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Title:  CD38 as a prognostic indicator in B cell chronic lymphocytic leukemia
United States Patent: 
7,316,906
Issued: 
January 8, 2008

Inventors: 
Chiorazzi; Nicholas (Tenafly, NJ), Damle; Rajendra N. (Lynbrook, NY), Wasil; Tarun (St John's, CA)
Assignee: 
The Feinstein Institute for Medical Research (Manhasset, NY)
Appl. No.: 
10/211,394
Filed: 
August 2, 2002


 

George Washington University's Healthcare MBA


Abstract

The subject invention discloses a method for determining the prognosis and probable clinical course of a subject diagnosed with B-CLL. Specifically, the invention involves comparing CD38 expression in a biological sample from the subject containing B-CLL cells to a baseline level of CD38 expression, wherein an elevated level of CD38 expression in relation to the baseline level of CD38 expression may indicate poor prognosis or aggressive course of disease in the subject. Also disclosed is a method for determining whether the Ig V genes of the B-CLL cells of a B-CLL patient are mutated, comprising comparing CD38 expression in a biological sample from the subject containing B-CLL cells to a baseline level of CD38 expression, wherein a lower level of CD38 expression in relation to the baseline level indicates IG V gene mutation.

Description of the Invention

BACKGROUND OF THE INVENTION

B cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world (Rai K, Patel D: Chronic Lymphocytic Leukemia, in Hoffman R, Benz E, Shattil S, Furie B, Cohen H, Silberstein L (eds): Hematology: Basic Principles and Practice (ed 2nd). New York, Churchill Livingstone, 1995, p 1308). Around 7,500 individuals develop and 5,000 die from this disease each year (Landis S H, et al., CA Cancer J Clin 48:6, 1998). Age is an important factor, since the incidence of B-CLL increases linearly with each decade above the age of 40 (Ries L, et al: SEER cancer statistics review 1973-1991: Tables and graphs., in Ries L, et al (eds). Bethesda, NIH, 1994; Rai K R, Clin Geriatr Med 13:245, 1997). In addition, gender is relevant, since men outnumber women by an approximate 2:1 ratio (Catovsky D, et al., Br J Haematol 72:141, 1989) and may have a worse clinical outcome (Id.; Mandelli F, et al., J Clin Oncol 5:398, 1987).

Patients with B-CLL follow heterogeneous clinical courses. Some survive for prolonged periods without definitive therapy, while others die rapidly despite aggressive treatment (Rai K, Patel D: Chronic Lymphocytic Leukemia, in Hoffman R, et al. (eds): Hematology: Basic Principles and Practice (ed 2nd). New York, Churchill Livingstone, 1995, p 1308; Zwiebel J A, Cheson B D, Semin Oncol 25:42, 1998). While various staging systems, most notably the Rai and Binet staging systems, have been developed to address this clinical heterogeneity (Rai K R, et al., Blood 46, 219, 1975; Binet J L, et al., Cancer 48:198, 1981; and Rai K: A critical analysis of staging in CLL, in Gail R, Rai K (eds): Chronic Lymphocytic Leukemia. Recent Progress and Future Directions. New York, Alan R Liss, 1987, p 253), they cannot accurately predict whether an early or intermediate stage patient will experience an indolent or aggressive course of disease. Specifically, since these systems consider gross manifestations of the disease, including the level of blood and marrow lymphocyte counts, the size and distribution of the lymph nodes, the spleen size, the degree of anemia and the patient's blood platelet count, they can only identify patients with poor prognostic outcome when the disease has progressed to a more advanced state.

At the present time, there is no known treatment for B-CLL which has been shown to definitively increase life expectancy. Consequently, only patients classified in the advanced stages of B-CLL have been considered for aggressive treatment such as chemotherapy, radiation therapy, surgery, immunotherapy or transplantation. These treatments may exact a severe physical and emotional toll on the patient without necessarily improving outcome; in some instances, B-CLL patients may even succumb from the rigors of treatment rather than from the effects of B-CLL. Patients classified in the early stages of B-CLL, who may be in better physical condition to receive more aggressive or experimental treatment, generally receive no treatment as long as the condition remains stable. This is for two reasons. First, currently available therapies do not extend life span. Second, there are currently no reliable indicators of which early stage patients will do well and which will do poorly. Further, the unpredictable course of the disease can make interpreting the results of clinical trials difficult, as some early stage patients will follow an indolent course even without the benefit of treatment.

