Means and methods for diagnosing, classifying and / or monitoring pediatric tumors - Patent Application 20070122997

Abstract

The present invention relates to the field of medical diagnostics, in particular to methods and reagents for the detection and monitoring of pediatric tumors by single-tube multiparameter flow cytometry (MFC). A kit of parts for the flow cytometric detection of pediatric tumor cells is provided, comprising fluorochrome-conjugated antibodies directed against the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271, the cytoplasmic marker cyCD3, and one or more nuclear markers nuMyogenin and / or nuMyoD1, wherein: (i) antibodies against the markers CD99 / CD8 are conjugated to the same fluorochrome and represent the first marker pair CD99 / CD8; (ii) an antibody against the marker EpCAM / CD4 is conjugated to the same fluorochrome, as well as representing the second marker pair EpCAM / CD4; (iii) antibodies against CD271 are conjugated to the same fluorochrome as antibodies against either cyCD3 or smCD3, as well as representing a third marker pair, CD271 / cyCD3 or CD271 / smCD3; wherein the fluorescent dyes are distinguishable between the first, second, and third marker pairs; and wherein the antibodies against the cytoplasmic marker and the nuclear marker are physically separated from the antibodies against the cell surface marker.

Description

[Technical Field]

[0001] The present invention relates to the field of medical diagnostics, particularly to diagnosing pediatric tumors using flow cytometry. The present invention provides a method and reagent for detecting and monitoring pediatric tumors using single-tube multiparameter flow cytometry (MFC). It also provides a tool for simultaneously characterizing tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating monocytes / dendritic cells (TIMs) infiltrating tumor tissue. The reagent composition of the present invention enables detection and characterization of tumor cells at the primary tumor site, as well as detection of tumor cells in tissues other than the primary tumor through analysis of peripheral blood, bone marrow, cerebrospinal fluid, metastatic tumor tissue, and any other tissue samples from patients, and evaluation of therapeutic efficacy. Kits and methods related to the reagent composition are also provided. [Background technology]

[0002] Pediatric solid tumors comprise a heterogeneous group of diseases that primarily occur in the 0-15 year old age group. Globally, it is estimated that there are more than 175,000 cases and 96,000 deaths per year in children. Solid tumors account for approximately 75% of all childhood cancers and include lymphoma, central nervous system (CNS) tumors, neuroblastoma, soft tissue sarcoma, nephroblastoma, bone tumor, retinoblastoma, hepatoblastoma, germ cell tumor, and carcinoma (Ward E, CA Cancer J CLIN 2014;64:83).

[0003] The correct diagnosis of childhood cancer is known to be complex and difficult. Although classic histological features generally strongly suggest a specific tumor type, most childhood tumors can be indistinguishable by light microscopy and immunohistochemistry alone. Because different diagnostic entities require different, highly specific treatments, early and accurate diagnosis, classification, and risk stratification are crucial, especially in childhood cancer patients.

[0004] Currently, conventional methods used for diagnostic screening and classification of pediatric solid tumors rely on morphological and immunohistochemical analysis of formalin-fixed, paraffin-embedded solid tumor specimens. The majority of pediatric solid tumors fall under the descriptive category of "small round cell tumors." Pediatric solid tumors include tumors formed by undifferentiated, small cells with scant cytoplasm and round, hyperchromatic nuclei. Due to the fact that these tumors exhibit similar morphological characteristics by light microscopy, accurate diagnosis of pediatric solid tumors is often difficult, potentially leading to misdiagnosis without further analysis (Magro G, Acta Histochem. 2015;117:397). Therefore, further immunohistochemical studies are essential. Despite this, there is no uniformly recommended antibody panel that can be applied to such immunohistochemical analysis for sensitive and specific diagnosis and classification of the majority of pediatric cancer subtypes. Furthermore, technical solutions for the simultaneous evaluation of all useful markers for the direct diagnosis and classification of tumors, based on immunohistochemistry or flow cytometry, are currently not available or have not been described.

[0005] Therefore, in contrast to the diagnostic workup of childhood leukemia, where MFC immunophenotyping is essential for rapid diagnosis, classification, and monitoring of the disease, MFC immunophenotyping is not routinely used for the diagnostic workup of childhood solid tumors. In fact, MFC immunophenotyping has so far been limited to the evaluation of a limited number of markers to identify specific diagnostic entities that express highly distinctive phenotypes, such as the CD45-CD56+ phenotypic profile in neuroendocrine tumors (Bryson GJ, J Clin Pathol. 2002;55:535), the CD45-CD56+GD2+ immunophenotype in neuroblastoma (Bozzi F, Anticancer Res. 2006;26:3281), the CD57+CD56+CD99+CD45- signature in primitive neuroectodermal tumors (Dubois SG, Pediatr Blood Cancer. 2010;54:13), and the CD45-CD56+nuMyogenin+ profile in rhabdomyosarcoma (Almazán-Moga A, Cytometry). A.2014;85:1020), including:

[0006] However, despite these phenotypic profiles being characteristic of specific diagnostic entities within pediatric cancers, they have systematically demonstrated relatively low efficacy when prospectively tested in clinical settings compared to conventional diagnostic procedures based on panels of four- to six-color antibody combinations due to their inability to accurately distinguish between different tumor types. Additionally, diagnostic MFC analysis of pediatric solid tumor tissues did not simultaneously provide information on the types and numbers of tumor-infiltrating inflammatory and immune cells coexisting within the tumor within the same sample.

[0007] In 2013, Ferreira-Facio et al. (Ferreira-Facio, PLoS One. 2013;8:e55534) examined the staining patterns of tumor cells from 52 pediatric solid tumors by MFC immunophenotyping using a panel of antibody combinations containing 33 different antibody specificities in multiple tubes with up to eight colors. In this study, all samples classified as reactive / inflammatory by conventional histopathological criteria were also correctly diagnosed by MFC alone. Similarly, tumor cells were detected by MFC in all but two rare lymphoma samples. Based on this study, it was concluded that the combined evaluation of CD45, CD56, CD81, CD99, EpCAM, GD2, nuclear nuMyoD1, nuMyogenin, and CD271, in addition to other specific B- and T-cell markers, could be a useful antibody panel for both tumor diagnosis and classification. However, the authors failed to provide a method for combining all useful markers into a single antibody combination that would allow i) identification of childhood cancer tumor cells, ii) their diagnostic classification, and iii) detailed analysis of tumor-infiltrating immune cells coexisting in the same sample. In addition, they systematically failed to identify some tumor subtypes, such as Hodgkin's lymphoma.

[0008] In parallel with this, in International Publication WO 2010 / 140885, van Dongen et al. reported a panel of antibody reagents conjugated with fluorescent compounds to be used for the immunophenotypic characterization of normal, reactive, regenerative, and neoplastic white blood cell populations. However, such panels of antibody combinations have been entirely limited to leukemia and lymphoma patients, and no similar attempts have been made in pediatric cancers for the diagnosis, classification, and monitoring of non-hematopoietic solid tumors and hematologic tumors (e.g., pediatric lymphoma).

[0009] Therefore, we set out to design a single combination of antibody reagents that would enable the systematic identification and classification of tumor cells in pediatric solid tumor samples and simultaneously provide detailed characterization of infiltrating immune cells coexisting in the same sample. Furthermore, we aimed to provide an MFC protocol comprising a single and unique combination of antibody reagents for diagnostic screening and classification of solid tumors in pediatric patients and simultaneous detailed analysis of the tumor's immune cell microenvironment.

