Patent Grant
9777332
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 415313SEQLIST.txt, created on Mar. 6, 2012, and having a size of 225,858 bytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
This invention relates generally to the detection of minimal residual disease in patients with acute lymphoblastic leukemia (ALL) and, more specifically, to a method to improve minimal residual disease monitoring for risk assignment and selection of therapeutic regimens.
Leukemia relapse is the major cause of treatment failure for patients with acute lymphoblastic leukemia (ALL) (Pui et al. (2009) N Engl J Med. 360:2730-2741; Gokbuget and Hoelzer (2009) Semin. Hematol. 46:64-75; and Faderl et al. (2010) Cancer. 116:1165-1176). Relapse originates from leukemic cells that are resistant to chemotherapy but become undetectable after initial treatment in most cases. Nevertheless, methods more sensitive than microscopic examination can demonstrate leukemic cells in a proportion of samples with no morphologic evidence of leukemia, a finding termed “minimal residual disease (MRD)” (Campana (2009) Hematol. Oncol Clin North Am. 23:1083-98, vii).
MRD is currently the most powerful prognostic indicator in childhood ALL (Cave et al. (1998) N Engl J Med. 339:591-598; Coustan-Smith et al. (1998) Lancet. 351:550-554; van Dongen et al. (1998) Lancet. 352:1731-1738; Coustan-Smith et al. (2000) Blood. 96:2691-2696; Dworzak et al. (2002) Blood. 99:1952-1958; Nyvold et al. (2002) Blood. 99:1253-1258; Zhou et al. (2007) Blood. 110:1607-1611; Borowitz et al. (2008) Blood. 111:5477-5485. Basso et al. (2009) J Clin Oncol. 27:5168-5174; Canter et al. (2010) Blood. 115:3206-3214; Stow et al. (2010) Blood. 115:4657-4663). There is strong evidence supporting its prognostic significance in adult ALL (Krampera et al. (2003) Br J Haematol. 120:74-79; Vidriales, et al. (2003) Blood. 101:4695-4700; Raff et al. (2007) Blood. 109:910-915; Holowiecki et al. (2008) Br. J. Haematol. 142:227-237; Bassan et al. (2009) Blood. 113:4153-4162).
Thus, MRD monitoring has been introduced into many contemporary treatment protocols for risk assignment and selection of therapeutic regimens (Pui et al. (2009) N Engl J Med. 360:2730-2741; Gokbuget and Hoelzer. (2009) Semin. Hematol. 46:64-75; and Faderl et al. (2010) Cancer. 116:1165-1176). MRD measurements are also clinically useful in patients with relapsed ALL who achieve a second remission (Coustan-Smith et al. (2004) Leukemia 18:499-504; Paganin et al. (2008) Leukemia. 22:2193-2200; Raetz et al. (2008) J Clin. Oncol. 26:3971-3978), can help optimize the timing of hematopoietic stem cell transplantation (Bader et al. (2009) J Clin Oncol. 27:377-384), and guide decisions about donor lymphocyte infusion post-transplant (Lankester et al. (2010) Leukemia. 24:1462-1469).
Among methods for detecting MRD in ALL, PCR amplification of antigen-receptor genes has proven to be valuable and has been extensively standardized (Bruggemann, M., et al. (2010) Leukemia. 24:521-535) but the technical expertise and instrumentation required limit its application to specialized centers. PCR amplification of fusion transcripts may also provide useful clinical information but its applicability in ALL is restricted by the fact that molecular targets currently adaptable to routine MRD studies are present in only a minority of patients. Id. Flow cytometric detection of leukemia-specific markers has been shown to predict outcome in numerous clinical correlative studies (Coustan-Smith et al. (1998) Lancet. 351:550-554; Coustan-Smith et al. (2000) Blood. 96:2691-2696; Dworzak et al. (2002) Blood. 99:1952-1958; Borowitz, et al. (2008) Blood. 111:5477-5485; Basso et al. (2009) J Clin Oncol. 27:5168-5174; Krampera et al. (2003) Br J Haematol 120:74-79; Vidriales et al. (2003) Blood 101:4695-4700; Holowiecki et al. (2008) Br. J. Haematol. 142:227-237). The method holds potential for wider applicability than molecular techniques because flow cytometric methods for leukemia diagnosis are already established at most cancer centers worldwide (Campana (2009) Hematol. Oncol Clin North Am. 23:1083-98, vii).
MRD studies by flow cytometry rely on panels of antibodies to define unique immunophenotypic signatures of leukemic cells which must distinguish leukemic blasts from their normal counterparts, the CD19+ CD10+ lymphoid progenitors of the bone marrow (“hematogones”) (Campana (2009) Hematol. Oncol Clin North Am. 23:1083-98, vii; Bruggemann et al. (2010) Leukemia. 24:521-535; McKenna et al. (2001) Blood. 98:2498-2507; Lucio et al. (2001) Leukemia. 15:1185-1192).
Standard four-color flow cytometry can detect one leukemic cell in up to 10,000 normal bone marrow or peripheral blood cells but this task typically requires considerable interpretative expertise. Therefore, identification of new leukemia markers that are easily detectable and are stably expressed in a large proportion of ALL cases could simplify the application of MRD studies, and help extend their benefit to all patients and enhance the sensitivity of MRD detection.
Methods and compositions are provided for identifying patients with minimal residual disease. The diagnostic methods generally comprise contacting a specimen from a patient with a plurality of probes, wherein each of said probes specifically binds to a distinct marker, wherein a first probe specifically binds to CD19, a second probe specifically binds to CD10, a third probe specifically binds to CD34, a fourth probe specifically binds to CD45, and at least two additional probes that specifically binds to any two of CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102, or CD300a. The complex formed between each of the probes and their marker is detected and a value is generated corresponding to an expression level of each of said marker. An expression profile is generated by combining the expression level values. In such methods, the expression of CD19, CD10, CD34 and CD45 and a modulated level of at least two of CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102, or CD300a relative to a normal control is indicative of minimal residual disease in acute lymphoblastic leukemia.
Additional methods for diagnosing minimal residual disease in a subject further include obtaining a specimen from a subject. The specimen is contacted with a plurality of probes, wherein each of the probes specifically binds to a distinct marker, wherein a first probe specifically binds to CD19 and a second probe specifically binds to CD10. A CD19+/CD10+ cell is isolated from the specimen. The expression level of at least two gene products expressed in said CD19+/CD10+ cell is determined, wherein the at least two gene products encode CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a. An expression profile is generated by combining the values generated of the expression levels of the indicated markers. The expression of CD19 and CD10 and a modulated level of at least two gene products encoding CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102, or CD300a relative to a normal control is indicative of minimal residual disease in acute lymphoblastic leukemia.
Kits for practicing the diagnostic methods of the invention are also provided, as well as, kits for evaluating the efficacy of a particular therapy for a subject with ALL.
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Methods and compositions are provided for identifying patients with minimal residual disease in acute lymphoblastic leukemia (ALL). As discussed in further detail herein, genes expressed at different levels in ALL cells were compared to their normal counterparts. Using genome-wide gene analysis, expression profiles of 270 cases of newly diagnosed B-lineage ALL were compared to expression profiles of highly purified normal CD19+ CD10+ cells. Genes that had a substantially abnormal expression in leukemic cells were tested by flow cytometry to assess levels of protein expression. Promising molecules were examined in detail for optimization as MRD markers. Use of newly identified markers allows for the identification of unique leukemia profiles in ALL patients. Moreover, the compositions and methods disclosed herein allow for the detection of 1 leukemic cell in 100,000 normal bone marrow cells, thus significantly enhancing the power of flow cytometric monitoring of MRD in ALL.
Acute Lymphocytic Leukemia and Disease
Methods and compositions are provided herein directed to detection of minimal residual disease in acute B-lymphoblastic leukemia.
In specific methods, a specimen is taken from a subject with cancer, wherein the cancer is leukemia. Leukemia is a cancer of the bone marrow and blood. The four major types of leukemia are acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Acute leukemia is a rapidly progressing cancer that produces blood cells that are not fully developed. Acute lymphocytic leukemia is often referred to as “acute lymphoblastic leukemia” because the leukemic cell that replaces the normal marrow cell is the (leukemic) lymphoblast. There are two principal ALL subtypes including a) the B-lymphocyte subtype—identified by finding cell surface markers on the leukemic blast cells common to normal B lymphocytes; and b) the T lymphocyte subtypes—identified by finding cell surface markers on the leukemic blast cells common to normal T lymphocytes. About 15% of cases are of the T-lymphocyte, and 85% of cases are of the precursor B-cell subtype.
The effects of ALL include uncontrolled and exaggerated growth and accumulation of lymphoblasts which fail to function as normal blood cells, and blockage of the production of normal marrow cells. The lack of production of normal marrow cells often leads to a deficiency of red cells (anemia), platelets (thrombocytopenia) and normal white cells, especially neutrophils (neutropenia), in the blood. ALL progresses rapidly without treatment, therefore the methods and compositions provided herein can be employed to allow for the early detection of acute lymphocytic leukemia.
“Diagnosing ALL” and/or “detecting minimal residual disease (MRD)” or “diagnosing minimal residual disease (MRD)” is intended to include, for example, diagnosing or detecting the presence of acute lymphoblastic leukemia (ALL) by identifying or detecting cells and/or cell products in specimens that are indicative of ALL, monitoring the progression of the disease, monitoring and/or detecting the recurrence of ALL disease in patients who had been previously treated for ALL, and monitoring and/or detecting minimal residual disease. The terms “diagnosing,” “detecting,” and “identifying” when used with acute lymphoblastic leukemia or minimal residual disease (MRD) are used interchangeably herein to refer to the identifying or detecting cells and/or cell products in specimens that are indicative of disease.
One method disclosed herein is directed to monitoring remission of leukemia. Remission is defined as the absence of outward signs of cancer, or in the case of ALL, the absence of detectable cancer cells in the body after a course of therapy. Remission in ALL can be characterized, for example, as a lack of detectable abnormal cells in the blood, bone marrow, and/or cerebrospinal fluid, and less than 5% blast cells in the bone marrow. Embodiments of the invention seek to detect cancer cells in instances where there is a relatively minimal amount of disease (minimal residual disease (MDR)) by phenotypic analysis. Standard detection methods define minimal residual disease as an incidence of less than one leukemic cell in 10,000 normal bone marrow/blood cells. The methods and compositions of the instant invention can detect minimal residual disease with an incidence of less than one in 100,000 cells.
Most ALL patients achieve at least an initial remission. However, some patients have residual leukemic cells in their marrow. Other patients achieve remission then “relapse” wherein they have a decrease in normal blood cells and a return of leukemia cells in the marrow. Embodiments of the invention detect leukemia and can help evaluate the risk for relapse after initial treatment. In addition to the detection of evidence of minimal residual disease, embodiments of the invention can further help to evaluate treatment regimens. For example, the detection and characterization of MRD can be indicative of the efficacy of certain treatment regimes, e.g., stem cell transplant.
In other embodiments of the invention, detection or diagnosing MRD can help determine whether additional treatment may be necessary. One of skill in the art will recognize that in these methods the term “therapy” can include any therapy for treating ALL, including but not limited to chemotherapy, radiation therapy, stem cell transplantation, and biological therapy (e.g., monoclonal antibody therapy). Depending on the subtype, specific drugs or drug combinations, drug dosages, duration of treatment, and other types of treatment, may be indicated to achieve optimal results.
In still other embodiments of the invention, methods for evaluating the efficacy of a therapy for treating ALL in a subject are provided. Embodiments of the invention can also be used to test specimens taken from a subject during the course of therapy to monitor the effects of treatment. Such methods typically comprise comparing the level of expression of a plurality of markers of the invention in a first specimen procured prior to the initiation of therapy with that from a second sample obtained following administration of at least a portion of the therapy. In some embodiments, a significantly lower and/or an undetectable level of expression of a marker in the second specimen relative to that of the first specimen obtained prior to the initiation of the therapy can be a positive indication of the efficacy of the therapy. In other embodiments, a significantly higher level of expression of a marker in the second sample can be a negative indication of the efficacy of the therapy. A positive indication of the efficacy of the therapy can mean that the therapy is producing beneficial results in the treatment of ALL and no minimal residual disease is detected. A negative indication of the efficacy of the therapy can mean that the therapy is not having beneficial effects with respect to treatment of ALL and minimal residual disease is detected.
