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WO2012075501A2 - Diagnostic et graduation de la leucémie lymphocytaire aiguë - Google Patents

Diagnostic et graduation de la leucémie lymphocytaire aiguë Download PDF

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Publication number
WO2012075501A2
WO2012075501A2 PCT/US2011/063320 US2011063320W WO2012075501A2 WO 2012075501 A2 WO2012075501 A2 WO 2012075501A2 US 2011063320 W US2011063320 W US 2011063320W WO 2012075501 A2 WO2012075501 A2 WO 2012075501A2
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patient
proteins
protein
expression
antibody
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WO2012075501A3 (fr
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Steven Kornblau
Nianxiang Zhang
Yi Hua Qiu
Kevin Coombes
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University of Texas System
University of Texas at Austin
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University of Texas at Austin
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes

Definitions

  • the present invention relates generally to the fields of protein biology and oncology. More particularly, it concerns the diagnosis, classification and grading of acute lymphocytic leukemia (ALL) based on the expression of various proteins identified as relevant to various ALL states.
  • ALL acute lymphocytic leukemia
  • Staging is a system that describes the size and extent that the cancer has spread, and different stages of cancer are treated differently. Thus, in order for a doctor or patient to make well informed decisions on treatment options, the correct stage of the cancer must be known.
  • TNM Staging is the most common to measure the extent of the spread of cancer: “T” refers to the size of the tumor; “N” refers to the number and location of lymph nodes involved; and “M” refers to metastasis.
  • T refers to the size of the tumor
  • N refers to the number and location of lymph nodes involved
  • M refers to metastasis.
  • T refers to the size of the tumor
  • N refers to the number and location of lymph nodes involved
  • M refers to metastasis
  • Another way to assess cancer is to examine the nature of the cancer cells or the cancer tissue. This can be performed a number of ways, but using a microscope is typical. This is often referred to as called grading the cancer. Different grading systems are used for various cancers, but generally, the more the cells differ in their appearance from normal cells, the
  • the cells of Grade 1 tumors resemble normal cells, and tend to grow and multiply slowly.
  • the cells of Grade 4 tumors do not look like at alj normal cells of the same type.
  • High-grade cancers tend to grow more rapidly and spread faster than low-grade tumors.
  • examining cells or tissues (histology) microscopically creates the opportunity for variation and inaccuracy. For example, it depends greatly on what part of the tumor is used for analysis, and how the samples are treated prior to examination.
  • An aspect of the present invention relates to a method for proteomic profiling of an acute lymphocytic leukemia (ALL) patient, the method comprising the steps of: (a) determining or assessing a relative expression level of a plurality of proteins in each of constellations 1 -15 in ALL cells of said patient; (b) assigning said patient to a protein expression signature group (ProExpSig) 1 through 7 based on said determination, to thereby profile said patient's ALL.
  • ALL acute lymphocytic leukemia
  • Step (a) may comprise determining, assessing, or measuring the expression/activity levels of a selection of the 128 proteins of constellations 1-15, and then comparing the expression/activity levels to control levels or expression levels of such proteins in non-neoplastic, non-ALL bone marrow or blood cells. More than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 1 10, more than 120 proteins or all 128 proteins may be examined.
  • the method may further comprise generating a prognosis, wherein if the patient is assigned to ProExpSig 1 , 2, 4 or 7, said patient has a favorable prognosis, and if said patient is assigned to ProExpSig 3, 5 or 6, said patient has an unfavorable prognosis.
  • the method may comprise examining each of the following proteins or protein classes: Myc, ARC, Survivin, activated STPs, activated Stats, PKCa, GAB2 Stats 3 and 6, mTOR and TSC2 axis, cyclins Dl and D3, pRB and cleaved caspases. Determining or assessing may comprise
  • the determining or assessing may comprise mass spectroscopy, flow cytometry, or mass cytometry.
  • the determining or assessing may comprise obtaining a sample from said subject and providing said sample to a third party for analysis of protein expression.
  • the method may comprise treating the patient, such as with a chemo-, immune- or radiotherapy. Treating may comprise not administering a therapy to said subject.
  • the method may further comprise performing steps (a)-(c) after treating said patient and determining the efficacy of the therapy based on modulation of proteins in constellations 1 -15.
  • the invention contemplates the provision of a kit for the proteomic profiling of ALL patients, the kit comprising a selection of antibodies that specifically recognize and bind to at least 30 proteins as listed in constellations 1-15, said selection including a plurality of proteins from each of the constellations.
  • the kit will comprise a selection of antibodies that specifically recognize and bind to at least 70 proteins as listed in constellations 1-15.
  • the kit will comprise a selection of antibodies that specifically recognize and bind to at least 100 proteins as listed in constellations 1-15.
  • the kit will comprise a selection of antibodies that specifically recognize and bind all 128 proteins as listed in constellations 1-15.
