RU2574987C2 - Methods for diagnosing and inducing leukaemia/lymphoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely a diagnostic technique for acute lymphoblastic leukaemia in a patient involving detection of leukaemia indicators, blood cell testing for leukaemia, blood cell incubation with a leukaemia inducing factor to induce expression of cell surface markers which are a leukaemia indicator, wherein the leukaemia inducing factor is Aspergillus flavus supernatant, EBV-infected CCL87 supernatant, purified EBV culture or a combination thereof. The blood cell testing for cell surface markers, wherein the level of cell surface markers is the leukaemia indicator, or blood plasma testing for leukaemia. Incubation with leukaemia inducing factor in a tray with leukaemia inducing factor, blood plasma incubation with leukaemia inducing factor. Blood plasma immune-sorbent assay and IgG responsiveness measurement in biological samples, wherein IgG responsiveness is the leukaemia indicator. A method for introducing leukaemia inducing factor into mononuclear cells.

EFFECT: using the declared methods enables diagnosing and detecting acute lymphoblastic leukaemia in the patients effectively.

14 cl, 24 dwg, 4 ex

Description

FIELD OF THE INVENTION

This invention relates to medical diagnostics and is used in diseases such as leukemia and diffuse lymphomas, as well as in vitro re-induction of leukemia in mononuclear leukocyte cells of patients in remission. In particular, the invention includes a procedure for inducing leukemic or leukemoid surface markers into mononuclear leukocytes, as well as a blood plasma test to identify and diagnose individuals at risk for developing leukemia / diffuse lymphomas.

BACKGROUND OF THE INVENTION

Leukemia is a heterogeneous group of cancers of the blood-forming organs, characterized by the production of abnormal white blood cells, their release into the bloodstream and organ infiltration. According to estimates by the US National Cancer Institute in 2010, 43,050 new cases of leukemia and 21,840 leukemia-related deaths were identified. Although leukemia can occur at any age, about 90% of cases have been diagnosed in adults. However, the cause of leukemia in most cases remains unknown (Ilakura H, Coutre SE: Acute lymphoblastic leukemia in adults. In: Greer JP, et al., Editors. Leukemia in adults. Philadelphia: Lippincott Williams and Wilkins. 2009. p. 1821; Faderel S, et al., Acute lymphoblastic leukemia. In: Hong WK, et al., Editors. Holland-Frei cancer medicine. Shelton, Connecticut: People's Medical Publishing House, 2010. p. 1591; MacArthur AC, et al., Risk of Childhood leukemia Associated with Vaccination, Infection, and Medication Use in Childhood. Am. J Epidemiol. 2008; 167 (5): 598-606). Among the alleged causes of leukemia are natural and artificial ionizing radiation (Maloney W. Leukemia in survivors of atomic bombing. N Eng J Med. 1955; 253: 88; Preston D, Kusumi S, Tomonaga M, et al. Cancer incidence in atomic Bomb survivors. Part III. Leukemia, lymphoma and myeloma, 1950-1987. Radiat Res. 1994; 137: S68-S97; Little MP, et al., The statistical power of epidemiological studies analyzing the relationship between exposure to ionizing radiation and cancer , with special reference to childhood leukemia and natural background radiation. Radiat Res. 2010; 174 (3): 387-402; Davies A, Modan B, Djaldetti M, et al. Epidemiological observations on leukemia in Israel. Arch Intern Med. 1961 ; 108 (1): 86-90), viruses such as Epstein-Barr virus (Tokunaga M, et al., Epstein-Barr virus in adult T-cell leukemia / lymphoma. Am J Pathol. 1993; 143 (5): 1263-1268), Human T-lymphotropic virus (HTLV-1) (Phillips AA, et al., A critical analysis of prognostic factors in North American patiensts with human T-cell lymphotropic virus type -1-associated adult T-cell leukemia / lymphoma: a multicenter clinicopathologic experience and new prognostic score. Cancer 2010; 116 (14): 3438-3446; Jeang KT. HTLV-1 and adult T-cell leukemia: insights into viral transformation of cells 30 years after virus discovery. J Formos Med Assoc. 2010; 109 (10): 688-93), infections (Greaves MF, Alexander FE. An infectious etiology for common acute lymphoblastic leukemia in childhood? Leukemia. 1993; 7: 349-360; Smith MA, et al., Investigation of leukemia cells from children with common acute lymphoblastic leukemia for genomic sequences of the primate polyomaviruses, JC virus, BK virus and simian virus 40. Med Pediatr Oncol. 1999; 33: 441-443; Smith MA, et al., Evidence that childhood acute lymphoblastic leukemia is associated with an infectious agent linked to hygiene conditions. Cancer Causes Control. 1998; 9: 285-298), some chemicals such as benzene and alkylating chemotherapy drugs (Wen WQ, et al., Paternal military service and risk for childhood leukemia in offspring. Am J Epidemiol. 2000; 151: 231-240; Momota H, et al., Acute lymphoblastic leukemia after temozolomide treatment for anaplastic astrocytoma in a child with a germline TP53 mutation Pediatr Blood Cancer. 2010; 55 (3): 577-9; Borgmann A, et al., Secondary malignant neoplasms after intensive treatment of relapsed acute lymphoblastic leukaemia in childhood. ALL-REZ BFM Study Group. Eur J Cancer. 2008; 44 (2): 257-68), family predisposition (Karakas Z, Tugcu D, Unuvar A, Atay D, Akcay A, Gedik H, Kayserili H, Dogan O, Anak S, Devecioglu O. Li-Fraumeni syndrome in a Turkish family. Pediatr Hematol Oncol. 2010; (4): 197-305; Buffer PA, et al., Environmental and genetic risk factors for childhood leukemia: Appraising the evidence. Cancer Investigation. 2005; 23 (1): 60-75) , drugs (Tebbi CK, et al., Dexrazoxane-associated risk for acute myeloid leukemia / myelodysplastic syndrome and other secondary malignancies in pediatric Hodgkin's disease. J Clin Oncol. 2007; 25 (5): 493-500), genetic factors, chromosomal and metabolic abnormalities (Fong CT, Brodeur GM. Down's syndrome and leukemia: epidemiology, genetics, cytogenetics and mechanisms of leukemogenesis. Cancer Genet Cytogenet. 1987; 28 (1): 55-76; Chao MM, et al., T- cell acute lymphoblastic leukemia in association wit h Borjeson-Forssman-Lehman syndrome due to a mutation in PHF6. Pediatr Blood Cancer. 2010; 55 (4): 722-4; Kato K, et al., Late recurrence of precursor B-cell acute lymphoblastic leukemia 9 years and 7 months after allogenic hematopoietic stem cell transplantation. J Pediatr Hematol Oncol. 2010; 32 (7): e290-3; Smith MT, et al., Low NAD (P) H: quinine oxidoreductase 1 activity is associated with increased risk of acute leukemia in adults. Blood 2001; 97: 1422-1426; Hengstler JG, et al., Polymorphisms of N-acetyltransferases, glutathione S-transferases, microsomal epoxide hydrolase and sulfotransferases: influence on cancer susceptibility. Recent Results Cancer Res. 1998; 154: 47-85; Schenk TM, et al., Multilineage involvement of Philadelphia chromosome positive acute lymphoblastic leukemia. Leukemia. 1998; 12: 666-674; Felix CA, et al., Immunoglobulin and T cell receptor gene configuration in acute lymphoblastic leukemia of infancy. Blood 1987; 70: 536-541), immune disorders (Vajdic CM, et al., Are antibody deficiency disorders associated with a narrower range of cancers than other forms of immunodeficiency? Blood. 2010; 116 (8): 1228-34), and factors environmental-related (Magnani C, et al., Parental occupation and other environmental factors in the etiology of leukemias and non-Hodgkin's lymphomas in childhood: a case-control study. Tumori. 1990; 76 (5): 413-9 ; Non-Ionizing Radiation, Part 1: Static and Extremely Low-Frequency (ELF) Electric and Magnetic Fields (IARC Monographs on the Evaluation of the Carcinogenic Risks). Geneva: World Health Organization, pp.332-333, 338, 2002; Kroll ME, et al., Childhood cancer and magnetic fields from high-voltage lines in England and Wales: a case-control study. Br J Cancer. 2010; 103 (7): 1122-7; Little, MP, et al. , The statistical power of epidemiological studies analyzing the relationship between exposure to ionizing rad iation and cancer, with special reference to childhood leukemia and natural background radiation. Radiat Res 2010; 174 (3): 387-402). None of the above factors can be consistently applied in most cases, in addition, it was not possible to prove with certainty that the presence of these factors predictably led to the development of leukemia in all individuals who were exposed to these factors.

