MEANS AND METHODS FOR THE DIAGNOSIS, CLASSIFICATION AND/OR MONITORING OF PEDIATRIC TUMORS
20240248091 ยท 2024-07-25
Inventors
- Cristiane De Sa Ferreira Facio (Rio de Janeiro, BR)
- Vitor Dessanti Botafogo Gon?alves (Salamanca, ES)
- Elaine Sobral Da Costa (Rio de Janeiro, BR)
- Jacobus Johannes Maria Van Dongen (Leiden, NL)
- Jos? Alberto Orfao De Matos Correia E Vale (Salamanca, ES)
Cpc classification
International classification
Abstract
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 the nuclear marker(s) nuMyogenin and/or nuMyoD1, wherein (i) the antibodies against the markers CD99/CD8 are conjugated to the same fluorochrome and representing a first marker pair CD99/CD8; (ii) the antibodies against the markers EpCAM/CD4 are conjugated to the same fluorochrome and representing a second marker pair EpCAM/CD4; (ii) the antibody against CD271 is conjugated to the same fluorochrome as the antibody against either cyCD3 or smCD3 and representing a third marker pair CD271/cyCD3 or CD271/smCD3; wherein between the first, second and third marker pairs the fluorochromes are distinguishable; and wherein the antibodies against the cytoplasmic and the nuclear markers are physically separated from the antibodies against the cell surface markers.
Claims
1. A kit-of-parts for the flow cytometric detection of pediatric tumor cells, the kit 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 the nuclear marker(s) nuMyogenin and/or nuMyoD1, wherein (i) the antibodies against the markers CD99/CD8 are conjugated to the same fluorochrome and representing a first marker pair CD99/CD8; (ii) the antibodies against the markers EpCAM/CD4 are conjugated to the same fluorochrome and representing a second marker pair EpCAM/CD4; (ii) the antibody against CD271 is conjugated to the same fluorochrome as the antibody against either cyCD3 or smCD3 and representing a third marker pair CD271/cyCD3 or CD271/smCD3; wherein the kit comprises antibodies conjugated to >8 distinguishable fluorochromes; wherein between the first, second and third marker pairs the fluorochromes are distinguishable; and wherein the antibodies against the cytoplasmic and the nuclear markers are physically separated from the antibodies against the cell surface markers.
2. Kit-of-parts according to claim 1, comprising a first reagent composition comprising the conjugated antibodies against the cell surface markers CD45, CD56, GD2, CD99, CD8, EpCAM, CD4, smCD3, CD19 and CD271 contained in a first container, and a second reagent composition comprising the conjugated antibodies against the cytoplasmic marker cyCD3 and the nuclear marker(s) nuMyogenin and/or nuMyoD1, contained in a second container.
3. Kit-of-parts according to claim 1, comprising the third marker pair CD271/cyCD3 and wherein the antibodies against the markers smCD3/CD19 are conjugated to the same fluorochrome to form a fourth marker pair smCD3/CD19, and wherein between different pairs the fluorochromes are distinguishable.
4. Kit-of-parts according to claim 1, comprising the third marker pair CD271/smCD3, and wherein the antibodies against the markers CD19 and cyCD3 are each conjugated to a distinct fluorochrome.
5. Kit-of-parts according to claim 1, comprising antibodies against the markers nuMyogenin and nuMyoD1, and wherein the antibodies against the markers nuMyogenin/nuMyoD1 are conjugated to the same fluorochrome to form a fifth marker pair nuMyogenin/nuMyoD1, and wherein between different pairs the fluorochromes are distinguishable.
6. Kit-of-parts according to claim 2, wherein the first reagent composition further comprises fluorochrome-conjugated antibodies against one or more of the Hodgkin lymphoma cell surface markers HLA-DR, CD30, CD71, CD40 and CD95.
7. Kit-of-parts according to claim 1, further comprising fluorochrome-conjugated antibodies against one or more of the germ cell tumor cell surface markers OCT-3/4, BAP and PLAP.
8. Kit-of-parts according to claim 7, comprising antibodies against the markers OCT-3/4 and PLAP, and wherein the antibodies against the markers OCT-3/4 /PLAP are conjugated to the same fluorochrome to form a marker pair OCT-3/4 /PLAP, and wherein between different pairs the fluorochromes are distinguishable.
9. Kit-of-parts according to claim 1, further comprising fluorochrome-conjugated antibodies against one or more of the bone tumor cell surface markers osteopontin and bone alkaline phosphatase.
10. Kit-of-parts according to claim 9, comprising antibodies against the markers osteopontin and bone alkaline phosphatase referred to as BAP, and wherein the antibodies against the markers osteopontin/BAP are conjugated to the same fluorochrome to form a marker pair osteopontin/BAP, and wherein between different pairs the fluorochromes are distinguishable.
11. (canceled)
12. Kit-of-parts according to claim 1, further comprising a nucleated cell integrity dye.
13. Kit-of-parts according to claim 1, further comprising reagents for fixing and permeabilizing cells, optionally together with instructions for use, buffer, and/or control samples.
14. A multi-color flow cytometric method for identification and classification of pediatric tumors, comprising the steps of: (a) Staining an aliquot of a biological sample comprising or suspected to comprise childhood tumor cells with the fluorochrome-conjugated antibodies against cell surface markers as comprised in a kit-of-part according to claim 1; followed by (b) Contacting the stained cells with a fixation solution, followed by (c) permeabilizing the fixed and stained cells with a permeabilizing solution; followed by (d) staining the permeabilized cells with the fluorochrome-conjugated antibodies against cytoplasmic and nuclear markers as comprised in a kit-of-part according to claim 1; (e) analyzing the stained cells in said aliquot in a flow cytometer; and (f) storing and evaluating the data obtained.
15. The method according to claim 14, wherein the biological sample is a primary tumor tissue sample, peripheral blood, bone marrow, tissue sample comprising one of lymph nodes, adenoid, spleen, or liver, or other type of body fluid comprising one of cerebrospinal fluid, vitreous fluid, synovial fluid, final needle aspirate, pleural effusions or ascites, said sample being obtained from a pediatric patient.