Specimens from Subjects
In embodiments of the invention, the method comprises obtaining a “specimen” from a subject. The term “specimen” is intended to include blood cells, bone marrow cells, and cellular products that are derived from blood and bone marrow cells. Cellular products can include, but are not limited to, expressed proteins, expressed RNA, and DNA. In embodiments of the invention, a specimen can include cells derived from a variety of sources including, but not limited to, single cells, a collection of cells, tissue, cell culture, bone marrow, blood, or other bodily fluids. A tissue or cell source may include a tissue biopsy sample, a cell sorted population, cell culture, or a single cell. Sources for the specimen of the present invention include cells from peripheral blood or bone marrow, such as blast cells from peripheral blood or bone marrow. The term “specimen” can be used interchangeably with the term “sample” or “patient sample.”
A specimen may be processed in another embodiment to release or otherwise make available a nucleic acid or a protein for detection as described herein. Such processing may include, in one embodiment, steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the specimen. Thus, the nucleic acid to be amplified in one embodiment by the methods of the invention may be DNA or RNA. Isolation of protein, RNA, and DNA from the aforementioned sources is known to those of skill in the art, and is discussed herein.
In one embodiment, the method comprises obtaining a peripheral blood sample from a subject and analyzing the expression level of specific markers in leukocytes from the blood sample taken from the subject. To do blood tests, blood samples are generally taken from a vein in the subject's arm.
In another embodiment, the method comprises obtaining a bone marrow sample from a subject and analyzing the expression level of specific markers combinations in leukocytes from the blood sample taken from the subject. Specimens of marrow cells are obtained by bone marrow aspiration and biopsy.
The obtaining of a specimen uses methods well known in the art, as is the means to analyze leukocyte populations. For example, leukocyte populations can be prepared from whole blood by differential centrifugation, or for example, by density gradient centrifugation. The method can be conducted on leukocytes in blood samples which have not undergone any leukocyte enrichment, on whole blood samples, or where red blood cells have been lysed. In other embodiments the method can be conducted on enriched and purified subpopulations of cells, using methods well known in the art.
Analyzing Specimens
In embodiments of the invention, the method comprises “contacting” the specimen with a plurality of probes. In one embodiment, the term “contacting” is in reference to probes that are antibodies and generally referring to methods of “cell staining.” In a method of the invention, an antibody is added to a specimen and the antibody recognizes and binds to a specific protein for example, on the surface of cells in the specimen. A complex is thereby formed between the probe and the expressed protein. The complex can be detected and visualized by various techniques, as will be discussed herein. Combinations of antibody probes can be collectively added to a specimen and thereby “stain” the cell for later analysis by visualization with a flow cytometer or microscope, for example. One of skill in the art could determine whether a cell expressed a specific protein based on the level of antibody that bound to the cell using standard methods.
In embodiments of the invention the term “contacting” in reference to probes that are nucleic acids, refers to methods of detecting expression of an mRNA of interest in a specimen. A detectable complex can be formed when a nucleic acid probe specific to an expressed gene of interest hybridizes and binds an mRNA/cDNA expressed by cells in a specimen. One of skill in the art could determine whether a cell expressed a specific mRNA based on the level of detectable PCR product, for example, using standard methods.
Detecting Expression of Markers for Minimal Residual Disease
As used herein a “marker” can be any gene or protein whose level of expression in a tissue or cell is used comparatively to evaluate the level of expression to that of a normal or healthy cell or tissue. In particular embodiments of the invention, antibodies are used to detect marker expression at the protein level. In other aspects of the invention, marker expression is detected at the nucleic acid level.
Markers of the invention may be referred to herein interchangeably as “markers,” “immunophenotypic markers,” “leukemia-associated phenotypic markers,” “phenotypic markers,” or “cell markers.” “Leukemia-associated markers” can refer to particular combinations of markers used to diagnosis a particular leukemia, for example, an expression profile of different combinations of markers may be particular to a patient with ALL. In particular embodiments of the invention, markers can refer to “antigenic markers,” “antigens,” or “cell surface antigens,” referring to proteins that are expressed on the cell surface. Combinations of markers of the invention are selective for ALL, and specifically minimal residual disease.
The various markers employed in the methods and compositions disclosed herein, can have a modulated level of expression when compared to an appropriate control. Alternatively, a given marker need not show a modulated level of expression, but rather must only be expressed in the given sample. Specific expression profiles of the given marker combinations that are predictive of the various states disclosed herein are discussed in further detail elsewhere herein.
As used herein, a “modulated level” of a marker can comprise any statistically significant increase (overexpression) or decrease (underexpression) of the given marker when compared to an appropriate control. The modulated level can be assayed by monitoring either the concentration of and/or activity of the marker polypeptide and/or the level of the mRNA encoding the marker polypeptide. In general, a modulate level of marker can include either an increase or a decrease of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher relative to an appropriate control.
By “overexpressed” it is intended that the marker of interest is overexpressed in ALL cells but is not overexpressed in conditions classified as nonmalignant, benign, and/or any conditions that are not considered to be indicative of clinical disease. In general, an overexpressed marker can include any statistically significant increase in expression when compared to an appropriate control, including for example, an increase of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher relative to an appropriate control.
By “underexpressed” it is intended that the marker of interest is underexpressed in ALL cells but is not underexpressed in conditions classified as nonmalignant, benign, and/or any conditions that are not considered to be indicative of clinical disease. Thus, detection of various combinations of markers of the invention permit the differentiation of specimens indicative of an increased likelihood of minimal residual disease associated with ALL as compared to those of normal control specimens that are indicative of nonmalignant and benign proliferation.
The level of expression of a particular marker that is sufficient to constitute “overexpression” will vary depending on the specific marker used. In particular embodiments of the invention, a “threshold level” of expression over a normal control is established for a particular marker, wherein expression levels above this value are deemed overexpression. Overexpression of a particular marker can refer to an increase in the percentage of a population detected as expressing a particular marker or marker combination. Overexpression can also refer to the level of expression on a population of cells as detected by an increase in the mean fluorescence intensity (MFI). For example, in one embodiment of the invention, “overexpression” may be determined if the marker MFI for the specimen is at least three-fold above the normal control, wherein a three-fold increase in MFI is the “threshold level.” In other embodiments, an overexpressed marker can include any statistically significant decrease in expression when compared to an appropriate control, including for example, an increase of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher relative to an appropriate control or at least at least a 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 10 fold or higher expression level relative to an appropriate control.
The level of expression of a particular marker that is sufficient to constitute “underexpression” will vary depending on the specific marker used. In particular embodiments of the invention, a “threshold level” of expression is established for a particular marker, wherein expression levels below this value are deemed underexpression. Underexpression of a particular marker can refer to a decrease in the percentage of a population detected as expressing a particular marker or marker combination. Underexpression can also refer to the level of expression on a population of cells as detected by a decrease in the mean fluorescence intensity (MFI). For example, in one embodiment of the invention, “underexpression” may be determined for that particular marker if the marker MFI for the specimen is less than the normal control by at least half, wherein a 50% reduction MFI is the “threshold level”. In other embodiments, an underexpressed marker can include any statistically significant decrease in expression when compared to an appropriate control, including for example, a decrease of at least at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or lower relative to an appropriate control or at least at least a 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 10 fold or lower expression level relative to an appropriate control.
The methods of the invention comprise diagnosing minimal residual disease in a sample taken from a subject by detecting the expression of a plurality of markers that are modulated in ALL. Markers CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, and CD49f were analyzed and found differentially expressed in up to 81.4% of ALL cases. Embodiments of the invention can include, but are not limited to, compositions and methods related to new markers for the detection of minimal residual disease (MRD) comprising: CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, and CD49f.
In yet other embodiments, diagnosing minimal residual disease in a sample taken from a subject can comprise the detection of combinations of markers including, but not limited to markers CD19, CD10, CD34, and CD45, in combination with markers comprising: CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In yet other embodiments, diagnosing minimal residual disease in a sample taken from a subject can comprise the detection of combinations of markers including, but not limited to: CD19, CD10, CD34, and CD45, in combination with marker comprising: CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
The methods of the invention can comprise MRD detection by flow cytometry with preferred combinations of probes to specific markers. MRD detection can be combined with at least 4 different probes, and can include in some embodiments at least 5, 6, 7, 8, 9, 10, 11, and 12 different probes. When incorporated with at least 6-probes, the new marker combinations afford the detection of one leukemic cell amongst 105 bone marrow cells. These new markers allow MRD studies in all B-lineage ALL patients, and increase the sensitivity of detecting minimal residual disease.
Probes to Detect Markers of Minimal Residual Disease
The term “probe” refers to any molecule that is capable of specifically binding to an intended target molecule, for example, a nucleotide transcript or a protein encoded by a marker gene. RNA/DNA probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Likewise, antibody probes to specific targets can be generated by one of skill in the art, or derived from appropriate sources. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
By “specifically binds,” it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. An epitope is a site on an antigen or marker where the antibody binds via its variable region. The epitope is therefore a part of the antigen or marker, but the epitope is only a portion of the marker recognized by the antibody. According to this definition, an antibody is said to “specifically bind” to an epitope or have “antigen specificity” when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. As used herein, therefore, “specifically binds” is used interchangeably with recognition of a defined epitope on an antigen or marker, or any epitope contained in the antigen or marker. For example the term “specifically binds” when used in conjunction with a particular antibody is used to indicate that there is recognition of a certain epitope of the antigen and the interaction between the antibody and epitope is a non-random interaction indicative of the presence or “expression” of the certain epitope. The term “specifically binds” when used in conjunction with a particular marker is used to indicate that there is recognition of a certain antigen or marker and the interaction between the antibody and antigen or marker is a non-random interaction indicative of the presence or “expression” of the certain antigen or marker.
Embodiments of the invention, include methods and kits comprising probes to detect markers and combinations of markers in TABLE 1 comprising genes overexpressed in B-lineage ALL.
Embodiments of the invention, include methods and kits comprising probes to detect markers and combinations of markers in TABLE 2 comprising genes underexpressed in B-lineage ALL.
Embodiments of the invention include methods and kits comprising probes to detect markers and combinations of markers comprising CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
Embodiments of the invention, include methods and kits comprising a plurality of probes to detect markers and combinations of markers comprising CD19, CD10, CD34, and CD45, and any two of CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a (See TABLE 3).
Embodiments of the invention include methods and kits comprising contacting a specimen with a plurality of probes to detect expression levels of markers comprising CD19, CD10, CD34, and CD45, and any two of CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In one embodiment of the invention, a method comprises obtaining a specimen from a subject and contacting the specimen with plurality of probes to detect expression levels of markers. As a first step, the specimen is contacted by a plurality of probes to CD19, CD10, CD34 and CD45 wherein a first probe specifically binds to CD19, a second probe specifically binds to CD10, a third probe specifically binds to CD34, and a fourth probe specifically binds to CD45, and at least two additional probes specifically bind to any two of CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a. As a third step, at least one complex formed between each of said probes and said markers is detected and a value generated, wherein the value corresponds to an expression level of each of said marker. As a fourth step, an expression profile is generated by combining said values generated. The expression profile displaying each of the markers from step one and those selected in step two is compared to a normal control expression profile. The expression of CD19 and CD10, a modulated level of CD34, and CD45, and a modulated level of at least one of CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a is indicative of minimal residual disease in acute lymphoblastic leukemia.
In yet another embodiment of the invention, the method further comprises contacting the specimen with at least three, four, five, six, seven, or eight additional probes to detect expression levels of markers comprising CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
In another embodiment of the invention, the method further comprises permeabilization of cells in the specimen prior to the contacting the specimen with a probe to detect expression levels of markers comprising CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
In yet another embodiment of the invention, said plurality of probes specifically bind distinct markers comprising: (a) CD10, CD19, CD34, CD45, CD38, CD24, and CD44; (b) CD10, CD19, CD34, CD45, CD38, CD58, and CD44; (c) CD10, CD19, CD34, CD45, CD38, CD73, and CD15; (d) CD10, CD19, CD34, CD45, CD38, CD200, and CD44; (e) CD10, CD19, CD34, CD45, CD66c, CD123, and CD86; (f) CD10, CD19, CD34, CD45, CD72, CD13, and CD33; or (g) CD10, CD19, CD34, CD45, CD79b, HSPB1, and Bcl-2.
In another embodiment of the invention said modulated level comprises the expression of CD34 of CD45.
In yet another embodiment of the invention said modulated level comprises the overexpression of CD44, CD58, CD73, CD200, CD86, HSPB1, BCL2, CD164, CD97, CD99, or CD300a and/or the underexpression of CD38, CD72, or CD79b, relative to a normal control.