Leukemia can be divided into subgroups, including acute and chronic leukemia. In the case of acute leukemia, there is a rapid increase in immature white blood cells (white blood cells) formed in the bone marrow, and a decrease in the production of normal blood cells. Acute leukemia include acute lymphoblastic leukemia (ALL) and acute myelocytic leukemia (AML). Chronic leukemia include chronic lymphocytic leukemia and chronic myeloid leukemia (CLL and CML). Due to the rapid development of the disease and the accumulation of cancer cells in acute leukemia, immediate treatment is required.

Acute leukemia is the most common form of cancer in children. Acute lymphoblastic leukemia (ALL) and diffuse lymphomas also make up a significant proportion of cancers in children. ALL is the most common form of leukemia in children (Pui CH. Acute lymphoblastic leukemia in children. Curr opin oncol. 2000; 12: 3-12), accounting for four of all cases of cancer in children and about 75% of all cases of leukemia in children moreover, the peak of leukemia disease occurs at the age of 2-5 years (Pui CH. Acute lymphoblastic leukemia in children. Curr opin oncol. 2000; 12: 3-12; Miller R. Acute lymphoblastic leukemia. In: CK Tebbi, editor. Major Topics in Pediatric and Adolescent Oncology. Boston: GK Hall Medical Publishers; 1982. p.3-43). Today, despite numerous studies, the cause of leukemia in children remains unknown, with the exception of individual cases. As indicated above, various etiologies have been proposed, such as viral infections, exposure to chemicals, drugs caused by ionizing radiation, chromosomal abnormalities / genetic factors, immune deficiency, environmental factors, etc., but none of these factors cannot be consistently applied in most cases, in addition, it was not possible to unequivocally prove that the presence of these factors predictably led to the development of cancer. The International Association for the Study of Cancer conducted a retrospective study on the dependence of the incidence of leukemia disease on the proximity of power lines; limited evidence has been obtained that high levels of magnetic fields of extremely low frequencies (ELF) can lead to a twofold increase in the risk of developing leukemia in children, but these findings were later refuted (Non-Ionizing Radiation, Part 1: Static and Extremely Low-Frequency (ELF ) Electric and Magnetic Fields (IARC Monographs on the Evaluation of the Carcinogenic Risks). Geneva: World Health Organization, pp. 323-333, 338, 2002; Kroll ME, et al., Childhood cancer and magnetic fields from high-voltage lines in England and Wales: a case-control study. Br J Cancer. 2010; 103 (7): 1122-7; Little MP, et al., The statistical power of epidemiological studies analyzing the relationship between exposure to ionizing radiation and cancer, with special reference to childhood leukemia and natural bac kground radiation. Radiat Res 2010; 174 (3): 387-402).

The leukemia diagnosis is usually made on the basis of a general blood test, bone marrow examination using light microscopy, flow cytometric determination of surface antigenic phenotypes of a cell, and other studies. In the case of lymphomas, a lymph node biopsy can be performed for diagnosis using flow cytometry and cytometry using pathological methods, as well as other tests. Typically, with leukemia and lymphomas, a cytogenetic examination is also performed, which can give a certain prognostic value. In case of acute leukemia and diffuse lymphomas, spinal puncture is required to exclude damage to the central nervous system.

Most types of leukemia are treated on the basis of combined drug treatment with chemotherapy regimens: such drugs as prednisone, lasparaginase, vincristine, daunorubicin, antimetabolites are used, both with the use of radiation therapy (radiotherapy) and without this therapy (Pui CH. Acute lymphoblastic leukemia in children. Curr opin oncol. 2000; 12: 3-12) when it comes to the central nervous system. In some cases, during remission or after relapse, bone marrow transplantation may be required. The main goal of the treatment is to achieve normal reproduction of the bone marrow, which is called remission, and also to eliminate the systematic infiltration of organs by cancer cells.

Modern methods for diagnosing leukemia require highly qualified specialists and equipment that can analyze various parameters, including bone marrow examination, flow cytometry and cytogenetics. However, the current proposed tests are characterized by an unrecoverable amount of uncertainty associated with the level of technical expertise of diagnostic doctors and technical personnel; in addition, carrying out these tests requires considerable time. To date, there are no laboratory tests that can confidently predict a predisposition to leukemia before the detection of this disease. In addition, there are no opportunities for systematic screening of the population, including infants and children. Also, methods for preventing leukemia and lymphomas in people predisposed to these diseases are unknown. Accordingly, there is an urgent need for a quick and reliable method for detecting the predisposition and diagnosis of leukemia and lymphomas, and screening to select vaccines and drugs to prevent leukemia.

SUMMARY OF THE INVENTION

In this invention, cell surface markers and enzyme-linked immunosorbent assay (ELISA) are used to establish a predisposition to leukemia. It was shown that the action of specific proteins on cells can lead to the restoration of cell surface markers in the peripheral mononuclear blood cells of patients who have previously undergone leukemia, which allows us to determine the potential for leukemia. Similarly, the invention provides recognition of the potential for developing leukemia based on the identification of antibodies present in the plasma of patients who have previously experienced leukemia, and not in "normal" people, which may make it possible to identify the cause of the disease. The proposed results show that the research methods presented here can be used to identify patients who suffer from leukemia, have had leukemia, or are predisposed to leukemia.

The detection of leukemic potential at the cell level is based on new research results suggesting that acute lymphoblastic leukemia (ALL) can be re-induced in patients previously suffering from leukemia, in patients in remission, and in patients undergoing therapy based on culture supernatant or synthetic a protein of a fungal agent, i.e., aspergillus flavus, with or without a virus, for example, Epstein-Barr virus (EBV), in vitro. In addition, the combination of aspergillus flavus (yellow aspergillus) and EBV induces a new protein that enhances the manifestation of cell surface markers characteristic of acute lymphoblastic leukemia (ALL), which is detected by flow cytometry in mononuclear leukocyte cells of patients who have undergone ALL in the past. This flow cytometry method may reveal a predisposition to the development of leukemia markers in “susceptible” individuals. A method based on the use of the supernatant of aspergillus flavus culture or EBV, or a combination thereof used in an enzyme-linked immunosorbent assay (ELISA), can distinguish between "normal" individuals from patients who have undergone ALL in the past. These research results differ from the inconsistent exposure to aflatoxins, which does not allow the separation of normal individuals from patients who have previously undergone leukemia. These research results are used for the etiology of leukemia and diffuse lymphomas. Potentially, this invention can be used for mass screening and identification of individuals with a predisposition to leukemia.

Mushrooms were isolated in virtually all parts of the globe. Species of aspergillus, such as aspergillus flavus, parasiticus, nomius, and others, are widespread in nature and have been isolated from various environmental sources, including homes and food products such as greens, corn, peanuts, soybeans (Roze, et al., Aspergillus volatiles regulate Aflatoxin synthesis and asexual sporulation in Aspergillus parasiticus. Applied and Environmental Micro. 2007; 73 (22): 7268-7216). Aspergillus (aspergillus) and other fungi are capable of producing various peptides and substances, including ergot, alkaloids, aflatoxins (Kosalec I, and Pepeljnjak S. Mycotoxigenicity of clinical and environmental Aspergillus fumigates and Aspergillus flavus isolates. Acta Pharm. 2005; 55 (4) 365-375; Roze, LV, et al., Aspergillus volatiles regulate Aflatoxin synthesis and a sexual sporulation in Aspergillus parasiticus. Applied and Environmental Micro. 2007; 73 (22): 7268-7276; Latge JP. Aspergillus fumigates and aspergillosis. Clin Microbiol Rev. 1999; 12: 310-350; Fischer G, and Dott, W. Relevance of airborne fungi and their secondary metabolites for envorinmental, occupational and indoor hygiene. Arch Microbiol. 2003; 179: 75-82; Fischer G, et al., Species-specific profiles of mycotoxins produced in cultures and associated with conidia of airborne fungi derived from biowaste. Int J Hyg_En viron Health. 2000; 203: 105-116; Raper KB, and Fennell DI. The genus Aspergillus. Baltimore: Williams &Wilkins;1965; Stack D, et al., Nonribosomal peptide synthesis in Aspergillus fumigates and other fungi. Microbiology. 2007; 153: 1297-1306; Yang CV, et al., Inhibition of ebselen on Aflatoxin B1-induced hepatocarcinogenesis in Fischer 355 rats. Carcinogenesis. 2000; 21 (12): 2237-2243; Ito, Y, et al., Aspergillus pseudotamarii, a new Aflatoxin producing species in Aspergillus section Flavi. Mycol Res. 2001; 105 (2): 233-239; Matsumura M, Mori T: Detection of aflatoxins in autopsied materials from a patient infected with Aspergillus flavus. Nippon Ishinkin Gakkai Zasshi. 1998; 39 (3): 167-71; Louria DB, et al., Aflatoxin-induced tumors in mice. Medical Mycology. 1974; 12 (3): 371-375; Cole RJ, et al., Mycotoxins produced by Aspergillus fumigates species isolated from molded silage. J Agric Food Chem. 1977; 25: 826-830). Aflatoxins are powerful, naturally-occurring mycotoxins produced by many varieties of aspergillus, including aspergillus flavus. At least thirteen subtypes of aflatoxins were identified, with B1 being the most toxic. Both aflatoxin B1 and aflatoxin B2 are produced by aspergillus flavus. People are often exposed to aspergillus species, which are widespread in the environment. Aflatoxins are known to be metabolized by the liver. The carcinogenesis and infections induced by various aspergillus strains are well known (Louria DB, et al., Aflatoxin-induced tumors in mice. Medical Mycology. 1974; 12 (3): 371-375; Yarborough, A, e al., Immunoperoxidase detection of 8-hydroxydeoxyguanosine in Aflatoxin B1-treated rat liver and human oral mucosal cells. Cancer Research. 1996; 56: 683-688).