  • compositions and kits of the invention can be used to achieve methods of the invention.
  • FIG. 1 Custom made Reverse Phase Protein Arrays were produced using 285 samples from 215 patients with ALL and probed with 128 different antibodies. The data presented in this poster is only for the 194 newly diagnosed ALL cases.
  • FIG. 2 Bootstrap clustering was performed using the 15 protein constellations and the resulting dendogram "cut” at different levels defining different numbers of signatures. The Gap statistic was used to suggest the optimal number of protein expression "signatures” and suggested that there were 7 signatures.
  • FIG. 3 Samples were clustered based on overall similarity into 7 Protein Expression
  • FIGS. 4A-B The prognostic implications of the 7 different Protein Expression Signatures on overall survival and remission duration is shown. It was apparent that these 7 Protein Expression Signatures reduced to 2 groups Favorable and Unfavorable as shown in the bottom two panels. Protein expression signatures in ALL and overall survival is shown in FIG. 4A. Protein expression signatures in ALL and remission duration are shown in FIG. 4B.
  • FIG. 5 Protein expression signatures separate PH+ ALL into good and poor prognosis groups.
  • FIG. 6 Characteristics of good vs. bad PH+ signatures.
  • RPPA reverse phase protein array
  • ALL patient derived acute lymphocytic leukemia
  • the invention is based in part on the general concept and idea that certain constellation activation patterns of proteins in key growth, survival and death (apoptotic) pathways determine the biological behavior of ALL.
  • key regulatory proteins/molecules can determine the aggressiveness of the disease.
  • the inventors were able to identify select proteins amongst many other proteins that influence ALL phenotype and/or prognosis.
  • the larger expression patterns may be equally important to assign a patient to a certain class. Therefore, once a protein expression pattern is identified, a more accurate assessment of a patient's response to therapy and outcome can be made.
  • several proteins may form a marker set.
  • Protein expression may be detected or analyzed in patients (e.g., via RPPA, or antibody array, etc.) prior to, during, and/or after treatment with an experimental or conventional therapeutic.
  • This approach may be used to identify targets and/or molecular signatures associated with the therapy and may be advantageously used to monitor for development of resistance during therapy. If a broad marker set would be available, patients could be selectively treated with new experimental or conventional chemotherapy agents based on expression of these markers specifically targeted by a drug/agent.
  • this approach may be used in choosing patients and/or the enrichment of or analysis of molecular defect(s) in patient populations participating in a clinical trial. This may save time, cost, and may improve reponses and ultimately clincial outcome. Drug development could be made more rational.
  • Acute lymphoblastic leukemia is a cancer of the white blood cells characterized by excess lymphoblasts. Malignant, immature white blood cells
  • ALL typically causes damage and death by crowding out normal cells in the bone marrow, and by spreading (infiltrating) to other organs. ALL is most common in childhood with a peak incidence at 2-5 years of age, and another peak in old age. The overall cure rate in children is about 80%, and about 45%-60% of adults have long-term disease-free survival.
  • Lymphocytic refers to the relatively short time course of the disease (being fatal in as little as a few weeks if left untreated) to differentiate it from the very different disease of Chronic Lymphocytic Leukemia which has a potential time course of many years. It is interchangeably referred to as Lymphocytic or Lymphoblastic. This refers to the cells that are involved, which if they were normal would be referred to as lymphocytes but are seen in this disease in a relatively immature (also termed 'blast') state.
  • ALL In the U.S., the incidence of ALL is roughly 6000 new cases per year (as of 2009), or approximately 1 in 50,000. ALL accounts for approximately 70 percent of all childhood (ages 0 to 19 years) leukemia cases, making it the most common type of childhood cancer. It has a peak incident rate of 2-5 years old, decreasing in incidence with increasing age before increasing again at around 50 years old. ALL is slightly more common in males than females. There is an increased incidence in people with Down Syndrome, Fanconi anemia, Bloom syndrome, Ataxia telangiectasia, X-linked agammaglobulinemia and Severe combined immunodeficiency.
  • ALL initial symptoms are not specific to ALL, but worsen to the point that medical help is sought.
  • the signs and symptoms of ALL are variable but generally follow from bone marrow replacement and/or organ infiltration: generalized weakness and fatigue, anemia, frequent or unexplained fever and infections, weight loss and/or loss of appetite, excessive and unexplained bruising, bone pain, joint pains (caused by the spread of "blast” cells to the surface of the bone or into the joint from the marrow cavity), breathlessness, enlarged lymph nodes, liver and/or spleen, pitting edema (swelling) in the lower limbs and/or abdomen, and petechiae, which are tiny red spots or lines in the skin due to low platelet levels.
  • ALL The signs and symptoms of ALL result from the lack of normal and healthy blood cells because they are crowded out by malignant and immature leukocytes (white blood cells). Therefore, people with ALL experience symptoms from malfunctioning of their erythrocytes (red blood cells), leukocytes, and platelets. Laboratory tests which might show abnormalities include blood count tests, renal function tests, electrolyte tests and liver enzyme tests.