Generating Expression Profiles
As used herein, an “expression profile” comprises one or more values corresponding to a measurement of the relative abundance of a gene expression product (i.e., a marker). Such values may include measurements of RNA levels or protein abundance. Thus, an expression profile can comprise values representing the measurement of the transcriptional state or the translational state of the gene. As is known to those of skill in the art, the transcriptional state and translational state are related.
In embodiments of the invention, an “expression profile” of a specimen can include the identities and relative abundance, or “expression level,” of the RNA species, especially mRNAs present in populations of cells in the specimen. Preferably, a sufficient fraction or mRNA is used generate an expression profile using combinations of markers predictive of minimal residual disease. An expression profile can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies.
In embodiments, an “expression profile” of a specimen can include the identities and relative abundance, or “expression level”, of the constituent protein species expressed in populations of cells in the specimen. Expression profiles according to the invention comprise one or more values representing the expression level of a gene having differential expression in minimal residual disease as compared to a normal control specimen. Each expression profile can contain a sufficient number of values such that the profile can be used to distinguish samples containing a minimal number of leukemic cells or minimal residual disease as compared to specimens taken from normal controls. In some embodiments, an expression profile can comprise four values. In other embodiments, an expression profile can comprise more than four values corresponding to differentially expressed genes, for example at least 5, 6, 7, 8, 9, 10, 11, or 12 values.
In other embodiments of the invention, an expression profile can comprise values corresponding to mRNA expression levels as detected by nucleic acid probes. In exemplary embodiments, it may be advantageous to use a greater number of probes and therefore analyze the expression of a greater number of genes simultaneously. Therefore, in other embodiments of the invention 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170. 180, 190, 200, or >200 probes are reasonable. Embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probe sets shown in TABLE 1 comprising genes overexpressed in B-lineage ALL. Other embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probe sets shown in TABLE 2 comprising genes underexpressed in B-lineage ALL.
laevis)
laevis)
Drosophila)
laevis)
Drosophila)
Drosophila)
Normal” Controls
In one embodiment, a “normal control” used in the methods and kits of the invention are taken from a subject, or pool of subjects diagnosed and validated as “normal.” As discussed elsewhere herein, the corresponding predictive markers which are assayed in these samples can include, but are not limited to, CD19, CD10, CD34, CD45, CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 and CD300a or combinations thereof. In embodiments of the invention, specimens from normal controls correspond to blood or bone marrow specimens classified as nonmalignant, benign, and/or other conditions that are not considered to be indicative of clinical disease.
In specific embodiments, it may be necessary to phenotypically differentiate “hematogones” in normal controls from leukemic cells in ALL specimens. Hematogones are benign lymphoid precursors whose morphology and immunophenotype are similar to the blasts found in ALL, but are considered distinctive from cells indicative of MRD or ALL. Hematogone cells are more commonly found in pediatric bone marrow aspirates following therapy.
It is recognized that the accuracy of detecting and diagnosing minimal residual disease in ALL leukemia will vary based on the strength of the correlation between the expression levels of the differentially expressed genes when compared to normal controls. When the values in the expression profiles represent the expression levels of genes whose expression is strongly correlated with the physiologic condition, it may be possible to use a fewer number of values in the expression profile and still obtain an acceptable level of diagnostic or prognostic accuracy.
RNA Expression Profiling
The values in the expression profiles of the invention are measurements representing the absolute or the relative expression level of differentially expressed genes. The expression levels of marker genes may be determined by any method known in the art for assessing the expression level of an RNA molecule in a specimen. For example, expression levels of RNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are expressly incorporated herein by reference. Gene expression detection may also comprise nucleic acid probes in solution. Expression levels of RNA may also be monitored using the reverse transcriptase polymerase chain reaction (e.g., TaqMan®).
In one embodiment, microarrays are used to measure the values to be included in the expression profiles. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, TABLE 1 and TABLE 2. See also, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
In one approach, total mRNA isolated from cells taken from the subject is converted to labeled cDNA and then hybridized to an oligonucleotide array. Each specimen is hybridized to a separate array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample.
Embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 1 comprising genes overexpressed in B-lineage ALL. Other embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 2 comprising genes underexpressed in B-lineage ALL.
In embodiments of the invention, an expression profile is generated by the detection of nucleic acid corresponding to the expression of mRNA from a specimen. As a first step, a specimen is contacted with a set of probes to CD19 and CD10, wherein a first probe specifically binds to CD19 and a second probe specifically binds to CD10. As a second step a CD19+/CD10+ cell is isolated from said specimen. As a third step, an expression level of at least two gene products encoding CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a is determined in said CD19+/CD10+ isolated cell population from step two. As a forth step, an expression profile is generated of the CD19+/CD10+ cells of step two. The expression profile of step four, displaying a modulated level of at least one gene product, is indicative of minimal residual disease in acute lymphoblastic leukemia. In embodiments of the invention, modulation of the gene product detected from gene products encoding CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 and CD300a, relative to a normal control, is indicative of minimal residual disease in acute lymphoblastic leukemia.
As is well understood in the art and in yet other embodiments of the invention, various subsets of cells can be isolated for analysis, for example, it may be advantageous to compare CD19+, CD10− cells. In yet other embodiments, other combinations of probes may be included in the first step, for example, CD19, CD10, CD45, and CD34. For example, the subpopulations of cells that can be isolated by contacting a specimen with probes to markers CD19, CD10, CD45, and CD34 can include, but are not limited to: CD19+, CD10+, CD45+, and CD34+ cells; CD19+, CD10−, CD45+, and CD34+ cells; CD19+, CD10−, CD45−, and CD34+ cells; CD19+, CD10−, CD45+, and CD34−; CD19+, CD10+, CD45−, and CD34+ cells; CD19+, CD10+, CD45+, and CD34−; and CD19+, CD10+, CD45−, and CD34− cells. All of the preceeding subpopulations of cell can be isolated by contacting the specimen with said plurality of probes, but in addition, modulated activity of any one probe of that combination could also be selected thereby increasing the number of subpoplations analysed. The design of assays to optimize detection conditions and determining cell sorting conditions is standard and well within the routine capabilities of those of ordinary skill in the art.
In embodiments of the invention, determining the expression profile of the specimen that is CD10+/CD19+ further comprises probe combinations comprising (a) CD38, CD24, and CD44; (b) CD38, CD58, and CD44; (c) CD38, CD73, and CD15; (d) CD38, CD200, and CD44; (e) CD66c, CD123, and CD86; (f) CD72, CD13, and CD33; or (g) CD79b, HSPB1, and Bcl-2. In embodiments of the invention said modulated level comprises overexpression of CD44, CD58, CD73, CD200, CD86, HSPB1, BCL2, CD164, CD97, CD99, or CD300a and/or the underexpression of CD38, CD72, or CD79b, relative to a normal control.
In embodiments of the invention, determining the expression level of a gene product in cells expressing each of CD19, CD10, CD34, and CD45, can further comprise probes to detect at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, or twenty-two markers comprising: CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
Protein Expression Profile and Antibody Detection
In other embodiments, the values in the expression profile are obtained by measuring the abundance of the protein products of the differentially-expressed genes. The abundance of these protein products can be determined, for example, using antibodies specific for the protein products of the differentially-expressed genes. The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments, which can be generated by treating the antibody with an enzyme such as pepsin.
The terms “antibody” and “antibodies” broadly encompass naturally occurring forms of antibodies and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site (e.g., Fab′, F(ab)2, Fv, single chain antibodies, diabodies). Antibody derivatives may comprise a protein or chemical moiety conjugated to the antibody.
In embodiments of the invention, the antibody can be a polyclonal, monoclonal, or recombinant, e.g., a chimeric or humanized, fully human, non-human (e.g., murine, or single chain antibody). The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
The term “polyclonal antibody” as used herein refers to an antibody obtained from a population of heterogeneous antibodies derived from a multiple B cell response to an antigen which will recognize a variety of epitopes on the antigen. Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a marker protein immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized biomarker protein. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, C. (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor, et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole, et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan, et al. eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) in Monoclonal Antibodies: A New Dimension In Biological Analyses (Plenum Publishing Corp., NY); and Lerner (1981) Yale J. Biol. Med., 54:387 402).
As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a marker protein to thereby isolate immunoglobulin library members that bind the marker protein. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.
Antigen-binding fragments and variants of the monoclonal antibodies disclosed herein are contemplated and within the scope of the present invention. Such variants, for example, will retain the desired binding properties of the parent antibody. Methods for making antibody fragments and variants are generally available in the art. For example, amino acid sequence variants of a monoclonal antibody described herein can be prepared by mutations in the cloned DNA sequence encoding the antibody of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the polypeptide of interest may be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.
Preferably, variants of an antibody to a reference marker will have amino acid sequences that have at least 70% or 75% sequence identity, preferably at least 80% or 85% sequence identity, more preferably at least 90%, 91%, 92%, 93%, 94% or 95% sequence identity to the amino acid sequence for the reference antibody molecule, or to a shorter portion of the reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence identity. For purposes of the present invention, percent sequence identity is determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, for example, differ from the reference antibody by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
In embodiments of the invention, an antibody can be used to detect the marker or protein product of a differentially expressed gene in order to evaluate the abundance and pattern of expression of the protein. These antibodies can also be used diagnostically to monitor protein expression levels over time as part of a clinical monitoring procedure, e.g., determine the efficacy of a given therapy and reoccurrence of disease.
In embodiments of the invention, antibodies specifically bind to and detect markers comprising CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In embodiments of the invention antibodies specifically bind to and detect markers comprising CD19, CD10, CD34, CD45, CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
Embodiments of the invention include antibodies described in TABLE 3, TABLE 4, and TABLE 5.
Optical Detection Methods:
Detection of antibodies can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials (fluorophores, flurochromes), luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of fluorophores/flurochromes, include phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), coumarin dye (Horizon v450), sulphonyl chloride (Texas Red), cyanine (CY3, CY5, Cy7), FAM, JOE, TAMRA, TET, VIC, rhodamine; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. The skilled artisan will understand that additional moieties may be suitable for the method of the invention.
A detectable moiety generally refers in one embodiment to a composition or moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical or chemical means such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. The terms “fluorophore” and “fluorochrome” are defined as a chemical group, or component of a molecule that causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. A fluorophore/fluorchrome can refer to various fluorescent substances, including dyes, used in fluorescence microscopy or flow cytometry to stain specimens. The terms fluorophore” and “fluorochrome” are herein used interchangeably.
Fluorochromes may be conjugated to antibodies, proteins, polypeptides, peptides, or nucleotide probes which specifically bind to antigens, proteins, polypeptides, peptides, polysaccharides, DNA, or RNA sequences. Thus, binding of an antibody, protein, polypeptide, peptide, or nucleotide probe to an antigen, protein, polypeptide, peptide, polysaccharide, DNA, or RNA may be detected by measuring a signal generated from a fluorochrome by flow cytometry, or any suitable optical imaging technique. Detection of a signal may indicate binding, whereas lack of detection of a signal may indicate lack of binding.
Methods and compositions for detectably labeling nucleic acid probes, such as oligonucleotides, DNA-RNA hybrids, etc. are well known in the art. See, e.g., U.S. Pat. Nos. 6,316,230; 6,297,016; 6,316,610; 6,060,240; 6,150,107; and 6,028,290, each of which is hereby incorporated by reference in their entirety.
The compositions of the invention further comprise monoclonal antibodies and variants and fragments thereof that specifically bind to marker proteins of interest, thereby forming a detectable complex. The monoclonal antibodies may be labeled with a detectable substance to facilitate marker protein detection in the sample. Such antibodies find use in practicing the methods of the invention. Monoclonal antibodies having the binding characteristics of the antibodies disclosed herein are also encompassed by the present invention. Compositions further comprise antigen-binding variants and fragments of the monoclonal antibodies.
In embodiments of the invention, a probe is an antibody, including but not limited to a whole antibody molecule, a F(ab′)2, Fab′, Fv, Fd′, or Fd fragment. In yet other embodiments, an antibody can be conjugated with a detectable moiety, wherein the detectable moiety can be, for example, a fluorophore, a chromophore, a radionuclide, or an enzyme. In embodiments of the invention a fluorophore can for example, can be, but is not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), and coumarin dye (Horizon v450). Detection of complexes formed between an antibody probe and marker can be achieved by an optical detection technique, including, but not limited to flow cytometry and microscopy.