Such drawbacks have led researchers to develop adjuvant prognostic criteria to be used in conjunction with the Rai and Binet staging systems, including several parameters such as lymphocyte doubling time (Montserrat E, et al., Br J Haematol 62:567, 1986), circulating levels of .beta.2-microglobulin (Di Giovanni S, et al., Acta Haematol 81:181, 1989; Keating M J, et al., Blood 86:606A, 1995 (Abstract)), circulating levels of soluble CD23 (Sarfati M, et al., Blood 88:4259, 1996), serum thymidine kinase levels (Kallander C F, et al., Cancer 54:2450, 1984; Hallek M, et al., Blood 93:1732, 1999), bone marrow histology (Rozman C, et al., Blood 64:642, 1984), and cytogenetic abnormalities (Juliusson G, et al., N Engl J Med 323:720, 1990).

An accurate prognostic indicator for B-CLL not related to the symptoms of advanced disease would be desirable in the treatment and case management of B-CLL patients. Specifically, a prognostic indicator that could be evaluated at the cellular level at the earliest stages of the disease (before onset of thrombocytopenia, anemia, spleen and liver enlargement, etc.) would help physicians identify which patients would progress to a more advanced state of the disease and allow the option of more aggressive or experimental treatment at a much earlier stage. Additionally, clinical trials of new drugs or experimental therapies could be directed to patients depending upon their prognostic outlook, thereby allowing for more relevant results in clinical trials. Ideally, the expression of such a prognostic indicator would remain constant over the course of disease.

B-CLL is characterized by the clonal accumulation of CD5.sup.+ cells (Caligaris-Cappio, et al., J Exp Med 155:623-8, 1982). Although these cells originally were considered antigen inexperienced "virgin" lymphocytes, recent data indicate that at least half of these cases represent expansions of previously-triggered, post germinal center "memory" B cells (Schroeder and Dighiero, Immunol Today 15:288-294, 1994; Fais, et al., J Clin Invest 102:1515-1525, 1998). This conclusion is based on the presence of significant numbers of somatic mutations in the immunoglobulin (Ig) heavy (H) chain variable region (V) genes. In a study of 83 (64 IgM.sup.+ and 19 non-IgM.sup.+) B-CLL cases, the inventor and colleagues found significant numbers of V.sub.H mutations in approximately 50% of the IgM.sup.+ and 75% of the non-IgM+ (IgG and IgA) cases (Fais, et al, supra, 1998). Taken together with newer studies undertaken by the inventor and colleagues, V.sub.H and V.sub.L sequencing data suggest that approximately 60% of B-CLL cases can be considered to be derived from post-germinal center (GC) memory B-cells. Thus, the inventor hypothesized that B-CLL cases can be divided into two categories, namely cells clonally derived from post-germinal center memory B-cells (hereinafter referred to as "post-GC B cells") and pre-germinal center B cells (hereinafter referred to as "pre-GC B cells"), some of which may be antigen inexperienced "virgin" lymphocytes or activated B cells that were transformed without entering a germinal center, and these categories may be relevant to prognosis.

The expression of specific cell surface markers distinguishes subsets of normal human B cells that differ in differentiation and activation stages and in biologic properties (Clark and Lane, Ann Rev Immunol 9:97-127, 1991). In particular, analyses of CD38 and IgD expression have been especially useful in distinguishing B-cells at various stages of differentiation from naive through memory cells (Pascual, et al., J Exp Med 180:329-339, 1994; Zupo, et al., Blood, 88:1365-1374, 1996).

Accordingly, the inventor sought to determine whether the distinctions based upon surface membrane phenotype of B-CLL cells (CD38.sup.+ or CD38.sup.-) or Ig V gene mutation status might predict different clinical courses and outcomes for B-CLL patients notwithstanding similar staging of these patients using conventional staging methods. Undertaking the experiments described herein, the inventor has discovered a strong correlation between CD38 expression and Ig V gene mutation, and a strong independent correlation between each of CD38 expression and IgV gene mutation and patient prognosis. Since CD38 expression in a subject's B-CLL cells may be easily and relatively inexpensively determined through various methods known and commonly used in the art, CD38 expression in particular may be a valuable prognostic indicator in B-CLL cases and should aid in the management of B-CLL patients.

SUMMARY OF THE INVENTION

The present invention discloses a method for determining the prognosis of a subject with chronic lymphocytic leukemia ("B-CLL"), comprising determining whether the subject's B-CLL cells have been clonally expanded from post-GC B cells (post-germinal center memory B-cells) or pre-GC B cells (pre-germinal center B cells, some of which may be antigen inexperienced "virgin" lymphocytes or activated B cells that were transformed without entering a germinal center), wherein clonal expansion from post-GC B cells may be indicative of an indolent course of B-CLL in the subject or favorable prognosis, and clonal expansion from pre-GC B cells may be indicative of poor prognosis or an aggressive course of disease.