Epstein-Barr virus (type 4 human herpes virus, HHV-4 or EBV), belonging to the herpes virus family, is one of the most common viral pathogens that affect humans. Various strains of this virus are common throughout the world. EBV infects B cells. In vitro (in vitro) infection leads to the transformation of lymphocytes, which express new proteins. EBNA-2, EBNA-3C and LMP-1 are important for the transformation of lymphocytes, while other proteins, such as EBNA-LP and EBERs, are not involved in this process. EBNA-1 protein is necessary to preserve the viral genome. In the case of acute infections, the signs of this virus are well known, chronic inactive persistence may occur with periodic relapses and reactivation. EBV nuclear proteins are produced by alternative splicing of the transcript, starting from the Cp promoter, or from the Wp promoter at the left end of the genome. Genes are EBNA-LP / EBNA-2 / EBNA-3A / EBNA-3B / EBNA-3C / EBNA-1 within the genome.

EBV has been associated with carcinogenesis (Tokunaga M, Imai S, Uemura T Tokudome, Osato T, and Sato E. Epstein-Barr virus in adult T-cell leukemia / lymphoma. Am J Pathol. 1993; 143 (5): 1263-1268) autoimmune disorders, infections, and even diabetes. Most people become infected with this virus when they reach puberty. In the United States, the previous exposure of the virus to adults over 35 years of age is 95%. When a disease occurs in adolescence or adulthood, it can cause symptoms of infectious mononucleosis in 50% of infected people (CDC Data 2010).

The effects of the simultaneous exposure of aspergillus flavus and the Epstein-Barr virus to humans, as well as the gene-environmental interaction with each agent and their combination, have not been previously described.

The studies described here showed that mononuclear cells obtained from the peripheral blood flow of patients with ALL produce a reaction when exposed to the supernatant of aspergillus flavus culture, or EBV sources, or a combination of these two factors, forming blast cells that are indistinguishable from the cell surface phenotype leukemic cells. This phenomenon can be observed in patients undergoing therapeutic treatment, and in those people who have already completed treatment, even if they had been treated for many years before this study. Exposed cells exhibit cell surface phenotypes (CD10 / 19, CD34 / CD19, CD34 / CD117) that are signs of acute lymphoblastic leukemia. This reaction is enhanced by the addition of a CCL87 culture supernatant that contains Epstein-Barr virus (EBV) or by the addition of purified EBV virus, with or without incubation. Control samples, including mononuclear white blood cells (leukocytes), taken from normal people and patients with sickle cell disease (SC), give a different reaction. Based on the ELISA technique, it was found that the plasma of patents suffering from ALL reacts with the supernatant of aspergillus flavus cultures. However, various results were obtained when the cells of "normal" people (control samples), that is, the cells of normal donors, and patients with sickle cell disease, or those with solid tumors, were treated in vitro (in laboratory conditions) on an identical basis ... a statistically clear separation of leukemia and "normal" control samples was obtained. Irradiation of cultures containing aspergillus flavus in combination with or without EBV led to an increase in the described effects. Replacing the supernatant of aspergillus flavus culture with mycocladus corymbifera varieties or purified available aflatoxin B1 (AT), as well as replacing EBV with avian leukemia virus (cV), does not lead to similar discriminant mutations in the cells of leukemia patients in remission and in cells of “normal” people ". Substitution with plasma (pl), or the supernatant of 1C3 B-lymphoblastic cell line (CRL-2312), with the marmoset of the monkey (OM) did not induce the development of ALL surface markers. Further experiments showed that the development of surface phenotypes of leukemia cells occurs gradually, begins two hours after incubation with aspergillus flavus, with or without Epstein-Barr virus, and ends after 24 hours of incubation. No changes were noted in the normal control samples.

In an analysis based on liquid rapid protein chromatography (FPLC), aspergillus flavus supernatant (X) shows three peak protein values. EBV produces one protein peak. Adding EBV with short incubation (less than four hours) or long incubation (seven days) increases the first peak value of aspergillus flavus protein and induces the formation of an additional new peak value. In addition, only irradiation of aspergillus flavus or irradiation of aspergillus flavus in combination with EBV induces a further increase in the inductive abilities of the aspergillus flavus fraction of the first peak in patients who have previously undergone leukemia, but not in control samples. After irradiation, the effects of fractions of peaks 2 and 3 remain unchanged. A comparison of aspergillus flavus peaks, EBV peaks, and combinations thereof are shown in Figures 1-3. While fractions of the first peak have the highest specific activity, the other two peaks are also effective in inducing leukemic cell markers in sensitized cells and in detecting diseases based on the ELISA technique. These substances can induce leukemia markers in patients previously undergoing leukemia, but not in control samples. Testing of these peaks shows that they are also useful for the serological detection of patients predisposed to leukemia, with the first peak having the highest activity.

The above experiments describe the induction of cell surface phenotypes characteristic of acute lymphoblastic leukemia or diffuse lymphomas upon exposure of the supernatant of aspergillus flavus culture, or EBV, or a combination thereof to patients in a state of prolonged remission or "cured" their disorders. The last-mentioned effect is enhanced when cells are exposed to a combination of these factors, or when a combination of these reagents is irradiated prior to incubation with mononuclear cells from patients in remission or "cured" of leukemia or lymphoma. These effects cannot be duplicated in the case of normal control samples or in patients with solid tumors. The discriminatory effect detected in the presence of these reagents is not observed in the case of purified aflatoxin B, avian leukemia virus or other agents used as control samples. The study also described the detection of antibodies against aspergillus flavus and EBV and their combination in the plasma of patients with acute lymphoblastic leukemia and diffuse lymphomas, but not in patients with solid tumors and not in control samples. These research results show that individuals with acute lymphoblastic leukemia or diffuse lymphomas can have a distinct genotype, which allows re-induction of leukemic markers with a certain exposure. It is assumed that gene-environmental interactions with aspergillus flavus in the presence of a viral pathogen or without this pathogen cause phenotypes characteristic of these diseases or their relapses. The research results may be applicable to the etiology of cancer in general and leukemia / diffuse lymphoma in particular.

The effect observed here is apparently unique to aspergillus flavus or to EBV, which clearly induces the expression of leukemic cell surface markers in people with leukemia, people who have recovered from leukemia, and people predisposed to leukemia. Other compositions do not distinguish between people with leukemia and those who have had leukemia in the past, as well as those who have no predisposition to leukemia. For example, aflatoxin induces the expression of leukemic markers on cell surfaces, but does not have this effect with respect to all samples, while other viruses similar to the monkey sovinolytic virus do not induce marker expression. As such, the tests conducted here are based on the new (previously unknown) ability of aspergillus flavus and / or EBV to induce markers on the cell surface.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of this invention, you should pay attention to the following detailed description in combination with the accompanying drawings, in which:

Figure 1 (A) - (D) is an FPLC analysis (analysis based on liquid flash chromatography) of fractional isolates from the supernatant of (A) aspergillus flavus culture (X), (B) purified (EBV), (C) the combination of cultured supernatant aspergillus flavus and EBV (X + EBV), and (D) - all charts superimposed on each other.

Figure 2 (A) - (D) shows the FPLC analysis of supernatant culture cultures of (A) aspergillus flavus (X), (B) incubated purified Epstein-Barr virus (EBV), (C) a combination of aspergillus flavus and Epstein-Barr virus ( X + EBV), incubated for seven days, and also (D) is an overhead graph showing the emergence of a new peak not shown during a separate incubation of aspergillus flavus (X) or Epstein-Barr virus (EBV).

Figure 3 (A) - (C) shows FPLC fractionation of the supernatant (A) of aspergillus flavus (C), (B) a combination of aspergillus flavus and Epstein-Barr virus (X + EBV) after 50 centiGray (cGy) irradiation, and (C ), where both graphs are superimposed.