“Cell staining” when used in reference to an antibody means that the antibody recognizes an marker and binds to marker in the specimen forming a complex, thereby “labeling” or otherwise “staining” the cell expressing the marker to make it visible and/or detectable by microscopy or flow cytometry. Combinations of antibodies can be collectively added a specimen and thereby “stain the cell” for later analysis by visualization with a flow cytometer or microscope, for example. One of skill in the art could determine whether a cell expressed a specific protein based on the level of antibody that bound to the cell using standard methods.
The methods of the invention can also be used in immunofluorescence histochemistry. This technique involves the use of antibodies labeled with various fluorophores to detect substances within a specimen. In exemplary embodiments a pathologist can derive a great deal of morphological information of diagnostic value by examining a specimen from a subject by microscope. Immunohistochemistry is particularly relevant to, for example, the early diagnosis of cancer or pre-acute states such as minimal residual disease in ALL. Combinations of fluorophores or other detectable labels can be used by the methods on this invention, thereby greatly increasing the number of distinguishable signals in multicolor protocols.
In another embodiment, the method employs flow cytometry. In another embodiment, in a peripheral blood sample or blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used in one embodiment to exclude debris and dead cells.
Flow cytometry is an optical technique that analyzes particles or cells in a fluid mixture based on their optical characteristics, via the use of a flow cytometer (See, for example, Shapiro, “Practical Flow Cytometry,” Third Ed. (Alan R. Liss, Inc., 1995); and Melamed et al. “Flow Cytometry and Sorting,” Second Ed. (Wiley-Liss 1990)). Flow cytometers hydrodynamically focus a fluid suspension of particles/cells into a thin stream so that they flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles/cells. Commonly used flow cytometers such as the Becton-Dickinson Immunocytometry Systems “FACSCAN” (San Jose, Calif.) can measure forward light scatter (generally correlated with the refractive index and size of the particle/cell being illuminated), side light scatter (generally correlated with the cell granularity), and particle fluorescence at one or more wavelengths. Data acquisition and analysis can be done using FASCALIBER® LSRII flow cytometers (Becton Dickinson), and CELLQUEST Pro™, BD FACSD
In embodiments of the invention, determining an expression profile of a specimen is generated using combinations of probes that bind specifically to CD10, CD19, CD34, and CD45, wherein combinations comprise (a) CD10, CD19, CD34, CD45, CD38, CD24, and CD44; (b) CD10, CD19, CD34, CD45, CD38, CD58, and CD44; (c) CD10, CD19, CD34, CD45, CD38, CD73, and CD15; (d) CD10, CD19, CD34, CD45, CD38, CD200, and CD44; (e) CD10, CD19, CD34, CD45, CD66c, CD123, and CD86; (f) CD10, CD19, CD34, CD45, CD72, CD13, and CD33; or (g) CD10, CD19, CD34, CD45, CD79b, HSPB1, and Bcl-2.
In other embodiments of the invention, antibodies can be directly conjugated for simultaneous detection. For example, a method of the invention can comprise antibodies directly conjugated to a detectable fluorochrome for simultaneous detection of a plurality of markers wherein: CD34 is conjugated to PerCP, CD19 is conjugated to APC, CD10 is conjugated to PE-Cy5, CD45 is coupled to APC-H7, CD38 is conjugated to FITC, CD24 is conjugated to PE, CD44 is conjugated to Horizon v450, CD58 is conjugated to PE, CD73 is conjugated to PE, CD15 is conjugated to Horizon v450, CD200 is conjugated to PE, CD66c is conjugated to FITC, CD123 is conjugated to PE, CD86 is conjugated to Horizon v450, CD72 is conjugated to FITC, CD13 is conjugated to PE, CD33 is conjugated to v450, CD79b is conjugated to FITC, HSPB1 is conjugated to PE, and Bcl2 is conjugated to Horizon V450. The skilled artisan will understand that any one antibody marker can be coupled to any fluorochrome for use in combination with any other antibody, and that preferred combinations can be used simultaneously with other antibody markers by the selection of different combinations of antibodies labeled with different flurochromes.
TABLE 3 shows exemplary marker combinations for detection of MRD in B-lineage ALL incorporating the top 10 differentially expressed markers including CD24, CD44, CD73, CD200, CD123, CD86, CD72, CD79b, HSPB1, and Bcl2.
CD24
CD44
CD44
CD73
CD200
CD44
CD123
CD86
CD72
CD79b
HSPB1
Bcl2
Cell Sorting and Selection of Subpopulations of Cells in a Specimen
Multiparameter flow cytometric cell analysis can be used as part of the methods of this invention. The simultaneous analysis of multiple predictive parameters using flow cytometry is known to those of skill in the art. In one embodiment, the population of cells to be analyzed is contacted with a panel of antibodies directed against distinct cell surface markers, under conditions effective to allow antibody probe binding. The antibodies employed can be monoclonal antibodies, and can, in another embodiment, be labeled in a manner to allow their subsequent detection.
In embodiments of the invention, fluorochromes can be excited by at least two different lasers to give off light of at least four different wavelengths, with the potential, for simultaneous analysis of at least four different markers. An additional two parameters include two light scattering parameters; direct and orthogonal, or side-scattering capability which can be analyzed concurrently with antibody detection, thereby allowing for cell analysis on the basis of at least 6 parameters. In embodiments of the invention at least five, six, seven, eight, nine, ten, eleven, or twelve different antibody probes, can be used simultaneously, thereby allowing for cell analysis on the basis of at least seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen different parameters.
Multiparameter cell sorting can be used in an embodiment of the invention to isolate cells based on a specific expression profile. For example, in one embodiment cell sorting analysis can be achieved using fluorescence-activated flow cytometry, by methods well described in the art. In one embodiment cells can be sorted based on the co-expression of markers CD19 and CD10, wherein in combination with the expression of CD19 and CD10 the expression of other markers can be interrogated. In another embodiment of the invention, mRNA expression profiles can be generated from a purified population of CD19+ and CD10+ cells isolated from a subject specimen for the purpose of diagnosing minimal residual disease.
As is well understood in the art and in yet other embodiments of the invention, various subsets of cells can be isolated for analysis, for example, it may be advantageous to compare CD19+, CD10+ cells and CD19+, CD10− cells. Both subpopulations can be isolated by contacting the specimen with probes to CD19 and CD10, whereas in one condition cells are positively selected (CD19+, CD10+), and in the later, are negatively selected (CD19+, CD10−). Likewise, in yet other embodiments, other combinations of probes may be included in a first step, for example, CD19, CD10, CD45, and CD34, wherein modulated levels of CD45 and CD34 may be reflected in the subsets that are isolated. For example, the subpopulations of cells that could be isolated by contacting a specimen with probes to markers CD19, CD10, CD45, and CD34 could include, but are not limited to: CD19+, CD10+, CD45+, and CD34+ cells; CD19+, CD10−, CD45+, and CD34+ cells; CD19+, CD10−, CD45−, and CD34+ cells; CD19+, CD10−, CD45+, and CD34−; CD19+, CD10+, CD45−, and CD34+ cells; CD19+, CD10+, CD45+, and CD34−; and CD19+, CD10+, CD45−, and CD34− cells. All the preceeding subpopulations of cell could be isolated from by contacting the specimen with said probes, but in addition, using flow cytometry for example, modulated activity could representative of a spectrum of expression to negative to positive, and also be selected. The number of potential subpopulations analyzed could thereby increase the number of subpoplations analysed. The design of assays to optimize detection conditions and determining cell sorting conditions to isolate specific subpopulations of cells is standard and well within the routine capabilities of those of ordinary skill in the art.
In yet other embodiments of the invention, enrichment of specific subpopulations of cells can be achieved by other methods as well. For example a wide variety of magnetic bead separation and isolation procedures can be used to selectively negatively and positively enrich samples for specific subpopulations of cells. For example, in some embodiments a mixture of magnetic beads coupled to lineage specific antibodies can be used to deplete, T cells, NK cells, monocytes, platelets, dendritic cells, granulocytes and erythrocytes, thereby negatively isolating B cells. The skilled artisan will understand that combinations of different antibodies can be used alone or in combination, and in multiple successive rounds of isolation, to positively and/or negatively select for subpopulations of cells.
One of skill in the art will recognize that optimization of reagents and conditions, for example, antibody titer and parameters for detection of antigen-antibody binding, is needed to maximize the signal to noise ratio for a particular antibody. Antibody concentrations that maximize specific binding to the markers of the invention and minimize non-specific binding (or “background”) will be determined. In particular embodiments, appropriate antibody titers are determined by initially testing various antibody dilutions on patient serum samples. The design of assays to optimize antibody titer and detection conditions is standard and well within the routine capabilities of those of ordinary skill in the art. Some antibodies require additional optimization to reduce background and/or to increase specificity and sensitivity.
The skilled artisan will recognize that optimization of multiparameter assays designed to detect a plurality of antibody probes simultaneously will be necessary. In embodiments of the invention, maximization of signal to noise ratio, as well an optimization of fluorochrome combinations will be necessary for each of the antibody probes combinations. Conjugated-antibody concentrations that maximize specific binding to the markers of the invention and minimize non-specific binding (or “background”) will be determined with other such conjugated antibody probes as is known in the art. The design of assays to optimize and compensate the signals detected for the various conjugated antibodies is standard and well within the routine capabilities of those of ordinary skill in the art. Some antibodies require additional optimization to reduce background and/or to increase specificity and sensitivity.
Antibody and Nucleic Acid Probes to Target Genes
The antibodies used to practice the invention are selected to have high specificity for the marker proteins of interest. Methods for making antibodies and for selecting appropriate antibodies are known in the art. In some embodiments, commercial antibodies directed to specific marker proteins may be used to practice the invention (see TABLE 4). The antibodies of the invention may be selected on the basis of desirable staining of cytological, rather than histological, samples. That is, in particular embodiments the antibodies are selected with the desired combination in mind and for binding specificity.
The markers and combinations of markers of the invention include genes or proteins that are selectively expressed, overexpressed or underexpressed in leukemia, and specifically in ALL, as defined herein above, and may be combined with known markers as well as those presently unknown in the art. In particular embodiments, markers are intracellular proteins, secreted proteins or proteins that are predicted to encode membranous proteins with transmembrane segments and extracellular domains. In some embodiments of the invention, probes can detect markers that are polypeptides expressed at the surface of the cell. In other embodiments, probes can detect markers that are polypeptides expressed intracellularly. In still other embodiments, probes detect markers that are polynucleotides. In still other embodiments, kits and methods of invention can comprise probes that can detect markers that include polypeptides and polynucleotides.
The methods of the invention can comprise MRD detection by gene array with preferred combinations of markers. MRD detection can be combined, for example, with at least 3 different marker combinations. Embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 1 comprising genes overexpressed in B-lineage ALL. Other embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 2 comprising genes underexpressed in B-lineage ALL.
Embodiments of the invention can include, but are not limited to, compositions and methods related to the detection of new markers for minimal residual disease (MRD) comprising: CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In yet other embodiments, diagnosing minimal residual disease in a sample taken from a subject can comprise the detection of combinations of markers including, but not limited to markers CD19, CD10, CD34, and CD45, in combination with markers comprising CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In yet other embodiments, diagnosing minimal residual disease in a sample taken from a subject can comprise the detection of combinations of markers including, but not limited to: CD19, CD10, CD34, and CD45, in combination with markers comprising CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
“CD” or “cluster designation” refers to a system to classify cell markers defining cells based on what molecules are present on their surface. These “cell markers” or “markers” are often used to associate cells with certain immune functions. While using one CD molecule to define populations is uncommon (though a few examples exist), combining markers is often used to identify cell types with very specific characteristics within the immune system. The system was originally intended for the classification of the monoclonal antibodies (mAbs) generated by different laboratories around the world against epitopes on the surface molecules of leukocytes (white blood cells). Since then, the use of the CD nomenclature has expanded to include many other cell types, and more than 320 CD unique clusters and subclusters have been identified. In some instances CD antigens are expressed only at certain stages of development or under certain conditions, for example after cell activation or in certain disease conditions. Unlike the morphological criteria used in classical hematology for the description of specific developmental stages of lymphocytes, the use of monoclonal antibodies, and the “CD”marker annotation, allows the objective and precise analysis and standardized typing of mature and immature normal and malignant cells of all hematopoietic cell lineages. Antibodies that recognize cell surface markers can also be referred to by the CD designation. The CD designation can help to delineate the biologic traits that distinguish normal immune and hematopoietic cells from their malignant counterparts, which is of fundamental importance in understanding hematological malignancies.
