based on the number of cells per milliliter of whole blood.

[0301] At 2.5×10 7 Tumor cells (MCF-7) were triple-diluted 10-fold in a leukocyte suspension, and changes in low-concentration cell recovery were measured, followed by identification as described in Example 1. Assuming this change is effective for all cell types, the results were used for all LoD calculations.

[0302] Research findings:

[0303] See Table 4 below

[0304] Table 4:

[0305]

[0306]

[0307] Example 4

[0308] Metastatic breast cancer patients

[0309] This describes the use of biomarker analysis with the listed cell types (see table) to identify aggressive breast cancer with bone metastases.

[0310] Materials and general procedures:

[0311] As described in Example 1.

[0312] Research Procedure:

[0313] The ratio between normal and abnormal EB is a requirement for excluding benign myelopathy. The cutoff value is calculated as follows: the count of class 1a cells per milliliter (see Table 4) is divided by the sum of the counts of all classes 1c to 4 cells per milliliter.

[0314] The patient had a history of early-stage breast cancer, which recurred 5 years later, and also had malignant tumors in the liver and bones. She had not received any treatment at the time of the liquid biopsy.

[0315] Research findings:

[0316] Liquid biopsy confirmed malignancy in the patient, detecting circulating epithelial cells above the abnormal threshold. The ratio of normal to abnormal EB was very low (measured at 0.83), giving a quantitative impression of significant bone marrow damage. The EB profile suggested severe bone marrow injury and bone metastasis. Class 1b-d cells are a common feature of myelopathy, therefore high levels of class 1b-d cells should have been found in the blood of the patient studied in this study. The threshold values ​​given in Table 6 only distinguish between normal and abnormal, and therefore may represent the minimum threshold values ​​for myelopathy. The patient's EB concentration was significantly higher than the abnormal threshold. Clinical indications were related to the patient's EB concentration.

[0317] Table 5:

[0318]

[0319] Table 6:

[0320]

[0321] Example 5

[0322] metastatic small cell lung cancer patients

[0323] The table describes the use of biomarker analysis with the listed cell types to identify bone marrow involvement in extensive small cell lung cancer (SCLC) without bone metastasis.

[0324] Materials and general procedures:

[0325] As described in Example 4.

[0326] Research Procedure:

[0327] A 59-year-old male patient with small cell lung cancer (SCLC) presented with hemoptysis. A chest CT scan revealed a 6.3 cm mass in the right upper lung, multiple mediastinal lymph nodes, and a 4 cm subcutaneous nodule in the mid-abdomen. Bronchoscopy and biopsy confirmed extensive-stage small cell lung cancer. He underwent liquid biopsy blood tests during three cycles of first-line therapy, receiving a regimen of cyclophosphamide, doxorubicin, and vincristine. A chemotherapeutic response was observed in the first cycle. Two months later, the patient developed multiple brain metastases and passed away.

[0328] Research findings:

[0329] In a retrospective analysis, malignancy was confirmed by cell-based liquid biopsy, with 47 circulating epithelial cells detected per milliliter of whole blood. Moderate bone marrow impairment supports a stage of systemic disease (see Table 6). The ratio of normal to abnormal EB was measured at 6.22, confirming bone marrow involvement and indicating the involvement of disseminated tumor cells. The EB spectrum (including a relatively low ratio) suggests moderate bone marrow impairment, does not support a diagnosis of bone metastasis, and is consistent with clinical findings. These results may support the general view that SCLC generates far less bone marrow tumor cell dissemination than, for example, breast cancer cells (see Example 4).