  • Diagnosing ALL begins with a medical history, physical examination, complete blood count, and blood smears. Because the symptoms are so general, many other diseases with similar symptoms must be excluded. Typically, the higher the white blood cell count, the worse the prognosis. Blast cells are seen on blood smear in a majority of cases (blast cells are precursors to immune cell lines). A bone marrow biopsy can provide conclusive proof of ALL. A lumbar puncture (also known as a spinal tap) can be used to determine if the spinal column and brain has been invaded.
  • TdT is a protein expressed early in the development of pre-T and pre-B cells while CALLA is an antigen found in 80% of ALL cases and also in the "blast crisis" of CML.
  • cancer is caused by damage to DNA that leads to uncontrolled cellular growth and spread throughout the body, either by increasing chemical signals that cause growth, or interrupting chemical signals that control growth. Damage can be caused through the formation of fusion genes, as well as the dysregulation of a proto-oncogene via juxtaposition of it to the promotor of another gene, e.g., the T-cell receptor gene. This damage may be caused by environmental factors such as chemicals, drugs or radiation.
  • ALL is associated with exposure to radiation and chemicals in animals and humans.
  • the association of radiation and leukemia in humans has been clearly established in studies of victims of the Chernobyl nuclear reactor and atom bombs in Hiroshima and Nagasaki.
  • exposure to benzene and other chemicals can cause leukemia.
  • Epidemiological studies have associated leukemia with workplace exposure to chemicals, but these studies are not as conclusive.
  • Ethnicity - Caucasians are more likely to develop acute leukemia than African- Americans, Asians and Hispanics and tend to have a better prognosis than non- Caucasians.
  • Cytogenetics the study of characteristic large changes in the chromosomes of cancer cells, is an important predictor of outcome. Some cytogenetic subtypes have a worse prognosis than others. These include:
  • chromosomes 9 and 22 known as the Philadelphia chromosome, occurs in about 20% of adult and 5% in pediatric cases of ALL; a translocation between chromosomes 4 and 1 1 occurs in about 4% of cases and is most common in infants under 12 months.
  • translocations of chromosomes carry a poorer prognosis. Some translocations are relatively favorable. For example, Hyperdiploidy (>50 chromosomes) is a good prognostic factor.
  • Genome-wide copy number changes can be assessed by conventional cytogenetics or virtual karyotyping.
  • SNP array virtual karyotyping can detect copy number changes and LOH status, while arrayCGH can detect only copy number changes.
  • Copy neutral LOH (acquired
  • ALL is not a solid tumor
  • TNM notation as used in solid cancers is of little use.
  • ALL-LI small uniform cells
  • ALL-L2 large varied cells
  • ALL-L3 large varied cells with vacuoles (bubble-like features)
  • Each subtype is then further classified by determining the surface markers of the abnormal lymphocytes, called immunophenotyping.
  • immunophenotyping There are 2 main immunologic types: pre-B cell and pre-T cell.
  • pre-B cell The mature B-cell ALL (L3) is now classified as Burkitt's lymphoma/leukemia. Subtyping helps determine the prognosis and most appropriate treatment in treating ALL.
  • Precursor B acute lymphoblastic leukemia/lymphoma is characterized by the following cytogenetic subtypes:
  • Burkitt's lymphoma is a cancer of the lymphatic system (in particular, B lymphocytes). It is named after Denis Parsons Burkitt, a surgeon who first described the disease in 1956 while working in equatorial Africa. Almost by definition, Burkitt's lymphoma is associated with a chromosomal translocation of the c-myc gene. This gene is found at 8q24. The most common variant is t(8;14)(q23;q32). This involves c-myc and IGH@. A variant of this, a three-way translocation, t(8;14;18), has also been identified. A rare variant is at t(2;8)(pl2;q24). This involves IGK@ and c-myc. Another rare variant is t(8;22)(q24;ql 1). This involves IGL@ and c-myc.
  • Burkitt's lymphoma can be divided into three main clinical variants: the endemic, the sporadic and the immunodeficiency-associated variants, which are all associated with HTV and AIDS. Burkitt's lymphoma is usually associated with over 90% of AIDS cases.
  • Burkitt's lymphoma All facial features exhibited by Burkitt's lymphoma are associated to HTV/AIDS. The endemic variant occurs in equatorial Africa. It is the most common malignancy of children in this area. Children affected with the disease often also had chronic malaria, which is believed to have reduced resistance to Epstein-Barr virus (EBV), allowing it to take hold. The disease characteristically involves the jaw or other facial bone, distal ileum, cecum, ovaries, kidney or the breast. The sporadic type of Burkitt lymphoma (also known as "non-African”) is another form of non-Hodgkin's lymphoma found outside of Africa.
