Patent Description:
Non-Hodgkin lymphomas (NHL) hold eighth position of all neoplasms among men and eleventh among women worldwide. It is estimated that lymphomas are diagnosed in more than <NUM>,<NUM> individuals annually. The number of deaths is about <NUM>,<NUM>. In Poland, non-Hodgkin lymphomas constitute about <NUM>% of cases and deaths due to neoplastic diseases among men and <NUM>% among women according to the <NPL>. Many years of more and more detailed studies have revealed that under the same name, there are different subtypes of diseases present. Despite the fact that lymphomas initially might have similar clinical symptoms, genetic and molecular profiles thereof vary, and hence they differ in terms of prognosis, course and treatment.

Current diagnostic procedures used in patients with suspected lymphoproliferative disorder include pathomorphological analysis of tissue samples collected from the affected organ and flow cytometry of biopsy specimens.

In lymphoma diagnostics, histopathological analysis based on various materials depending on the clinical picture is performed as a standard. Sections collected surgically most frequently come from lymph nodes or extranodal tissues: skin, gastrointestinal tract, bone marrow, spleen, thymus and tonsils. In most cases, in order to specify the type of lymphoma, it is necessary to perform at least one additional testing: immunohistochemical, genetic or molecular.

Immunohistochemical staining is the differential diagnostics of lymphomas based on the National Comprehensive Cancer Network. The interpretation of immunophenotype should correlate with clinical picture and morphological features of lymphomas.

In flow cytometry, a material from fine-needle aspiration biopsy of a lymph node, peripheral blood or bone marrow is subjected to immunophenotyping.

Lymphoma diagnostics also includes cytogenetic testing (in situ hybridisation/FISH) and cytology testing, in which a material from fine-needle aspiration biopsy of a lymph node, peripheral blood or bone marrow is analysed, as well as diagnostic imaging which employs tomographic techniques.

In the state of art, some other attempts of providing an effective diagnostic test which could be used in case of lymphoproliferative disorders are known.

<CIT> provides a method for diagnosing lymphomas based on the detection of LY6G6F, VSIG10, TMEM25 and LSR proteins expression.

<CIT> discloses a diagnostic method in which the mixed lymphocyte reaction (MLR) is used in tissue culture to detect the tumour-induced autoimmune reaction characteristic of lymphoid neoplasms. Lymphocytes from blood, spleen and lymph nodes are used in combination. The mixed lymphocyte reaction resulting from any of three possible combinations indicates the presence of a lymphoid neoplasm.

<CIT> discloses a diagnostic method for detecting lymphomas, comprising the following steps: determining the hGSTA1 expression level in a tumor sample obtained from a patient, ii) comparing the expression level determined in step i) with a predetermined reference value, iii) concluding that the patient is affected when the hGSTA1 expression level is lower than the predetermined reference value.

<CIT> relates to methods for diagnosing hematological cancers. Particularly, the invention relates to a method comprising the steps of i) detecting the presence of CD45RARO NK cells in a sample obtained from a patient, ii) concluding that the patient suffers from a haematological cancer when the presence of CD45RARO NK cells is detected in the sample, wherein the presence of at least one phenotypic marker indicates the type of the cancer.

<CIT> provides an antibody with the ability to bind the ED-A isoform of fibronectin, wherein the antibody binding with the protein indicates the presence of neoplastic cells of lymphoid tissue.

<CIT> discloses a method for diagnosing neoplasms, including lymphomas, based on algorithm taking into account a measurement of adhesion forces between abnormal cells using atomic force microscope (AFM) or optical tweezers.

<CIT> provides a diagnostic method in which mechanical properties of tumour cells and reference cells are analyzed under a mechanical load which leads to linear or non-linear deformation of said cell. Cells expansion, which results from the application of mechanical stress, is used to determine the risk of tumour metastases, as well as, where appropriate, the presence of uncontrollably proliferating invasive cells or tissue of tumour origin. In case of non-linear deformation of the cell, the risk of the presence of abnormally proliferating cells is determined, based on the mean value of the expansion in the direction of stressing of cells in the sample. The method is realised using optical tweezers.