[0330] Table 7:

[0331]

[0332] Table 8:

[0333] 1a 77 17 General bone marrow diseases 1b 2 3 No statistical significance 1c 0.8 2 No statistical significance 1d 0 2.5 No damage 2a 7.2 1.6 Moderate injury 2b 0 1.6 No damage 3a 1.2 1.6 No statistical significance 3b 0.4 1.6 No statistical significance 4 0.8 1.6 No statistical significance Normal / abnormal ratio 6.22 in conclusion Moderate bone marrow injury

[0334] Example 6

[0335] Healthy donors with underlying chronic diseases

[0336] The use of listed cell types for biomarker analysis (see Table 3) is described to identify bone marrow effects in a healthy donor with potentially confirmed stable thrombocytosis and another donor with diabetes.

[0337] Materials and general procedures

[0338] As described in Example 4.

[0339] Research Procedure:

[0340] Two healthy female donors (aged 60 (D1), with underlying thrombocytosis; and aged 43 (D2), with type II diabetes) donated 10 mL of blood for EB profile analysis. The first donor's platelet count had remained stable for the past 6 years, and she was asymptomatic, with a count of approximately 3 × 10⁻⁶. 5 Compared to the healthy platelet count / μL, the measured platelet count was three times higher. The diabetic patient exhibited mild, stable disease.

[0341] Research findings:

[0342] No circulating epithelial cells were found in either donor, ruling out any malignant tumors. In donor 1, one type 3a cell was identified in 2.5 mL of blood. To support the non-malignant nature, a high ratio of normal to abnormal EB was measured. However, a significant increase in type 1a EB cells suggested bone marrow abnormalities. Type 3a cells were found, but below the level of disability (LOD), leading to and supporting a conclusion of low-grade injury. Donor 2 showed no statistically significant erythroblastic abnormalities, but the presence of both type 2a and 3a cells indicated mild bone marrow injury.

[0343] Table 9:

[0344]

[0345] Table 10:

[0346]

[0347] Example 7

[0348] Mild residual disease in early-stage breast cancer

[0349] The study describes the use of the listed cell types for biomarker profiling analysis (see Table 3) to identify minimal residual disease in patients with stage II breast cancer after surgery.

[0350] Materials and general procedures

[0351] As described in Example 4.

[0352] Research Procedure:

[0353] The donor, a 37-year-old female, was diagnosed with stage II low-risk hormone-positive breast cancer. Four months after tumor resection, during adjuvant therapy, she donated 10 mL of peripheral blood for analysis of her EB and CTC profiles.

[0354] Research findings:

[0355] The presence of a small number of circulating epithelial cells indicates residual malignancy in the donor. The ratio of normal to abnormal EB was low. Therefore, an assessment of low-grade bone marrow injury was given, supported by the presence of class 2a and 3a cells, but below the level of disease (LOD). These findings suggest that the likely origin of CTCs can be attributed to bone marrow dissemination at this site, resulting in low-grade bone marrow injury, concluding with low-grade distant active minimal residual disease.

[0356] Table 11:

[0357]

[0358] Table 12:

[0359] 1a 12.4 17 Mild bone marrow injury 1b 0.6 3 No statistical significance 1c 1.2 2 Mild bone marrow injury 1d 0 2.5 No damage 2a 0.2 1.6 Mild bone marrow injury 2b 0 1.6 No damage 3a 0.4 1.6 Mild bone marrow injury 3b 0 1.6 No damage 4 0 1.6 No damage Normal / abnormal ratio 7.2 in conclusion Low-grade bone marrow injury

[0360] Example 8

[0361] Solid tissue cancer staging

[0362] This demonstrates the potential to translate these biomarkers into clinical cancer care, serving as complementary diagnostic tests to histopathological staging of lesions, particularly carcinoma, melanoma, and sarcoma.

[0363] Materials and general procedures

[0364] As described in Example 1.