  • EBV Epstein-Barr virus
  • the tumor cells have a similar appearance to the cancer cells of classical African or endemic Burkitt lymphoma. Again it is believed that impaired immunity provides an opening for development of the Epstein-Barr virus.
  • Non-Hodgkin lymphoma which includes Burkitt's, accounts for 30-50% of childhood lymphoma. The jaw is less commonly involved, compared to the endemic variant. The ileo-cecal region is the common site of involvement.
  • Immunodeficiency- associated Burkitt lymphoma is usually associated with HIV infection or occurs in the setting of post-transplant patients who are taking immunosuppressive drugs. Burkitt lymphoma can be one of the diseases associated with the initial manifestation of AIDS.
  • Immunodeficiency-associated Burkitt lymphoma may demonstrate more plasmacytic appearance or more pleomorphism, but these features are not specific.
  • Treatment includes dose-adjusted EPOCH with Rituxan (rituximab). Effect of the chemotherapy, as with all cancers, depends on the time of diagnosis. With faster growing cancers, such as Burkitt's, the cancer actually responds faster than with slower growing cancers. This rapid response to chemotherapy can be hazardous to patient, as a phenomenon called "tumor lysis syndrome" could occur. Close monitoring of patient and adequate hydration is essential during the process.
  • Chemotherapy for Burkitt's includes cyclophosphamide, doxorubicin, vincristine, methotrexate, cytarabine, ifosfamide, etoposide and rituximab.
  • l lq23 is a genetic abnormality that is characteristic of childhood lymphoblastic leukemia, which is characterized by rearrangements of the MLL gene and may be rearranged despite normal cytogenetics. It has a poor prognosis. It also is often accompanied by a T(4;l 1) abnormality as well.
  • T(4;l 1 ) is a genetic abnormality that is characteristic of adult lymphoblastic leukemia, which is characterized by rearrangements of the MLL gene and may be rearranged despite normal cytogenetics. It has a poor prognosis.
  • Philadelphia chromosome or Philadelphia translocation is a specific chromosomal abnormality that is normally associated with chronic myelogenous leukemia (CML). It is the result of a reciprocal translocation between chromosome 9 and 22, and is specifically designated t(9;22)(q34;ql 1). The presence of this translocation is a highly sensitive test for CML, since 95% of people with CML have this abnormality (the remainder have either a cryptic translocation that is invisible on G-banded chromosome preparations, or a variant translocation involving another chromosome or chromosomes as well as the long arm of chromosomes 9 and 22). However, the presence of the Philadelphia (Ph) chromosome is also found in acute lymphoblastic leukemia (ALL, 25-30% in adult and 2-10% in pediatric cases) and occasionally in acute myelogenous leukemia (AML).
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • T cell ALL is an aggressive (fast-growing) type of leukemia (blood cancer) in which too many T-cell lymphoblasts (immature white blood cells) are found in the bone marrow and blood. Also called precursor T-lymphoblastic leukemia and T-cell acute lymphoblastic leukemia. 6.
  • B cell ALL is an aggressive (fast-growing) type of leukemia (blood cancer) in which too many B-cell lymphoblasts (immature white blood cells) are found in the bone marrow and blood. It is the most common type of acute lymphoblastic leukemia (ALL). Also called B-cell acute lymphoblastic leukemia and precursor B-lymphoblastic leukemia.
  • the inventors have generated 1 5 protein constellations that, when taken as groups, can help define characteristic protein signatures. These proteins, representing groups, do not each need to be interrogated, but a sufficient plurality from each group must be examined ⁇ e.g., at least 50%, 60%, 75%, 80%, 90% or all). Data regarding survival and remission duration of each of the seven protein signatures is shown in FIGS. 4A- B.
  • Table 1 below shows data for each of the seven protein signatures for each of the genes in each constellation.
  • a positive value in Table 1 indicates an overexpression of the specific protein in a constellation ("ProteinCon", for example " 1 " refers to protein constellation 1 ) in one of the seven protein signatures (e.g. , "SG2" refers to protein signature 2, "SG3” refers to protein signature 3, etc.)
  • a negative value in Table 1 indicates an underexpression of the specific protein in the constellation in one of the seven protein signatures.
  • the expression pattern of: underexpression of (P27 and NF2), and overexpression of (AMPKa, sall4, and STAT3) is the expected expression pattern of constellation 4 for protein signature 2.
  • HDAC3 -0.3639 -0.19568 0.156898 -0.14252 0.293613 -0.12749 -0.01603 9 caspase9 -0.49612 -0.13529 -0.14074 0.080021 0.377492 -0.24025 -0.22445 9
  • Constellation 1 proteins include MEK , Bid, Caspase3, P38, AIF, Statl .p, Stat5, Bax and ERK2.
  • Constellation 2 proteins include GSK3, AKT, PDK l .p, TSC2, PDK1 , RafB, AMPKa.p, NFkB.p65 and SHIP l .