Figure 4 is a graph showing an example of the relative percentage of cells labeled with one of the leukemic (ALL) cell surface markers CD10 / CD19 on day 2 of incubation. X is the supernatant of aspergillus flavus fungal culture; E is the supernatant from the EBV-infected CCL-87 culture; pl is human plasma; eV is a separately used Epstein-Barr virus (2 × 10 ⁶ PFU); eV is a bird leukemia virus at 2 × 10 ⁶ PFU / ml; AT is aflatoxin; OM is the supernatant of cell culture with the monkey owl. Mononuclear leukocytes were obtained from a group of patients who had undergone acute lymphoblastic leukemia (ALL) in the past and were compared with control samples of “normal” people. The control samples were mononuclear cells from a group of sickle cell patients undergoing routine blood exchange transfusion (blood from a blood bank and plasma). Cellular surface phenotypes (CD10 / 19, CD34 / 19, CD34 / CD117) were analyzed daily for four days using a flow cytometer (BD FACS Canto I, Becton, Dickinson, & Co., Franklin Lakes, NJ). The effects of all cell surface markers were similar; thus, the results of CD10 / 19 are demonstrated. These results are presented as the percentage of control samples (a more detailed analysis and deviations from the standard are presented in Figure 15).

Figures 5 (A) to (C) are graphs showing the cell surface phenotype of mononuclear leukocytes upon exposure to the supernatant of aspergillus flavus culture or EBV. Cells of long-term leukemia patients (ALL) and cells of "normal" sickle cell patients (SC) (control samples) were incubated with either supernatant of aspergillus flavus fungal culture (X) or supernatant from EBV-infected CCL-87 cultures (E) and were fixed 1-4 days after incubation.

фильтр (Corning Inc, Corning, NY) (X), либо с супернатантом клеточной культуры совинолицей мартышки (CRL-2312) (от); причем клеточные поверхностные фенотипы были проанализированы на 1-4 день после инкубации. Клеточные поверхностные фенотипы, характерные для острого лимфобластного лейкоза (ALL), оценивались ежедневно в течение четырех дней после инкубации, используя поточный цитометр (BD FACS Canto II, Becton, Dickinson, & Co., Franklin Lakes, NJ). Результаты представлены как процентное соотношение контрольных образцов.Figures 6 (A) - (C) are graphs illustrating cell surface phenotypes. Cells of patients who had previously undergone leukemia (ALL), and cell samples of "normal" patients / sickle cell disease (SC) patients (control samples) were incubated either with supernatant of aspergillus flavus fungal culture, filtered through 2.5 μm $$$$

a filter (Corning Inc, Corning, NY) (X), or with a cell culture supernatant with a monkey sovinolyticus (CRL-2312) (from); moreover, cell surface phenotypes were analyzed 1-4 days after incubation. The cell surface phenotypes characteristic of acute lymphoblastic leukemia (ALL) were evaluated daily for four days after incubation using a flow cytometer (BD FACS Canto II, Becton, Dickinson, & Co., Franklin Lakes, NJ). The results are presented as the percentage of control samples.

Figures 7 (A) - (C) are graphs showing cell surface phenotyping. Mononuclear cells from long-term leukemic patients (ALL) and cell samples of "normal" patients / sickle cell disease (SC) patients (control samples) were incubated either with supernatant of aspergillus flavus fungal culture (X), or with 10 μg / ml aflatoxin B1 (AT), and were compared with “normal” control samples. Cultures were evaluated and fixed at 1-4 days after incubation.

Figures 8 (A) to (C) are graphs showing cell surface phenotyping of cells of individuals who have long suffered leukemia under the influence of the supernatant of aspergillus flavus culture (X) as opposed to purified Epstein-Barr virus (eV) compared to "normal" control samples. The study was repeated daily, starting from 1-4 days after incubation.

Figures 9 (A) - (C) are graphs showing cell surface phenotyping of cells of individuals who have long suffered leukemia under the influence of the supernatant of aspergillus flavus culture (X) compared to avian leukemia virus (cV). Cultures were evaluated daily, starting from 1-4 days after incubation. Control samples are mononuclear cells from "normal" patients / sickle cell patients.

Figures 10 (A) - (C) are graphs showing cell phenotyping of cells of people who have long suffered leukemia under the influence of the supernatant of aspergillus flavus culture (X) compared to autologous plasma (pl). Cultures were evaluated daily, starting from 1-4 days after incubation.

Figures 11 (A) to (C) are graphs showing cell phenotyping of cells of individuals who have experienced leukemia in the past under the influence of the supernatant of aspergillus flavus culture (X) compared with a combination of aspergillus flavus (X) and Epstein-Barr virus (emitted from CCL87 cell culture) (E) (X + E). Cultures were evaluated daily, starting from 1-4 days after incubation. Control samples are mononuclear cells from "normal" patients / sickle cell patients.

Figures 12 (A) - (C) are graphs showing cell surface phenotyping of individuals who have experienced past leukemia (ALL) exposed to Epstein-Barr virus derived from a CCL-87 cell culture (E) compared to leukemia virus birds (cV). Cultures were evaluated daily, starting from 1-4 days after incubation. Control samples are mononuclear cells from "normal" patients / sickle cell patients.

Figures 13 (A) to (C) are graphs showing cellular surface phenotypes of leukemic patient cells (ALL) and cell samples of “normal” sickle cell (SC) patients (control samples). Cells were incubated with either purified Epstein-Barr virus at 2 × 10 ⁶ PFU / ml (eV) or avian leukemia virus at 2 × 10 ⁶ PFU / ml (cV) and recorded 1-4 days after incubation.

Figures 14 (A) - (C) are graphs comparing the effects of culture supernatant aspergillus flavus (X) and mycocladus corymbifera (MC), another fungal pathogen and aflatoxin (AT) on the development of the cell surface phenotype characteristic of acute lymphoblastic leukemia ( ALL) in mononuclear leukocytes of patients in a state of prolonged remission (ALL) and in the cells of "normal" patients / patients with sickle cell disease ("normal control samples"). The results are presented in columns with standard deviation.

Figures 15 (A) - (C) are graphs showing summarized cell surface phenotyping data for (A) CD10 / CD19, (B) CD34 / CD19, and (C) CD34 / CD117 mononuclear leukocytes of people who have long had leukemia incubated with medium, plasma (pl), avian leukemia virus (cV), mycocladus corymbifera (SB), aflatoxin (AT), monkey owl cells (CRL-2312 culture) (OM), aspergillus flavus (X), CCL87 (E) , aspergillus flavus + CCL87 (X + E), and purified EBV (eV). Cultures were evaluated daily, starting from 1-4 days after incubation. Control samples are mononuclear cells from "normal" patients / sickle cell patients.

Figures 16 (A) to (C) are graphs showing an ELISA-based difference between cell samples of patients who have long had leukemia and cell samples of people not suffering from leukemia. (A) The supernatant from aspergillus flavus fungal culture (X) was incubated with the plasma of patients undergoing leukemia (ALL) and compared with SC (that is, with “normal” control samples); (B) The supernatant of aspergillus flavus fungal culture (X) was incubated with samples from non-ill ALL patients (solid tumors) compared to SC ("normal" control samples); (C) The supernatant from aspergillus flavus fungal culture (X) was incubated with normal human samples from normal blood donors (blood taken from a blood bank and plasma), and compared with the plasma of sickle cell disease (SC) patients.

Figures 17 (A) to (C) are graphs showing ELISA-based detection showing the difference between cell samples of patients suffering from leukemia, cell samples of people not suffering from leukemia. (A) The supernatant from the EBV-infected CCL-87 culture (E) was incubated with the plasma of patients who had long suffered acute lymphoblastic leukemia and were now in remission and compared with SC ("normal" control samples); (B) The supernatant from an EBV-infected CCL-87 culture (E) was incubated with non-cancerous patient cell samples (ALL) (solid tumors) and compared with SC (“normal” control samples); (C) the supernatant from the EBV-infected CCL-87 culture (E) was incubated with the plasma of normal blood donors (plasma from blood blank and plasma) and compared with SC ("normal" control samples).

Figures 18 (A) to (F) are graphs demonstrating that the ELISA technique using the supernatant of 1C3 B-lymphoblastic cell line (CRL-2312) of the cell culture with the monkey sovinolytic (OM) does not distinguish the plasma of patients with acute lymphoblastic leukemia ( ALL) from the plasma of "normal" patients / sickle cell patients (A), patients with solid tumors (B), or normal plasma obtained from a blood bank and plasma, that is, from "normal" donors (C).

The combination of aspergillus flavus culture (X) supernatant added to CCL-87 culture containing EBV (CCL87) can distinguish patients with acute lymphoblastic leukemia in a state of prolonged remission from patients with normal control samples / patients with sickle cell disease (D ) Solid patient plasma (non-ALL) and normal control samples cannot be differentiated using this X + CCL87 combination (E). When using X + CCL-87, the plasma of sickle cell patients is indistinguishable from the plasma of randomly selected healthy blood donors (F).