In one method of the present invention, CD38 expression of B-CLL cells in a biological sample from the subject is compared to a baseline level of CD38 expression of B-CLL cells, wherein an elevated level of CD38 expression in relation to the baseline level of CD38 expression may indicate poor prognosis or aggressive course of disease in the subject. In one embodiment, the percentage of total B-CLL cells which are CD38.sup.+ in the biological sample is compared to a baseline percentage of CD38.sup.+ B-CLL cells, wherein an elevated percentage of CD38.sup.+ B-CLL cells in relation to the baseline percentage of CD38.sup.+ B-CLL cells is indicative of poor prognosis. In another embodiment of the invention, the density of CD38 surface membrane expression on the B-CLL cells in a biological sample from the subject is compared to a baseline density of CD38 surface membrane expression of B-CLL cells, wherein an elevated density of CD38 surface membrane expression in relation to the baseline density of CD38 surface membrane expression may indicate poor prognosis.

Also disclosed is a method for determining whether the Ig V genes of the B-CLL cells of a B-CLL patient are mutated, comprising comparing CD38 expression of B-CLL cells (either as a function of relative percentage of CD38.sup.+ B-CLL cells, or as a function of the relative density of CD38 surface membrane expression on the B-CLL cells) in a biological sample from the subject to a baseline level of CD38 expression, wherein a lower level of CD38 expression in relation to the baseline level of CD38 expression indicates IG V gene mutation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining the prognosis or projected clinical course in a subject with B cell chronic lymphocytic leukemia ("B-CLL"). In particular, the method of the present invention discloses an immunophenotypic prognostic indicator which predicts whether the course of disease in a specific B-CLL patient will be aggressive or indolent, thereby aiding the clinician in managing the patient and evaluating the modality of treatment to be used.

Since at the current time there are no known treatments that will definitively increase the life expectancy of persons diagnosed with B-CLL, clinicians must balance the rigors of aggressive or experimental treatment with the likelihood that such treatment will result in tangible benefit to the patient. In fact, some B-CLL patients succumb to the combined effects of treatment and B-CLL rather than to the effects of B-CLL alone. Accordingly, more aggressive treatment, such as radiation therapy, chemotherapy, transplants and immunotherapy, has traditionally been reserved for those B-CLL patients already in the advanced stages of B-CLL who stage higher in the conventional Rai and Binet staging systems. However, these patients may be the most ill equipped to handle the rigors of such treatment.

Additionally, the heterogeneous course of B-CLL complicates the evaluation of clinical trials, as it is difficult to distinguish patients who are effectively responding to the therapy being administered from patients who would have never progressed to a more advanced stage of the disease regardless of treatment. Accordingly, an immunophenotypic prognostic indicator which is predictive of a patient's clinical course, notwithstanding the conventional stage of the disease, will aid clinicians in better evaluating treatment options, as well as greatly enhancing the value of clinical studies by better distinguishing the effects of treatment.

The present invention generally disclose a method for determining the prognosis of a subject with chronic lymphocytic leukemia ("B-CLL"), comprising determining whether the subject's B-CLL cells (B-CLL B cells) have been clonally expanded from post-GC B cells (post-germinal center memory B-cells) or pre-BC B cells (pre-germinal center B cells, some of which may be antigen inexperienced "virgin" lymphocytes or activated B cells that were transformed without entering a germinal center). Clonal expansion from post-GC B cells may be indicative of an indolent course of B-CLL in the subject or favorable prognosis, while clonal expansion from pre-GC B cells may be indicative of poor prognosis or an aggressive course of disease.

Specifically, in the preferred embodiment of the present invention, CD38 expression of B-CLL cells in a biological sample from the subject is compared to a baseline level of CD38 expression of B-CLL cells, wherein an elevated level of CD38 expression in relation to the baseline level of CD38 expression may indicate poor prognosis or aggressive course of disease in the subject. The method may be performed using any tissue containing B-CLL cells, including but not limited to spleen, lymph nodes, bone marrow, lymph, a whole blood sample from the subject or a whole blood sample that has been treated and processed to isolate the peripheral blood mononuclear cells ("PBMC").

In one embodiment, the percentage of total B-CLL cells in the biological sample which are CD38.sup.+ is compared to a baseline percentage of CD38.sup.+ B-CLL cells, wherein an elevated percentage of CD38.sup.+ B-CLL cells in relation to the baseline percentage of CD38.sup.+ B-CLL cells is indicative of poor prognosis, and a lower percentage of CD38.sup.+ B-CLL cells in relation to the baseline percentage is indicative of a favorable prognosis or indolent course of disease. Alternatively, the density of CD38 surface membrane expression on the B-CLL cells in a biological sample from the subject is compared to a baseline density of CD38 surface membrane expression on B-CLL cells, wherein an elevated density of CD38 surface membrane expression in relation to the baseline density surface membrane expression may indicate poor prognosis, and a lower density of CD38 surface membrane expression in relation to the baseline density of CD38 surface membrane expression may indicate a favorable prognosis or a more indolent course of disease.