The CD designation of molecules is often used to identify cells and expression patterns detected by various applications including flow cytometry. Very generally, cell populations can be defined using a ‘+’ or ‘−’ a symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. For example, a “CD34+, CD31−” cell is one that expresses CD34, but not CD31. This exemplary CD combination typically corresponds to a stem cell, opposed to a fully-differentiated endothelial cell. Therefore, generally speaking, use of CD with a number X, as in “CDX,” can refer to a protein that in humans is encoded by the CDX gene, for example. Markers can be referred to by their CD designation, but can also be referred to by the gene or encoded protein name. Antibodies recognizing specific proteins can also be referred to by the CD number of the marker that is recognized.
CD34 (cluster designation 34) encompasses the human gene at locus 1q32 corresponding to sequences having GENBANK® Accession Nos: NM_001773, NP_001764.1. The protein is a monomeric cell surface antigen with a molecular mass of approximately 110 kDa that is selectively expressed on human hematopoietic progenitor cells.
CD19 (cluster designation 19) encompasses the human gene at locus 16p11.2 corresponding to sequences having GENBANK® Accession Nos: NM_01178098 and NP_001171569.1. CD19 is a cell surface molecule expressed only by B lymphocytes and follicular dendritic cells of the hematopoietic system. It is the earliest of the B-lineage-restricted antigens to be expressed and is present on most pre-B cells and most non-T-cell acute lymphocytic leukemia cells and B-cell type chronic lymphocytic leukemia cells (Tedder and Isaacs, 1989. J. Immun. 143: 712-717).
CD10 (cluster designation 10) encompasses the human gene at locus 3q21-q27 corresponding to sequences GENBANK® Accession Nos: NM_000902 and NP_000893.2. The protein is also known as “membrane metallo-endopeptidase (MME)” or “common acute lymphocytic leukemia antigen (CALLA).” The common acute lymphocytic leukemia antigen (CALLA) is an important cell surface marker in the diagnosis of human acute lymphocytic leukemia (ALL). It is present on leukemic cells of pre-B phenotype, which represents 85% of cases of ALL. The CALLA gene encodes a 100 kDa type II transmembrane glycoprotein Barker et al. (1989) J. Immun. 142: 283-287.
CD45 (cluster designation 45), encompasses the human gene at locus 1q31-q32 corresponding to sequences having GENBANK® Accession Nos: NM_002838, NP_002829.2. CD45 is also called B220; CD45R; GP180; LCA; LY5; and T200. It is a membrane-bound protein tyrosine phosphatase expressed in various isoforms, of between 180 and 220 kDa in size. CD45 is expressed on virtually all leukocytes, including myeloid and lymphoid precursors in bone marrow and mature lymphocytes in lymph nodes. CD45 (T200) is expressed on all hematopoietic cells except mature red cells and their immediate progenitors. It is not found, however, on other differentiated tissues; thus, it can be used as an antigenic marker with which to identify undifferentiated hematopoietic tumors.
CD38 (cluster designation 38) encompasses the human gene at locus 4p15.32 corresponding to sequences having GENBANK® Accession Nos: NM_001775.2, NP_001766.2, as set forth in SEQ ID NO: 1 and SEQ ID NO:2, respectively. CD38 is commonly used as a human leukocyte differentiation marker and is a novel multifunctional ectoenzyme widely expressed in cells and tissues especially in leukocytes. CD38 also functions in cell adhesion, signal transduction and calcium signaling. It has been demonstrated to be involved in the synthesis and hydrolysis of Cyclic ADP-ribose (cADPR) (Takasawa et al. (1993). J. Biol. Chem. 268: 26052-26054, 1993). Cyclic ADP-ribose is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca(2+) mobilization in the endoplasmic reticulum for secretion of insulin (Takasawa et al. (1993) Science 259: 370-373).
CD24 (cluster designation 24) encompasses the human gene at locus 6q21 corresponding to sequences having GENBANK® Accession Nos: NM_013230, NP_037362.1, as set forth in SEQ ID NO: 3 and SEQ ID NO:4, respectively. CD24 is also known as “small cell lung carcinoma cluster 4 antigen” Studies with monoclonal antibodies indicate that most other hematopoietic cells, including T cells, monocytes, red blood cells, and platelets, seem not to express the CD24 antigen. CD24 has been implicated in both activation and differentiation of B lymphocytes because its expression pattern changes at critical times during B-cell development. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone ML5 which reacts with a 35-45 kDa two-chain glycoprotein (see TABLE 4, BD Pharmingen™ Cat. NO. 555428) (Schlossman et al. ed. (1995) Leukocyte Typing V: White Cell Differentiation Antigens. New York: Oxford University Press).
CD44 (cluster designation 44) encompasses the human gene at locus 11p13 corresponding to sequences having GENBANK® Accession Nos: NM_000610, NP_000601.3, as set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. It is also known as “Indian blood group” antigen. It is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs). This integral cell membrane glycoprotein participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis (Aruffo et al. (1990) Cell 61: 1303-1313). In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone 515 which reacts with a 85 kDa glycoprotein expressed on leukocytes, erythrocytes, epithelial cells and weakly on platelets (see TABLE 4, BD Pharmingen™, Cat. NO. 5500989) (Patel et al. “CD44 Workshop Panel Report” In: Kishimoto et al, ed. (1997) Leukocyte Typing Vi: White Cell Differentiation Antigens. New York: Garland Publishing Inc: 373-375).
CD58 (cluster designation 58) encompasses the human gene at locus 1p13 corresponding to sequences having GENBANK® Accession Nos: NM_001779, NP_001770.1, as set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. CD58 is also known as “lymphocyte function-associated antigen (LFA3)”. The CD58 gene encodes a CD2 receptor. The presence of these both CD2 and CD58 antigens on opposing cells optimizes immune recognition, facilitating contacts between helper T lymphocytes and antigen-presenting cells as well as between cytolytic effectors and target cells (Wang et al. (1999) Cell 97: 791-803).
CD73 (cluster designation 73) encompasses the human gene at locus 6q14-q21 corresponding to sequences having GENBANK® Accession Nos: NM_002526 and NP_002517.1, as set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively. CD73 is a membrane-bound enzyme also known as “ecto-5-prime-nucleotidase,” and is expressed on subsets of B-cells and T-cells as well as other cell types. The enzyme is used as a marker of lymphocyte differentiation. CD73 is thought to function as a cell adhesion molecule and to mediate lymphocyte binding to endothelial cells (Airas, L. et al. (1995) J. Exp. Med. 182(2): 1603-8) and adhesion between B-cells and follicular dendritic cells (Airas and Jalkanen (1996) Blood. 88(5):1755-64). In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone AD2 which reacts with a 70 kDa glycosyl phosphatidylinositol (GPI)-anchored glycoprotein expressed on subsets of T and B lymphocytes, follicular dendritic cells, epithelial cells, and endothelial cells (see TABLE 4, BD Pharmingen™, Cat. NO. 550257) (Schlossman et al. ed. (1995) Leukocyte Typing V: White Cell Differentiation Antigens. New York: Oxford University Press).
CD15 (cluster designation 15) encompasses the human gene at locus 11q12-qter corresponding to sequences having GENBANK® Accession Nos: NM_002033 and NP_002024.1, as set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively. CD15 expresses a protein also known as “fucosyltransferase 4 (alpha (1,3),” “myeloid-specific fucosyltransferase,” “ELAM ligand fucosyltransferase,” and “galactoside 3-L-fucosyltransferase.” The product of this gene transfers fucose to N-acetyllactosamine polysaccharides to generate fucosylated carbohydrate structures. CD15 catalyzes the synthesis of the non-sialylated antigen, Lewis x (CD15).
CD200 (cluster designation 200) encompasses the human gene located at locus 3q12-q13 corresponding to sequences having GENBANK® Accession Nos: NM_001004196 and NP_001004196.2, as set forth in SEQ ID NO:13 and SEQ ID NO:14, respectively. CD200 was identified as a type II membrane glycoprotein expressed on a number of cell types relevant to the inflammatory and immune cascade, including dendritic cells, endothelial cells, and activated T cells. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone MRC OX-104 which recognizes a 40-45 kDa membrane glycoprotein expressed on resting and activated T cells and B cells, as well as a subset of CD34+ progenitor cells, but not on NK cells, monocytes, granulocytes, or platelets (see TABLE 4, BD Pharmingen™, Cat. NO. 552475) (Mason et al. ed. (2002) Leukocyte Typing VII. New York: Oxford University Press).
CD66c (cluster designation 66c) encompasses the human gene at locus 19q13.1-q13.2 corresponding to sequences having GENBANK® Accession Nos: NM_002483 and NP_002474.3, as set forth in SEQ ID NO:15 and SEQ ID NO:16, respectively. CD66c is also known as “carcinoembryonic antigen-related cell adhesion molecule.” CD66c is one of the most widely used tumor markers in serum immunoassay determinations of carcinoma.
CD123 (cluster designation 123) encompasses the human gene at loci Xp22.3 and Yp13.3 corresponding to sequences having GENBANK® Accession Nos: NM_002183 and NP_002174.1, as set forth in SEQ ID NO:17 and SEQ ID NO:18, respectively. CD123 is also known as the low affinity receptor for IL-3 encoding an interleukin 3 specific subunit of a heterodimeric cytokine receptor. The receptor is comprised of a ligand specific alpha subunit and a signal transducing beta subunit shared by the receptors for interleukin 3 (IL3), colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5 (IL5). CD123 is expressed on a subset of peripheral blood dendritic cells, progenitor cells, monocytes, eosinophils, basophils, and hematopoietic cells, such as erythroid cells and B cells. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone 9F5 which reacts to the α-chain of the interleukin-3 receptor (IL-3Rα) expressed on a subset of peripheral blood dendritic cells, on a subset of progenitor cells, monocytes, eosinophils, and basophils (see TABLE 4, BD Pharmingen™, Cat. NO. 303545) (Sun et al. (1996) Blood. 87: 88-92).
CD86 (cluster designation 86) encompasses the human gene at locus 3q21 corresponding to sequences having GENBANK® Accession Nos: NM_006889 and NP_008820.3, as set forth in SEQ ID NO:19 and SEQ ID NO:20, respectively. CD86 encodes a type I membrane protein that is a member of the immunoglobulin superfamily. This protein is expressed by antigen-presenting cells, and it is the ligand for two proteins at the cell surface of T cells: CD28 antigen and cytotoxic T-lymphocyte-associated protein 4. Binding of this protein with CD28 antigen is a co-stimulatory signal for activation of the T-cell. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone 2331 (FUN-1) which recognizes a 75 kDa cell surface protein expressed primarily on monocytes and activated B cells (see TABLE 4, BD Pharmingen™, Cat. NO. 555658) (Nozawa et al. (1993) J Path. 169(3):309-315).
CD72 (cluster designation 72) encompasses the human gene at locus 9p13.3 corresponding to sequences having GENBANK® Accession Nos: NM_001782 and NP_001773.1, as set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. CD72 is expressed on B cells and is the ligand for CD5. CD5 and/or CD72 engagement delivers critical co-stimulatory signals to B cells. It has been suggested that CD72 is a key molecule in regulating mature B cell differentiation, particularly in preventing the differentiation of naïve B cells into plasma cells (Yamazaki et al. (2005) Eur. J. Immunol.). In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone JA-117 which reacts with a 39-43 kDa type II integral membrane protein expressed on B cells from early precursor stages through mature B cells, although not on plasma cells (see TABLE 4, BD Pharmingen™, Cat. NO. 555918) (Schlossman et al. ed. (1995) Leukocyte Typing V. White Cell Differentiation Antigens. New York: Oxford University Press).
CD13 (cluster designation 13) encompasses the human gene at locus 15q25-q26 corresponding to sequences having GENBANK® Accession Nos: NM_001150 and NP_001141.2, as set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. CD13 encodes a surface antigen glycoprotein of about 150 kDa, also known as “aminopeptidase-N.” CD13 is an enzyme thought to be involved in the metabolism of regulatory peptides expressed by diverse cell types, including small intestinal and renal tubular epithelial cells, macrophages, granulocytes, and synaptic membranes from the CNS. Defects in this gene appear to be a cause of various types of leukemia or lymphoma.