  • Constellation 3 proteins include GSK3.p, CREB.pl 33, MEK.p, erk.p,
  • PKC.delta.p664, P38.p EEF2alpha.p, AKT.p473, AKT.p308, S6RP.p240.244, S6RP.p235.236, PKCalpha.p657 and PKCa.
  • Constellation 4 proteins include P27, AMPKa, sall4, STAT3 and NF2.
  • Constellation 6 proteins include Survivin, c-myc, ARC, PKC.beta.lI, Bim and SMAD1.
  • Constellation 6 proteins include PTEN, PTEN.p, Smad4, BAD.pl 36, CREB, MTOR, Smac, Bad, SSBP2, bcl.2, GAB2 and PI3Kp85.
  • Constellation 7 proteins include XIAP, LKB1 , JUNB, cyclin.Dl, BAK, sall4.LiChai, bcl.xl, PKC.beta.1, IGFBP2, RB.p807.81 1, RB and cyclin.D3.
  • Constellation 8 proteins include caspase7, caspase3.dvdl 75, PARP.clvd214, caspase8, capsase9.dvd330 and caspase9.dvd315.
  • Constellation 9 proteins include YAP, Mcl. l , P53.p, cyclin.Bl , CDC2, JAB 1 , Nurr77, IRS. l .p, HDAC3, capsase9, HIF.alpha, eaten in. beta, p, Smad6, C.Met.p, and NF2.p518.
  • Constellation 10 proteins include MTOR.p, MDM2, TRIM24, S6RP, SHIP2, FAK, P21 , PPAR.gamma, PKC.delta.p645, Notch3 and catenin.alpha.
  • Constellation 1 1 proteins include catenin.beta, FOX03A.p, YAP.pl27, LYN, PBKpl lO and Racl 23.
  • Constellation 12 proteins include TCF4, PP2A, P C.delta, FOX03A, CIAP and
  • Constellation 13 proteins include cyclin, CDK4, P53, P70S6K, PI 6, and P70S6K.p
  • Constellation 14 proteins include stat6.p641, GAB2.p, stat5.p694, stat3.p727 and stat3.p705
  • Constellation 15 proteins include integrin3.beta, SRC, PARP, src.p527, statl , LCK, and src.p416.
  • the seven protein signatures could be assigned into two prognostic groups - favorable and unfavorable. If the patient is assigned to ProExpSig 1, 2, 4 or 7, the patient has a favorable prognosis, and if said patient is assigned to ProExpSig 3, 5 or 6, the patient has an unfavorable prognosis. Survival and remission duration data is presented for each of the groups in FIGS. 4A-B.
  • methods are provided for the assaying of protein expression in patients suffering from gliomas.
  • the principle applications of this assay are to: (a) determine what grade of glioma a given patient suffers from; and (b) determine the likelihood and extent of patient survival.
  • the expression of a particular set of target proteins, set forth in the preceding sections, will be measured.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • antibody also refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • immunodetection methods are provided.
  • immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot to mention a few.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle & Ben-Zeev, 1999; Gulbis & Galand, 1993; De Jager et ai, 1993; and Nakamura et al, 1987, each incorporated herein by reference.
  • the immunobinding methods include obtaining a sample suspected of containing a relevant polypeptide, and contacting the sample with a first antibody under conditions effective to allow the formation of immunocomplexes.
  • the biological sample analyzed may be any sample that is suspected of containing an antigen, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, or even a biological fluid.
  • 95340726.1 18 generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection designed by Charles Cantor uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the target antigen(s).
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • streptavidin or avidin
  • biotinylated DNA or complementary biotinylated DNA
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • immunoassays are in essence binding assays.
  • Certain immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
  • the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the antigen are immobilized onto the well surface and then contacted with the anti-ORF message and anti- ORF translated product antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-ORF message and anti-ORF translated product antibodies are detected. Where the initial anti-ORF message and anti-ORF translated product antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-ORF message and anti-ORF translated product antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the antigens are immobilized involves the use of antibody competition in the detection.
  • labeled antibodies against an antigen are added to the wells, allowed to bind, and detected by means of their label.
  • the amount of an antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen during incubation with coated wells.
  • the presence of an antigen in the sample acts to reduce the amount of antibody against the antigen available for binding to the well and thus reduces the ultimate signal.
  • This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen- coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • Under conditions effective to allow immune complex (antigen antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • the "suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et ai, 1999; Allred ei a/., 1990).
  • immunohistochemistry uses antibodies to detect and quantify antigens in intact tissue samples.
  • frozen-sections are prepared by rehydrating frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • An antibody microarray such as a capture phase microarray or a forward phase microarray may be used to detect a plurality of proteins ⁇ e.g., Labaer et ai, 2005; Hultschig et ai, 2006).