[0010] These goals have been surprisingly met with the development of novel fluorochrome-antibody combinations of over eight colors for the simultaneous detection of over 12 different proteins that display diverse but distinctive expression profiles in individual tumor cells present in pediatric solid tumor samples, allowing for the identification and classification of pediatric cancers into distinct diagnostic entities as well as the simultaneous assessment of tumor-infiltrating immune cells. More particularly, it has been found that this is achieved by staining for (i) the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19, and CD271; (ii) the cytoplasmic marker cyCD3; and (iii) one or more nuclear markers nuMyogenin and / or nuMyoD1, where an antibody against the markers CD99 / CD8 is conjugated to a first fluorescent dye and represents the first marker pair, CD99 / CD8; an antibody against the markers EpCAM / CD4 is conjugated to a second fluorescent dye and represents the second marker pair, EpCAM / CD4; and an antibody against CD271 is conjugated to a third fluorescent dye as is an antibody against either cyCD3 or smCD3 and represents the third marker pair, CD271 / cyCD3 or CD271 / smCD3. To allow for stepwise staining of cell surface markers on intact cells in an aliquot of the test sample, and then permeabilizing the same aliquot and staining of intracellular and nuclear markers, the antibodies for the cytoplasmic and nuclear markers are physically separated from the antibodies against the cell surface markers (i.e., not mixed with the antibodies against the cell surface markers). Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, the present invention provides a kit of parts for the flow cytometric detection of pediatric tumor cells, comprising fluorochrome-conjugated antibodies directed against the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271, the cytoplasmic marker cyCD3, and one or more nuclear markers nuMyogenin and / or nuMyoD1, wherein: (i) antibodies against the markers CD99 / CD8 are conjugated to the same fluorochrome and represent the first marker pair CD99 / CD8; (ii) an antibody against the marker EpCAM / CD4 is conjugated to the same fluorochrome, as well as representing the second marker pair EpCAM / CD4; (iii) antibodies against CD271 are conjugated to the same fluorochrome as antibodies against either cyCD3 or smCD3, as well as representing a third marker pair, CD271 / cyCD3 or CD271 / smCD3; wherein the kit comprises antibodies conjugated to eight or more distinguishable fluorescent dyes (a combination of antibodies of at least eight colors); and wherein the fluorescent dyes are distinguishable between the first, second, and third marker pairs; and wherein the antibodies against the cytoplasmic marker and the nuclear marker are physically separated from the antibodies against the cell surface marker.

[0012] This kit for staining aliquots of single-cell suspensions contains a unique panel of fluorochrome-conjugated antibody combinations for a set of 12 "backbone markers," where the antibodies for some selected markers are each conjugated to a unique, different fluorochrome, while the antibodies for other selected markers are "paired," i.e., the antibodies for the different markers are conjugated to the same fluorochrome.

[0013] For example, in the kit of the present invention, antibodies against four selected markers, CD45, CD56, GD2, and nuMyogenin, are each conjugated to a different fluorochrome (i.e., fluorochromes 1 to 4, respectively), while antibodies against each of the marker pairs, CD99 / CD8, EpCAM / CD4, and CD271 / cy / smCD3, are combined with another, but different, fluorochrome (i.e., fluorochromes 5 to 8, respectively) (i.e., antibody combination 1 in Table 1).

[0014] Various kit designs are encompassed, each comprising at least two containers (reagent tubes). For example, the kit may include two or more containers, the total number of containers constituting a unique antibody panel as provided herein. In one embodiment, the bound antibodies to the cell surface markers are divided across two or more containers. Similarly, the bound antibodies to the cytoplasmic marker cyCD3 and the one or more nuclear markers nuMyogenin and / or nuMyoD1 may be present in separate containers. For convenience, the kit preferably comprises a first reagent composition contained in a first container, the first reagent composition comprising a mixture of bound antibodies to the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19, and CD271, and a second reagent composition contained in a second container, the second reagent composition comprising bound antibodies to the cytoplasmic marker cyCD3 and the one or more nuclear markers nuMyogenin and / or nuMyoD1.

[0015] According to the present invention, both the cytoplasmic (cy) and surface membrane (sm) versions of CD3 are used as markers, and the marker CD271 is always paired with either cyCD or smCD3. The marker cyCD3 can be a unique marker or form a marker pair with CD271, while the marker smCD3 can form a marker pair with either CD271 or CD19.

[0016] In one embodiment, the kit of parts comprises a third marker pair, an antibody against CD271 / cyCD3, and an antibody against the markers smCD3 / CD19, conjugated to the same fluorochrome to form a fourth marker pair, smCD3 / CD19, and wherein the fluorochromes are distinguishable between different pairs. See, for example, any one of combinations 1-3, 7-16, and 18-29 in Table 1. In another embodiment, the kit of parts comprises a third marker pair, CD271 / smCD3, and wherein the antibodies against the markers CD19 and cyCD3 are conjugated to different fluorochromes, respectively. See, for example, any one of combinations 4-6, 17, and 30 in Table 1.

[0017] The kits of the present invention are further characterized by the presence of antibodies against one or both of the nuclear markers nuMyogenin and nuMyoD1. In one embodiment, only the marker nuMyogenin is used. See, for example, any one of combinations 1, 4, 7, 9, 11, 13, 15, 18, 23-26 in Table 1. Thus, the 12 markers can be coupled to a total of nine fluorochromes: six markers (CD45, CD56, GD2, nuMyogenin, CD19, and cyCD3) are each coupled to a different fluorochrome, and the other six markers are arranged in multiple antibody pairs (CD99 / CD8, EpCAM / CD4, and smCD3 / CD271), with each pair coupled to a different fluorochrome. In yet another embodiment, only the marker nuMyoD1 is used. See, for example, any one of combinations 2, 5, 8, 10, 16, 17, 19, 21, 27, 29-30 in Table 1. In yet another embodiment, the present invention provides a kit of parts comprising antibodies to the markers nuMyogenin and nuMyoD1, wherein the antibodies to the markers nuMyogenin / nuMyoD1 are conjugated to the same fluorochrome to form a further marker pair nuMyogenin / nuMyoD1, and between different pairs (i.e., CD99 / CD8 pair, EpCAM / CD4 pair, CD271 / (cy / sm)CD3 pair, and nuMyogenin / nuMyoD1 pair), the fluorochrome is distinguishable. Antibodies against the markers nuMyogenin / nuMyoD1 can be conjugated to the same fluorochrome to form additional marker pairs, such as nuMyogenin / nuMyoD1, and the fluorochromes can distinguish between different pairs (i.e., CD99 / CD8 pair, EpCAM / CD4 pair, CD271 / (cy / sm)CD3, and nuMyogenin / nuMyoD1 pair). See, for example, any one of combinations 3, 6, 12, 14, 20, 22, and 28 in Table 1.

[0018] Further embodiments of the present invention relate to any of the above antibody combinations extended to include additional markers coupled with up to five different additional fluorochromes, for a total of 12-14 colors, for further identification and characterization of Hodgkin's lymphoma cells, germ cell tumors, and / or bone tumors. For Hodgkin's lymphoma cells, one or more markers may be selected from the following proteins: HLADR, CD30, CD71, CD40, and CD95. For further characterization of germ cell tumors, OCT-3 / 4, BAP, and / or PLAP markers may be used, while for bone tumors, osteopontin and / or bone alkaline phosphatase may be used in addition to the 12 backbone markers.

[0019] Thus, in some embodiments, the kit of parts according to the invention further comprises fluorochrome-conjugated antibodies against one or more of the Hodgkin's lymphoma cell surface markers HLA-DR, CD30, CD71, CD40, and CD95. As shown by exemplary antibody combinations 11-14, 17-30 in Table 1, a unique fluorophore is used for each of the one or more additional antibodies. In the kit, they may be suitably combined in admixture with antibodies against the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19, and CD271.

[0020] Alternatively or additionally, a kit of parts according to the invention may further comprise fluorochrome-conjugated antibodies to one or more of the germ cell tumor cell surface markers OCT-3 / 4, BAP, and PLAP. In one aspect, the kit comprises antibodies to the markers OCT-3 / 4 and PLAP, wherein the antibodies to the markers OCT-3 / 4 / PLAP are conjugated to the same fluorochrome to form the marker pair OCT-3 / 4 / PLAP, and wherein the fluorochromes are distinguishable between different pairs.

[0021] See exemplary antibody combinations 7, 8, 25-29 in Table 1.

[0022] Alternatively or additionally, the kit of parts may further comprise fluorochrome-conjugated antibodies against one or both of the bone tumor cell surface markers osteopontin and bone alkaline phosphatase (BAP).

[0023] In one aspect, the kit includes antibodies to the markers osteopontin and BAP, and wherein the antibodies to the marker osteopontin / BAP are conjugated to the same fluorescent dye to form the marker pair osteopontin / BAP, and wherein the fluorescent dyes are distinguishable between different pairs. See exemplary antibody combinations 7, 8, 21, 22, 24, 26-29 in Table 1.

[0024] In certain embodiments, four Hodgkin's lymphoma markers (i.e., HLADR, CD30, CD40, and CD95), one or a mixture of two or three of the above germ cell tumor markers (e.g., OCT-3 / 4, BAP, and / or PLAP), and one or both of the bone tumor markers are combined with the backbone markers described in combinations 1, 3, and 5 (e.g., antibody combinations 18-29 in Table 1) at six additional fluorochrome positions.

[0025] A preferred aspect of the present invention relates to a kit of parts comprising one or more antibody combinations selected from antibody combinations 1 to 30 in Table 1.

[0026] Any of the above antibody combinations can be combined with a reagent to remove debris and / or unlysed cells, such as a dye capable of detecting nucleated cells. Thus, a kit of parts is also provided that further comprises a nucleated cell integrity dye.