16. The method according to claim 14, further comprising selecting an appropriate targeted therapy.
17. The method of claim 14 wherein the evaluating of the data obtained is used in the diagnosis and classification of one or more pediatric tumors, selected from: i) neuroectodermal neoplasias; ii) tumors with myofibroblastic cell differentiation; iii) identification of commitment into multiple cell lineages; and iv) T and B-lymphoblastic lymphoma/leukemia.
18. A combination for the flow cytometric detection of pediatric tumor cells, the combination 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 the nuclear marker(s) nuMyogenin and/or nuMyoD1, wherein (i) the antibodies against the markers CD99/CD8 are conjugated to the same fluorochrome and representing a first marker pair CD99/CD8; (ii) the antibodies against the markers EpCAM/CD4 are conjugated to the same fluorochrome and representing a second marker pair EpCAM/CD4; (ii) the antibody against CD271 is conjugated to the same fluorochrome as the antibody against either cyCD3 or smCD3 and representing a third marker pair CD271/cyCD3 or CD271/smCD3; wherein the combination comprises antibodies conjugated to ?8 distinguishable fluorochromes; wherein between the first, second and third marker pairs the fluorochromes are distinguishable; and wherein the antibodies against the cytoplasmic and the nuclear markers are physically separated from the antibodies against the cell surface markers.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0063]
[0064]
[0065]
DETAILED DESCRIPTION
[0066] More specifically, in an example, a tissue aliquot is placed in phosphate buffered saline (PBS) and subjected to mechanical disaggregation to prepare a single-cell suspensions suitable for further flow cytometry analysis aimed at maximum cell viability and cell recovery. For this purpose, tissue is suitably placed (e.g. into a Petri dish) in PBS containing 0.5% w/v bovine serum albumin, then minced into small (2-4 mm) pieces and mechanically disaggregated with sterile needles. The resulting tumor cell suspension may be sequentially filtered (e.g. using a sterile syringe of 120 mm pore size) to eliminate cell clumps and debris, centrifuged and re-suspended in PBS/0.5 w/v % BSA, at a final concentration of ?5?10.sup.5 cells/tube. Aliquots of 50 ?L (i.e. 50,000 cells) of the single cell suspension can then be placed in separate tubes.
[0067] As another example, e.g. in order to immunophenotype malignant cells present in metastatic sites, an ascitic fluid sample, a pleural fluid sample and/or a urine sample is collected. The sample (cell suspension) is sequentially filtered through a sterile syringe to eliminate cell clumps and debris; centrifuged and resuspended at a final concentration of ?5?10.sup.5 cells/tube.
[0068] Then, an aliquot of the single cell suspension is contacted with each of the fluorochrome-conjugated antibodies directed against cell surface markers comprises in a kit as defined herein above. The cells and antibody reagents are mixed well, and incubated e.g. for 30 min at RT, protected from light, to allow for antibody binding to the cell surface marker(s), if present on the cells. After this incubation, cells are washed and centrifuged to remove unbound antibodies, and approximately 50 ?L of residual volume is left in each tube for further staining of intracellular (i.e. cytoplasmic and nuclear) markers according to the invention.
[0069] To that end, the cell pellet is resuspended by gentle mixing with a fixative reagent, for example Reagent A (a fixative containing PFA) of the Fix&Perm? reagent kit (An der Grub, Vienna, Austria), followed by another incubation for 15 min at RT protected from light. Subsequently, cells are washed and the cell pellet resuspended by gentle mixing in a permeabilizing solution (for example Reagent B of the Fix&Perm? kit containing a permeabilizing agent like saponin). After gently mixing, the appropriate volume of each of the antibodies against the intracellular markers comprised in a kit of the invention is added, mixed and incubated for 15 min at RT protected from light.
[0070] Unbound antibodies are removed by washing using for example PBS containing 0.09% NaN.sub.3 and 0.5% w/v BSA, and approximately 50 ?L residual volume is left in each tube. Upon mixing well, the residual volume of the cell pellet is suitably resuspended in 200 ?L PBS with 0.09% NaN.sub.3 and 0.5% w/v BSA and immediately measured in a multicolor flow cytometer, for example a flow cytometer equipped with 4 lasers and 13 fluorescence detectors such as a BD LSRFortessa X-20 flow cytometer equipped with 4 lasers and 13 fluorescence detectors.
[0071] For data analysis, conventional manual Boolean gating strategies or automated clustering analyses in combination or not with a direct comparison with software databases can be used via flow cytometry software programs that allow for manual and/or automatic gating and analysis of FCS files.
[0072] For example, firstly a gate (G1) on SSClo/FSClo/CD45hi cells is performed for the identification of lymphocytes. This G1-cell population may then be further subsetted by drawing 4 additional gates for the identification of lymphocyte subsets in the following manner: CD45.sup.hi/smCD3.sup.+/cyCD3.sup.+ for T cells (G1A), CD45.sup.hi/CD19.sup.+ for B cells (G1B), CD45.sup.+lo/CD19.sup.+ for plasma cells (G1C) and CD45.sup.?/smCD3.sup.?/cyCD3.sup.?/CD56.sup.+ for NK cells (G1D). T-cells can be further subdivided into CD4.sup.+/CD8.sup.?, CD4.sup.?/CD8.sup.+, CD4.sup.+/CD8.sup.+ and CD4.sup.?/CD8.sup.? T-cell subsets.
[0073] A second gate (G2) to include SSC.sup.int/hi/FSC.sup.int/hi/CD45.sup.+ cells can be drawn for the identification of myeloid cells. Further subsets of myeloid cell populations may be achieved by establishing a gate on CD45.sup.?/CD4.sup.+ myeloid cells to identify monocytes and dendritic cells vs neutrophils and eosinophils (SSC.sup.hi/CD45.sup.+).