  • Capture microarrays may use direct labeling or a sandwich assay approach. In direct labeling, the entire experimental sample is subjected to a labeling procedure that modifies all analytes with some detectable marker, such as a fluorescent tag. The advantage of this approach is that it allows the simultaneous measurement of many analytes.
  • ASRs analyte-specific reagents, such as antibodies
  • sandwich immunoassay avoids the need for labeling and yields a highly specific signal.
  • This approach generally requires the existence of two independent ASRs for each analyte.
  • a useful hybrid approach is to print multiple different capture ASRs for the same analyte at different features on the array and then detect using the labeling approach. Concordant signals from the multiple ASRs can confirm the specificity of the signal while still allowing simultaneous measurement of multiple analytes.
  • capture microarrays ⁇ e.g., Sreekumar et ai, 2001 ; Miller et ai, 2003; Nielsen et ai, 2003; Haab, 2003.
  • (Fab) 2 fragments may be included on an antibody microarray and may provide improved microarray readout (Song et ai, 2004).
  • Reverse Phase Proteins Array is a technique where very small amounts of protein are printed onto a slide. Hundreds to thousands of protein samples can be printed on a single slide.The slide is then probed with an antibody and the quantity of that protein is determined. By this method the level of expression of a protein as well as its activation state (e.g. phosphorylation) can be determined provided a suiTable lntibody exists. Becauses small amounts of proteins are needed hundreds of proteins can be measured quantitatively on a
  • the assay permits for high throughput so that large numbers of patient samples can be analysed rapidly.
  • the inventors have optimized the technique for use with leukemia samples, allowing for screening of large numbers of samples from patients with Acute Lymphhocytic Leukemia (ALL).
  • ALL Acute Lymphhocytic Leukemia
  • the implication is that patients could be analyzed at the time of diagnosis and categorized into different groups with prognostic implications. This could be used to allocate patients to different therapies or to predict prognosis. For example patients with low probablities of responding to conventional therapy might be treated with novel agents or sent for stem cell transplant arlier. As more targeted therapies become available, determination of which pathways are active could be utilized to select targeted therapies most likely to be effective against an individual patients leukemia.
  • MS mass spectrometry
  • mass spectrometry may be used to look for the levels of these proteins particularly.
  • the mass cytometry or flow cytometry may be used to detect proteins.
  • a "CyTOF" or mass cytometry method may be used and allows for real time single cell multitarget immunoassays based on inductively coupled plasma time-of- flight mass spectrometry (Bandura et ai, 2009). This approach may be used for the detection of proteins and other molecules in individual cells. The approach is based on attaching
  • ESI is a convenient ionization technique developed by Fenn and colleagues (Fenn et al, 1989) that is used to produce gaseous ions from highly polar, mostly nonvolatile biomolecules, including lipids.
  • the sample is injected as a liquid at low flow rates (1-10 ⁇ 7 ⁇ ) through a capillary tube to which a strong electric field is applied.
  • the field generates additional charges to the liquid at the end of the capillary and produces a fine spray of highly charged droplets that are electrostatically attracted to the mass spectrometer inlet.
  • the evaporation of the solvent from the surface of a droplet as it travels through the desolvation chamber increases its charge density substantially. When this increase exceeds the Rayleigh stability limit, ions are ejected and ready for MS analysis.
  • a typical conventional ESI source consists of a metal capillary of typically 0.1-0.3 mm in diameter, with a tip held approximately 0.5 to 5 cm (but more usually 1 to 3 cm) away from an electrically grounded circular interface having at its center the sampling orifice, such as described by abarle et al. (1993).
  • a potential difference of between 1 to 5 kV (but more typically 2 to 3 kV) is applied to the capillary by power supply to generate a high electrostatic field (10 6 to 10 7 V/m) at the capillary tip.
  • a sample liquid carrying the analyte to be analyzed by the mass spectrometer is delivered to tip through an internal passage from a suitable source (such as from a chromatograph or directly from a sample solution via a liquid flow controller).
  • a suitable source such as from a chromatograph or directly from a sample solution via a liquid flow controller.
  • the liquid leaves the capillary tip as a small highly electrically charged droplets and further undergoes desolvation and breakdown to form single or multicharged gas phase ions in the form of an ion beam.
  • the ions are then collected by the grounded (or negatively charged) interface plate and led through an the orifice into an analyzer of the mass spectrometer. During this operation, the voltage applied to the capillary is held constant.
  • ESI tandem mass spectroscopy In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to simultaneously analyze both precursor ions and product ions, thereby monitoring a single precursor product reaction and producing (through selective reaction monitoring (SRM)) a signal only when the desired precursor ion is present.
  • SRM selective reaction monitoring
  • the internal standard is a stable isotope-labeled version of the analyte, this is known as quantification by the stable isotope dilution method.
  • This approach has been used to accurately measure pharmaceuticals (Zweigenbaum et al, 2000; Zweigenbaum et al, 1999) and bioactive peptides (Desiderio et al, 1996; Lovelace et al, 1991).