[0027] The specific fluorochromes to which antibodies for use in the present invention are conjugated can be selected from a wide variety of fluorochromes known in the art and / or from fluorochromes yet to be developed. In one embodiment, the following fluorochrome(s) or any other combination of fluorochromes and / or fluorochrome tandems that can be measured simultaneously in a greater than 8-color flow cytometer are used: i) Pacific Blue (PacB), brilliant violet (BV) 421, Cascade blue, Alexa fluor (AF) 405, eFluor (EF) 450, Horizon V450 (HV450), Vio-Blue, Dylight 405, Super Bright 436, BV480; ii) Pacific Orange (PacO), OC515, BV510, BV480, Cascade yellow, BV570, VioGreen, AF430, Amcyan, HV500, khrome orange (KO), BUV496, EF506, Qdot 525, Qdot 545, BUV563, Qdot 565; iii) Super Bright 600, BV605, eVolve 605 Qdot 585, Qdot 605; iv) BV650, EF625NC, EF700NC, Super Bright 645, eVolve 655, Qdot 655; v) Qdot 700, Super Bright 702; vi) BV711, Qdot 705; vii) BV750, BV785, BV786, Qdot 800; viii) fluorescein isothiocyanate (FITC), AF488, BB515, VioBright FITC, VioBright 515, Cy2, Oregon Green 488, Dylight 488, AF500, EF 525NC, AF514, AF532; ix) phycoerythrin (PE); x) Cy3, AF555, AF546, Delight (Dylght) 550; xi)PE-CF594, PE-EF610, PE-Vio615, PE / Dazzle 594, AF568, PE-alexa 594; xii)EF585NC, EF605NC, PE-Texas red(ECD), PE-AF610; xiii) Peridinin chlorophyll protein (PerCP), PerCP-EF710, PERCP-Vio700, PerCPCy5.5, peridinin Peridinin cyanin 5 (PE-cy5), BB660, BB700; xiv) PEcy7, PE-Vio770, PE-AF750, PE-AF700, PERCP-EF710, BB790; xv) allophycocyanin (APC), AF647, Cy5, EF660, AF660, AF633, EF625NC, APC-Cy5.5, EF700NC; APC-R700; xvi) AF680, APC-A680, AF700, APC-A700; xvii) APC-hilite7 (APCH7), APC-R700, APC / Fire 750, APC-Vio 770, AF680, APC-A750, APC-C750, AF700, APC-EF780, APC-cy7, Cy7, AF750, AF790.

[0028] The kits of parts provided herein may further include reagents or solutions for fixing and permeabilizing cells, optionally along with instructions, buffers, and / or control samples. In one embodiment, the fixation reagent comprises about 0.3 to 1.5% w / v, e.g., 0.5% or 1% w / v, paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.4). An exemplary permeabilization reagent comprises about 0.1% to about 0.5% w / v saponin diluted in PBS.

[0029] A further aspect of the present invention is a multicolor flow cytometry method for the identification and classification of pediatric (solid) tumors, comprising: (a) staining an aliquot of a biological sample containing or suspected of containing pediatric tumor cells with a fluorochrome-conjugated antibody against a cell surface marker contained in a kit of parts according to the invention; then (b) contacting the stained cells with a fixative; and then (c) permeabilizing the fixed and stained cells with a permeabilization solution; then (d) staining the permeabilized cells with fluorochrome-conjugated antibodies against intracellular markers (cytoplasmic and nuclear markers) contained in the kit of parts according to the invention; (e) analyzing the stained cells in the aliquot in a flow cytometer; and (f) storing and evaluating the data obtained; The present invention relates to the above method, which comprises the steps of:

[0030] Although the kit and proposed antibody combinations are exemplified herein below by the study of primary tumor tissue, the kit can also be administered by analysis of bone marrow, peripheral blood, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid, vitreous fluid, synovial fluid, bronchoalveolar lavage, urine, and any other type of biological sample obtained from pediatric patients under investigation for pediatric tumors, such as non-Hodgkin's lymphoma, neuroblastoma, Wilms' tumor, germ cell tumors, soft tissue sarcomas (e.g., rhabdomyosarcoma, Ewing's sarcoma family of tumors, chondrosarcoma, osteosarcoma), and epithelial cell tumors.

[0031] In one embodiment, the biological sample is a primary tumor tissue sample; peripheral blood; bone marrow; a tissue sample, such as a lymph node, adenoid, spleen, or liver; or other types of body fluids, such as cerebrospinal fluid, vitreous fluid, synovial fluid, final needle aspirate, pleural effusion, or peritoneal fluid, wherein the sample is obtained from a pediatric patient.

[0032] For example, an aliquot of tissue is placed in phosphate-buffered saline (PBS) and subjected to mechanical dissociation to prepare a single-cell suspension suitable for further flow cytometry analysis for maximum cell viability and recovery. To this end, the tissue is preferably placed (e.g., in a Petri dish) in PBS containing 0.5% w / v bovine serum albumin, then minced into small pieces (2-4 mm) and mechanically dissociated with a sterile needle. The resulting tumor cell suspension is subsequently filtered (e.g., using a sterile syringe with a 120 mm pore size) to remove cell clumps and debris, centrifuged, and resuspended in PBS / 0.5% w / v BSA to a final concentration of 5x10 cells. 5 A 50 μL (i.e., 50,000) aliquot of the single cell suspension can be placed in a separate tube.

[0033] In another example, to examine the immunophenotype of malignant cells present in, for example, metastatic sites, ascites, pleural, and / or urine samples are collected. The samples (cell suspensions) are sequentially filtered through a sterile syringe to remove cell clumps and debris; centrifuged; and then diluted to a final concentration of 5x10 5 Resuspend at 10 or more cells / tube.

[0034] An aliquot of the single cell suspension is then contacted with each of the fluorochrome-conjugated antibodies against cell surface markers comprised in the kit as defined hereinabove. The cells and antibody reagents are thoroughly mixed and incubated, protected from light, for example, at room temperature for 30 minutes to allow the antibodies to bind to one or more cell surface markers, if present on the cells. After this incubation, the cells are washed and centrifuged to remove unbound antibody, leaving a residual volume of approximately 50 μL in each tube for further staining of intracellular markers (i.e., cytoplasmic and nuclear markers) in accordance with the present invention.

[0035] To this end, the cell pellet is fixed with a fixative, e.g., Fix&Perm 商標 The cells are resuspended by gentle mixing with reagent A (a fixative containing PFA) from the reagent kit (An der Grub, Vienna, Austria), followed by a further incubation for 15 minutes at room temperature in the dark. Subsequently, the cells are washed, and the cell pellet is resuspended in a permeabilization solution (e.g., Fix & Perm, which contains a permeabilizing agent, such as saponin). 商標 The cells are resuspended by gentle mixing in Reagent B) of the kit. After gentle mixing, an appropriate amount of each antibody against an intracellular marker included in the kit of the present invention is added, mixed, and incubated for 15 minutes at room temperature in the dark.

[0036] Unbound antibody was removed by washing with, for example, PBS containing 0.09% NaN3 and 0.5% w / v BSA, leaving a residual volume of approximately 50 μL in each tube. After thorough mixing, the residual volume of the cell pellet was appropriately resuspended in 200 μL of PBS containing 0.09% NaN3 and 0.5% w / v BSA and immediately measured in a multicolor flow cytometer, for example, a flow cytometer equipped with four lasers and 13 fluorescence detectors, such as a BD LSRFortessa X-20 flow cytometer.

[0037] For data analysis, conventional manual Boolean gating strategies or automated clustering analysis, with or without direct comparison to software databases, can be used via flow cytometry software programs that allow manual and / or automated gating and analysis of FCS files.

[0038] For example, first, a gate (G1) on SSClo / FSClo / CD45hi cells is performed to identify lymphocytes. This G1 cell population can then be further subsetted by drawing four additional gates to identify lymphocyte subsets in the following manner: CD45 for T cells; hi / smCD3 + / cyCD3 + (G1A), CD45 for B cells hi / CD19 + (G1B), CD45 for plasma cells +lo / CD19 + (G1C) and CD45 for NK cells + / smCD3 - / cyCD3 - / CD56 + (G1D). T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 +and CD4 - / CD8 - These can be further subdivided into T cell subsets.

[0039] SSC int / hi / FSC int / hi / CD45 + A second gate (G2) to contain cells can be drawn to identify myeloid cells. + / CD4 + Gates were established for myeloid cells to separate monocytes and dendritic cells from neutrophils and eosinophils (SSCs). hi / CD45 + ), further subsets of myeloid cell populations can be achieved.