Figures 19 (A) to (C) are graphs showing an ELISA detection technique using peaks from an enzyme assay of exclusively the supernatant of aspergillus flavus, Epstein-Barr virus, and combinations thereof during incubation.

Figures 20 (A) - (C) are graphs showing the effects of isolated peaks 1, 2, 3 of the aspergillus flavus supernatant alone, or in combination with Epstein-Barr virus (EBV), with 50 centiGrey endowment, or without irradiation, on development cell surface markers characteristic of acute lymphoblastic leukemia in patients previously undergoing leukemia and in control samples (SC). All data are presented as percentages of control samples.

Figures 21 (A) to (C) are graphs showing cellular surface phenotyping of mononuclear blood cells. Cells were obtained from long-term leukemia patients who were exposed to the supernatant of aspergillus flavus culture (X) compared to EBV-infected CCL-87 culture (CCL87), purified EBV, or avian leukemia virus (cV)). Cultures were evaluated daily, starting from 1-4 days after incubation. Control samples are mononuclear cells from "normal" patients / sickle cell patients.

Figures 22 (A) - (B) show the gradual development of the leukemic cell surface phenotype CD10 / CD19 after one, two, three, four, five, six and 24 hours of incubation with aspergillus flavus (X), Epstein-Barr virus (eV), a combination of X and eV (X + eV), compared with control samples (medium only). Cell surface markers CD10 / CD19 or (B) CD19 / CD34 were evaluated for patients in the state of long-term remission of leukemia (ALL) (A) compared with control samples of "normal" patients / sickle cell disease (SC) (B). All data are presented as a percentage of control samples with standard deviation.

Figures 23 (A) - (B) show the gradual development of the leukemic cell surface phenotype CD19 / CD34 after one, two, three, four, five, six and 24 hours of incubation with aspergillus flavus (X), with Epstein-Barr virus (eV) , a combination of X and eV (X + eV), compared to control samples (medium only). Cell surface markers CD19 / CD34 were evaluated for patients in the state of long-term remission of leukemia (ALL) (A) compared with control samples of "normal" patients / patients with sickle cell disease (SC) (B). All data are presented as percentages of control samples.

Figures 24 (A) - (B) show the continuous development of the CD34 / CD117 leukemic cell surface phenotype after one, two, three, four, five, six and 24 hours of incubation with aspergillus flavus (X), with Epstein-Barr virus (eV) , a combination of X and eV (X + eV), compared with control samples (medium only). Cell surface markers CD34 / CD117 were evaluated for patients in the state of long-term remission of leukemia (ALL) (A) compared with control samples of “normal” patients / patients with sickle cell disease (SC) (B). All data are presented as percentages of control samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

These studies show that exposure to the supernatant of aspergillus flavus and EBV, alone or in combination with each other, can in vitro (in vitro) reinduce the leukemic phenotype in the mononuclear leukocytes of patients in a state of long remission, with ALL and non-ALL, in control samples, including patients with solid tumors. These studies shed light on the role of these pathogens in the occurrence of acute lymphoblastic leukemia in patients genetically predisposed to this disease.

In this context, “diagnosing” acute lymphoblastic leukemia means classifying a medical condition, predicting or predicting whether a particular abnormal condition is likely to occur or return after evidence-based treatment, identifying cases of illness in individuals, determining the severity of the disease, monitoring the development of the disease.

As used herein, “individual” means mammalian species, including humans, primates, mice, and domestic animals, such as cattle and sheep.

As used herein, “leukemia patient” means any individual who has been diagnosed or previously diagnosed with acute lymphoblastic leukemia / diffuse lymphoma. This term includes individuals who have previously been treated for acute lymphoblastic leukemia / diffuse lymphoma, and have demonstrated long-term remission after treatment for leukemia.

As used herein, the term “non-leukemic patients” means any individuals who have been diagnosed or have previously been diagnosed with solid tumors other than leukemia / diffuse lymphoma.

As used herein, “detection” means the establishment or identification of the presence of a detectable antibody based on an ELISA technique or the induction of cell surface phenotypes characteristic of acute lymphoblastic leukemia on mononuclear leukocytes.

In this context, an “antibody” or “antibodies” is used broadly and includes proteins, as described above, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and antibody fragments.

Standard methodologies were used for all cultures, techniques, ELISA, flow cytometry, FPLC, gel electrophoresis and protein determination.

Example 1

Four aspergillus flavus isolates were collected from individual patient homes for which ALL was diagnosed for 45 years. All four isolates have been shown to have identical characteristics and a similar effect of non-cellular transformation. Thus, the filtered supernatant of only one isolate (UGB) with a high growth rate was used for all the studies described. The "UGB" isolate was cultured in a glass bottle containing a lower layer of 1% solid agarose (Aneresco, Solon, Ohio) in water with an upper layer of 3.5% Chapek-Doks broth (Difco, Becton Dickenson, Sparks, Md). The cultures were incubated at 37 ° C in atmospheric air. The culture supernatant was selected for research when it was possible to achieve confluent growth of aspergillus flavus, usually this happened every two weeks. The supernatant was filtered in a 0.25 µm filter (Corning Inc. Corning, NY) and stored in a refrigerator at 4 ° C until use.

EBV type 2 Jijoye Burkitt's lymphoma cell line (CCL-87) and clone 13C (EBV transformed) B-lymphoblastic cell line (CRL-2312) owl-faced monkey were obtained from the American type culture collection (ATCC, Manassas, VA) and cultivated in accordance with standard explantation protocols and sterile technology. CCL-87 and CRL-2312 cells were cultured in RPMI 1640 medium (GIBCO laboratories) supplemented with 10% bovine fetal serum (Atlanta Biologicals), 10 mM HEPES (GIBCO Laboratories) and 1 mM sodium pyruvate (Sigma-Aldrich). For experiments, the supernatant from these cell lines, which were grown to confluence, was used. The supernatant of CRL-2312 cell lines was used as a negative control.

The mycocladus corymbifera species was selectively cultivated from normal environmental sources (strawberries), cultivated identically to that described for aspergillus flavus, selected for analysis, and then the supernatant was filtered and properly maintained.

Aflatoxin B1 was purchased (Sigma Chemicals, St. Louis, MO) and was used in control samples.

Aliquots of aspergillus flavus, the supernatant of EBV-infected CCL-87 culture and purified Epstein-Barr virus were analyzed based on liquid rapid protein chromatography (FPLC). The protein fractions of aspergillus flaws (X), Epstein-Barr virus (EBV, eV) and the supernatant of their combination were obtained on the basis of FPLC systems (Bio-Rad Laboratories, Inc., Hercules, California) with BioScale ™ Macro-Prep Column High Q cartridge . The BioLogic ™ LP system with the following accessories was used: BioLogic ™ LP controller gradient mixer, MV-6 manual injection valve, BioLogic ™ LP flow conductivity cuvette of the optical module in a conductivity stand, SV-5 switching valve for diverter / bypass valves, Econo gradient pump, EP-1 model, Econo UV monitor, fraction collector, model 2110, and enhanced accuracy data reporting software. Purification of each peak was made possible through the use of Sephadex columns and standard protein purification techniques.

After two weeks of incubation, the supernatant of aspergillus flavus culture (X) shows three distinct peaks shown in Figure 1 (A), where EBV at the same time shows a separate broad peak, shown in Figure 1 (B). Incubation of aspergillus flavus (X) with EBV generates a new peak after four hours of incubation, as shown in Figure 1 (C).

The overhead graph shown in Figure 1 (D) better demonstrates the occurrence of this peak (marked with black squares). The protein fraction indicated by this peak is active in the transformation of sensitized cells into cells with a leukemic phenotype. This technique allows the identification of individuals in remission / individuals who have experienced leukemia in the past. Interestingly, the fraction of the first type shows a drop in the same region, i.e., 50-75KD, as the Epstein-Barr virus, as shown in Figures 1 (A), (C), and (D). The consequences of incubation last up to 7 days; this is confirmed by the similarity of the results obtained after 4 hours and 7 days, as shown in Figure 2 (A) - (D). However, the signal generated by the fraction at the first peak of the aspergillus flavus culture supernatant was significantly stronger and broader, as shown in Figure 2 (A), and applied to extracts that were incubated with EBV, as shown in Figure 2 (C). Thus, incubation of the supernatant of the aspergillus flavus culture and EBV leads to the appearance of a new peak that was not previously present in the supernatant of aspergillus flavus culture or when incubating only EBV.