[0074] Finally, a gate (G3) can be performed on CD45.sup.? cells to select tumor cells, erythroblasts, endothelial and mesenchymal stromal cells, where Wilms tumor cells (
[0075] As will be appreciated by a person skilled in the art, an antibody panel, kit or kit according to the invention is advantageously used in the field of pediatric cancer diagnosis; for example in early diagnosis, diagnostic subclassification, staging and/or monitoring of pediatric cancer both at the primary tumor tissue site and at metastatic sites (e.g. ascitic fluid, pleural effusions, urine, bone marrow, cerebrospinal fluid, lymph node and bronchoalveolar lavage, among other tumor specimens) and peripheral blood. It allows both to accurately distinguish between the tumor cells and other residual cells in the sample, and to characterize the distinct subsets of immune cells coexisting with the tumor cells in the same sample. In addition, it provides tools for fast classification of pediatric solid tumors with high sensitivity (detection of 10.sup.?1 to 10.sup.?5 tumor cells among other cells in the sample) and it further provides numerical and phenotypic information about the tumor-associated microenvironment, as it allows identification and enumeration of the major lymphocyte, neutrophil, monocyte/macrophage and dendritic cell populations and mesenchymal cells, in the infiltrated or non-infiltrated patient specimens.
[0076] The procedure includes the following sequential steps: i) to obtain a biological sample from a patient suspected of suffering from a pediatric cancer; ii) to stain such biological sample with a panel of ?12-antibodies (e.g. a combination according to Table 1) conjugated with 8-distinct fluorochromes in 8-color antibody stainings, aimed at the identification and classification of the tumor cells as well as of the infiltrating immune cells coexisting in the sample; iii) to measure the stained cells in a conventional >8-color flow cytometry instrument, and; iv) to analyze the flow cytometric data obtained using dedicated software tools in order to distinctly identify tumor cells vs normal cells coexisting in the sample, further enumerate such tumor cells, define the levels of expression per cell of each marker being expressed on it, classify the tumor cells into distinct WHO diagnostic entities according to their immunophenotypic profile, and, identify and enumerate the distinct populations of immune cells and their major subsets that coexist with the tumor cells in the sample.
[0077] The procedure here described can be used for the diagnosis and classification of the most common types of pediatric tumors including: i) neuroectodermal neoplasias, such as neuroblastoma, ganglioneuroblastoma, ganglioneuroma, extraosseous Ewing sarcoma and classical Ewing sarcoma for which most useful antigens are CD45.sup.?, CD56.sup.++, CD99.sup.?or+, GD2.sup.++ and CD271.sup.+; ii) tumors with myofibroblastic cell differentiation as assessed by a CD45.sup.?, CD56.sup.+, anti-.sub.nuMyogenin.sup.+, anti-.sub.nuMyoD1.sup.+ and CD271.sup.+ phenotype; iii) identification of commitment into multiple cell lineages as defined by the expression pattern of e.g. CD45.sup.?, CD56.sup.++, CD271.sup.+ and EpCAM.sup.+ in Wilms tumor; and iv) T and B-lymphoblastic lymphoma/leukemia which characteristically show expression of cyCD3.sup.+ and CD45.sup.++CD19.sup.?, vs CD19.sup.+ CD45.sup.?/+ cyCD3.sup.? tumor cells.
[0078] Furthermore, the invention also permits simultaneous flow cytometric identification of lymphocytes (CD45.sup.++/SSC.sup.lo cells) including cyCD3.sup.+/CD3.sup.+ T-cells, CD19.sup.+ B-cells and CD19.sup.?/CD3.sup.?/CD56.sup.+ NK cells, CD19.sup.+/CD45.sup.lo plasma cells and CD4.sup.+/SSC.sup.int monocytes and dendritic cells, plus CD271++ mesenchymal cells and endothelial cells.
[0079] Based on the expression of CD56, CD4 and CD8, T-cells can be further divided into CD4.sup.+/CD8.sup.?, CD4.sup.?/CD8.sup.+, CD4.sup.+/CD8.sup.+, CD4.sup.?/CD8.sup.? T cells; each of these T-cell subsets can be further divided into subsets that express or not CD56. Simultaneously, CD56.sup.+ NK cells can be further subdivided into CD56.sup.+hi, CD56.sup.+lo/CD8.sup.?, CD56.sup.+lo/CD8.sup.+ NK subsets.
[0080] Notably, each of the kits and antibody combinations provided herein to identify and classify the tumor cell population, also provide means to quantify protein expression levels per tumor cell; therefore, it provides critical information, not only for the diagnosis, classification and monitoring of pediatric solid tumors, but also for selecting appropriate targeted therapies (e.g. anti-GD2 in GD2.sup.+ neuroblastoma and other GD2.sup.+ tumors).
[0081] The invention might also be used to detect and count tumor cells in bone marrow and peripheral blood samples prior to autologous stem cell transplantation, or for monitoring purposes after any type of therapy had been given to the patient. Furthermore, this invention can also be used to identify neoplastic and non-neoplastic cells in small and/or paucicellular samples such as vitreous fluid, cerebrospinal fluid and fine needle aspirated tissue samples for staging purposes, diagnosis of disease recurrence or patient monitoring at residual disease levels.
[0082] The diagnostic outcome of a flow cytometric method of the invention is advantageously used to aid in selecting an appropriate (targeted) therapy such as anti-GD2 antibody-based or chimeric antigen receptor (CAR) T-cell therapy.
[0083] The invention also provides the use of a kit-of-parts as herein disclosed in the diagnosis and classification of one or more pediatric tumors. For example, the pediatric tumor is selected from: i) neuroectodermal neoplasias, such as neuroblastoma, ganglioneuroblastoma, ganglioneuroma, extraosseous Ewing sarcoma and classical Ewing sarcoma ; ii) tumors with myofibroblastic cell differentiation; iii) identification of commitment into multiple cell lineages; and iv) T and B-lymphoblastic lymphoma/leukemia.
[0084] The invention is exemplified by the examples below, which are provided for illustrating purposes and in no manner limit the scope.