[0040] Finally, gate (G3) was designed to select for tumor cells, erythroblasts, endothelial cells, and mesenchymal stromal cells by CD45 - This can be performed in cells, where Wilms' tumor cells (Figure 1) express CD45 - / CD56 +hi / CD271 + / GD2 - / EpCAM - / + / CD99 - / nu Myogenin - Rhabdomyosarcoma cells are characterized by their CD45 phenotype (Figure 1). - / CD56 +hi / CD271 +hi / GD2 - / EpCAM - / CD99 - / nu Myogenin + Neuroblastoma cells are characterized by a CD45 - / CD56 +hi / CD271 - / + / GD2 +hi / EpCAM - / CD99 - / nu Myogenin -(Figure 3), and PNET cells are characterized by CD45 - / CD56 + / CD271 +hi / GD2 +lo / EpCAM - / CD99 + / nu Myogenin - Figure 1 illustrates all exemplary data analysis steps in Wilms tumor samples. Table 2 shows the percentages of neoplastic and immune cells in the various pediatric solid tumor samples analyzed.

[0041] As will be appreciated by those skilled in the art, the antibody panel, kit or kits according to the present invention can be advantageously used in the field of childhood cancer diagnosis, for example, in the early diagnosis, diagnostic subclassification, staging and / or monitoring of childhood cancers, both at the primary tumor tissue site and at metastatic sites (e.g., ascites, pleural effusion, urine, bone marrow, cerebrospinal fluid, lymph nodes and bronchoalveolar lavage, among other tumor specimens) and in peripheral blood. It allows to accurately distinguish tumor cells from other residual cells in a sample and to characterize different subsets of immune cells coexisting with tumor cells in the same sample. In addition, it can be used as a tool for sensitive and rapid classification of childhood solid tumors (e.g., to identify 10 or more of the remaining cells in a sample). -1 ~10 -5 It further provides numerical and phenotypic information about the tumor-associated microenvironment, as it provides a comprehensive analysis of tumor tissue (detecting tumor cells) and allows for the identification and enumeration of major lymphocytes, neutrophils, monocytes / macrophages, dendritic cell populations, and mesenchymal cells in infiltrating and non-infiltrating patient samples.

[0042] The procedure includes the following sequential steps: i) collecting a biological sample from a patient suspected of having a childhood cancer; ii) staining the biological sample with a panel of 12 or more antibodies coupled to 8 individual fluorochromes in an 8-color antibody stain (e.g., a combination according to Table 1) for the purpose of identifying and classifying coexisting tumor cells and infiltrating immune cells in the biological sample; iii) measuring the stained cells in a conventional 8+ color flow cytometry device; and iv) analyzing the obtained flow cytometry data using a dedicated software tool to unambiguously identify coexisting tumor and normal cells in the sample, further enumerate such tumor cells, define the per-cell expression level of each marker expressed thereon, classify the tumor cells into WHO diagnostic entities according to their immunophenotypic profile, and identify and enumerate different populations of immune cells and their major subsets coexisting with the tumor cells in the sample.

[0043] The procedures described herein can be used for the diagnosis and classification of the most common types of pediatric tumors, including: i) neuroectodermal neoplasms, such as neuroblastoma, ganglioneuroblastoma, ganglioneuromatosis, extraskeletal Ewing's sarcoma, and classical Ewing's sarcoma, whose most useful antigen is CD45 - , CD56 ++ , CD99 -or+ , GD2 ++ and CD271 + ii) CD45 - , CD56 + , anti nu Myogenin + , anti nu MyoD1 + and CD271 + iii) tumors with myofibroblastic differentiation, as assessed by phenotype; and iii) Wilms' tumor, e.g., CD45 - , CD56 ++ , CD271 + and EpCAM + Identification of multiple cell lineage commitments defined by expression patterns of cyCD3; and iv) cyCD3+ and CD45 ++ CD19 - Tumor cells and CD19 + CD45 - / + cyCD3 - Expression on tumor cells is characteristic of T and B lymphoblastic lymphoma / leukemia.

[0044] Furthermore, the present invention also provides a method for detecting lymphocytes (CD45 ++ / SSC lo cells), e.g., cyCD3 + / CD3 + T cells, CD19 + B-cells and CD19 - / CD3 - / CD56 + NK cells, CD19 + / CD45 lo Plasma cells and CD4 + / SSC int It allows the simultaneous identification by flow cytometry of the above lymphocytes, including monocytes and dendritic cells, as well as CD271++ mesenchymal and endothelial cells.

[0045] Based on the expression of CD56, CD4, and CD8, T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + , CD4 - / CD8 - Each of these T cell subsets can be further divided into CD56-expressing and CD56-nonexpressing subsets. + NK cells express CD56 +hi , CD56 +lo / CD8 - , CD56 +lo / CD8 + can be further subdivided into NK subsets.

[0046] Of note, each of the kits and antibody combinations provided herein for identifying and classifying tumor cell populations also provides a means for quantifying protein expression levels per tumor cell, which is therefore useful not only for the diagnosis, classification, and monitoring of pediatric solid tumors, but also for the selection of targeted and appropriate therapies (e.g., GD2 + Neuroblastoma and other GD2 + The present invention also provides important information for the selection of anti-GD2 antibodies in tumors. The present invention can also be used to detect and enumerate tumor cells in bone marrow and peripheral blood samples before autologous stem cell transplantation, or for monitoring purposes after a patient has received any type of treatment. Furthermore, the present invention can also be used to identify tumor and non-tumor cells in small and / or non-mucous samples, such as vitreous humor, cerebrospinal fluid, and fine needle aspirate tissue samples, for disease staging purposes, diagnosing disease recurrence, or monitoring patients for residual disease levels.

[0047] The diagnostic results of the flow cytometry method of the present invention are advantageously used to aid in selecting an appropriate (targeted) therapy, such as an anti-GD2 antibody-based or chimeric antigen receptor (CAR) T-cell therapy.