Irradiation of the supernatant fraction (50 cGy) of aspergillus flavus caused a slight shift in the elution point and led to a decrease in the signal of peaks 2 and 3, which was shown in Figure 3 (A). This effect was not observed in the case of X + EBV culture, as shown in Figure 3 (B). The shift of the supernatant of aspergillus flavus culture is especially evident when this schedule is superimposed on the EBV schedule, and a new peak appears that reflects in further studies increased activity in the transformation of sensitized cells into cells with leukemic surface markers, which allows the identification of individuals with a history of leukemia.

For studies involving incubation of a combination of various leukemia inducing substances, unfiltered supernatants of aspergillus flavus cultures are used. Twenty milliliters of this supernatant were incubated in 100 ml culture flasks (Sarsted, Inc., Newton, NC) together with 2 ml of either isolated EBV containing 2 × 10 ⁶ PFU virus or CCL-87 culture supernatant. The cultures were incubated at 37 ° C with 5% CO ₂ for 7 days. Cultures were shaken daily during incubation and filtered in a 2.5 μm filter (Corning Inc. Corning, NY), and then stored in a refrigerator at 4 ° C until use.

Example 2

If patient / parent agrees, approximately 15 ml of blood was obtained from leukemic patients in remission, patients who have long had leukemia, and from normal volunteers. Additional “normal” control samples were taken from the first blood sampling for patients with sickle cell disease who underwent manual exchange blood transfusion, or from blood samples taken from a blood bank and plasma from normal donors. The patient's blood was placed in heparin (1000 USP units / ml). Peripheral mononuclear blood cells (PBMCs) were isolated by density gradient centrifugation (400 × g, 40 minutes, and 18 ° C) with Ficoll Pak Plus (GE Healthcare, Amersham Biosciences; Uppsala, Sweden), followed by phosphate washing -saline buffer solution. Plasma was also simultaneously collected and stored at -80 ° C until use. The PBMCs obtained by centrifugation in a density gradient were resuspended in ice cold bovine fetal serum with 10% dimethyl sulfoxide (DMSO) at 10 ⁷ cells / ml. Aliquots of cell suspension (1.0 ml) were prepared in cryovials and immediately transferred to a pre-chilled (4 ° C) Nalgene Cryo 1 ° C cryostat container (Nalge Nunc International, Rochester, NY) and placed in a -70 ° C overnight refrigeration . Frozen samples were then placed in a liquid nitrogen refrigeration unit for 24 hours. Samples remained in liquid nitrogen until they were thawed and analyzed.

Frozen samples were thawed in a 37 ° C water bath with continuous stirring. Each 1 ml of thawed cell suspension was slowly diluted with RPMI 1640 medium (Gibco Laboratories, Grand Island, N) supplemented with 10% bovine fetal serum (Atlanta Biologicals, Norcross, GA), 10 mM HEPES (Gibco Laboratories) and 1 mM sodium purivate (Sigma -Aldrich, St. Louis, MO) at room temperature. Cells were centrifuged and washed twice in 10 ml of medium. These cells (frozen / thawed PBMCs) were then evaluated for viability using the trypan blue displacement method, counted and resuspended in the assay medium.

Epstein-Barr virus, aflatoxin and CCL-87 cultures were acquired and stored as described above. Several control samples were used, including the supernatant of mycocladus corymbifera culture samples, which were selectively cultured from normal environmental sources (strawberries), filtered identically as described for aspergillus flavus. Other control samples included avian leukemia virus, aflatoxin and supernatant 1C3 B-cell line lymphoblast (CRL-2312) monkey sovinolitsa. Standard methods were used for all cultures, including flow cytometry, ELISA, FPLC, gel electrophoresis and protein determination. The radiation dose, when used, was 50 centi Gray.

Control samples and cells of test patients isolated on the basis of a density gradient were subjected to various in vitro treatments. About 1 × 10 ⁶ cells were incubated with the supernatant of aspergillus flavus cultures, CCL-87 supernatant (with EBV genome) or patient plasma (cells were taken) or with various combinations of CCL-87 supernatant + plasma; X + CCL-87 supernatant; supernatant of aspergillus flavus cultures + plasma or supernatant of mycocladus corymbifera cultures; CCL-87 supernatant + plasma, all in a ratio of 1: 1 and amounted to less than 10 ml with the addition of RPMI 1640 medium in 25 cm ² flasks with tissue cultures. Cell samples were also exposed to aflatoxin, or avian leukemia virus, or the CRL-2312 supernatant, which serve as positive and negative control samples. Flasks with samples subjected to various processing were incubated for 4 days at 37 ° C and 5% CO ₂ .

At time intervals of 24, 48, 72 and 96 hours, cell surface phenotyping was performed based on flow cytometry. Briefly, the cell suspension from each treatment was centrifuged, and cell clots were incubated with anti-CD34-FITC (clone 581, BD Biosciences, Oxford, UK), anti-CD10-PE (clone HI10A, BD Biosciences), anti-CD19- APC (clone HIB19, BD Biosciences), anti-CD45-APC-Cy7 (clone 2D1, BD Biosciences) and anti-CD-117-PerCP-Cy5.5 (clone 104D2, BD Biosciences) for 45 minutes at 4 ° C. Cells were washed and resuspended in staining buffer (BSA, BD Biosciences). Acquisition and analysis was performed using activated cell sorting based on fluorescence (FACS) BD FACS Canto II using FACSDiva v 6.1.2. Samples were selected based on direct light scattering. Dead cells were excluded by setting appropriate thresholds. The percentage of cells that tested positive for CD10CD19, CD34CD19 and CD34CD117 was recorded.

The results of incubation of mononuclear blood cells from normal subjects, “normal” control samples, such as patients with sickle cell disease, patients previously treated for acute lymphoblastic leukemia (ALL cells from leukemic patients), and patients not suffering from leukemia (leukemia patients) were analyzed for cell surface markers, while establishing the leukemia status of the cells, as shown in Figure 4. Only in comparison with the medium, which was used as a control about Samples were when cells or from leukemic patients in remission on chemotherapy, or from patients who in the past suffered leukemia exposed supernatant aspergillus flaws cultures, CCL-87 containing EBV, or exposed to purified EBV culture, there was a significant increase in cell surface markers. This is shown in Figures 4 and 15. These results show that the supernatant of aspergillus flavus, EBV / EBV containing cultures, and combinations of aspergillus flavus and EBV selectively induce leukemic cell surface markers in leukemic patients, rather than in control samples. Other substances used do not exhibit differentiating effects. Aflatoxin randomly stimulates cell surface markers in leukemia patients and in control samples.

The effects of inducing substances, such as aspergillus flavus supernatant and EBV, on mononuclear leukocytes from leukemic patients began after four hours of incubation and became completely apparent after 1 day, as shown in Figures 21-23, and the exposure data was saved after that day. Exposure to aspergillus flavus was evident in samples where combinations of CD10 / CD19, CD34 / CD19 and CD34 / CD117 were detected. The addition of aspergillus flavus or aspergillus flavus and CCL-87 containing Epstein-Barr virus stimulates the development of cell surface phenotypes characteristic of acute lymphoblastic leukemia in former leukemia patients, and not in control samples, as shown in Figures 11 (A) - (C ), or with purified Epstein-Barr virus, as shown in Figures 8 (A) - (C). The supernatant of aspergillus flavus cultures and Epstein-Barr virus (eV) showed the same effect on the development of cell surface phenotyping for leukemia-prone individuals, and did not show any effect on cells from sickle cell patients (normal control samples). Reactions similar to those observed in leukemia patients were not observed in the "normal" control samples, as shown in Figures 5 (A) - (C). Further, cells from past leukemia individuals can be re-induced to exhibit cell surface markers characteristic of acute lymphoblastic leukemia (ALL), with aspergillus flavus supernatant with or without Epstein-Barr virus. Figure 11 (A) - (C). This effect was characteristic of individuals predisposed to leukemia, since neither the supernatant of the aspergillus flavus culture, nor the supernatant of the aspergillus flavus culture with EBV had an effect on the control samples, as shown in Figures 15 (A) - (C).