EXAMPLE 1
Analysis of a Tumor Mass Sample
[0085] Sample collection. Solid tumor specimens were collected at the surgical room from fifty-five pediatric patients; tumor samples were sent to pathology and they were divided into two aliquots by an experienced pathologist: one was used for conventional pathology and the second for flow cytometry. The tissue aliquot used for flow cytometry was immediately placed in phosphate buffered saline (PBS) in wet ice and transported to the flow cytometry laboratory. Once the specimen arrived it was weighted, a physical description was recorded, and the sample was further divided into two small fragments: one for fresh-frozen storage at ?80? C. and the other for immediate mechanical disaggregation.
[0086] Mechanical disaggregation of the tumor specimen. Fifty-five tumor tissue specimens were immediately disaggregated into single-cell suspensions suitable for further flow cytometry analysis aimed at maximum cell viability and cell recovery. For this purpose, the tissue was placed into a Petri dish in 2ml PBS containing 0.5% bovine serum albumin (BSA; Calbiochem, La Jolla, CA). The tumor specimen was then minced into small pieces (2-4 mm) with a scalpel blade and mechanically disaggregated with sterile needles. Afterward, the tumor cell suspension was sequentially filtered through a sterile Filcon syringe (120 mm pore size) to eliminate cell clumps and debris, centrifuged (10 min at 540 g) and re-suspended in 500 ?l of PBS containing 0.5% BSA, at a final concentration of ?5?105 cells/tube. Aliquots of 50 ?L (i.e. 50,000 cells) of the single cell suspension were then placed in a different tube. A total of 4 distinct aliquots/per sample were stained per tube to be acquired.
[0087] Staining of the sample. Fifty ?l of sample (single cell suspension of the disaggregated tissues) was added to each of the 4 tube aliquots, followed by adding the appropriate volumes (saturating concentrations) of each of the corresponding reagent compositions, comprising antibodies directed against cell surface markers, as recommended for this single tube panels [0088] 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); [0089] 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); [0090] 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) [0091] 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).
[0092] Subsequently, the cells and antibody reagents were mixed well, and they were incubated for 30 min at RT protected from light. After this incubation, 2 mL of PBS with 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540?g. Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L of residual volume was left in each tube. The cell pellet was resuspended by gentle mixing and 100 ?L of Reagent A (fixative containing PFA) of the Fix&Perm? reagent kit (An der Grub, Vienna, Austria) was added, followed by another incubation for 15 min at RT protected from light. Subsequently, 2 mL of PBS with 0.09%NaN.sub.3 containing 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540 g.
[0093] Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L of residual volume was left in each tube; the cell pellet was resuspended by gently mixing and 100 ?L of Reagent B (permeabilizing solution containing saponin) of the Fix&Perm? kit was added. After gently mixing, the appropriate volume of each of the antibodies against intracellular markers (nuMyoD1, nuMyogenin, OCT3 and cyCD3) was added, mixed and incubated for 15 min at RT protected from light. Afterward, 2 mL of PBS containing 0.09% NaN.sub.3 and 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540 g, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L residual volume was left in each tube. Upon mixing well, the residual volume the cell pellet was resuspended in 200 ?L PBS with 0.09% NaN3 and 0.5% BSA and immediately measured in a BD LSRFortessa X-20 flow cytometer equipped with 4 lasers and 13 fluorescence detectors.
[0094] Data analysis. Firstly, a gate (G1) on SSC.sup.lo/FSC.sup.lo/CD45.sup.hi cells was performed for the identification of lymphocytes; and selected then, this G1-cells were further subsetted by drawing 4 additional gates for the identification of lymphocyte subsets as follows: CD45.sup.hi/smCD3.sup.+/cyCD3.sup.+ for T cells (G1A), CD45.sup.hi/CD19.sup.+ for B cells (G1B), CD45.sup.+lo/CD19.sup.+ for plasma cells (G1C) and CD45.sup.+/smCD3.sup.?/cyCD3.sup.?/CD56.sup.+ for NK cells (G1D). T-cells were further subdivided into CD4.sup.+/CD8.sup.?, CD4.sup.?/CD8.sup.+, CD4.sup.+/CD8.sup.+ and CD4.sup.?/CD8.sup.? T-cell subsets (panel E in
EXAMPLE 2
Analysis of Ascitic Fluid, Pleural Fluid and Urine Sample
[0095] Sample collection. In order to immunophenotype malignant cells present in metastatic sites, samples from 5 children previously diagnosed with pediatric cancer were studied: 1 ascitic fluid, 3 pleural fluid and 1 urine sample. Samples were collected at the surgical room or at the intensive care unit and processed either at diagnosis or at relapse, by following the sequential steps described below. Firstly, the sample (cell suspension) was sequentially filtered through a sterile Filcon syringe (120 mm pore size) to eliminate cell clumps and debris; then, it was centrifuged (10 min at 540 g) and resuspended in 500 ?l of PBS containing 0.5% BSA, at a final concentration of >5?105 cells/tube. Four aliquots of 50 ?L (i.e. 50,000 cells) of the single cell suspension were then made and placed in different tubes.
[0096] Staining of the sample. Fifty ?l of sample (single cell suspension of the distinct body fluids) was added to each of the 4 tube aliquots, followed by the appropriate volumes (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). Afterward, the cells and antibody reagents were mixed well and incubated for 30 min at RT 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 well and centrifuged for 5 min at 540 g. Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50?L of residual volume was left in each tube. The cell pellet was resuspended by gentle mixing and 100 ?L of Reagent A (fixative containing PFA) of the Fix&Perm? reagent kit (An der Grub, Vienna, Austria) was added, followed by another incubation for 15 min at RT protected from light. Subsequently, 2 mL of PBS with 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540 g. Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L of residual volume was left in each tube; the cell pellet was resuspended by gently mixing and 100 ?L of Reagent B (permeabilizing solution containing saponin) of the Fix&Perm? kit was added. After gently mixing, the appropriate volume of each of the intracellular antibodies (nuMyoD1, nuMyogenin, Osteopontin, BAP, PLAP and cyCD3 APC-H7) was added, mixed and incubated for 15 min at RT protected from light. Afterward, 2 mL of PBS with 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540g, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L residual volume was left in each tube. Upon mixing well, the residual volume, the cell pellet was resuspended in 200 ?L PBS containing 0.09% NaN3 and 0.5% BSA and immediately measured in a BD Symphony X-20 flow cytometer equipped with 5 lasers and 48 fluorescence detectors.