[0365] Diagnostic testing principles and procedures:

[0366] In this study, cellular blood tests, performed concurrently with tissue biopsies as part of a liquid biopsy donation, were conducted for histopathological studies to increase the certainty of cancer metastasis status. Conventional staging methods, such as in breast cancer diagnosis, rely on tumor tissue and nearby lymph nodes as the first impression of the tumor system. However, they often fail to adequately diagnose distant tumor cell dissemination (especially very low-level dissemination, such as to the bone marrow), leading to inadequate understanding. Liquid biopsy scores bone marrow damage, thus aiding in routine staging and potentially improving the accuracy of metastatic cancer detection to refine treatment decisions. Compared to tissue biopsies or molecular-based studies, liquid biopsy tests do not rely on predictions of distant tissue invasiveness but constitute a real-time assessment of ongoing tumor evolution. Therefore, liquid biopsy tests may not necessarily depend on confirmation of malignancy through histopathology; they can produce a diagnosis of malignancy on their own and provide results regarding bone marrow damage. By combining (liquid or tissue biopsy) methods for malignancy diagnosis, bone marrow damage can be interpreted as a result of DTC invasion.

[0367] According to liquid biopsy, bone marrow tumor cell infiltration (and therefore systemic spread) will signal through abnormalities in the number and / or type of circulating red blood cells. Therefore, these tests interpret bone marrow damage as “excluding distant invasion,” “suspected invasion,” and “distant invasive cancer.” The latter is indicated by severe and moderate bone marrow damage in the presence of EB abnormalities. Moderate damage (classes 1ab only) and mild damage (class 1cd) indicate “suspected invasion,” and “non-invasive” can be diagnosed based on biomarker concentrations below a critical value. Liquid biopsy strongly suggests the presence of metastatic disease in the diagnosis of “invasive cancer,” regardless of histopathological staging. On the other hand, suspected invasion or non-invasiveness should follow histopathological staging. Therefore, liquid biopsy tests cannot reproduce all other aspects of conventional staging and can therefore be used as supplementary or adjunctive tests.

[0368] Figure 2 and Figure 3 The diagnostic procedure is illustrated. This procedure begins with parallel tissue and liquid biopsies in individuals suspected of having cancer. In treatment-naïve individuals, consistency between the two is necessary for a diagnosis of malignancy. In the liquid biopsy, the presence of more than a given threshold of CTCs reflects, but is not definitively identified as, malignancy (see Example 3). If consistent with the tissue biopsy, the invasiveness status should be compared. The procedure can benefit the patient in cases where a positive distant invasive cancer is detected by liquid biopsy, while a negative distant invasive cancer is detected by conventional methods. Therefore, re-staging is recommended, potentially requiring stage 4 treatment. In any other case, such as a liquid biopsy yielding "no invasion" or "suspected invasion," the outcome is independent of conventional staging, which relies solely on histopathological prediction and / or a diagnosis specifically reflecting metastasis.