To a greater extent, the selective effect on cell surface markers was manifested in samples from former leukemia patients during the induction of leukemic cell surface phenotypes. Previously, a similar selective effect on the re-induction of leukemic cell surface markers was either unknown or simply not reported. Further, cell incubation with other compounds, such as cell culture supernatant of the 1C3 B-lymphoblastic cell line (CRL-2312) of the monkey owen, as shown in Figures 6 (A) - (C), avian leukemia virus, as shown in Figures 9 ( A) - (C) and Figures 12 (A) - (C), and human plasma, as shown in Figures 10 (A) - (C), did not give an increase in cell surface markers in any of the samples. In contrast to supernatant of aspergillus flavus cultures or EBV, cell incubation with purified aflatoxin (AT) randomly induced increased development of CD10 / CD19, CD34 / CD19 and CD34 / CD117 in both normal and leukemic cells, as shown in Figures 7 (A) - (C). Adding plasma to the mixture reduces this effect, but does not eliminate it. Figures 10 (A) - (C) and 15 (A) - (C). Also, the combination of CCL-87 or EBV with the supernatant of aspergillus flavus leads to the development of the aforementioned cell surface phenotypes as a percentage of the control samples, regardless of whether the cells were incubated with CCL-87 / aspergillus flavus or purified EBV / aspergillus flavus, as shown in Figure 15 (A) - (C). This effect remained stable for all patients with acute lymphoblastic leukemia and patients with diffuse lymphomas. However, the combination of CCL-87 / aspergillus flavus or EBV / aspergillus flavus did not affect the cell surface phenotype of the "normal" control samples or patients with solid tumors, which reproduces the results of exposure to EBV or only aspergillus flavus supernatant on "normal" cells. Mycocladus corymbifera was compared with the supernatan of an aspergillus flavus culture to determine whether all fungal pathogens could actually induce similar effects in the aforementioned leukemic patients, with aflatoxin (AT) being used as an indiscriminate positive control. The supernatant aspergillus flavus (X) used in this study made it possible to distinguish between former leukemic patients and control samples with the development of cell surface phenotyping, which made it possible to correctly identify former leukemic patients. By comparison, mycocladus corymbifera (MC) did not allow for the identification or distinction between former leukemia patients and “normal” sickle-cell / cell samples, showing that this effect is typical of aspergillus flavus culture supernatant in our experiment, as shown in Figures 14 (A) - (C).

A summary of the results is presented in Figures 15 (A) to (C), demonstrating that incubation of aspergillus flavus, supernatant of EBV-infected CCL-87 culture, purified Epstein-Barr virus, and combinations of aspergillus flavus with supernatant of EBV-infected CCL-87 culture or aspergillus flavus with crude Epstein-Barr virus, selectively increased cell surface markers in former leukemia patients. When purified aflatoxin was added to either aspergillus flavus supernatant, the cells of both leukemic patients and normal individuals showed leukemic cell surface phenotypes, confirming that aflatoxin was nonspecific, as seen in Figures 15 (A) - (C). The supernatant (CRL-2312) of the 1C3 culture of the B-lymphoblastic cell line of the monkey owl, which was used as a nonspecific negative control, and human plasma did not show a significant effect on the cell surface phenotype in relation to leukemia or "normal" cells of the control samples, which is seen in Figures 15 (A) - (C). Thus, in the framework of our experiment, only the supernatant of aspergillus flavus (X) cultures and EBV-containing cultures (E and eV) stimulate the development of the cell surface phenotype characteristic of acute lymphoblastic leukemia (ALL) in previous leukemia patients, and not in control samples . Other viral pathogens, such as avian leukemia virus, plasma, or a fungal pathogen such as mycocladus corymbifera (cV, pl, (MC), respectively, do not stimulate the development of any cell surface phenotype in leukemia and control samples, while AT stimulates the development of such surface markers in all cells, non-selectively inducing cells from previous leukemia patients and from control samples.

Radiation effects: When only aspergillus flavus cultures or these cultures incubated with EBV, as described above, were irradiated at 50 Santi Gray, the supernatant of the latter culture showed increased activity in the field of leukemic phenotypes in cells from leukemic patients in remission, which is established on the basis of flow cytometry, but did not induce any leukemic phenotypes in normal control samples. This may indicate that irradiation may induce the induction of leukemia-producing proteins in the presence of these organisms.

Data are presented as arithmetic mean ± standard deviation (SD). The results were analyzed using the two-tailed paired student test for assessing statistical significance. Statistical discrepancies are present at probability levels p <0.05.

Example 3

For ELISA-based studies, with parental / patient consent, approximately 15 ml of blood was obtained from leukemic patients in remission, from patients who had long had leukemia, and from normal volunteers. Additional “normal” control samples were collected, upon agreement, from the first blood sampling for analysis in sickle cell disease patients who underwent a manual partial exchange blood transfusion. In addition, blood taken from a blood and plasma bank was used as a control sample. The patient's blood was placed in heparin (1000 USP units / ml). Plasma was also collected simultaneously, which was stored at -80 ° C until use.

A high-quality ELISA was performed based on the “sandwich” method in order to detect antibodies in plasma samples for antigens in various conditioned media. Briefly, 96-well microtiter plates were filled with 100 μl of any medium, XRT, CCL-87 supernatant, or CRL-2312 supernatant, or a combination of XRT and CCL-87 supernatant, and then incubated overnight at 4 ° C. The microplates were then blocked by adding 2% BSA in PBS for 2 hours at 37 ° C. Serum samples (100 μl) were immersed in triplicate and incubated for 2 hours at room temperature. Finally, a goat anti-human immunoglobulin of class G (IgG) conjugated with alkaline phosphatase (Promega, Madison, WI) was dissolved in a ratio of 1: 5000 in block buffer and added (100 µl) to each well. Microplates were incubated for an additional 2 hours at room temperature, and the reaction was visualized by adding 50 μl of chromogenic (pigment-forming) substrate (PNPP, Thermo Fisher Scientific, Lafayette, CO) for 30 minutes. The reaction was stopped with 100 μl H ₂ SO ₄ and the absorbance at 450 nm was measured with reduction at 630 nm using an ELISA plate reader. The plates were washed five times with wash buffer (PBS, pH 7.4, containing 0.1% (v / v) Tween 20) after each step.

Qualitative ELISA revealed a significant difference when the supernatant of aspergillus flavus was tested in relation to the plasma of patients with ALL, to normal control samples or to samples of non-cancerous cancer patients (solid tumors). Based on the ELISA method, using the supernatant of an aspergillus flavus culture, or CCL-87 of a culture containing EBV, or a combination thereof, it became possible to distinguish the plasma of patients with leukemia / diffuse lymphoma from the plasma of "normal" control samples, as shown in Figures 16 (A) , 17 (A), and 19 (A).

After incubation with aspergillus flavus fungal culture, IgG immunoreactivity for the leukemic patient increases significantly, forming a group different from the "normal" samples. This made it easy to distinguish the plasma of former leukemia patients from the plasma of control samples, including patients with sickle cell disease (SC) and normal blood donors (blood obtained from a blood bank and plasma), as shown in Figure 16 (A). This group of leukemic patients did not overlap with normal samples. In addition, incubation of leukemic patient samples with EBV-infected CCL-87 culture or supernatant from aspergillus flavus fungal culture and CCL-87 (X + CCL-87), Figure 19 (D) - (F), led to a similar arrangement of two distinct groups of leukemic patients versus "normal" samples, as shown in Figures 17 (A) and 19 (A). The supernatant of aspergillus flavus or CCL-87 culture containing EBV, did not allow to recognize or separate the plasma of patients with other cancers (for example, solid tumors), as shown in Figures 16 (B) and 17 (B). The stratification of leukemic patient samples from non-leukemic patient samples depends on the induction factor used, since the supernatant of the cell culture 1C3 B-lymphoblastic cell line (CRL-2312) with the monkey owl did not increase IgG immunoreactivity of the samples, as shown in Figures 18 (A) - (C). Thus, a patentable ELISA test using the supernatant of aspergillus flaws, or EBV, or a combination thereof, can distinguish the plasma of normal individuals from the plasma of leukemic patients. Similar results were obtained when one CCL-87 supernatant containing Epstein-Barr virus, or the same composition with the addition of aspergillus flavus supernatant, was tested in the plasma of normal patients, patients with ALL, or patients with solid tumors (Figures 17 (A ) - (C) and 18 (D) - (F). It was found that the fraction that showed the first peak of aspergillus flavus, separated using FPLC, is most effective in the separation of plasma from previous leukemia patients compared to control samples, as shown in Figure 20 (A) and (B). Peak 2 shows significantly lower activity and seems to overlap control samples, while peak 3 is significantly less active in identifying former leukemia patients when compared with control samples, as shown in Figure 19 (A). Incubation of aspergillus flavus peak 1 fractions using EBV showed increased stratification, and peak 2 fractions showed only a slight increase, and no changes were observed for peak 3 fractions, as shown in Figures 19 (B) and (C). However, none of these peaks made it possible to distinguish patients with solid tumors from patients with sickle cell disease or from normal individuals.

The source of EBV did not significantly alter the induction of the cell surface phenotype characteristic of former leukemic patients with ALL, as shown in Figures 21 (A) to (C). Both EBV sources make it possible to clearly anticipate and separate patients with leukemia cells from normal individuals or patients with solid tumors.

Data are presented as arithmetic mean ± standard deviation (SD). The results were analyzed using the two-tailed paired student test for assessing statistical significance. Statistical discrepancies are present at probability levels p <0.05.