[0048] The present invention also provides for the use of the kits of parts disclosed herein in the diagnosis and classification of one or more pediatric tumors, for example, selected from i) neuroectodermal neoplasms, such as neuroblastoma, ganglioneuroblastoma, ganglioneuromatosis, extraskeletal Ewing's sarcoma, and classical Ewing's sarcoma; ii) tumors with myofibroblastic differentiation; iii) identification of multiple lineage commitments; and iv) T and B-lymphoblastic lymphoma / leukemia. The present invention may be configured as follows. [Section 1] 1. A kit of parts for flow cytometric detection of pediatric tumor cells, the kit comprising fluorochrome-conjugated antibodies directed against cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271, a cytoplasmic marker cyCD3, and one or more nuclear markers nuMyogenin and / or nuMyoD1, wherein: (i) antibodies against the markers CD99 / CD8 are conjugated to the same fluorochrome and represent the first marker pair CD99 / CD8; (ii) an antibody against the marker EpCAM / CD4 is conjugated to the same fluorochrome, as well as representing the second marker pair EpCAM / CD4; (iii) antibodies against CD271 are conjugated to the same fluorochrome as antibodies against either cyCD3 or smCD3, as well as representing a third marker pair, CD271 / cyCD3 or CD271 / smCD3; wherein the kit comprises antibodies conjugated to eight or more distinguishable fluorescent dyes; wherein the fluorescent dyes are distinguishable between the first, second, and third marker pairs; and wherein the antibodies against the cytoplasmic marker and the nuclear marker are physically separate from the antibodies against the cell surface marker. [Section 2] Item 1. The kit of parts according to Item 1, comprising: a first reagent composition contained in a first container, the first reagent composition comprising the conjugated antibodies against the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19, and CD271; and a second reagent composition contained in a second container, the second reagent composition comprising the conjugated antibodies against the cytoplasmic marker cyCD3 and the one or more nuclear markers nuMyogenin and / or nuMyoD1. [Section 3] Item 3. The kit of parts according to item 1 or 2, further comprising a third marker pair, CD271 / cyCD3, wherein the antibody against the marker smCD3 / CD19 is bound to the same fluorescent dye to form a fourth marker pair, smCD3 / CD19, and wherein the fluorescent dyes are distinguishable between different pairs. [Section 4] Item 3. The kit of parts according to item 1 or 2, further comprising a third marker pair, CD271 / smC3, wherein the antibodies against the markers CD19 and cyCD3 are each conjugated to a different fluorescent dye. [Section 5] The kit of parts according to any one of items 1 to 4, further comprising antibodies against the markers nuMyogenin and nuMyoD1, wherein the antibodies against the markers nuMyogenin / nuMyoD1 are bound to the same fluorescent dye to form a fifth marker pair nuMyogenin / nuMyoD1, and wherein the fluorescent dyes are distinguishable between different pairs. [Section 6] Item 6. The kit of parts according to any one of Items 1 to 5, wherein the first reagent composition further comprises a fluorescent dye-conjugated antibody against one or more of Hodgkin's lymphoma cell surface markers: HLA-DR, CD30, CD71, CD40, and CD95. [Section 7] Item 7. The kit of parts according to any one of Items 1 to 6, further comprising a fluorescent dye-conjugated antibody against one or more germ cell tumor cell surface markers, OCT-3 / 4, BAP, and PLAP. [Section 8] Item 8. The kit of parts according to Item 7, comprising antibodies against the markers OCT-3 / 4 and PLAP, wherein the antibodies against the markers OCT-3 / 4 / PLAP are bound to the same fluorescent dye to form a marker pair OCT-3 / 4 / PLAP, and wherein the fluorescent dyes are distinguishable between different pairs. [Section 9] Item 9. The kit of parts according to any one of Items 1 to 8, further comprising a fluorescent dye-conjugated antibody against one or more of osteopontin and bone alkaline phosphatase, which are bone tumor cell surface markers. [Section 10] Item 10. The kit of parts according to Item 9, comprising antibodies against the markers osteopontin and BAP, and wherein the antibodies against the marker osteopontin / BAP are bound to the same fluorescent dye to form a marker pair osteopontin / BAP, and wherein the fluorescent dye is distinguishable between different pairs. [Section 11] Item 11. The kit of parts according to any one of Items 1 to 10, comprising one or more antibody combinations selected from antibody combinations 1 to 30 in Table 1. [Section 12] Item 12. The kit of parts according to any one of Items 1 to 11, further comprising a nucleated cell integrity dye. [Section 13] Item 13. The kit of parts according to any one of Items 1 to 12, further comprising a reagent for fixing and permeabilizing cells, and optionally, an instruction manual, a buffer, and / or a control sample. [Section 14] 1. A multicolor flow cytometry method for the identification and classification of pediatric tumors, comprising: (a) staining an aliquot of a biological sample containing or suspected of containing pediatric tumor cells with a fluorochrome-conjugated antibody against a cell surface marker contained in the kit of parts described in any one of paragraphs 1 to 13; and then (b) contacting the stained cells with a fixative; and then (c) permeabilizing the fixed and stained cells with a permeabilization solution; then (d) staining the permeabilized cells with fluorescent dye-conjugated antibodies against cytoplasmic and nuclear markers contained in the kit of parts according to any one of Items 1 to 13; (e) analyzing the stained cells in the aliquot in a flow cytometer; and (f) storing and evaluating the data obtained; The method, comprising the steps of: [Section 15] 15. The method of claim 14, wherein the biological sample is a primary tumor tissue sample, peripheral blood, bone marrow, a tissue sample such as a lymph node, adenoid, spleen or liver, or other type of body fluid such as cerebrospinal fluid, vitreous fluid, synovial fluid, final needle aspirate, pleural effusion or ascites, wherein the sample is obtained from a pediatric patient. [Section 16] Item 16. The method of item 14 or 15, further comprising selecting an appropriate targeted therapy. [Section 17] 14. Use of the kit of parts according to any one of items 1 to 13 in the diagnosis and classification of one or more pediatric tumors, preferably one or more selected from: i) neuroectodermal neoplasms, such as neuroblastoma, ganglioneuroblastoma, ganglioneuromatosis, extraskeletal Ewing's sarcoma and classical Ewing's sarcoma; ii) tumors with myofibroblastic differentiation; iii) identification of commitment to multiple cell lineages; and iv) T and B lymphoblastic lymphoma / leukemia. [Brief explanation of the drawings]

[0049] [Figure 1] Figure 1 shows sequential gating strategy analysis to identify major subset populations in tumor bulk samples using two-dimensional dot plots. Panel A shows the identification of three groups of cell populations: G1 (SSC1 / CD45+hi) represents lymphocytes, G2 (SSCint / hi / CD45+) represents myeloid cells, and G3 (SSCint-hi / CD45-) represents tumor cells. Within the G1 cell gate (panel B), four additional gates allowed the identification of the following major lymphocyte populations: C) CD45+hi / CD19+ B cells (G1B), and D) CD45+lo / CD19+ plasma cells (G1C); and F) CD45+ / smCD3- / cyCD3- / CD56+ NK cells (G1D). Further, T cells (CD45+hi / smCD3+ / cyCD3+ T cells G1A) were subdivided in panel E) as follows: (CD45+hi / smCD3+ / cyCD3+ T cells G1A), where T cells were subdivided in panel E as follows: CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, and CD4- / CD8- T cell subsets. In the G2 cell gate (panel G), three additional gates based on one bivariate dot plot (SSC / CD45) allowed for the identification of eosinophils (SSChi / CD45+), neutrophils (SSCint / CD45+lo), and monocytes (SSCint / CD45+); further, in panel H, a bivariate dot plot (SSC / CD4) allowed for better identification of neutrophils / eosinophils (SSCint / CD4-) and monocytes / macrophages / dendritic cells (SSCint / CD4+) in the G2 cell gate. Finally, in the G3 cell gate, tumor cells were characterized as CD45- / CD56+hi / CD271+ / GD2- / EpCAM- / + / CD99- / nuMyogenin phenotype (Wilms tumor). [Figure 2] Figure 2 shows the sequential gating strategy for identifying major subset populations in pleural fluid samples using two-dimensional dot plots. Panel A shows the identification of three groups of cell populations: G1 (SSC1 / CD45+hi) represents lymphocytes, G2 (SSCint / hi / CD45+) represents myeloid cells, and G3 (SSChi / CD45-) represents tumor cells. Within the G1 cell gate (panel B), four additional gates allowed the identification of the following major lymphocyte populations: C) CD45+hi / CD19+ B cells (G1B) and CD45+lo / CD19+ plasma cells (G1C); and D) CD45+ / smCD3- / cyCD3- / CD56+ NK cells (G1D). Further, T cells (CD45hi / smCD3+ / cyCD3+ T cells G1A) were subdivided in panel E into the following T cell subsets: CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, and CD4- / CD8-. G) After selection of G2 cells, three additional gates based on one bivariate dot plot (SSC / CD45) allowed for the identification of eosinophils (SSChi / CD45+), neutrophils (SSCint / CD45+lo), and monocytes (SSCint / CD45+); H) Further bivariate dot plot (SSC / CD4) analysis of the G2 population allowed for better identification of neutrophils (SSCint / CD4-) and monocytes / dendritic cells (SSCint / CD4+). Finally, the selected G3 population was analyzed to select and identify the most relevant cell markers to classify tumor cells characterized by the CD45- / CD56+hi / CD271+hi / GD2- / EpCAM- / CD99- / nuMyoD1+ phenotype (rhabdomyosarcoma). [Figure 3]Figure 3 shows the sequential gating strategy for identifying major subset populations in bone marrow using two-dimensional dot plots. Panel A shows the identification of three groups of cell populations: G1 (SSC1 / CD45+hi) represents lymphocytes, G2 (SSCint / hi / CD45+) represents myeloid cells, and G3 (SSChi / CD45-) represents tumor cells. Within the G1 cell gate (panel B), four additional gates allowed the identification of the following major lymphocyte populations: C) CD45+hi / CD19+ B cells (G1B) and CD45+lo / CD19+ plasma cells (G1C); and D) CD45+ / smCD3- / cyCD3- / CD56+ NK cells (G1D). Further, T cells (CD45hi / smCD3+ / cyCD3+ T cells G1A) were subdivided as follows in panel E) into CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, and CD4- / CD8- cell subsets. G) After selection of G2 cells, three additional gates based on one bivariate dot plot (SSC / CD45) allowed for the identification of eosinophils (SSChi / CD45+), neutrophils (SSCint / CD45+lo), and monocytes (SSCint / CD45+); H) Further bivariate dot plot (SSC / CD4) analysis of the G2 population allowed for better identification of neutrophils (SSCint / CD4-) and monocytes / dendritic cells (SSCint / CD4+). Finally, in the G3 cell gate, tumor cells were characterized as CD45- / CD56+hi / CD271- / + / GD2++ / EpCAM- / CD99- / nuMyoD1 phenotype (neuroblastoma), while nucleated red blood cells were presented as SSClo / CD45- / CD56- and mesenchymal cells as SSChi / CD45- / CD56- / +lo / CD271+hi. DETAILED DESCRIPTION OF THE INVENTION

[0050] Experimental section

[0051] The present invention is illustrated by the following examples, which are offered for purposes of illustration and not to limit the scope in any way.