Claims

1. Use of a product for determining the presence of abnormal erythroblasts in a subject's body fluid sample in the preparation of a product for detecting, diagnosing, monitoring, or predicting a pathological state in a subject's bone marrow, wherein the steps for determining the presence of abnormal erythroblasts in a subject's body fluid sample include at least: The presence of abnormal erythrocytes in a subject's body fluid sample is determined using a product that determines the presence of abnormal erythrocytes in the subject's body fluid sample. The abnormal erythroblasts described therein include one or more phenotypic features that are significantly different from those of normal erythroblasts; The determination includes determining the ploidy of the erythroblasts and the potential presence of (i) nuclear budding or lobulation phenotype, (ii) internuclear bridges, (iii) two or more nuclei in a single cell, (iv) megaloblasts, (v) giant erythroblasts, (vi) erythroblasts undergoing synchronous cytoplasmic division, (vii) erythroblast aggregates containing at least three adherent erythroblasts, and (viii) increased intranuclear ploidy of erythroblasts. The determination also includes determining that at least CD71 and / or GPA are present in the cells of the subject sample, as well as one or more of CD44, CD45, VAV1, Kell blood group protein, and nucleic acid; The determination also includes determining the presence of EpCam and cytokeratin, as well as vimentin, in the cells of the subject sample; The determination also includes morphological analysis of the cells in the sample, wherein the cell morphology analysis includes classifying the cells in the sample as: -1 class, wherein the sample comprises round or oval cells containing a single nucleus; -2, wherein the sample comprises round or oval cells containing at least two nuclei; -3 categories, wherein the samples include cell pairs containing contracted cells; and -4, wherein the sample comprises an aggregate of at least three round or oval cells having one or more nuclei; The first category includes further subdividing the cells into the following subcategories: -1a category, wherein the sample comprises normal erythroblasts with a diameter of 6.5 µm to 12.4 µm, having a dense nucleus and a high nucleocytoplasmic ratio; -1b class, wherein the sample contains macrocirculatory erythroblasts with a diameter >12.5 µm, having at least one low-density nucleus and low nucleocytoplasmic ratio with a diameter of 6 to 10 µm; -1c class, wherein the sample contains megaloblasts with asynchronous nucleocytoplasmic and medium to high density chromatin and a high nucleocytoplasmic ratio; and -1d class, wherein the sample contains giant erythroblasts with a diameter of 4 µm to 6 µm, having a total condensed nucleus without nucleoplasmic asynchrony and a low nucleoplasmic ratio; Among the findings: -Class 1a cells present in the sample at a rate of 10 to 500 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a rate of 1 to 10 cells per milliliter, indicate mild bone marrow damage; or -Class 1a cells present in the sample at a rate of 1000 to 5000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a rate of 10 to 50 cells per milliliter, indicate moderate bone marrow injury; or -Class 1a cells present in the sample at a level greater than 10,000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a level greater than 10 cells per milliliter, indicate severe bone marrow injury.

2. The use according to claim 1, wherein the second category includes further subdividing the cells into the following subcategories: Class -2a, wherein the cells are binucleated; and -2b class, wherein the cell contains at least 3 nuclei; The detection of 0.5 to 10 cells of type 2a and type 2b per milliliter of sample indicates moderate bone marrow injury; or the detection of 10 to 50 cells of type 2a and type 2b per milliliter of sample indicates severe bone marrow injury.

3. The use according to claim 1, wherein the third category includes further subdividing the cells into the following subcategories: Class -3a, wherein the cells do not show nuclear bridging; and -3b class, wherein the cells exhibit nuclear bridges, and wherein the nuclei are not equivalent in size, shape and / or chromatin density; The presence of 1 to 10 class 3a cells per milliliter of sample indicates moderate bone marrow injury; or the presence of 10 to 50 class 3a cells per milliliter of sample and / or 1 to 10 class 3b cells per milliliter of sample indicates severe bone marrow injury.

4. The use according to claim 1, wherein the amount of at least one of the four cell types identified per milliliter of sample indicates severe bone marrow injury.

5. The use according to claim 1, wherein mild or moderate bone marrow injury is identified, EpCam-positive and CD45-negative circulating tumor cells are identified in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm and / or EpCam-negative and vimentin-positive circulating tumor cells are identified in the sample, and at least one of (i) class 1a cells to class 1c / 1d cells, (ii) class 1a cells to class 2 cells and (iii) class 1a cells to class 3 cells is identified in a ratio of 50 or 50 to 0, indicating bone marrow injury, minimal residual cancer, or micrometastases associated with invasive solid tissue cancer in the bone marrow, which are related to cancer treatment-related non-invasive or dormant cancer.

6. The use according to claim 2 or 3, wherein moderate bone marrow injury is identified in the absence of type 2b cells per milliliter of sample, EpCam-positive and CD45-negative circulating tumor cells are identified in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm and / or EpCam-negative and vimentin-positive circulating tumor cells are identified in the sample, and the ratio of at least one of (i) type 1a cells to type 1c / 1d cells, (ii) type 1a cells to type 2 cells and (iii) type 1a cells to type 3 cells is identified as 50 or 50 to 0, indicating the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow.