CD79b (cluster designation 79b) encompasses the human gene at locus 17q23 corresponding to sequences having GENBANK® Accession Nos: NM_000626 and NP_000617.1, as set forth in SEQ ID NO:25 and SEQ ID NO:26, respectively. CD79b is a B lymphocyte antigen receptor that is a multimeric complex that includes the antigen specific component, surface immunoglobulin (Ig). Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta, which are necessary for expression and function of the B-cell antigen receptor. The CD79b gene encodes the Ig-beta protein of the B-cell antigen component. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone AT107-2 which recognizes an immunogen peptide containing 20 amino acid residues derived from murine CD79b (NP_032365.1, NM_008339.2). The antibody is cross reactive with human, rat, pig, and dog CD79b (see TABLE 4, AbDserotec, Cat. NO. MCA2209FT) (Dornan et al. (2009) Blood. 114:2721-9; Vendel et al. (2009) J. Immunol. 182: 1509-17).
CD33 (cluster designation 33) encompasses the human gene at locus 19q13.41 corresponding to sequences having GENBANK® Accession Nos: NM_001082618.1 and NP_001076087.1, as set forth in SEQ ID NO:27 and SEQ ID NO:28, respectively. CD33, or Siglec-3 is a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells. CD33 has been implicated in the negative regulation of cell proliferation of normal or leukemic myeloid cells. (Vitale et al. (1999) PNAS. 96, 15091-15096).
HSPB1 encompasses the human gene at locus 7q11.23 corresponding to sequences having GENBANK® Accession Nos: NM_001540 and NP_001531.1. HSPB1, as set forth in SEQ ID NO:29 and SEQ ID NO:30, respectively, is a gene that encodes the intercellular heat shock 27 kDa protein. Expression of this gene is induced by environmental stress and developmental changes. The encoded protein is involved in stress resistance and actin organization and translocates from the cytoplasm to the nucleus upon stress induction. Synthesis of the small (27-kDa) HSP has been shown to be correlated with the acquisition of thermotolerance. In some embodiments, the variants detected encompass an eptitope recognized by antibody HSP27, clone G3.1 conjugated to biotin that recognizes a 24-27 kDa hsp27 protein (early called 24K) expressed on approximately 50% of breast carcinomas and drug resistant cancer cells (See TABLE 4, Thermo Scientific, Cat. NO. MS-101-BO, -B1, or -B, for biotin labeled). In other embodiments, the variants encompass the eptitope recognized by the antibody HSPB1 derived from G3.1, conjugated to PE (see TABLE 4, Stressgen, Cat. NO. SPA-800PE) (Edward et al. (1980) Biochem Biophys Research Commun. 93:804-812; Ciocca et al. (1991) Breast Cancer Research and Treatment. 20:33-42).
Bcl-2 (B-cell lymphoma 2) encompasses the human gene at locus 18q21.3 corresponding to sequences having GENBANK® Accession Nos: NM_000633 and NM_000657, and NP_000624.2 and NP_000648.2, as set forth in SEQ ID NO:31 and SEQ ID NO:33, and SEQ ID NO:32 and SEQ ID NO:34, respectively. This gene encodes an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes. Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described in chromosomal translocations involving chromosomes 14 and 18 in follicular lymphomas. Constitutive expression of BCL2, such as in the case of translocation of BCL2 to Ig heavy chain locus, is thought to be the cause of follicular lymphoma. The Bcl-2 gene has been implicated in a number of cancers, including melanoma, breast, prostate, and lung carcinomas. It is also thought to be involved in resistance to conventional cancer treatment. Two transcript variants, produced by alternate splicing, differ in their C-terminal ends. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone 100 which is expressed in lymphocytes and monocytes but not appreciably in granulocytes (see TABLE 4, Invitrogen, Cat. NO. MHBLC014) (Hill et al. (1996) Blood. 88:1046).
CD164 (cluster designation 164) encompasses the human gene at locus 6q21 corresponding to sequences having GENBANK® Accession Nos: NM_006016 and NP_006007.2, as set forth in SEQ ID NO:35 and SEQ ID NO:36, respectively. CD164 is a type-I integral transmembrane sialomucin that functions as an adhesion receptor. Sialomucins are a heterogeneous group of secreted or membrane-associated mucins that appear to play two key but opposing roles in vivo: first as cytoprotective or anti-adhesive agents, and second as adhesion receptors. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone N6B6 which recognizes a 80-90 kDa mucin-like molecule present on CD34+ progenitor cells during the early stages of B cell, erythroid cell and myelomonocytic cell development. It is also expressed on epithelial cells, peripheral blood monocytes and weakly on lymphocytes (see TABLE 4, BD Pharmingen™, Cat. NO. 551298) (Watt et al. (1998). Blood 92 (3):849-66; and Zannettino et al. (1998). Blood. 92 (8):2613-28).
CD304 (cluster designation 304) encompasses the human gene at locus 10p12 corresponding to sequences having GENBANK® Accession Nos: NM_001024628 and NP_001019799.1, as set forth in SEQ ID NO:37 and SEQ ID NO:38, respectively. CD304 is also known as “NRP1,” “Neuropilin-1 (NP-1),” and “BDCA-4,” and encodes a protein with numerous functions. On neurons, it is a receptor for axon growth guidance class-3 semaphorins SEMA3A and plexin-1. On endothelial and some tumor cells it is a VEGF165 receptor, and on plasmacytoid dendritic cells it has a similar role to CD303 but does not decrease interferon production upon activation. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone AD5-17F6 (see TABLE 4, Miltenyi Biotec Cat. NO. 130-090-533) (Dzionek, A., et al. (2000) J. Immunol. 165:6037-6046.)
CD97 (cluster designation 97) encompasses the human gene at locus 19p13 corresponding to sequences having GENBANK® Accession Nos: NM_078481 and NP_510966.1, as set forth in SEQ ID NO:39 and SEQ ID NO:40, respectively. The encoded product is a glycoprotein present on the surface of most activated leukocytes and spans the membrane seven times, which is a defining feature of G protein-coupled receptors. The protein has an extended extracellular region with several N-terminal epidermal growth factor (EGF)-like domains, which mediate binding to its cellular ligand, decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade. The presence of structural features characteristic of extracellular matrix proteins and transmembrane proteins suggests that this protein is a receptor involved in both cell adhesion and signaling processes early after leukocyte activation. Alternative splicing has been observed for this gene and three variants have been found. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone VIM3b which recognizes a triplet of membrane proteins of 74, 80 and 89 kDa strongly expressed on phytohemagglutinin (PHA)-activated T and B cells, virtually all monocytes and granulocytes, and several cell lines including HL-60, THP-1, and K562. It is weakly expressed on resting lymphocytes (see TABLE 4, BD Pharmingen™, Cat. NO. 555774) (Schlossman, et al. ed. (1995) Leukocyte Typing V. New York: Oxford University Press).
CD99 (cluster designation 99) encompasses the human gene at loci Xp22.32 and Yp11.3 corresponding to sequences having GENBANK® Accession Nos: NM_001122898 and NP_001116370.1, as set forth in SEQ ID NO:41 and SEQ ID NO:42, respectively. CD99 is a cell surface glycoprotein involved in leukocyte migration, T-cell adhesion, ganglioside GM1 and transmembrane protein transport, and T cell death by a caspase-independent pathway. Two transcript variants encoding different isoforms have been found for this gene. This variant 2 lacks an alternate in-frame exon compared to variant 1. The resulting isoform 2 has the same N- and C-termini but is shorter compared to isoform 1. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone TU 12 which recognizes a 32 kDa sialoglycoprotein expressed on all leukocyte lineages, although is differentially expressed during T and B-lymphoid and granulocytic development, with higher densities being expressed during early hematopoietic stages (see TABLE 4, BD Pharmingen™, Cat. NO. 555689) (Knapp et al, ed. (1989) Leucocyte Typing IV. New York: Oxford University Press; 1989).
CD102 (cluster designation 102) encompasses the human gene at locus 17q23-q25 corresponding to sequences having GENBANK® Accession Nos: NM_000873 and NP_000864.2, as set forth in SEQ ID NO:43 and SEQ ID NO:44, respectively. CD102 is also known as intercellular adhesion molecule 2 (ICAM-2). This variant (5) differs in the 5′ UTR compared to variant 1. All five variants encode the same protein. The protein encoded by this gene is a member of the intercellular adhesion molecule (ICAM) family. All ICAM proteins are type-I transmembrane glycoproteins, contain 2-9 immunoglobulin-like C2-type domains, and bind to the leukocyte adhesion LFA-1 protein. This protein may play a role in lymphocyte recirculation by blocking LFA-1-dependent cell adhesion. CD102 mediates adhesive interactions important for antigen-specific immune response, NK-cell mediated clearance, lymphocyte recirculation, and other cellular interactions important for immune response and surveillance. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone CBR-IC/2 which recognizes a type-I membrane glycoprotein with an approximate molecular weight of 55-65 kDa expressed on vascular endothelial cells, lymphocytes, monocytes, but not on granuloctyes. (see TABLE 4, BD Pharmingen™, Cat. NO. 558080) (Barclay et al. ed. (1997) The Leukocyte Antigen FactsBook. San Diego: Academic Press).
CD300a (cluster designation 300a) encompasses the human gene at locus 17q25.2 corresponding to sequences having GENBANK® Accession Nos: NM_007261 and NP_009192.2, as set forth in SEQ ID NO:45 and SEQ ID NO:46, respectively. CD300a also known as “inhibitory receptor protein (IRp60)” is a surface molecule of 60 kDa expressed by all human natural killer (NK) cells. The IRp60 gene, encodes a molecule that is highly O- and N-glycosylated, belonging to the immunoglobulin superfamily (Ig-SF). The ligand of the CD300a is still unknown but IRp60 does not appear to recognize HLA-class I molecules. In some embodiments, the variants detected encompass an eptitope recognized by the antibody derived from clone E59.126 expressed on T lymphocyte subsets, monocytes, and granulocytes (see TABLE 4, Beckman Coulter Cat. NO. A22328).
Intracellular Protein Targets
In some embodiments of the invention, the expression of intracellular proteins, for example, BCL2 and HSPB1, are detected using flow cytometry by first permeablizing the cell surface membrane to allow access of antibody through the membrane. In one embodiment a permeabilization reagent, such as those containing various surfactants (e.g., saponin, Triton X-100, Tween-20, N-acyl sarcosine, etc) or organic solvents (e.g., alcohols, acetone) or other similar solution, is used. A permeabilization reagent is optimally used in a sufficient amount enabling penetration of antibodies to the intercellular space, while substantially preserving the cellular membrane. Ideally, the permeabilizing agent creates apertures in the cell membrane without affecting the gross morphology of the cell such that flow cytometric light scattering characteristics of the cell are not affected. Such methods of permeabilizing cells are well known in the art.
In embodiments of the invention, the cell may be fixed prior to or during permeabilization to maintain the integrity of the cell. Methods of fixation are also well known in the art. In some embodiments, fixation and permeabilization can be combined. An example of a fixation/permeabilizing agent is INTRAPREP™ (Beckman Coulter, Inc.) which comprises 5.5% v/v formaldehyde as a fixation reagent and a phosphate buffered saline (PBS)-saponin-based permeabilization reagent.
RNA Expression Profiling:
In other embodiments, the expression of a marker of interest is detected at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, determining the level of marker mRNA in a specimen taken from a patient. Many expression detection methods use isolated RNA. Generally, blood, serum, or tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a marker of the present invention. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
An alternative method for determining the level of marker mRNA in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al. U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, marker expression is assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System). Such methods typically utilize pairs of oligonucleotide primers that are specific for the marker of interest. Methods for designing oligonucleotide primers specific for a known sequence are well known in the art.
Marker expression levels of RNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The detection of marker expression may also comprise using nucleic acid probes in solution.
In one embodiment of the invention, microarrays are used to detect marker expression. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 herein incorporated by reference.
In one approach, total mRNA isolated from a specimen is converted to labeled cRNA and then hybridized to an oligonucleotide array. Each specimen is hybridized to a separate array. Relative transcript levels may be calculated by reference to appropriate controls present on the array and in the sample. In one embodiment of the invention, RNA can be isolated from a subpopulation of cells with a characteristic expression profile, for example, cells co-expressing markers CD19 and CD10.