[0052] Example 1: Analysis of tumor mass samples Sample Collection. Solid tumor specimens were collected in the surgical suite from 55 pediatric patients; tumor samples were sent to pathology, and an experienced pathologist divided the tumor samples into two aliquots: one for routine pathology examination and one for flow cytometry. The tissue aliquot used for flow cytometry was immediately placed in phosphate-buffered saline (PBS) on wet ice and transported to the flow cytometry laboratory. Upon arrival, the specimen was weighed, physical characteristics recorded, and the sample was subdivided into two pieces: one for fresh-frozen storage at −80°C and one for immediate mechanical disaggregation.

[0053] Mechanical disaggregation of tumor specimens. 55 tumor tissue specimens were immediately disaggregated into single-cell suspensions suitable for further flow cytometry analysis to maximize cell viability and recovery. To this end, the tissues were placed in a Petri dish in 2 ml of PBS containing 0.5% bovine serum albumin (BSA; Calbiochem, La Jolla, CA). Next, the tumor specimens were minced into small pieces (2-4 mm) with a scalpel blade and mechanically disaggregated with a sterile needle. The tumor cell suspension was then sequentially filtered through a sterile Filcon syringe (120 mm bore) to remove cell clumps and debris, centrifuged (540 x g for 10 min), and resuspended in 500 μl of PBS containing 0.5% BSA to a final concentration of 5 x 10 cells. 5 The cells were then stained in a single tube, with a total of 4 different aliquots per sample.

[0054] Sample Staining: 50 μl of sample (single cell suspension of disaggregated tissue) was added to each of four tube aliquots, followed by the addition of an appropriate amount (saturating concentration) of each of the corresponding reagent compositions containing antibodies directed against the cell surface markers recommended for this single-tube panel. i) CyCD3 BV421+CD271 BV421 / CD45 BV510 CD99 FITC+CD8 FITC / nuMyogenin PE / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 AF647 / csmD3 APC-H7+CD19 APC-H7 (i.e., antibody combination 1 in Table 1); ii) CyCD3 BV421+CD271 BV421 / CD45 PO / CD99 FITC+ CD8 FITC / nuMyoD1 PE / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 AF647 / cyCD3 APC-H7+CD19 APC-H7 (antibody combination 2 in Table 1); iii) CyCD3 BV786+CD271 BV786 / HLADR APC / CD45 AF700 / CD30 APCH7 / CD71 BV650 / CD95 BV421 / CD99 FITC+CD8 FITC / nuMyoD PE / CD40 BV711 / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 BV510 / smCD3 APC-H7+CD19 APC-H7 (antibody combination 17 in Table 1); iv) CyCD3 BV786+CD271 BV786 / HLADR PECF594 / CD45 AF700 / CD30 BV650 / CD99 FITC+CD8 FITC / GD2 BV510 / osteopontin APC / numyogenin PE / CD40 BV711 / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / OCT3 APCH7 / CD95 BV421 / smCD3 BV605+CD19 BV605 (antibody combination 18 in Table 1).

[0055] The cells and antibody reagent were then thoroughly mixed and incubated at room temperature for 30 minutes, protected from light. After this incubation, 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed thoroughly, and centrifuged at 540 x g for 5 minutes. The supernatant was then discarded using a Pasteur pipette or vacuum device without disturbing the cell pellet, leaving approximately 50 μL of residual volume in each tube. The cell pellet was resuspended by gentle vortexing and resuspended in Fix & Perm 商標 100 μL of Reagent A (fixative containing PFA) from the reagent kit (An der Grub, Vienna, Austria) was added and further incubated for 15 min at room temperature in the dark. Subsequently, 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed thoroughly, and centrifuged at 540 × g for 5 min.

[0056] The supernatant is then discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and a residual volume of approximately 50 μL is left in each tube, and the cell pellet is resuspended by gentle mixing and resuspended in Fix&Perm 商標100 μL of Reagent B (saponin-containing permeabilization solution) from the kit was added. After gentle mixing, an appropriate amount of each antibody against intracellular markers (nuMyoD1, nuMyogenin, OCT3, and cyCD3) was added, mixed, and incubated at room temperature for 15 minutes, protected from light. 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was then added, mixed thoroughly, and centrifuged at 540 x g for 5 minutes. The supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μL in each tube. After thorough mixing, the cell pellets were resuspended in 200 μL of PBS containing 0.09% NaN3 and 0.5% BSA and immediately analyzed on a BD LSRFortessa X-20 flow cytometer equipped with four lasers and 13 fluorescence detectors.

[0057] Data analysis. First, SSC lo / FSC lo / CD45 hi A gate on cells (G1) was performed and selected to identify lymphocytes; the G1 cells were then further subsetted by drawing four additional gates to identify lymphocyte subsets in the following manner: CD45 for T cells; hi / smCD3 + / cyCD3 + (G1A), CD45 for B cells hi / CD19 + (G1B), CD45 for plasma cells +lo / CD19 + (G1C), and CD45 for NK cells + / smCD3 - / cyCD3 - / CD56 + (G1D). T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + and CD4 - / CD8 -These were further subdivided into T cell subsets (panel E in Figure 1). int / hi / FSC int / hi / CD45 + A second gate (G2) was drawn to include cells that were myeloid. + / CD4 + Gates were established to separate monocytes and dendritic cells from neutrophils and eosinophils (SSCs) hi / CD45 + Further subsetting of myeloid cell populations was achieved by identifying CD45+ / CD45+ cells. Finally, gate (G3) was used to select tumor cells, erythroblasts, endothelial cells, and mesenchymal stromal cells. - The study was performed in Wilms' tumor cells, where the CD45 - / CD56 +hi / CD271 + / GD2 - / EpCAM - / + / CD99 - / nu Myogenin - Rhabdomyosarcoma cells are characterized by a CD45 phenotype (Figure 1). - / CD56 +hi / CD271 +hi / GD2 - / EpCAM - / CD99 - / nu Myogenin + Neuroblastoma cells are characterized by a CD45 - / CD56 +hi / CD271 - / + / GD2 +hi / EpCAM - / CD99 - / nu Myogenin - (Figure 3), and PNET cells are characterized by CD45 - / CD56 + / CD271 +hi / GD2 +lo / EpCAM - / CD99 + / nu Myogenin -Figure 1 illustrates all the data analysis steps in Wilms tumor samples, and Table 2 shows the percentages of neoplastic and immune cells in the various pediatric solid tumor samples analyzed.

[0058] Example 2: Analysis of ascites, pleural fluid and urine samples Sample collection. To investigate the immunophenotype of malignant cells present at metastatic sites, samples from five children previously diagnosed with childhood cancer were examined: one ascites sample, three pleural fluid samples, and one urine sample. These samples were collected in the operating room or intensive care unit and processed in the following order at the time of diagnosis or recurrence: First, the sample (cell suspension) was sequentially filtered through a sterile Filcon syringe (120 mm pore size) to remove cell clumps and debris; then, it was centrifuged (540 × g for 10 min) and resuspended in 500 μl of PBS containing 0.5% BSA to a final concentration of 5 × 10 5 Next, four aliquots of 50 μL (i.e., 50,000 cells) of the single cell suspension were made and placed in different tubes.