Example 4

The studies described here show that purified mononuclear leukocytes obtained from the peripheral blood flow of patients with ALL or diffuse lymphoma who are undergoing a therapeutic study or have already completed treatment, including those who have been treated many years before this study, respond when they are the supernatant of aspergillus flavus culture or EBV sources, or a combination of these two substances, forming blast cells that are indistinguishable by cell surface phenotyping s of leukemic cells. Exposed cells showed cell surface phenotypes that are signs of acute lymphoblastic leukemia and diffuse lymphomas. This reaction is enhanced by the addition of the supernatant of CCL-87 culture containing Epstein-Barr virus (EBV), or by adding purified EBV. Moreover, irradiation of aspergillus flavus at 50 Santi Gray led to increased stimulation. The combined culture of aspergillus flavus and EBV resulted in the additional protein peak shown in Figures 1 (C) and 2 (C). This protein was highly effective in identifying individuals with a history of acute lymphoblastic leukemia / diffuse lymphoma, but not normal samples, using an ELISA technique. This substance also induced ALL leukemic phenotypes in the mononuclear peripheral blood cells of former leukemic patients, but not in uncontrolled samples.

Using an ELISA technique, the plasma of patients with ALL or diffuse lymphoma reacted with the supernatant of aspergillus flavus cultures. A clear separation of leukemia and “normal” control samples could be ensured. However, similar results were not obtained when plasma was used from normal control samples or from individuals with solid tumors that were identically treated in vitro.

Irradiation of cultures containing aspergillus flavus with or without EBV caused protein peaks, as shown in Figures 1 (A) - (C) compared to Figures 3 (A) - (C). Also, regardless of irradiation, peak 1 fraction, with or without incubation, showed the greatest activity in the development of CD10 / CD19, CD34 / CD19, and CD34 / CD117 cell surface phenotypes characteristic of leukemia cells in previous leukemia patients, as shown in the Figures 20 (A) - (C). The combination of aspergillus flavus (X) and Epstein-Barr virus (EBV), with or without irradiation, is significant in activity, while the supernatant 1C3 of the B-lymphoblastic cell line (CRL-2312) with the marmoset monkey (CRL-2312) (OM) does not had an effect on the cell surface phenotypes of former leukemia patients or on control samples.

Temporal analysis showed the gradual development of a leukemic cell surface phenotype in former leukemia patients after one, two, three, four, five, six and 24 hours of incubation with aspergillus flavus (X), Epstein-Barr virus (eV), or a combination of X and eV, compared with control samples (medium only). Moreover, the analysis shows that cells reduced the expression of cell surface markers within the first 3 hours of incubation with aspergillus flavus (X), Epstein-Barr virus (eV), or a combination of X and eV, as shown in Figures 22 (A), 23 (A), and 24 (A). After 4 hours, the cells transmit almost the same signals as the cells treated with the medium, and then surface markers begin to constantly increase up to at least 24 hours. This effect is not observed in the cells of the "normal" control samples / control samples with sickle cells, as shown in Figures 22 (B), 23 (B), and 24 (B). Without being tied to any particular theory, it is assumed that these results indicate the initial resistance of the cells to return to leukemia, and then reinduction of leukemia markers in former leukemia patients followed.

Replacing the supernatant of aspergillus flavus culture with mycocladus corymbifera varieties or purified purchased aflatoxin and substitution of EBV virus for bird leukemia did not lead to similar discriminant changes in normal samples and samples of patients with leukemia / diffuse lymphoma. This shows that the effects of the supernatant of aspergillus flavus and EBV are unlikely to be due to general, species-specific organisms.

The conclusion that EBV and the supernatant of aspergillus flavus reinduce phenotypic changes of the ALL / diffuse lymphoma type in the cells of patients with ALL and diffuse lymphoma, and not in the “control samples”, including patients with solid tumors, indicates a potential genetic predisposition of these individuals. Moreover, the fact that cells from patients suffering from ALL and diffuse lymphoma react similarly is really important. Clinically, these two groups are clearly related because diffuse lymphomas can transform into leukemia. Obtaining identical results using either cell surface phenotyping or an ELISA technique is really important. These studies may shed light on the role of EBV and aspergillus in the generation of leukemia in genetically predisposed patients. Moreover, the fact that irradiation changes the model of protein production using only aspergillus flavus or after incubation with EBV can shed light on the mechanism of carcinogenesis as a result of irradiation.

Previously, the regeneration of genomes that control the cell surface phenotypes of former leukemia patients was unknown. The above studies show that despite the seemingly complete morphological remission, the cells of patients with acute lymphoblastic leukemia, when subjected to certain conditions, demonstrate the ability to transform into a cell surface phenotype similar to the phenotype of acute lymphoblastic leukemia. Such effects do not induce phenotypic changes in normal individuals or patients with solid tumors. ELISA testing can be used as a tool to identify and isolate patients with a history of leukemia, post-ex-facto, from normal individuals and patients with solid tumors. This test can potentially be used for mass screening of normal individuals to establish their predisposition to the development of leukemia. Such work requires a large population. The results of the above experiments can also be used for the etiology of leukemia.

In the preceding specification, all disclosed documents, acts or information do not imply recognition that the specified document, act or information in any combination thereof was publicly available, known to the public, formed part of general knowledge in this area, or it was known that this information contributed to the decision any problems during priority.

All disclosed publications cited above are hereby incorporated by reference in their entirety, each publication in its entirety, to the extent that each publication was individually incorporated here by reference.

Since specific embodiments of the methods for diagnosing and vaccinating individuals against leukemia have been described and illustrated here, it will be apparent to those skilled in the art that variations and modifications are possible without departing from the spirit and principle of the invention in a broad sense. It is also understood that the following claims are intended to encompass all generic and specific features of the invention described herein, as well as all wording of the scope of this invention.

Claims (14)

1. A method for diagnosing acute lymphoblastic leukemia in a patient, comprising:
collecting a biological sample of a patient, where the biological sample is blood plasma or blood cells;
detecting signs of leukemia, which includes
blood cell testing for leukemia, also including:
incubating blood cells with a leukemia inducing factor in order to induce the expression of cell surface markers that are a sign of leukemia, where the leukemia inducing factor is Aspergillus flavus supernatant, EBL-infected CCL87 supernatant, purified EBV culture, or combinations thereof;
testing blood cells for cell surface markers by analyzing surface markers for blood cells, where the level of cell surface markers is a sign of leukemia; or
blood plasma testing for leukemia, also including:
receiving blood plasma from leukemia patients or potentially leukemic patients;
incubating the leukemia inducing factor on a tablet, where the leukemia inducing factor is the supernatant of Aspergillus flavus, EBV-infected CCL87 supernatant, purified EBV culture, or combinations thereof;
incubation of blood plasma inducing leukemia factor;
conducting immunosorbent analysis of blood plasma; and
determination of IgG immunoreactivity in biological samples, where IgG reactivity is an indicator of leukemia. 2. The method of claim 1, wherein the cell surface markers for flow cytometry are CD34, CD10, CD19, CD45, CD117, and a combination thereof. 3. The method of claim 2, wherein the cell surface markers for flow cytometry are a combination of CD10 / CD19, CD34 / CD19, or CD34 / CD117. 4. The method of claim 3, wherein more than one combination of cell surface markers is analyzed. 5. The method according to claim 1, where the diagnosis of blood cells using cell surface markers and analysis based on flow cytometry is carried out at least 24 hours after incubation of blood cells with a factor inducing leukemia. 6. The method according to p. 1, where the staining of blood cells using cell surface markers and analysis based on flow cytometry is carried out at least 72 hours after incubation of blood cells with a factor inducing leukemia. 7. The method according to claim 1, further comprising only a fungal pathogen or the causative agent in combination with a virus, such as EBV, without radiation or with radiation, to cause cancer, including the induction of leukemia or factor inducing leukemia. 8. The method of claim 1, wherein the IgG immunoreactivity is determined using anti-human IgG conjugated to alkaline phosphatase. 9. The method of claim 8, wherein alkaline phosphatase reacts with a chromogenic substrate to give a chromatic signal for recognition. 10. The method of claim 1, wherein the method is used for mass screening of patients. 11. A method for introducing a leukemia inducing factor into mononuclear cells obtained from a patient;
where the leukemia inducing factor is Aspergillus flavus supernatant, EBV-infected CCL87 supernatant, purified EBV culture, or combinations thereof;
where mononuclear cells are taken from a patient with leukemia, a patient who is undergoing treatment for leukemia, or a patient who has previously been treated for leukemia; and
where the introduction of a leukemia inducing factor forms blast cells in mononuclear cells. 12. The method of claim 11, further comprising re-inducing leukemia into mononuclear cells of patients predisposed to leukemia using a fungal pathogen. 13. The method according to p. 12, where the fungal pathogen is the supernatant of Aspergillus flavus. 14. The method of claim 13, wherein the blast cells have a surface phenotype homologous to leukemia cells.

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