[0097] Data analysis. Firstly, a gate (G1) on SSC.sup.lo/FSC.sup.lo/CD45.sup.+hi cells was performed for the identification of lymphocytes and selected; then, this G1-cells were further subseted by drawing 4 additional gates for the identification of lymphocyte subsets as follows: CD45.sup.+hi/smCD3.sup.+/cyCD3.sup.+ for T cells (G1A), CD45.sup.+hi/CD19.sup.+ for B cells (G1B), CD45.sup.+lo/CD19.sup.+ for plasma cells (G1C) and CD45.sup.+/smCD3.sup.?/cyCD3.sup.?/CD56.sup.+ for NK cells (G1D). T-cells were further subdivided into CD4.sup.+/CD8.sup.?, CD4.sup.?/CD8.sup.+, CD4.sup.+/CD8.sup.+ and CD4.sup.?/CD8.sup.? T-cell subsets (panel E in
EXAMPLE 3
Analysis of a Bone Marrow Sample
[0098] Sample collection. Twenty-three bone marrow and ten peripheral blood samples collected from 23 cancer patients were investigated for the presence of metastatic dissemination during staging procedures. At least 10?10.sup.6 nucleated cells from each sample were stained in 4 distinct tubes/aliquots per peripheral blood and bone marrow sample using the EUROFLOW bulk lysis protocol, as previously described (Flores-Montero et al., Leukemia. 2017 October; 31(10):2094-2103) for acquisition of >5?10.sup.6 cells/tube. Briefly, 2 ml of each sample plus 50 mL of ammonium chloride lysing solution were mixed in a 50 ml Falcon tube and incubated for 15 min in a roller or a sample-shaker device. Afterward, the sample was centrifuged at 800 g for 10 min and the supernatant discarded using a Pasteur pipette without disturbing the cell pellet. Upon discarding the supernatant, the tube was refilled with PBS containing 0.09% NaN.sub.3 and 0.5% BSA to a final volume of 50 ml and centrifuged once again at 800 g (5 min). Without disturbing the cell pellet, the supernatant was discarded, and the cell pellet was resuspended in 2 mL of PBS with 0.09% NaN.sub.3 and 0.5% BSA. Then, the cells were transferred to a 5 mL polystyrene round-bottom Falcon tube (FACS tube) in a volume of 300 ?l/tube. Such volume was completed with PBS containing 0.09% NaN.sub.3 and 0.5% BSA to reach 2 ml (final volume), gently mixed and centrifuged at 540 g for 5min; then, the supernatant was removed using a Pasteur pipette without disturbing the cell pellet. This procedure was repeated twice. The final cell concentration was adjusted with PBS with 0.09% NaN.sub.3 and 0.5% BSA to 5?105 cells/?L and around 100 ?L (i.e. 10 million cells) of the final cell suspension/sample were used per tube to be stained and measured in the flow cytometer.
[0099] Staining of the sample. One hundred ?l of the above processed cell suspensions were added to each of the 4 tube aliquots prepared per sample, followed by the appropriate volumes (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+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 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 mixed well, they were incubated for 30 min at RT 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 well and centrifuged for 5 min at 540 g. Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L of residual volume was left in each tube.
[0100] The cell pellet was resuspended by gentle mixing, and 100 ?L of Reagent A (fixative containing PFA) of the Fix&Perm? reagent kit (An der Grub, Vienna, Austria) was subsequently added, followed by another incubation for 15 min at RT protected from light. Subsequently, 2 mL of PBS with 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mix well and centrifuged for 5 min at 540 g. Then, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L of residual volume was left in each tube; the cell pellet was resuspended by gently mixing and 100 ?L of Reagent B (permeabilizing solution containing saponin) of the Fix&Perm? kit was subsequently added. After gently mixing, the appropriate volume of each of the intracellular antibodies (nuMyoD1, nuMyogenin PE, Osteopontin, BAP, OCT-3/4, PLAP and cyCD3) was added, mixed and incubated for 15 min at RT protected from light. Afterward, 2 mL of PBS with 0.09% NaN3 and 0.5% BSA was added to the cell pellet, mixed well and centrifuged for 5 min at 540 g, the supernatant was discarded using a Pasteur pipette or vacuum system without disturbing the cell pellet, and approximately 50 ?L residual volume was left in each tube. Upon mixing well the residual volume, the cell pellet was resuspended in 200 ?L PBS with 0.09% NaN3 and 0.5% BSA, and immediately measured in a BD LSRFortessa X-20 flow cytometer equipped with 4 lasers and 13 fluorescence detectors.