, <NUM> "Cell adhesion manipulation through single cell assembly for characterization of initial cell-to-cell interaction" reports a case study of characterizing initial interaction between leukemia cancer cells and bone marrow stromal cells through the use of an optical tweezers-based cell manipulation tool. The document does not disclose a diagnostic assays.

<NPL>" reports that CLL and other indolent lymphoma are not curable and usually relapse after treatment, a process in which the tumor microenvironment plays a pivotal role. The authors dissect the consecutive steps of CXCR5-dpendent tumor cell lodging and LTβR-dependent stroma-leukemia cell interaction. The publication reports that leukemia B cells need a longer time to form stable adhesion with stromal cells but there the investigation period is <NUM> minutes. No diagnostic assay is disclosed.

<NPL>" shows a procedure for mapping the trap stiffness in optical tweezers over the whole sample area. Such a map allows development of a real -time procedure for force mesarument at any point of the sample area suitable for measuring forces in living cells samples. The document does not disclose a diagnostic assays.

In <NPL>" multiple optical trapping to study the mechanism of red cell (dis)aggregation is used. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells. It is reported that cross-bridging occurred in normal reversible aggregation as binding and the occurrence of small tethers between opposite cell membranes was observed. The publication discloses that the formation of adhesion is in the order of seconds rather than minutes. The document does not disclose a diagnostic assays.

The percentage of patients with reactive changes among all patients diagnosed with a proliferative disorder is about <NUM>%. The diagnostic process requires more and more complicated diagnostic methods to accurately classify as well as quickly and effectively differentiate between neoplastic changes and non-malignant reactive changes.

Despite available diagnostic methods for lymphoid tissue neoplasms in the state of art, there is still a need to provide a simple and highly specific method for identifying abnormal cells and normal cells in patients with suspected neoplasm.

The aim of the invention is to provide an effective method enabling the efficient identification of abnormal cells formed in the process of carcinogenesis in patients with a suspected neoplastic disease of lymphoid tissue.

The aim has been achieved by the present invention.

The object of the invention is a method for diagnosing neoplasms of lymphoid tissues including the steps of:.

The cell manipulation technique in steps b) and c) is optical tweezers. Preferably, before bringing the B lymphocyte into contact with the bone marrow stromal cell, the B lymphocyte is captured into an optical trap by optical tweezers.

Preferably, the B lymphocyte is captured into the optical trap with the force of <NUM> pN. Preferably, the bond is considered as stable, when the B lymphocyte cannot be pulled away from the bone marrow stromal cell using the optical trap generated with a laser power of <NUM> mW.

Preferably, the neoplasm of lymphoid tissue is B-cell non-Hodgkin lymphoma (NHL). Preferably, the neoplasm of lymphoid tissue is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), MALT lymphoma, Burkitt lymphoma (BL), and high grade B-cell lymphoma (HGBL).

Preferably, the B lymphocyte cell is brought into contact with the central part of the bone marrow stromal cell.

Preferably, the bone marrow stromal cell is a fibroblast.

Preferably, bone marrow stroma cells are of HS-<NUM> line.

Optical tweezers are a device that generates optical forces able to manipulate with objects sized between <NUM> to <NUM> on a microscope slide. The appropriate shape of the laser beam with the wavelength of infrared or visible light range enables the analysis of mechanical properties of cells, the determination of elasticity of cell membranes, intercellular interactions in co-cultures of healthy and neoplastic cells, biological materials, DNA strands. The optical trap generated by the laser beam shaping system, coming from a highly focused laser beam, captures objects present on the slide in the focal plane of the microscopic lens. The system of optical tweezers allows direct or indirect (through special dielectric nanoparticles) measurements of shifts, forces affecting biological structures, or adhesive properties of captured cells.

In the method according to the invention, other cell manipulation techniques can be used.

The essence of the invention relates to the identification of normal and abnormal B lymphocytes based on a time necessary for a lymphoma cell to form a stable adhesion with bone marrow stromal fibroblasts. The method according to the invention enables the effective diagnostics of the neoplasm by differentiating between normal cells and abnormal cells of a stable co-culture of fibroblasts and B lymphocytes in optical tweezers. Thus, the method according to the invention based on the interactions between B lymphocytes and bone marrow stroma fibroblasts enables the distinction between normal B lymphocytes and abnormal cells with high effectiveness. Therefore, the diagnostic procedure at the first step thereof is already shorter and cheaper.