7. The use according to claim 1, wherein severe bone marrow injury is identified, EpCam-positive and CD45-negative circulating tumor cells are identified in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm and / or EpCam-negative and vimentin-positive circulating tumor cells are identified in the sample, and at least one of (i) class 1a cells to class 1c / 1d cells, (ii) class 1a cells to class 2 cells and (iii) class 1a cells to class 3 cells is identified in a ratio of 50 or 50 to 0, indicating the presence of active micrometastases associated with invasive solid tissue cancer in the bone marrow.

8. The use according to claim 2, 3 or 4, wherein severe bone marrow injury is identified in the absence of type 2b cells per milliliter of sample, EpCam-positive and CD45-negative circulating tumor cells are identified in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm and / or EpCam-negative and vimentin-positive circulating tumor cells are identified in the sample, and the ratio of at least one of (i) type 1a cells to type 1c / 1d cells, (ii) type 1a cells to type 2 cells and (iii) type 1a cells to type 3 cells is 50 or 50 to 0, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

9. The use according to claim 1, wherein the sample is a peripheral blood sample.

10. The use according to claim 1, wherein the sample is a venous blood sample.

11. The use according to claim 1, wherein the sample is an inferior vena cava sample or a portal vein sample.

12. The use according to claim 1, wherein morphological analysis is performed after Maggi staining of the cells.

13. A system for detecting, diagnosing, monitoring, or predicting pathological conditions in the bone marrow of a subject, comprising at least: A determining element used to determine the presence of abnormal erythroblasts in a subject's bodily fluid sample; and Identification element; The determining element is further configured to determine the ploidy of the erythroblast and the potential presence of (i) nuclear budding or lobulation phenotype, (ii) internuclear bridge, (iii) two or more nuclei in a single cell, (iv) megaloblast, (v) giant erythroblast, (vi) erythroblast with synchronous cytoplasmic division, (vii) erythroblast aggregate containing at least 3 erythroblasts adhering to it, and (viii) increased ploidy of the erythroblast nucleus. The determining element is further configured to determine the presence of at least CD71 and / or GPA in the cells of the subject sample, as well as one or more of CD44, CD45, VAV1, Kell blood group protein, and nucleic acid; The determining element is further configured to determine the presence of EpCam and cytokeratin, as well as vimentin, in the cells of the subject sample; The determining element is further configured to determine the morphological analysis of cells in the sample, the cell morphology analysis including classifying the cells in the sample as: -1 class, wherein the sample comprises round or oval cells containing a single nucleus; -2, wherein the sample comprises round or oval cells containing at least two nuclei; -3 categories, wherein the samples include cell pairs containing contracted cells; and -4, wherein the sample comprises an aggregate of at least three round or oval cells having one or more nuclei; The first category includes further subdividing the cells into the following subcategories: -1a category, wherein the sample comprises normal erythroblasts with a diameter of 6.5 µm to 12.4 µm, having a dense nucleus and a high nucleocytoplasmic ratio; -1b class, wherein the sample contains macrocirculatory erythroblasts with a diameter >12.5 µm, having at least one low-density nucleus and low nucleocytoplasmic ratio with a diameter of 6 to 10 µm; -1c class, wherein the sample contains megaloblasts with asynchronous nucleocytoplasmic and medium to high density chromatin and a high nucleocytoplasmic ratio; and -1d class, wherein the sample contains giant erythroblasts with a diameter of 4 µm to 6 µm, having a total condensed nucleus without nucleoplasmic asynchrony and a low nucleoplasmic ratio; The identification element identified that: -Class 1a cells present in the sample at a rate of 10 to 500 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a rate of 1 to 10 cells per milliliter, indicate mild bone marrow damage; or -Class 1a cells present in the sample at a rate of 1000 to 5000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a rate of 10 to 50 cells per milliliter, indicate moderate bone marrow injury; or -Class 1a cells present in the sample at a level greater than 10,000 cells per milliliter, and class 1b / 1c / 1d cells present in the sample at a level greater than 10 cells per milliliter, indicate severe bone marrow injury.