[0101] Data analysis. Firstly, a gate (G1) on SSC.sup.lo/FSC.sup.lo/CD45.sup.+hi cells was performed for the identification of lymphocytes and selected; then, this G1-cells were further subsetted by drawing 4 additional gates for the identification of lymphocyte subsets as follows: CD45.sup.+hi/smCD3.sup.+/cyCD3.sup.+ for T cells (G1A), CD45.sup.+hi/CD19.sup.+ for B cells (G1B), CD45.sup.+lo/CD19.sup.+ for plasma cells (G1C) and CD45.sup.+/smCD3.sup.?/cyCD3.sup.?/CD56.sup.+ for NK cells (G1D). T-cells were further subdivided into CD4.sup.+/CD8.sup.?, CD4.sup.?/CD8.sup.+, CD4.sup.+/CD8.sup.+ and CD4.sup.?/CD8.sup.? T-cell subsets (panel E in
TABLE-US-00001 TABLE 1 Panel of antibody combination of 12/13 backbone markers in 8 or 9 distinct fluorochromes and for 14 colors combinations: FLUOROCHOME POSITION F8 F1 F2 F3 F4 F5 F6 F7 e.g. F9 F10 F11 F12 F13 F14 e.g. e.g. e.g. e.g. e.g. e.g. e.g. PerCP e.g. e.g. e.g. e.g. e.g. e.g. BV421 BV510 BV605 BV650 BV711 BV786 FITC Cy5.5 PE PECF594 PECy7 APC AF700 APCH7 1 cyCD3/ CD45 CD99/ CD4/ nuMyogenin CD56 GD2 smCD3/ CD271 CD8 Epcam CD19 2 cyCD3/ CD45 CD99/ CD4/ nuMYOD1 CD56 GD2 smCD3/ CD271 CD8 Epcam CD19 3 cyCD3/ CD45 CD99/ CD4/ nuMyogenin/ CD56 GD2 smCD3/ CD271 CD8 Epcam nuMYOD1 CD19 4 smCD3/ CD45 CD19 CD99/ CD4/ nuMyogenin CD56 GD2 CyCD3 CD271 CD8 Epcam 5 smCD3/ CD45 CD19 CD99/ CD4/ nuMYOD1 CD56 GD2 CyCD3 CD271 CD8 Epcam 6 smCD3/ CD45 CD19 CD99/ CD4/ nuMyogenin/ CD56 GD2 CyCD3 CD271 CD8 Epcam nuMYOD1 7 cyCD3/ GD2 smCD3/ CD99/ CD4/ nuMyogenin CD56 Osteo- CD45 OCT3/4/ CD271 CD19 CD8 Epcam pontin/ PLAP BAP 8 cyCD3/ GD2 smCD3/ CD99/ CD4/ nuMYOD1 CD56 Osteo- CD45 OCT3/4/ CD271 CD19 CD8 Epcam pontin/ PLAP BAP 9 cyCD3/ GD2 smCD3/ CD99/ CD4/ nuMyogenin CD56 Osteo- CD45 OCT3/4 CD271 CD19 CD8 Epcam pontin 10 cyCD3/ GD2 smCD3/ CD99/ CD4/ nuMYOD1 CD56 Osteo- CD45 OCT3/4 CD271 CD19 CD8 Epcam pontin 11 cyCD3/ GD2 CD71 CD40 smCD3/ CD99/ CD4/ numyogenin CD56 CD45 CD30 CD271 CD19 CD8 Epcam 12 cyCD3/ GD2 CD71 CD40 smCD3/ CD99/ CD4/ numyogenin/ CD56 CD45 CD30 CD271 CD19 CD8 Epcam nuMYOD1 13 cyCD3/ GD2 CD71 CD40 smCD3/ CD99/ CD4/ numyogenin CD56 HLADR CD45 CD30 CD271 CD19 CD8 Epcam 14 cyCD3/ GD2 CD71 CD40 smCD3/ CD99/ CD4/ numyogenin/ CD56 HLADR CD45 CD30 CD271 CD19 CD8 Epcam nuMYOD1 15 cyCD3/ GD2 BAP smCD3/ CD99/ CD4/ nuMyogenin PLAP CD56 Osteo- CD45 OCT3/4 CD271 CD19 CD8 Epcam pontin 16 cyCD3/ GD2 BAP smCD3/ CD99/ CD4/ nuMYOD1 PLAP CD56 Osteo- CD45 OCT3/4 CD271 CD19 CD8 Epcam pontin 17 CD95 GD2 cyCD3/ CD71 CD40 CD19 CD99/ CD4/ nuMYOD1 CD56 HLADR CD45 CD30 CD271 CD8 Epcam 18 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin HLADR CD56 Osteo- CD45 OCT3/4 CD19 CD271 CD8 Epcam pontin 19 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ nuMYOD1 HLADR CD56 Osteo- CD45 OCT3/4 CD19 CD271 CD8 Epcam pontin 20 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin/ HLADR CD56 Osteo- CD45 OCT3/4 CD19 CD271 CD8 Epcam nuMYOD1 pontin 21 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ nuMYOD1 HLADR CD56 Osteo- CD45 PLAP CD19 CD271 CD8 Epcam pontin/ BAP 22 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin/ HLADR CD56 osteo- CD45 PLAP CD19 CD271 CD8 Epcam nuMYOD1 pontin/ BAP 23 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin HLADR CD56 BAP CD45 PLAP CD19 CD271 CD8 Epcam 24 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin HLADR CD56 Osteo- CD45 PLAP CD19 CD271 CD8 Epcam pontin/ BAP 25 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin HLADR CD56 BAP CD45 OCT3/4/ CD19 CD271 CD8 Epcam PLAP 26 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin HLADR CD56 Osteo- CD45 OCT3/4/ CD19 CD271 CD8 Epcam pontin/ PLAP BAP 27 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ nuMYOD1 HLADR CD56 Osteo- CD45 OCT3/4/ CD19 CD271 CD8 Epcam pontin/ PLAP BAP 28 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ numyogenin/ HLADR CD56 Osteo- CD45 OCT3/4/ CD19 CD271 CD8 Epcam nuMYOD1 pontin/ PLAP BAP 29 CD95 GD2 smCD3/ CD30 CD40 cyCD3/ CD99/ CD4/ nuMYOD1 HLADR CD56 Osteo- CD45 OCT3/4/ CD19 CD271 CD8 Epcam pontin/ PLAP BAP 30 CD95 GD2 cyCD3/ CD71 CD40 CD19 CD99/ Epcam/ nuMYOD1 HLADR CD56 CD45 CD30 CD271 CD8 CD4