The aim of the inventors was to obtain cells in a state as close as possible to the state thereof in in vivo conditions. The method according to the invention utilised a specifically defined clinical material in a form of normal and neoplastic B lymphocytes obtained for routine cancer diagnostics purposes. The clinical material comprises neoplastic cells obtained from patients diagnosed with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa associated lymphoid tissue lymphoma (MALT) , Burkitt lymphoma (BL), and high grade B-cell lymphoma (HGBL). Information on the origin and type of the clinical material included in the study are collected in Tables <NUM> and <NUM>, wherein Table <NUM> shows clinical and pathological data for control patients included in the study. The study included <NUM> individuals aged between <NUM> and <NUM> years old (mean M = <NUM>; standard deviation SD = <NUM> years). Basic statistics of traits characteristic for both groups are presented in Table <NUM> (age and sex of patients in test and control group; results of Mann-Whitney U test). The statistical analysis of results was performed in Statistica <NUM> software, StatSoft Poland. Both groups of patients did not differ significantly in terms of age (<NUM> vs. <NUM> years; p = <NUM>), nor sex structure (percentage of women: <NUM>% vs. <NUM>%; p = <NUM>).

Cell suspension is collected under USG control to PBS solution pH=<NUM> (Life Technologies, cat. No. <NUM>-<NUM>), and subsequently transferred sterilely to a probe with EDTA (Becton Dickinson, EDTA <NUM>, cat. No. <NUM>). After <NUM> minutes, the material is deep-frozen in <NUM>% foetal bovine serum (Life Technologies, FBS, cat. No. <NUM>-<NUM>) and <NUM>% DMSO (BioShop, cat. No. DMS555). Cells are kept in liquid nitrogen.

Following the surgical lymph node collection, the material is transferred to sterile PBS solution with <NUM>% antibiotic and antimycotic (Life Technologies, cat. No. <NUM>). B lymphocytes are isolated from the lymph node up to <NUM> hours post collection. The cells are aspirated using a biopsy needle to a syringe with the anticoagulant and sterile PBS solution. After <NUM> minutes of incubation at the room temperature, the material is frozen in <NUM>% FBS and <NUM>% DMSO. Cells are kept in liquid nitrogen.

The holographic system of tweezers used in the present method has the following parameters: trapping lasers: <NUM> 4W, <NUM> <NUM> mW; microscopic lens 100x NA <NUM>; holographic generation of optical traps using Hamamatsu LCoS modulator with the resolution of 800x600; laser beam control using galvano mirrors; optical path for spectroscopic analysis; video camera: CMOS max frame rate <NUM> fps (frames per second).

The sample is thawed, centrifuged twice in RPMI medium (<NUM> × g for <NUM>). The number and viability of cells is determined, and then the cells are incubated for <NUM> at <NUM>, <NUM>% CO<NUM>.

In cases where the percentage of abnormal B lymphocytes in immunophenotyping (flow cytometry) was lower than <NUM>%, the depletion of T lymphocytes and NK cells was performed using magnetic beads coated with anty-CD3 antibody (CD3+ Manual MACS® Cell Separation, Miltenyi Biotech). Control samples are subjected to this procedure each time. Samples of patients No. <NUM>, <NUM>, <NUM> due to a large percentage of normal B lymphocytes were subjected to additional magnetic isolation using beads coated with CD5+ and CD10+ antibodies. The effectiveness of magnetic isolation was confirmed with flow cytometry. Just before measurements in optical tweezers, cells are centrifuged and a suspension with density of <NUM>×<NUM><NUM>/ml is prepared.