  • Newer methods are performed on widely available MALDI-TOF instruments, which can resolve a wider mass range and have been used to quantify metabolites, peptides, and proteins.
  • Larger molecules such as peptides can be quantified using unlabeled homologous peptides as long as their chemistry is similar to the analyte peptide (Duncan et al, 1993; Bucknall et al, 2002). Protein quantification has been achieved by quantifying tryptic peptides (Mirgorodskaya et al, 2000). Complex mixtures such as crude extracts can be analyzed, but in some instances sample clean up is required (Nelson et al, 1994; Gobom et a/., 2000).
  • Secondary ion mass spectroscopy is an analytical method that uses ionized particles emitted from a surface for mass spectroscopy at a sensitivity of detection of a few parts per billion.
  • the sample surface is bombarded by primary energetic particles, such as electrons, ions ⁇ e.g., O, Cs), neutrals or even photons, forcing atomic and molecular particles to be ejected from the surface, a process called sputtering. Since some of these sputtered particles carry a charge, a mass spectrometer can be used to measure their mass and charge. Continued sputtering permits measuring of the exposed elements as material is removed. This in turn permits one to construct elemental depth profiles. Although the majority of secondary ionized particles are electrons, it is the secondary ions which are detected and analysis by the mass spectrometer in this method. 4.
  • LD-MS Laser desorption mass spectroscopy
  • LD-MS When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred to as LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy).
  • LDLPMS Laser Desorption Laser Photoionization Mass Spectroscopy
  • the LDLPMS method of analysis gives instantaneous volatilization of the sample, and this form of sample fragmentation permits rapid analysis without any wet extraction chemistry.
  • the LDLPMS instrumentation provides a profile of the species present while the retention time is low and the sample size is small.
  • an impactor strip is loaded into a vacuum chamber. The pulsed laser is fired upon a certain spot of the sample site, and species present are desorbed and ionized by the laser radiation. This ionization also causes the molecules to break up into smaller fragment-ions.
  • the positive or negative ions made are then accelerated into the flight tube, being detected at the end by a microchannel plate detector.
  • Signal intensity, or peak height, is measured as a function of travel time.
  • the applied voltage and charge of the particular ion determines the kinetic energy, and separation of fragments are due to different size causing different velocity. Each ion mass will thus have a different flight-time to the detector.
  • Positive ions are made from regular direct photoionization, but negative ion formation require a higher powered laser and a secondary process to gain electrons. Most of the molecules that come off the sample site are neutrals, and thus can attract electrons based on their electron affinity. The negative ion formation process is less efficient than forming just positive ions. The sample constituents will also affect the outlook of a negative ion spectra.
  • MALDI-TOF-MS Since its inception and commercial availability, the versatility of MALDI-TOF-MS has been demonstrated convincingly by its extensive use for qualitative analysis. For example, MALDI-TOF-MS has been employed for the characterization of synthetic polymers (Marie et al, 2000; Wu et al, 1998). peptide and protein analysis (Roepstorff et al, 2000;
  • MALDI-TOF-MS The properties that make MALDI-TOF-MS a popular qualitative tool— its ability to analyze molecules across an extensive mass range, high sensitivity, minimal sample preparation and rapid analysis times— also make it a potentially useful quantitative tool.
  • MALDI-TOF-MS also enables non-volatile and thermally labile molecules to be analyzed with relative ease. It is therefore prudent to explore the potential of MALDI-TOF-MS for quantitative analysis in clinical settings, for toxicological screenings, as well as for environmental analysis.
  • the application of MALDI-TOF-MS to the quantification of peptides and proteins is particularly relevant. The ability to quantify intact proteins in biological tissue and fluids presents a particular challenge in the expanding area of proteomics and investigators urgently require methods to accurately measure the absolute quantity of proteins.
  • the properties of the matrix material used in the MALDI method are critical. Only a select group of compounds is useful for the selective desorption of proteins and polypeptides. A review of all the matrix materials available for peptides and proteins shows that there are certain characteristics the compounds must share to be analytically useful. Despite its importance, very little is known about what makes a matrix material "successful" for MALDI. The few materials that do work well are used heavily by all MALDI practitioners and new molecules are constantly being evaluated as potential matrix candidates. With a few exceptions, most of the matrix materials used are solid organic acids. Liquid matrices have also been investigated, but are not used routinely.
  • the aim is to induce a lasting remission, defined as the absence of detectable cancer cells in the body (usually less than 5% blast cells on the bone marrow).
  • Treatment for acute leukemia can include chemotherapy, steroids, radiation therapy, intensive combined treatments (including bone marrow or stem cell transplants), and growth factors.
  • Chemotherapy is the initial treatment of choice. Most ALL patients will receive a combination of different treatments. There are no surgical options, due to the body-wide distribution of the malignant cells. In general, cytotoxic chemotherapy for ALL combines multiple antileukemic drugs in various combinations. Chemotherapy for ALL consists of three phases: remission induction, intensification, and maintenance therapy.