TABLE-US-00002 TABLE 2 Percentage of neoplastic cells and immune cells present in tumor mass biopsies at diagnosis from pediatric cancer patients (n = 55) classified according to the tumor diagnostic subtypes. Tissue cell distribution(%) % IN TOTAL CELULARITY % IN TOTAL LEUCOCYTES Diagnostic group (no Tumor Immune T Ratio T CD4 T CD8 of samples) Viabilitiy cells cells lymphocytes CD4:CD8 lymphocytes lymphocytes HEMATOLOGICAL MALIGNANCIES Diffuse large B cell 61 30.9 69 54.3 0.98 22.4 27.2 lymphoma (4) (39- (0- (7.4- (2.5- (0.44- (0.8- (1.5- 81.6) 92.6) 100) 91.2) 1.89) 41.2) 58.3) Burkitt Lymphoma 45.8 60 39.8 72.9 0.78 31.3 36.8 (9) (1.1- (14.5- (3.6- (35.3- (0.06- (3.8- (10.8- 83.9) 96.4) 85.5) 98.8) 2.00) 56) 67.9) NON-HEMATOLOGICAL MALIGNANCIES Neuroblastoma and 33.7 34.8 65.1 48.6 1.70 21.7 16 ganglioneuroblastoma (2- (6- (3.2- (7.4- (0.51- (2.8- (1.5- (10) 92.6) 96.8) 94) 88) 4.13) 49) 39.6) Nephroblastoma 37 64.3 34.6 35.5 0.56 11.6 22.6 (10) (4.3- (1.5- (1.7- (13.6- (0.16- (3.3- (6.8- 83.9) 98.3) 98.5) 67.7) 1.50) 31.7) 44.4) Ewing tumor and 29.5 68.3 31.6 41.6 0.48 12.4 24.9 related sarcomas of (9.4- (51.8- (8- (10.9- (0.16- (1.4- (8.8- bone (4) 52.7) 92) 48.2) 73) 1.00) 31.6) 46.8) Germ cell tumors 17.6 34.3 65 23.9 1.50 11.05 8.8 (11) (6- (0.9- (2.8- (2.7- (0.51- (0.7- (1.4- 45) 97.2) 99.1) 63.5) 3.80) 27.5) 25.6) Nasopharyngeal 55 32 60.4 37 1.05 11.9 12.3 carcinoma (3) (21- (10- (30.8- (22- (0.70- (4.2- (4.5- 65) 69.2) 90) 46.2) 1.55) 23.4) 18.2) Soft tissue 25.8 66.6 33.3 28 1.70 12.9 9.6 Sarcomas* (4) (3.6- (11- (1.5- (14.4- (0.98- (7.1- (3.4- 50) 98.4) 89) 64.8) 3.10) 25.9) 23.6) Tissue cell % IN TOTAL LEUCOCYTES distribution(%) Monocytes/ Diagnostic group (no B T lymphocytes NK Dendritic of samples) lymphocytes CD56+ CD56? cell Neutrophils cells Eosinophils HEMATOLOGICAL MALIGNANCIES Diffuse large B cell 5.1 7.9 46.3 3 25.8 8 2.1 lymphoma (4) (0.2- (0.4- (1.9- (0.4- (0.8- (1.5- (0- 14.4) 28.8) 88.8) 7.9) 84.4) 18) 5.9) Burkitt Lymphoma 5 3.5 72.5 1 6 3.2 1.5 (9) (0- (0.17- (23.7- (0- (0.1- (0- (0- 28.1) 11.6) 97.7) 2.5) 31.2) 14.9) 8.9) NON-HEMATOLOGICAL MALIGNANCIES Neuroblastoma and 10.2 5 43.5 2.1 27.4 13.2 1.8 ganglioneuroblastoma (0.1- (0.02- (6- (0- (0.93- (0- (0- (10) 36.5) 31.2) 87.8) 6.46) 78.7) 50.3) 8.2) Nephroblastoma 3 2.7 33.4 17.3 23.9 22 0.9 (10) (0- (0.05- (12.5- (1.6- (1.7- (2.4- (0- 9) 9.6) 62.4) 65.4) 59.6) 41.4) 2.5) Ewing tumor and 5.2 36.4 5.8 33.8 12.5 0.52 related sarcomas of 3.8 (.02- (10.9- (3- (7.8- (5.2- (0- bone (4) (0.7-8.1) 13) 60) 10.4) 60.3) 17.2) 1.1) Germ cell tumors 2.5 1.0 22.8 3.5 52.2 12.9 5 (11) (0.05- (0- (2.6- (0.17 (22.7- (1.0- (0.4- 5.8) 3.1) 61.3) 13.9) 98) 39.3) 38.7) Nasopharyngeal 35 2.3 30.2 2.4 1.6 3.7 0.02 carcinoma (3) (10- (1.9- (20.2- (0.4- (0.6- (0.5- (0- 54) 2.7) 40.2) 3.9) 3.3) 6.2) 0.04) Soft tissue 1.9 1.7 26.3 4.3 38.2 24.7 3.6 Sarcomas* (4) (1.5- (0.4- (13.2- (2.5- (14.5- (4.6- (0- 2.3) 4.6) 60.2) 6.3) 65) 63.6) 10.2) *Includes two undifferentiated sarcomas, one chondrosarcoma and one clear cell sarcoma distribution(%)
TABLE-US-00003 TABLE 3 Percentage of neoplastic cells and the different immune cells in fluidic samples. Fluidic cell distribution(%) Diagnostic group (no of samples) NON- % IN TOTAL CELULARITY % IN TOTAL LEUCOCYTES HEMATOLOGICAL Tumor Immune T Ratio T CD4 T CD8 MALIGNANCIES Viabilitiy cells cells lymphocytes CD4:CD8 lymphocytes lymphocytes Soft tissue 52.6 15.8 84 36.2 1.20 5.3 4.6 sarcoma (3)* (45.3- (0- (52.5- (10.5- (0.9- (4.9- (3.8- 57.7) 47.5) 100) 80.2) 1.5) 5.7) 5.4) Germ cell tumor 52.5 17.4 73 42 1.70 15.5 16.4 (2) .sup.? (25- (7.8- (73- (10.9- (0.84- (5.4- (2- 80) 27) 92.2) 73) 2.7) 25.7) 30.8) Fluidic cell distribution(%) Diagnostic group (no of samples) % IN TOTAL LEUCOCYTES NON- Monocytes/ HEMATOLOGICAL B T lymphocytes Dendritic MALIGNANCIES lymphocytes CD56+ CD56? NK cell Neutrophils cells Eosinophils Soft tissue 1.8 1.3 34.6 2.1 41.5 17.2 0 sarcoma (3)* (0.6- (1.1- (9.1- (1.3- (3.8- (1.6- 3.8) 1.46) 78.6) 4.7) 78.1) 42.8) Germ cell tumor 6 5.4 36.5 6.1 35.2 13.3 0.5 (2) .sup.? (0.3- (1.2- (9.7- (0.3- (1.5- (0.6- (0.2- 11.7) 9.6) 63.4) 12) 69) 26) 0.8) *Includes two pleural effusion and one urine samples; .sup.? Includes, one ascitic fluid and one pleural effusion sample.