In order to obtain optimal and replicable experimental conditions, a protocol has been developed which determines the method for starting a culture of HS-<NUM> bone marrow stromal cells. For HS-<NUM> cell line culture (ATCC, cat. No. ATCC® CRL-<NUM>™), DMEM medium is recommended (ATCC, Dulbecco's modified Eagle's medium, cat. No. ATCC® <NUM>-<NUM>™). The medium was additionally supplemented with foetal bovine serum (ATCC, FBS, cat. No. ATCC® <NUM>-<NUM>™). Cell culture was maintained in <NUM> at humid atmosphere in the presence of <NUM>% CO<NUM>.

HS-<NUM> cells after reaching the confluence of <NUM>% in a culture flask were passaged and their quantity and viability were determined using Trypan Blue (Invitrogen, The Countess™ automated cell counter). Subsequently, the cells in experimentally defined quantity (<NUM>,<NUM> cells in <NUM> ul of DMEM medium) were seeded on a sterile Petri dish with a glass bottom (Greiner bio-one, cat. No. <NUM>). After <NUM> from starting the culture, <NUM> of culture medium was added to cells. After <NUM> from starting the culture, the confluence of morphologically mature cells on dishes was about <NUM>%. Just before measurements in optical tweezers, cells not adhering to the substrate were washed off.

A measurment dish with HS-<NUM> fibroblasts was placed on a table of the optical manipulator. <NUM> ul of B lymphocyte suspension was transferred directly into the dish. Time of cell sedimentation was about <NUM> minutes. Subsequently, the B lymphocyte was captured into the optical trap with the force of <NUM> pN and brought into contact with the central part of the stromal cell, such that the cells remained in a direct contact until the stable co-culture formation (<FIG>).

Experimentally determined time intervals for cell trapping were, respectively: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for normal cells and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for abnormal cells.

It has been assumed that a stable co-culture is a state in which the B lymphocyte cannot be pulled away from HS-<NUM> stromal cell using the optical trap generated with a laser power of <NUM> mW.

The dish with cell substrate was changed every <NUM> minutes of manipulation while not allowing for the temperature of the culture medium to drop below <NUM>. The experiment was repeated at least twice for each case.

Tables <NUM> and <NUM> show the measurement results of time required for the co-culture formation in optical tweezers for abnormal and normal cells, respectively. Table <NUM> shows the time required for the formation of a stable bond between abnormal B lymphocytes and HS-<NUM> stromal cells using optical tweezers. Table <NUM> shows the time required for the formation of a stable bond between normal B lymphocytes and HS-<NUM> stroma cells using optical tweezers.

Basic statistics for the time of the stable adhesion formation for cells isolated from affected (T) and healthy individuals (C) together with the significance test are presented in Table <NUM>.

The obtained measurements indicate that lymphoma cells form a stable bond with HS-<NUM> stroma fibroblasts from <NUM> to <NUM> times slower than normal cells. The time required for the stable bond formation in abnormal cells is significantly longer than in normal cells (<NUM> vs. <NUM>). The result of the comparison between mean time values is statistically significant at the level of p < <NUM>. The power of the test is close to one (<FIG>).

Tables <NUM> and <NUM> show the percentage of B lymphocytes forming stable co-cultures with stromal cells in particular time intervals. Table <NUM> comprises the % distribution of abnormal cells forming a stable bond with stroma cells in particular time intervals. Table <NUM> comprises the % distribution of normal cells forming a stable bond with stroma cells in particular time intervals.

In order to estimate the cut-off value (cut-off) for the test differentiating between abnormal and normal cells, the analysis of ROC curves was preformed (<FIG>).

For a cut-off value of t > <NUM>, test sensitivity is Sens. = <NUM>% and specificity Spec. = <NUM>% (<FIG>).

Claim 1:
A method for in vitro diagnosing neoplasms of lymphoid tissues comprising the steps of:
a) providing B lymphocytes previously collected and isolated from a patient,
b) preparing a suspension of B lymphocytes as well as bone marrow stromal cells for measuring by optical tweezers technique,
c) by optical tweezers technique bringing a B lymphocyte to contact with a bone marrow stroma cell, such that the B lymphocyte and the bone marrow stromal cell remain in a direct contact until a stable adhesion is formed,
d) measuring time of the formation of the stable connection (t) between the B lymphocyte and the bone marrow stroma cell, wherein the stable adhesion formation of t > <NUM> indicates the presence of neoplastic cells.