In embodiments of the invention, an expression profile can comprise values corresponding to gene expression detected by mRNA expression levels where the expression of many genes can be analyzed simultaneously and interpreted in one sample. In embodiments of the invention, an mRNA probe set can include probes corresponding to those in TABLE 1 and TABLE 2. Embodiments of the invention, include but are not limited to the detection of markers and combinations of markers in TABLE 1 comprising genes overexpressed in B-lineage ALL. Embodiments of the invention, include but are not limited to the detection of markers and combinations of markers in TABLE 2 comprising genes underexpressed in B-lineage ALL.
Embodiments of the invention can include, but are not limited to, compositions and methods related to the detection of new markers for the detection of minimal residual disease (MRD) comprising: CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
In embodiments of the invention, an expression profile is generated by the detection of nucleic acid corresponds to the expression of mRNA. Embodiments of the method comprise a method of diagnosing minimal residual disease in a subject by obtaining a specimen from the subject. The specimen is contacted with a plurality of probes, wherein each of the probes specifically binds to a distinct marker, wherein a first probe specifically binds to CD19 and a second probe specifically binds to CD10. A CD19+/CD10+ cell is isolated from the specimen. An expression level of at least one, two, three, four, five, six or more gene products expressed in said CD19+, CD10+ cell is determined, wherein the at least one, two, three, four, five, six or more gene products encode CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a. An expression profile is then generated by combining the expression values of the gene products. In such methods, the expression of CD19 and CD10 and a modulated level of at least one gene products encoding CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102, or CD300a relative to a normal control is indicative of minimal residual disease in acute lymphoblastic leukemia.
Other embodiments of the method comprise contacting a specimen with a plurality of probes to detect expression levels of markers comprising CD19 and CD10 and at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, or twenty-two markers comprising: CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a.
In embodiments of the invention, determining the expression profile of a specimen that is CD10+/CD19+ further comprises probe combinations comprising (a) CD38, CD24, and CD44; (b) CD38, CD58, and CD44; (c) CD38, CD73, and CD15; (d) CD38, CD200, and CD44; (e) CD66c, CD123, and CD86; (f) CD72, CD13, and CD33; or (g) CD79b, HSPB1, and Bcl-2.
In yet other embodiments of the invention, a kit for detecting minimal residual disease in a subject is disclosed. The kit can comprise a plurality of antibodies, antibody fragments, or molecular probes wherein each antibody, antibody fragment, or molecular probe is reactive to each of markers CD10, CD19, CD34, and CD45, and at least one additional of antibody, antibody fragment, or molecular probe that specifically binds to at least one additional marker comprising CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a. The kit can include at least one antibody, antibody fragment, or molecular probe that is detectably labeled. Embodiments of the invention include detectable labels, which can be, but are not limited to a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof.
In embodiments of the invention, a kit can comprise combinations of probes comprising (a) CD10, CD19, CD34, CD45, CD38, CD24, and CD44; (b) CD10, CD19, CD34, CD45, CD38, CD58, and CD44; (c) CD10, CD19, CD34, CD45, CD38, CD73, and CD15; (d) CD10, CD19, CD34, CD45, CD38, CD200, and CD44; (e) CD10, CD19, CD34, CD45, CD66c, CD123, and CD86; (f) CD10, CD19, CD34, CD45, CD72, CD13, and CD33; or (g) CD10, CD19, CD34, CD45, CD79b, HSPB1, and Bcl-2.
Data and Expression Profile Analysis
Any method known in the art for comparing two or more data sets to detect similarity and/or variance between them may be used to compare a subject expression profile to a normal expression profile. To determine whether two or more expression profiles show statistically significant similarity and/or variance, statistical tests may be performed to determine whether any differences between the expression profiles are likely to have been achieved by a random event. Methods for comparing gene expression profiles to determine whether they share statistically significant similarity or variance are known in the art and also reviewed in Holloway et al. (2002) Nature Genetics Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl. 32:490-95, Quackenbush (2002) Nature Genetics Suppl. 32: 496-501; Slonim (2002) Nature Genetics Suppl. 32:502-08; and Chuaqui et at (2002) Nature Genetics Suppl. 32:509-514; each of which is herein incorporated by reference in its entirety.
An expression profile is “distinguishable” or “statistically distinguishable” from a normal control expression profile according to the invention if the two expression profiles do not share statistically significant similarity.
Kits for Detection of Minimal Residual Disease
Kits for practicing the screening and diagnostic methods of the invention are further provided. The kits of the invention may also include methods for use in diagnosing minimal residual disease in ALL, detecting or diagnosing ALL, monitoring disease status in a patient for the recurrence of ALL, or monitoring the efficacy of a treatment for ALL. These methods are described elsewhere herein.
As used herein, “kit” refers to a set of reagents for the purpose of performing the method embodiments of the invention, more particularly, the detection of minimal residual disease in patient specimens. The term “kit” is intended to mean any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an antibody, a nucleic acid probe, etc. for specifically detecting the expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use.
In embodiments of the invention, expression of markers can be assessed at the protein level or nucleic acid level, or both in combination. In some embodiments, expression of protein expression is detected using specific antibody probes. Expression of identified markers can also be detected by nucleic acid based techniques, including, for example, hybridization and RT-PCR. Expression can be evaluated in a variety of specimens taken from the body including, but not limited to, blood cells or bone marrow cells, and cellular products extracted from blood and bone marrow cells, including, but not limited to protein and RNA extracted from blood and bone marrow cells.
The kit can comprise a plurality of antibodies, antibody fragments, or molecular probes wherein each antibody, antibody fragment, or molecular probe is specific for CD10, CD19, CD34, CD45, CD38, CD58, CD15, CD66c, CD13, CD33, CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
The kit can comprise a plurality of antibodies, antibody fragments, or molecular probes wherein each antibody, antibody fragment, or molecular probe is reactive to each of markers CD10, CD19, CD34, and CD45, and at least one additional antibody, antibody fragment, or molecular probe that specifically binds to at least one additional marker comprising CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, or CD49f.
The kit can comprise a plurality of antibodies, antibody fragments, or molecular probes wherein each antibody, antibody fragment, or molecular probe is reactive to each of markers CD10, CD19, CD34, and CD45, and at least one additional antibody, antibody fragment, or molecular probe that specifically binds to at least one additional marker comprising CD38, CD24, CD44, CD58, CD73, CD15, CD200, CD66c, CD123, CD86, CD72, CD13, CD33, CD79b, HSPB1, BCL2, CD164, CD304, CD97, CD99, CD102 or CD300a. The kit can include at least one antibody, antibody fragment, or molecular probe that is detectably labeled. Embodiments of the invention include detectable labels, which can be, but are not limited to a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof.
In embodiments of the invention, a kit can comprise combinations of probes comprising (a) CD10, CD19, CD34, CD45, CD38, CD24, and CD44; (b) CD10, CD19, CD34, CD45, CD38, CD58, and CD44; (c) CD10, CD19, CD34, CD45, CD38, CD73, and CD15; (d) CD10, CD19, CD34, CD45, CD38, CD200, and CD44; (e) CD10, CD19, CD34, CD45, CD66c, CD123, and CD86; (f) CD10, CD19, CD34, CD45, CD72, CD13, and CD33; or (g) CD10, CD19, CD34, CD45, CD79b, HSPB1, and Bcl-2.
In yet another embodiment, a kit can comprises an array having a substrate with a plurality of addresses, where each address has a capture probe that can specifically bind a nucleic acid molecule comprising CD38, CD58, CD66c, CD79b, CD164, CD44, BCL2, HSPB1, CD72, CD73, CD24, CD123, CD86, CD200, CD304, CD97, CD99, CD102 or CD300a.
The methods of the invention can comprise MRD detection by gene array with preferred combinations of probes to specific markers. MRD detection can be combined, for example, with at least 3 different probes. Embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 1 comprising genes overexpressed in B-lineage ALL. Other embodiments of the invention, can include, but are not limited to, the detection of mRNA expression with probes specific for genes shown in TABLE 2 comprising genes underexpressed in B-lineage ALL.
One of skill in the art will further appreciate that any or all steps in the screening and diagnostic methods of the invention could be implemented by personnel or, alternatively, performed in an automated fashion. For example, the methods can be performed in an automated, semi-automated, or manual fashion, and as one-step or multi-step processes. Furthermore, the methods disclosed herein can also be combined with other methods known or later developed to permit a more accurate identification of patients having an increased likelihood of having minimal residual disease or a more reliable diagnosis of ALL.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.
Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Embodiments of the present invention are further defined in the following Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
Patients
Bone marrow samples were collected at presentation from 470 patients aged <1 to 18 years with newly diagnosed B-lineage ALL; 270 samples were included in genome-wide gene expression studies, and 200 were tested to validate the gene expression findings by flow cytometry. Bone marrow samples were obtained during therapy in 51 patients and samples obtained at relapse in 9. The diagnosis of B-lineage ALL was unequivocal and based on morphology, cytochemistry and cell marker expression. To determine gene expression and immunophenotype of normal lymphoid progenitors, bone marrow samples were collected from 22 healthy donors aged 2-25 yrs (median, 10 yrs) during the harvest of bone marrow for stem cell transplantation, and bone marrow samples obtained from 27 patients with B-lineage ALL, 7 with T-lineage ALL and 5 with acute myeloid leukemia (AML) during therapy.
Sample Preparation
Leukemic and normal mononuclear cells were collected after centrifugation on a density gradient (A
Flow Cytometric Analysis and MRD Studies
Flow cytometry using the antibodies listed in TABLES 3-5 were used to verify the differential expression of gene products in ALL cells. In most tests, the antibodies were used in combination with anti-CD19 conjugated to allophycocyanin (APC), anti-CD10 conjugated to either FITC or PE, and anti-CD34 conjugated to peridinin chlorophyll protein (PerCP). Isotype-matched nonreactive antibodies were used as controls. For cell staining, monunucleated cells were washed in PBS containing 0.5% bovine serum albumin and 0.5% sodium azide (PBSA), mixed with rabbit serum to block surface Fc receptors, incubated with the antibodies for 10 minutes at 20° C. in the dark, washed twice in PBSA and fixed with 0.5% paraformaldehyde. For intracellular markers, cells were permeabilized and fixed before exposure to antibodies using a permeablization reagent 8E. Measurements of antibody labeling were performed by multiparameter flow cytometry, using a FACSCALIBER® flow cytometer (Becton Dickinson, San Jose, Calif.).
Studies of MRD by flow cytometry were performed using combinations of monoclonal antibodies that identified leukemia-associated immunophenotypes determined at diagnosis. Cell staining was essentially performed as described above, using a combination of 4 markers simultaneously (see TABLE 4) and, where indicated, with 6-marker combinations (SEE TABLE 3). Data acquisition and analysis was done as previously described, using BD FACSCALIBER™ LSRII flow cytometers (Becton Dickinson), BD CELLQUEST Pro™, BD FACSD
Gene Expression Arrays Studies
A first-generation gene array study was previously performed (Chen et al. (2001) Blood. 97:2115-2120) and as previously described (Ross et al. (2003) Blood. 102:2951-2959). In Chen et al., diagnostic ALL samples from 4 patients were compared to CD19+ CD10+ bone marrow immature B cells taken from 2 healthy donors. The array contained probes for approximately 4000 genes.
In an embodiment of the invention, a gene expression array probing over 23,000 genes was used to screen a larger set of samples, including 270 cases of B-lineage ALL. The B-lineage ALLs encompassed the spectrum of genetic abnormalities occurring in ALL and were included to specifically identify other prognostic markers. Briefly, total RNA was isolated from freshly thawed B-lineage ALL cells and flow sorted normal CD19+CD10+ cells using the TRIZOL® reagent (Invitrogen, Carlsbad, Calif.). After generating cDNA, biotin-labeled cRNA was prepared according to the protocols of Affymetrix (Santa Clara, Calif.). The solutions were hybridized to HG-U133A oligonucleotide microarrays (Affymetrix). After staining with phycoerythrin-conjugated streptavidin, the arrays were read with a laser confocal scanner (Agilent, Palo Alto, Calif.). Signal values were computed from the image files using Affymetrix G
Expression data was generated using genome-wide expression data detected from 270 B-lineage ALL samples and CD19+CD10+ lymphoid progenitors obtained from the bone marrow of 4 healthy donors. Of the 23,000 genes compared, a total of 133 probe sets (corresponding to 112 genes, TABLE 1) had signals higher than 2-fold of the highest value obtained among normal CD19+CD10+ cells in 75% or more of ALL cases studied. Conversely, 192 probe sets (corresponding to 165, TABLE 2) had signals at least 50% lower than the lowest signal among the normal CD19+CD10+ cells in 75% or more of ALL cases. When the criteria for inclusion was extended to genes that were differentially expressed in at least 25% of ALL cases, 1405 probe sets were over-expressed and 1474 were under-expressed. The results are summarized in TABLES 1 and 2.