TABLE-US-00004 TABLE 4 Percentage of neoplastic cells and different immune cells present in bone marrow and peripheral blood samples from children diagnosed with pediatric solid tumors. Tissue cell distribution(%) % IN TOTAL CELULARITY % IN TOTAL LEUCOCYTES Diagnostic group (number of Tumor Immune T Ratio T CD4 T CD8 samples) Viabilitiy cells cells lymphocytes CD4:CD8 lymphocytes lymphocytes Neuroblastoma and ganglioneuroblastoma (30) Non-infiltrated bone 84.8 0 100 6 1.21 2.8 2.4 marrow (13) (70- (2.3- (0.24- (0.3- (1.5- 95) 12.1) 2.40) 5.5) 5.2) Infiltrated bone marrow 76.6 13.5 86.4 15.8 1.3 7.9 5.6 (10) (67- (0.2- (58.7- (3.7- (0.86- (1.5- (1.6- 87.8) 41.3) 99.8) 45.3) 2.00) 22.9) 15.2) Peripheral blood (7) 77.4 0.014 99.8 19.9 1.65 8.9 8.7 (54.6- (0- (99- (8.2- (.35- (4- (2.5- 96.7) 0.09) 100) 38.63) 4.6) 17.1) 20.5) Ewing tumor and related sarcomas of bone (2) Peripheral blood (2) 71.6 0 100 14.3 1.4 7.1 4.7 (60- (5.2- (1.3- (2.2- (1.6- 83.3) 23.4) 1.5) 12.1) 7.8) Rhabdomyosarcoma (1) Peripheral blood (1) 78 0 100 5.8 1.69 3.3 2.0 % IN TOTAL LEUCOCYTES Tissue cell distribution(%) Monocytes/ Diagnostic group (number of B T lymphocytes Dendritic samples) lymphocytes CD56+ CD56? NK cell Neutrophils cells Eosinophils Neuroblastoma and ganglioneuroblastoma (30) Non-infiltrated bone 13.3 0.04 6 0.89 50.8 6.7 2.8 marrow (13) (0.2- (.01- (2.3- (0.2- (20.3- (2.6- (1.6- 51.4) 0.1) 12.1) 1.6) 79.7) 12.5) 4) Infiltrated bone marrow 9.4 6.3 9.4 1.7 51.7 4.6 2.6 (10) (1.5- (0.6- (0.1- (0.02- (28.5- (0.6- (0.6- 23.5) 28) 17.6) 7.4) 78.2) 10.4) 5) Peripheral blood (7) 6.4 0.7 17.9 2.1 57.8 11.8 1.6 (1.6- (0.01- (7.46- (0.1- (23.2- (4.01- (0.2- 11.7) 2.7) 38.17) 8.4) 84.3) 24.4) 4.7) Ewing tumor and related sarcomas of bone (2) Peripheral blood (2) 3.9 1.3 12.9 1.5 74.3 3.9 1.8 (0.6- (0.5- (4.7- (1.1- (60- (1.9- (1.5- 7.3) 2.2) 21.2) 2) 88.6) 6) 2.1) Rhabdomyosarcoma (1) Peripheral blood (1) 1.8 0.34 5.5 0.89 82.8 8.2 0.02
REFERENCES
[0102] Almaz?n-Moga A, Roma J, Molist C, Vidal I, Jubierre L, Soriano A, Segura MF, Llort A, S?nchez de Toledo J, Gallego S. Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry. Cytometry A. 2014 December; 85(12):1020. [0103] Bozzi F, Gambirasio F, Luksch R, Collini P, Brando B, Fossati-Bellani F. Detecting CD56+/NB84+/CD45? immunophenotype in the bone marrow of patients with metastatic neuroblastoma using flow cytometry. Anticancer Res. 2006 September-October; 26(5A):3281. [0104] Bryson GJ, Lear D, Williamson R, Wong RC. Detection of the CD56+/CD45? immunophenotype by flow cytometry in neuroendocrine malignancies. J Clin Pathol. 2002 July; 55(7):535. [0105] Dubois SG, Epling CL, Teague J, Matthay KK, Sinclair E. Flow cytometric detection of Ewing sarcoma cells in peripheral blood and bone marrow. Pediatr Blood Cancer. 2010 January; 54(1): 13. [0106] Ferreira-Facio CS, Milito C, Botafogo V, Fontana M, Thiago LS, Oliveira E, da Rocha-Filho AS, Werneck F, Forny DN, Dekermacher S, de Azambuja AP, Ferman SE, de Faria PA, Land MG, Orfao A, Costa ES. Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer. PLOS One. 2013; 8(3): e55534. [0107] Magro G, Longo FR, Angelico G, Spadola S, Amore FF, Salvatorelli L. Immunohistochemistry as potential diagnostic pitfall in the most common solid tumors of children and adolescents. Acta Histochem. 2015 May-June; 117(4-5):397. [0108] Van Dongen JJM, Orfao de Matos Correia e Vale JA, Flores-Montero J, Almeida PJM, Van der Velden VHJ, Bottcher S, Rawstron AC, De Tute RM, Lhermitte LBS, Asnafi V, Mejstrikova E, Szczepanski T, Da Silva Lucio PJM, Marin Ayuso M, Pedreira CE. Methods, reagents and kits for flow cytometric immunophenotyping. US 2012/0165213 A1 June 2012. [0109] Ward E, DeSantis C, Robbins A, Kohler B, Jemal A. Childhood and adolescent cancer statistics, 2014. CA Cancer J Clin. 2014 March-April; 64(2):83.