[0059] Staining of samples. 50 μl of sample (single cell suspensions of different body fluids) was added to each of four tube aliquots, followed by the addition of appropriate amounts (saturating concentrations) of each of the corresponding antibodies directed against cell surface markers, as recommended for the following single-tube fluorochrome-conjugated antibody combinations: i) CyCD3 BV421 + CD271 BV421 / CD45 BV510 / CD99 FITC + CD8 FITC / nuMyogenin PE / EpCAM PERCPcy5.5 + CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 APC / smCD3 APCH7 / CD19 APC-H7 (antibody combination 1 in Table 1); ii) smCD3 BV421 + CD271 BV421 / CD45 BV510 / CD99 FITC + CD8 FITC / nuMyoD1 PE / EpCAM PERCPcy5.5 + CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 APC / cyCD3 APCH7 / CD19 BV786 (i.e., antibody combination 4 in Table 1); iii) CyCD3 BV421+CD271 BV421 / HLADR APC / CD45 AF700 / CD30 APCH7 / CD71 BV650 / CD99 FITC+CD8 FITC / numyogenin PE / CD40 BV711 / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 AF647 / smCD3 APC-H7+CD19 APC-H7 (i.e., antibody combination 13 in Table 1); and iv) CyCD3 BV786+CD271 BV786 / HLADR PECF594 / CD45 AF700 / CD30 BV650 / CD99 FITC+CD8 FITC / GD2 BV510 / osteopontin APC+BAP APC / nuMyogenin PE+nuMyoD1 PE / CD40 BV711 / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / PLAP APCH7 / CD95 BV421 / smCD3 BV605+CD19 BV605 (i.e., antibody combination 22 in Table 1). The cells and antibody reagents were then thoroughly mixed, and the mixture was incubated at room temperature for 30 minutes in the dark.After this incubation, 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed thoroughly, and centrifuged at 540 x g for 5 minutes. The supernatant was then discarded using a Pasteur pipette or vacuum device without disturbing the cell pellet, leaving approximately 50 μL of residual volume in each tube. The cell pellet was resuspended by gentle agitation and resuspended in Fix & Perm. 商標 100 μL of Reagent A (fixative containing PFA) from the reagent kit (An der Grub, Vienna, Austria) was added, and further incubated for 15 minutes at room temperature, protected from light. Subsequently, 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed thoroughly, and centrifuged at 540 × g for 5 minutes. Next, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 μL of residual volume was left in each tube. The cell pellet was resuspended by gentle mixing and then resuspended in Fix&Perm 商標 100 μL of Reagent B (saponin-containing permeabilization solution) from the kit was added. After gentle mixing, an appropriate volume of each intracellular antibody (nuMyoD1, nuMyogenin, osteopontin, BAP, PLAP, and cyCD3 APC-H7) was added, mixed, and incubated at room temperature for 15 minutes, protected from light. 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was then added, mixed, and centrifuged at 540 x g for 5 minutes. The supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μL in each tube. Upon thorough mixing, the remaining volume of the cell pellet was resuspended in 200 μL of PBS containing 0.09% NaN3 and 0.5% BSA and immediately measured on a BD Symphony X-20 flow cytometer equipped with 5 lasers and 48 fluorescence detectors.

[0060] Data analysis. First, SSC lo / FSClo / CD45 +hi A gate on cells (G1) was performed and selected to identify lymphocytes; the G1 cells were then further subsetted by drawing four additional gates to identify lymphocyte subsets in the following manner: CD45 for T cells; +hi / smCD3 + / cyCD3 + (G1A), CD45 for B cells +hi / CD19 + (G1B), CD45 for plasma cells +lo / CD19 + (G1C), and CD45 for NK cells + / smCD3 - / cyCD3 - / CD56 + (G1D). T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + and CD4 - / CD8 - Second, SSCs were further subdivided into T cell subsets (Figure 2, Panel E). int / hi / FSC int / hi / CD45 + A gate containing cells (G2) can be performed to identify myeloid cells. A further subset of the myeloid cell population expresses CD45 + / CD4 + This was achieved by establishing a gate on myeloid cells to distinguish between neutrophils and eosinophils from leukocytes and dendritic cells. Finally, gate (G3) was established to differentiate CD45 - / CD56 + / CD271 + / GD2 - / EpCAM - / + / CD99 - / nu Myogenin - CD45 was used to select tumor cells and Wilms' tumor cells that exhibited the phenotype - The present invention was performed on rhabdomyosarcoma cells, in which the rhabdomyosarcoma cells express CD45 - / CD56 + / CD271 + / GD2 - / EpCAM - / CD99 - / nu Myogenin + (Figure 2), and neuroblastoma cells express CD45 - / CD56 + / CD271 - / GD2 +hi / EpCAM - / CD99 - / nu Myogenin - and PNET cells express CD45 - / CD56 + / CD271 +hi / GD2 +lo / EpCAM - / CD99 + / nu Myogenin - Figure 2 illustrates all the gating steps performed during data analysis to identify both tumor cells and residual normal reactive immune cells in pleural fluid samples infiltrated with rhabdomyosarcoma cells; Table 3 then shows the percentage of neoplastic cells identified in the five fluid samples analyzed.

[0061] Example 3: Analysis of bone marrow samples Sample collection. Twenty-three bone marrow samples and ten peripheral blood samples taken from 23 cancer patients were investigated for the presence of metastatic dissemination during the staging procedure. At least 10 x 10 6 5x10 nucleated cells were lysed using the EUROFLOW bulk lysis protocol as previously described. 6To obtain more than 10 cells / tube, staining was performed in four different tubes / aliquots for each peripheral blood and bone marrow sample (Flores-Montero et al., Leukemia. 2017 Oct;31(10):2094-2103). Briefly, 2 ml of each sample was mixed with 50 ml of ammonium chloride lysis solution in a 50 ml Falcon tube and incubated for 15 minutes on a roller or sample shaker. The sample was then centrifuged at 800 x g for 10 minutes, and the supernatant was discarded using a Pasteur pipette without disturbing the cell pellet. Depending on the supernatant discarded, 0.09% NaN 3 The tube was refilled with PBS containing 0.09% NaN3 and 0.5% BSA to a final volume of 50 ml and centrifuged again at 800 x g (5 min). The supernatant was discarded without disturbing the cell pellet, and the cell pellet was resuspended in 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA. The cells were then transferred to 5 mL polystyrene round-bottom Falcon tubes ("FACS tubes") at a volume of 300 μl per tube. The tube was gently mixed with PBS containing 0.09% NaN3 and 0.5% BSA to a final volume of 2 mL and centrifuged at 540 x g for 5 min; the supernatant was then discarded using a Pasteur pipette without disturbing the cell pellet. This procedure was repeated twice. The final cell concentration was 5 x 10 5 The cell concentration was adjusted to 100 cells / μL in PBS containing 0.09% NaN3 and 0.5% BSA, and the final cell suspension / sample of approximately 100 μL (i.e., 10 million cells) per tube was stained and measured in a flow cytometer.

[0062] Staining of samples. 100 μl of the treated cell suspension was added to each of four tube aliquots prepared per sample, followed by the addition of an appropriate amount (saturating concentration) of the corresponding antibody directed against the cell surface marker recommended for the following single-tube fluorochrome-conjugated antibody combinations: i) CyCD3 BV421 + CD271 BV421 / CD45 BV510 / CD99 FITC + CD8 FITC / nuMyogenin PE + nuMyoD1 PE / EpCAM PERCPcy5.5 + CD4 PERCPcy5.5 / CD56 PEcy7 / GD2 APC / smCD3 APC-H7 + CD19 APC-H7 (i.e., antibody combination 3 in Table 1); ii) CyCD3 BV421 + CD271 BV421 / CD45 AF700 / CD99 FITC + CD8 FITC / GD2 BV510 / Osteopontin APC + BAP iii) APC / nuMyogenin PE / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / OCT-3 / 4 / APCH7+PLAP APCH7 / smCD3 BV786+CD19 BV786 (i.e., antibody combination 7 in Table 1); iii) CyCD3 BV421+CD271 BV421 / HLADR APC / CD45 AF700 / CD30 APCH7 / GD2 BV510 / CD99 FITC+CD8 FITC / nuMyogenin PE / CD40 BV711 / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / CD56 PEcy7 / smCD3 APC-H7+CD19 APC-H7 (i.e., antibody combination 13 in Table 1); and iv) CyCD3 BV786+CD271 BV786 / CD45 AF700 / PLAP APCH7 / CD99 FITC+CD8 FITC / GD2 BV510 / Osteopontin APC+BAP APC / nuMyogenin PE / CD95 BV421 / CD30 BV650 / CD40 BV711 / / EpCAM PERCPcy5.5+CD4 PERCPcy5.5 / HLADR PECF594 / CD56 PEcy7 / smCD3 BV605+CD19 BV605 (i.e., antibody combination 24 in Table 1).After the cells and antibody reagents directed against cell surface markers were thoroughly mixed, they were incubated for 30 minutes at room temperature, protected from light. After this incubation, 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed thoroughly, and centrifuged at 540 x g for 5 minutes. The supernatant was then discarded using a Pasteur pipette or vacuum device without disturbing the cell pellet, leaving approximately 50 μL of residual volume in each tube.

[0063] The cell pellet was resuspended by gentle mixing and subsequently added to Fix&Perm 商標 100 μL of Reagent A (fixative containing PFA) from the reagent kit (An der Grub, Vienna, Austria) was added, and the cells were further incubated for 15 minutes at room temperature, protected from light. 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was then added to the cell pellet, mixed thoroughly, and centrifuged at 540 × g for 5 minutes. The supernatant was then discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving a residual volume of approximately 50 μL in each tube; the cell pellet was resuspended by gentle mixing, followed by Fix&Perm 商標100 μL of Reagent B (saponin-containing permeabilization solution) from the kit was added. After gentle mixing, an appropriate volume of each intracellular antibody (nuMyoD1, nuMyogenin PE, osteopontin, BAP, OCT-3 / 4, PLAP, and cyCD3) was added, mixed, and incubated at room temperature for 15 minutes, protected from light. 2 mL of PBS containing 0.09% NaN3 and 0.5% BSA was then added to the cell pellet, mixed thoroughly, and centrifuged at 540 × g for 5 minutes. The supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 μL of residual volume in each tube. Upon thorough mixing, the remaining volume was resuspended in 200 μL of PBS containing 0.09% NaN3 and 0.5% BSA and immediately measured on a BD LSRFortessa X-20 flow cytometer equipped with four lasers and 13 fluorescence detectors.