Among the genes that were differentially expressed, some had been previously identified as abnormally expressed in ALL cells. For example, CD58 was over-expressed in 81.9% of cases (See also Chen J. S., et al. (2001) Blood. 97:2115-2120), and WT1 in 32.6% of cases (See also Ogawa et al. (2002) Blood. 101:1698-1704) whereas PAX5 was under-expressed in 86.7% and CD38 in 73.7% of cases (See also Coustan-Smith et al. (2002) Blood. 100:52-58). Among the 23 ALL specimens with MLL gene rearrangements, 100% over-expressed galectin-1 and 91.3% under-expressed CD10, characteristic features of this subset of ALL (See also Juszczynski et al. (2010) Clin Cancer Res. 16:2122-2130; Pui et al. (1994) J. Clin. Oncol. 12:909-915); most of these cases also over-expressed a cohort of genes (e.g., FLT3, LMO2, ADAM10, MEIS1) previously reported to be associated with MLL-rearranged leukemia (see also Ferrando et al. (2003) Blood. 102:262-268).
All 26 cases with TCF3-PBX1 over-expressed PBX1 (See also Kamps et al. (1991). Genes Dev. 5:358-368), and, among the 62 cases with ETV6-RUNX1, 46.8% over-expressed CD13 and 29.0% CD33 (Baruchel et al. (1997) Br J HaematoL 99:101-106).
Among the genes differentially expressed by gene array analysis, there were some already widely used for MRD studies by flow cytometry, i.e., CD58, CD38, CD13, and CD34, suggesting the possibility that other useful markers could be present among the remaining genes (Basso et al. (2009) J Clin Oncol. 27:5168-5174; Lucio et al. (2001) Leukemia. 15:1185-1192; Chen et al. (2001) Blood. 97:2115-2120; Coustan-Smith et al. (2002) Blood. 100:52-58).
To prioritize genes for validation by flow cytometry, an initial inclusion criteria was applied: a) differential expression in at least 25% of cases of ALL, or 40% of cases of a genetic subtype of ALL; b) over-expression in leukemic cells by at least 3-fold of the maximum value in normal cells, or under-expression by 3-fold of the minimum value in normal cells; and c) commercial availability of specific antibodies conjugated to fluorochromes suitable for flow cytometry. Guided by these criteria, 30 genes (25 overexpressed in ALL and 5 underexpressed) were selected (TABLE 6) and compared to determine whether the differential expression measured by microarray analysis at the mRNA level corresponded to differential expression of the encoded proteins.
After confirming the specificity of the antibodies with known positive and negative target cells (TABLE 6), the antibodies were tested for reactivity using ALL samples obtained at diagnosis (n=200) and leukemia-free bone marrow samples (n=61). Importantly, comparisons included not only bone marrow specimens from healthy donors (n=22) but also bone marrow from children with ALL (MRD-negative according to PCR amplification of antigen-receptor genes) and AML (MRD negative by flow cytometry) during chemotherapy (n=39), some with a high proportion of hematogones. When the overall expression in the ALL versus normal CD19+ CD10+ B-cell progenitor groups were compared the difference in overall expression was statistically significant (P<0.05) for 13 of the 30 markers tested. Additionally, 11 of the proteins encoded by the over-expressed genes were expressed at a significantly higher level in B-lineage ALL cells, while 2 of the 5 proteins encoded by under-expressed genes had a significantly lower expression (
1Data expressed as units calculated by MAS5.0 to a median target intensity of 500.
For the remaining 17 markers, the difference in overall expression between ALL cells and normal B-cell progenitors was not statistically significant (
However, some these later markers were found to be overexpressed when the mean fluorescent intensity MFI values were compared to normal controls. For example, in some cases the MFI values of a marker in ALL specimens was higher than the highest mean fluorescence intensity (MFI) value recorded among normal B-cell progenitors. In other ALL specimens, markers were found to be underexpressed in ALL cells. For markers that were under-expressed in ALL cells, for example, specimens had at least 50% lower MFI values compared to the lowest MFI measured in normal B-cell progenitors. Using MFI levels as a criteria for differential expression, a substantial proportion of ALL cases expressed markers whose MFI values that were higher or lower than that of normal B cell progenitors. Overall, 22 of the 30 markers were found differentially expressed in 5%-81.4% (median, 35%) of cases These results are summarized in TABLE 6.
For example, CD44 was differentially expressed in 81.4% cases studied, BCL2 was over-expressed in 76.6%, HSPB1 (heat shock protein 27) in 63.4% and CD73 in 54.5%. Notably, CD44 and CD24 were over-expressed in some cases and under-expressed in others. Importantly, some of the markers, such as CD97, CD99 and CD102 appeared to be over-expressed in a much larger proportion of cases when the comparisons included only resting bone marrow samples. However, when regenerating specimens rich in hematogones were included, it became clear that expression in leukemic cells for many of these cases was not outside the range of normality. Of the 30 markers studied, only 8 (EPOR, CD1c, CD120a, CD37, CD62L, CD132, CD83 and IL13RA1) showed no clear differential expression by the above criteria and were excluded from further studies.
To determine the reliability of the new markers to identify leukemic cells in clinical samples, 128 bone marrow samples were collected during treatment (46 during or at the end of remission induction therapy and 82 during post-remission therapy) from 51 patients with B-lineage ALL in whom expression of the markers on the leukemic cells had been measured at diagnosis. The markers included in these studies were the top 16 differentially expressed markers listed in TABLE 7, for a total of 258 tests.
Newly identified markers (TABLE 3, 4) were compared to standard marker combinations presently used in for MRD detection (TABLE 5). Using a threshold of 0.01% ALL cells to define MRD positivity, no discordant results were observed except for one comparison in which MRD was negative with the standard markers and 0.012% with the new markers. Overall, there was an excellent correlation in MRD estimates between new and standard markers (r=0.9816, P<0.0001 by Spearman's regression analysis of the MRD-positive tests;
In a subset of 52 samples from 18 patients studied with some of the markers (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD86, CD200, CD304, CD97, CD99,
11 Number of ALL cases that expressed the indicated marker at levels higher than the highest mean fluorescence intensity (MFI) value recorded among normal B-cell progenitors, and had an MFI higher than 10.
2Number of ALL cases that expressed the indicated marker at levels lower than the lowest MFI measured in normal B-cell progenitors, excluded cases in which a marker had an MFI lower than 10 on normal B-cell progenitors.
CD102 and CD300a), MRD estimates by PCR amplification of clonally rearranged immunoglobulin and T-cell receptor genes were also available. Using the threshold of 0.01% ALL cells to define MRD positivity, MRD was negative (<0.01%) by flow cytometry and PCR analysis in 35 of the 52 samples studied. By contrast, MRD was ≧0.01% according to both methods in 15 samples. Two additional samples had MRD≧0.01% by flow cytometry while PCR showed detectable signals but below the 0.01% threshold: 0.007% and 0.003%. Among the 101 tests performed, MRD was <0.01% by flow cytometry in 63 and ≧0.01% in 38, with a good correlation in the MRD positive estimates by the two techniques (r=0.8178, P<0.0001). Two samples had MRD≧0.01% by flow cytometry (1 tested with two markers) and also had positive PCR but below the 0.01% threshold.
To determine stability of the new markers during the course of the disease, a prerequisite for reliable MRD tracking (Gaipa et al. (2005) Leukemia. 19:49-56) the level of expression was compared to that recorded during MRD monitoring in 10 patients who had persistent MRD during remission induction therapy (day 19 and 42 from diagnosis).
The long-term stability of the markers was assessed by comparing the immunophenotype of paired samples collected at diagnosis and relapse from nine patients whose leukemic cells at diagnosis expressed some of the newly identified markers identified. In
As shown in TABLE 8, abnormal marker expression at diagnosis reverted to expression within the normal range at relapse in 7 of 55 (12.7%) of comparisons. In the remaining cases, marker expression remained abnormal. Importantly, in all 9 patients studied at least one marker remained abnormally expressed at relapse. In 58 additional comparisons where the markers at diagnosis were within the normal range, there were 10 instances (17.2%) in which expression became abnormal at relapse. These results suggest that false-negative results due to phenotypic shifts affecting these markers would be unlikely.
743
3333
2773
773
253
723
293
543
1003
1363
3293
1Values indicate MFI as determined by flow cytometry. Dash indicates MFI within the normal range
2Significant change in expression: from abnormal expression at diagnosis to expression within the normal range at relapse
3Significant change in expression: from expression within the normal range at diagnosis to abnormal expression at relapse
The new markers used in embodiments of the invention were associated with known genetic subtypes of ALL, including hyperdiploidy (51-65 chromosomes), ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, or MLL gene rearrangements.
Expression of some markers was clearly related to ALL genetic subtype. For example, among hyperdiploid (51-65 chromosomes) ALL cases, there was a significantly higher prevalence of CD123 (P<0.0001 by Fisher's exact test), CD86 (P<0.0001), CD200 (P=0.0003) and CD97 (P<0.0001) overexpression as compared to the other cases without this genetic abnormality. Among cases with ETV6-RUNX1, there was a higher prevalence of CD200 over-expression (P<0.0001), and of CD44 (P<0.0001), CD72 (P=0.0073) and CD79b (P=0.0109) under-expression. Finally, abnormal expression of CD164 was most prevalent among cases lacking all the genetic abnormalities analyzed (P=0.002).
In a proportion of patients with ALL, MRD cannot currently be monitored because of the lack of suitable immunophenotypes differentiating ALL from normal controls. (Campana (2009) Hematol. Oncol Clin North Am. 23:1083-98, vii; Bruggemann et al. (2010) Leukemia. 24:521-535) In other patients, leukemic cells express only one set of markers, and have an increased risk of false-negative MRD results due to immunophenotypic shifts (Coustan-Smith et al. (1998) Lancet. 351:550-554). The availability of the additional markers of embodiments of the invention enables MRD studies in patients whose ALL cells currently lack suitable leukemia-associated immunophenotypes and minimize the risk of false-negative results.
Using a method of the invention, expression profiling was used to evaluate 171 ALL diagnostic samples including: 11 subjects lacking detectable leukemia-associated immunophenotypes with the standard panel of antibodies; and another 37 subjects having only one immunophenotypic abnormality detectable with the standard panel.
In contrast to the standard method, the method of the present invention detected a phenotypic abnormality indicative of MRD in all of the 11 cases previously lacking a leukemia-associated immunophenotype. Moreover, the method of the present invention identified additional abnormalities in 36 of the 37 cases with one standard abnormality. Thus, the new markers substantially improved the potential for flow cytometric monitoring of MRD in 47 of 48 cases that could not be adequately studied previously. The number of new markers differentially expressed ranged from two to twelve (median, six). In 45 of the 47 cases, one or more markers were expressed at levels that exceeded by at least two-fold the normal range, allowing a particularly clear identification of ALL cells.
The availability of additional markers of leukemia improved the resolution of leukemic and normal cells during flow cytometric analysis. This was clearly demonstrated in experiments in which mixtures of leukemia and normal cells were analyzed using either one standard phenotypic abnormality (e.g., under-expression of CD38), or additional abnormalities revealed by the new markers. An example of such an experiment is shown in
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
This application is a 35 U.S.C. §273 U.S. National Stage of International Application PCT/US2012/028993 filed Mar. 14, 2012, which designates the U.S. and was published by the International Bureau in English on Oct. 4, 2012, and which claims the benefit of U.S. Provisional Application No. 61/470,056, filed Mar. 31, 2011, both of which are hereby incorporated by reference in their entirety.
This invention was made with support under United States Government Grant CA60419 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/028993 | 3/14/2012 | WO | 00 | 10/2/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/134813 | 10/4/2012 | WO | A |
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| 20060240434 | Gabert et al. | Oct 2006 | A1 |
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| 20120202202 | Wang et al. | Aug 2012 | A1 |
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|---|---|---|
| WO 2010140885 | Dec 2010 | BR |
| 2 259 065 | Aug 2010 | EP |
| WO 2007008759 | Jan 2007 | WO |
| WO 2010140885 | Dec 2010 | WO |
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| Number | Date | Country | |
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| 20140148354 A1 | May 2014 | US |
| Number | Date | Country | |
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| 61470056 | Mar 2011 | US |