14. The system of claim 13, wherein the second category comprises further subdividing the cells into the following subcategories: Class -2a, wherein the cells are binucleated; and -2b class, wherein the cell contains at least 3 nuclei; The identification element identifies 0.5 to 10 cells of type 2a and type 2b per milliliter of sample, indicating moderate bone marrow injury; or identifies 10 to 50 cells of type 2a and type 2b per milliliter of sample, indicating severe bone marrow injury.

15. The system of claim 13, wherein the three categories include further subdividing the cells into the following subcategories: Class -3a, wherein the cells do not show nuclear bridging; and -3b class, wherein the cells exhibit nuclear bridges, and wherein the nuclei are not equivalent in size, shape and / or chromatin density; The identification element identifies 1 to 10 class 3a cells per milliliter of sample, indicating moderate bone marrow injury; or identifies 10 to 50 class 3a cells per milliliter of sample and / or 1 to 10 class 3b cells per milliliter of sample, indicating severe bone marrow injury.

16. The system of claim 13, wherein the identification element identifies at least one cell of four cell types per milliliter of sample, indicating severe bone marrow injury.

17. The system of claim 13, wherein the identification element identifies mild or moderate bone marrow injury, identifies EpCam-positive and CD45-negative circulating tumor cells in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm, and / or EpCam-negative and vimentin-positive circulating tumor cells in the sample, and identifies a ratio of at least one of (i) class 1a cells to class 1c / 1d cells, (ii) class 1a cells to class 2 cells, and (iii) class 1a cells to class 3 cells of 50 or 50 to 0, indicating bone marrow injury, minimal residual cancer, or micrometastases associated with invasive solid tissue cancer in the bone marrow, which are related to cancer treatment-related non-invasive or dormant cancer.

18. The system of claim 14 or 15, wherein the identification element identifies moderate bone marrow injury in the absence of type 2b cells per milliliter of sample, identifies EpCam-positive and CD45-negative circulating tumor cells in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm, and / or EpCam-negative and vimentin-positive circulating tumor cells in the sample, and identifies at least one of (i) type 1a cells to type 1c / 1d cells, (ii) type 1a cells to type 2 cells, and (iii) type 1a cells to type 3 cells in a ratio of 50 or 50 to 0, indicating the presence of progressive micrometastases associated with invasive solid tissue cancer in the bone marrow.

19. The system of claim 13, wherein the identification element identifies severe bone marrow injury, identifies EpCam-positive and CD45-negative circulating tumor cells in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm, and / or EpCam-negative and vimentin-positive circulating tumor cells in the sample, and identifies at least one of (i) class 1a cells to class 1c / 1d cells, (ii) class 1a cells to class 2 cells, and (iii) class 1a cells to class 3 cells in a ratio of 50 or 50 to 0, indicating the presence of active micrometastases associated with invasive solid tissue cancer in the bone marrow.

20. The system of claim 14, 15 or 16, wherein the identification element identifies severe bone marrow injury in the absence of type 2b cells per milliliter of sample, identifies EpCam-positive and CD45-negative circulating tumor cells in the sample, circulating tumor cell clusters in the sample showing EpCam-positive and CD45-negative circulating tumor cells with a single cell diameter of 6-20 µm, and / or EpCam-negative and vimentin-positive circulating tumor cells in the sample, and identifies at least one of (i) type 1a cells to type 1c / 1d cells, (ii) type 1a cells to type 2 cells, and (iii) type 1a cells to type 3 cells in a ratio of 50 or 50 to 0, indicating bone metastases or primary bone cancer associated with invasive solid tissue cancer.

21. The system of claim 13, wherein the sample is a peripheral blood sample.

22. The system of claim 13, wherein the sample is a venous blood sample.

23. The system of claim 13, wherein the sample is an inferior vena cava sample or a portal vein sample.

24. The system of claim 13, wherein morphological analysis is performed after Maggi staining of the cells.

Images