[0064] Data analysis. First, SSC lo / FSC lo / CD45 +hi A gate on cells (G1) was performed and selected to identify lymphocytes; the G1 cells were then further subsetted by drawing four additional gates to identify lymphocyte subsets in the following manner: CD45 for T cells; +hi / smCD3 + / cyCD3 + (G1A), CD45 for B cells +hi / CD19 + (G1B), CD45 for plasma cells +lo / CD19 + (G1C), and CD45 for NK cells + / smCD3 - / cyCD3 - / CD56 + (G1D). T cells are CD4 + / CD8 - , CD4 - / CD8 + , CD4 + / CD8 + and CD4- / CD8 - These were further subdivided into T cell subsets (panel E in Figure 3). int / hi / FSC int / hi / CD45 + A second gate (G2) containing cells was drawn to identify myeloid cells. + / CD4 + Subsets of myeloid cell populations were achieved by establishing a gate on myeloid cells to identify monocytes and dendritic cells versus neutrophils and eosinophils. Finally, gate (G3) was established using CD45 IgG to select tumor cells, erythroblasts, endothelial cells, and mesenchymal stromal cells. - The tumor cells (Figure 1) express CD45 - / CD56 +hi / CD271 + / GD2 - / EpCAM - / + / CD99 - / nu Myogenin - Rhabdomyosarcoma cells are characterized by a CD45 phenotype (Figure 1). - / CD56 +hi / CD271 +hi / GD2 - / EpCAM - / CD99 - / nu Myogenin + Neuroblastoma cells are characterized by a CD45 - / CD56 +hi / CD271 - / + / GD2 +hi / EpCAM - / CD99 - / nu Myogenin - (Figure 3), and PNET cells are characterized by CD45 - / CD56 + / CD271 +hi / GD2 +lo / EpCAM - / CD99 + / nu Myogenin -Figure 3 illustrates all gating steps applied during data analysis in bone marrow samples infiltrated by neuroblastoma cells. Table 4 shows the percentages of neoplastic and immune cells in bone marrow and peripheral blood samples of all patients diagnosed with various pediatric solid tumors analyzed according to the present invention.

[0065] [Table 1] JPEG0007741825000002.jpg255168JPEG0007741825000003.jpg255166

[0066] [Table 2]

[0067] [Table 3]

[0068] [Table 4]

[0069] References JPEG0007741825000007.jpg237170JPEG0007741825000008.jpg82170

Claims

1. 1. A kit of parts for flow cytometric detection of pediatric tumor cells, the kit comprising fluorochrome-conjugated antibodies directed against cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271, a cytoplasmic marker cyCD3, and one or more nuclear markers nuMyogenin and / or nuMyoD1, wherein: (i) antibodies against the markers CD99 / CD8 are conjugated to the same fluorochrome and represent the first marker pair CD99 / CD8; (ii) an antibody against the marker EpCAM / CD4 is conjugated to the same fluorochrome and represents the second marker pair EpCAM / CD4; (iii) an antibody against CD271 is conjugated to the same fluorochrome as an antibody against either cyCD3 or smCD3, representing a third marker pair, CD271 / cyCD3 or CD271 / smCD3; wherein the kit comprises antibodies conjugated to eight or more distinguishable fluorescent dyes; wherein the fluorescent dyes are distinguishable between the first, second, and third marker pairs; and wherein the antibodies against the cytoplasmic marker and the nuclear marker are physically separate from the antibodies against the cell surface marker.

2. 2. The kit of parts of claim 1, comprising a first reagent composition contained in a first container, the first reagent composition comprising the conjugated antibodies to the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271, and a second reagent composition contained in a second container, the second reagent composition comprising the conjugated antibodies to the cytoplasmic marker cyCD3 and the one or more nuclear markers nuMyogenin and / or nuMyoD1.

3. 3. The kit of parts of claim 1 or 2, further comprising a third marker pair, CD271 / cyCD3, wherein the antibodies against the markers smCD3 / CD19 are conjugated to the same fluorescent dye to form a fourth marker pair, smCD3 / CD19, and wherein the fluorescent dyes are distinguishable between different pairs.

4. 3. The kit of parts of claim 1 or 2, further comprising a third marker pair, CD271 / smC3, wherein the antibodies against the markers CD19 and cyCD3 are each conjugated to a different fluorescent dye.

5. 5. The kit of parts of claim 1, further comprising antibodies against the markers nuMyogenin and nuMyoD1, wherein the antibodies against the markers nuMyogenin / nuMyoD1 are bound to the same fluorescent dye to form a fifth marker pair nuMyogenin / nuMyoD1, and wherein the fluorescent dyes are distinguishable between different pairs.

6. 6. The kit of parts according to claim 2 or any one of claims 3 to 5 when dependent on claim 2, wherein the first reagent composition further comprises a fluorochrome-conjugated antibody against one or more of Hodgkin's lymphoma cell surface markers HLA-DR, CD30, CD71, CD40 and CD95.

7. The kit of parts according to any one of claims 1 to 6, further comprising a fluorochrome-conjugated antibody against one or more of germ cell tumor cell surface markers OCT-3 / 4, BAP and PLAP.

8. 8. The kit of parts of claim 7, comprising antibodies against the markers OCT-3 / 4 and PLAP, and wherein the antibodies against the markers OCT-3 / 4 / PLAP are bound to the same fluorescent dye to form a marker pair OCT-3 / 4 / PLAP, and wherein the fluorescent dyes are distinguishable between different pairs.

9. The kit of parts according to any one of claims 1 to 8, further comprising a fluorochrome-conjugated antibody against one or more of the bone tumor cell surface markers osteopontin and bone alkaline phosphatase.

10. 10. The kit of parts of claim 9, wherein the kit of parts comprises antibodies against the markers osteopontin and BAP, and wherein the antibodies against the marker osteopontin / BAP are bound to the same fluorescent dye to form a marker pair osteopontin / BAP, and wherein the fluorescent dye is distinguishable between different pairs.

11. The kit of parts according to any one of claims 1 to 10, further comprising a nucleated cell integrity dye.

12. The kit of parts according to any one of claims 1 to 11, further comprising reagents for fixing and permeabilising cells, optionally accompanied by instructions for use, buffers and / or control samples.

13. 1. A multicolor flow cytometry method for the identification and classification of pediatric tumors, comprising: (a) staining an aliquot of a biological sample containing or suspected of containing pediatric tumor cells with a fluorochrome-conjugated antibody against a cell surface marker contained in a kit of parts according to any one of claims 1 to 12; and then (b) contacting the stained cells with a fixative; and then (c) permeabilizing the fixed and stained cells with a permeabilization solution; and then (d) staining the permeabilized cells with fluorochrome-conjugated antibodies against cytoplasmic and nuclear markers contained in the kit of parts according to any one of claims 1 to 12; (e) analyzing the stained cells in the aliquot in a flow cytometer; and (f) storing and evaluating the data obtained; The method, comprising the steps of:

14. 14. The method of claim 13, wherein the biological sample is a primary tumor tissue sample, peripheral blood, bone marrow, a tissue sample, or other type of body fluid, wherein the biological sample is obtained from a pediatric patient.

15. The method described in claim 13 or 14, wherein the pediatric tumor is selected from: i) neuroectodermal neoplasms; ii) tumors with myofibroblastic differentiation; iii) identified commitment to multiple cell lineages; and iv) T and B lymphoblastic lymphoma / leukemia.

16. The method described in claim 15, wherein the neuroectodermal neoplasm is neuroblastoma, ganglioneuroblastoma, ganglioneuromatosis, extraosseous Ewing's sarcoma, and classical Ewing's sarcoma.

17. A kit of parts according to any one of claims 1 to 12 for use in the diagnosis and classification of one or more pediatric tumours.

18. The kit of parts described in claim 17, wherein the pediatric tumor is selected from: i) neuroectodermal neoplasms; ii) tumors with myofibroblastic differentiation; iii) identified commitment to multiple cell lineages; and iv) T and B lymphoblastic lymphoma / leukemia.

19. The kit of parts described in claim 18, wherein the neuroectodermal neoplasm is neuroblastoma, ganglioneuroblastoma, ganglioneuromatosis, extraosseous Ewing's sarcoma, and classical Ewing's sarcoma.

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