  • Radiation therapy is used on painful bony areas, in high disease burdens, or as part of the preparations for a bone marrow transplant (total body irradiation). Radiation in the form of whole brain radiation is also used for central nervous system prophylaxis, to prevent recurrence of leukemia in the brain.
  • Whole brain prophylaxis radiation used to be a common method in treatment of children's ALL. Recent studies showed that CNS chemotherapy provided results as favorable but with less developmental side effects. As a result, the use of whole brain radiation has been more limited. Most specialists in adult leukemia have abandoned the use of radiation therapy for CNS prophylaxis.
  • EXAMPLE 1 Proteomic Profiling of 128 Proteins in 194 Acute Lymphocytic Leukemia (ALL) Patient Samples Using Reverse Phase Proteins Arrays (RPPA)
  • Protein expression signatures (ProExpSig), based on the activation state of cell cycle, apoptosis and signal transduction (STP) regulating proteins, existed and were prognostic in AML, the inventors extended this approach to evaluate protein expression patterns in ALL.
  • the inventors have generated RPPA using protein derived from the leukemia- enriched fraction of 283 primary ALL samples from 216 cases with the goal of providing comprehensive proteomic based classification of ALL. Included are 253 samples from 194 newly diagnosed ALL patients (179 Marrow (BM), 95 blood (PB) samples, 41 with concurrent PB and BM specimens), and 14 relapse (REL) samples from these same cases. Phenotypes included 163 pre-B, 22 T-Cell and 9 Burkitt's cases. There were 42 PH1+ cases but only the 28 treated with TKIs were included for outcome analysis (FIG. 1). All protein preps were made from fresh cells on the day of collection as we previously observed that protein expression changes dramatically in ALLs cells with cryopreservation.
  • Proteins were clustered using absolute Pearson correlation and Ward linkage. Based on the Gap statistic Fifteen "constellations" of proteins with correlated expression were identified. Samples were clustered based on overall similarity and this demonstrated that PH1+, I lq23, T(4: l l) and Burkitt's had similar protein expression patterns clustering together by either WHO classification or by cytogenetics. Many formed expected associations, e.g., cleaved caspases, phosphorylated STATs, activated STPs. Based on the combination of expression of these 15 constellations, 7 distinct ProExpSig were defined containing between 18 and 37 cases (FIG. 2, FIG. 3; raw data is shown in Table 1).
  • Signatures is shown below in Table 2. Features that were overrepresented in a group are shown in bold in Table 2. Features that were underrepresented in a group are shown in italics in Table 2. Restated, variables with statistically unequal distribution between the signatures are highlighted in bold if higher or italics if lower than expected.
  • Perturbation bootstrap clustering was performed on the 15 protein constellations and the 7 Protein Expression Signatures and demonstrated the reproducibility of the clustering.
  • the phenotype was highly unbalanced with T cell and Burkitt's cases significantly overrepresented, and B cell underrepresented in Signature 3 (O/E 5.7,4.2 and .3) and T cell ALL underrepresented in signatures 2, 4,5 and 6 (O/E .47, .76, .4 and .28).
  • PH+ disease was strongly associated with signatures 5 (18 of 22) and 2 (1 1 of 375 cases).
  • the PH+ dominated Sig5 was characterized by high expression of Myc, ARC, Survivin as well as high levels of activated STPs and Stats.
  • Sig5 had high expression of phosphor PKCa, GAB2 and Stat 3 and 6, while Sig 2 had higher expression of proteins in the mTOR and TSC2 axis.
  • Sig6 Almost all t(4: l 1) cases (9 of 1 1) were in Sig6, which was characterized by high levels of expression of most proteins excluding those that were highly expressed in the PH+ cases.
  • Cases with Burkitt's were characterized by high expression of cyclins Dl and D3, pRB and by high levels of cleaved caspases. The WBC was markedly higher in Sig 3 and 6.
  • ALL is characterized by 7 distinct protein expression signatures which form a favorable and an unfavorable prognostic groupings.
  • the adverse ProExpSig are associated with significantly shorter remission duration and survival. Differences within these ProExpSig can be used to direct the rational application of various targeted therapies to those patients most likely to benefit from them thereby improving outcome.

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Abstract

La présente invention concerne une approche protéomique au diagnostic, à la classification et à la graduation de la leucémie lymphocytaire aiguë (ALL), ainsi que pour la prédiction de survie de patients. Cette approche utilise des motifs d'expression protéiques globaux pour identifier des groupes de gènes dont l'expression est altérée dans diverses leucémies lymphocytaires aiguës et peut donc être utilisée pour classifier le type de leucémie lymphocytaire aiguë, le stade de la leucémie lymphocytaire aiguë, et également indiquer si un patient donné va être un survivant à court terme ou à long terme.
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