In both embodiments, i.e., comparing the relative percentage of CD38.sup.+ B-CLL cells (as a percentage of the B-CLL population in total, such as a percentage of CD5.sup.+/CD19.sup.+ lymphocytes) or comparing the relative density of CD38 surface membrane expression on B-CLL cells in the biological sample, the level of CD38 expression may be determined by any method currently known in the art, including any applicable direct or indirect immunofluorescence technique. Further, where relative density of CD38 surface membrane expression on the B-CLL cells in the biological sample is being determined, mean channel fluorescence may be used. In a preferred embodiment of the invention, the level of CD38.sup.+ B-CLL expression is determined using flow cytometry where the cells have been labeled with monoclonal antibodies conjugated with fluorescent dyes or enzymes, although visual immunofluorescence or other methods may also be used. In a specific embodiment, PBMCs are analyzed for surface expression of CD19/CD5/CD38 by triple color immunofluorescence using anti-CD19-APC, anti-CD5-FITC (CD5 being specific to B-CLL cells and CD19 being specific to B lymphocytes, although other combinations of antigens/labeled antibodies or enzymes may be used that are specific to narrow the analyzed pool to B-CLL cells) and anti-CD38-PE antibody conjugates. The preferred antibody conjugate is anti-CD38-PE (Simultest LeucoGATE, from Becton Dickinson Immunocytometry Systems, San Jose, Calif.).

Additionally, it is well within the skill of one of ordinary skill in the art to devise either direct or indirect immunoassay kits (i.e., ELISA or other kits) which use similar antigen/labeled antibody or enzyme combinations to detect levels of CD38 expression. The relative percentage of CD38.sup.+ B-CLL cells in relation to a percentage baseline of CD38.sup.+ B-CLL cells, or the relative density of CD38 surface membrane expression on B-CLL cells in relation to a baseline density of CD38 surface membrane expression, may be determined by comparing the resulting color, fluorescence or equivalent reaction with a control sample having a predetermined percentage of CD38.sup.+ B-CLL cells or density of CD38 (or the relevant epitope of CD38), as appropriate.

While the exact relative percentage of CD38.sup.+ B-CLL cells or density of CD38 surface membrane expression that defines poor or favorable prognosis, i.e., the baseline level of CD38 expression of the B-CLL cells, is somewhat arbitrary (as the numerical cut off value may be shifted upward or downward with an attendant loss of accuracy in the prognostic utility of the test), in a preferred embodiment of the invention using the disclosed antibody or one of equivalent avidity and specificity, as well as the disclosed anti-CD38-PE fluorochrome, the relative percentage of CD38.sup.+ B-CLL cells indicating poor prognosis is greater than 15%, more preferably is greater than 20%, even more preferably is greater than 25% and most preferably is greater than 30% of the total B-CLL cells in the biological sample. The preferred range may be affected by the specific anti-CD38 monoclonal antibody (mAB) used, as different mABs will have different binding affinities (avidity) for CD38 and may bind to different epitopes of CD38, as well as by the specific fluorochrome used. Further, similar variable parameters exist in isolating the B-CLL population in the biological sample. That is, the exact preferred baseline may vary depending upon the specific anti-CD5 and anti-CD19 mABs used, and the anti-CD5 and anti-CD19 fluorochrome conjugates used, for the reasons noted above regarding variations in avidity and specificity, as well as the efficiency of the mAB conjugated fluorochrome. Using the mABs and fluorochromes disclosed in the Experimental Details below will yield the preferred baseline range of CD38 expression as disclosed herein. However, all other elements being the same, mABs with a lower avidity will have a correspondingly increased baseline range, so that higher densities of CD38 surface membrane expression, or increased relative percentages of CD38.sup.+ B-CLL cells, will be required to establish a poor prognosis or aggressive course of disease.

However, it is well within the skill of one of ordinary skill in the art to determine the appropriate CD38 baseline level, by either using the experimental method disclosed herein (that is, comparing levels of CD38 expression among a characterized group of B-CLL patients with a known clinical course), or by comparing the relative avidity and specificity of the mABs disclosed herein and the mABs used in any particular instance, as well as the relative efficiency of the particular fluorochrome used, and thereafter deducing the appropriate baseline.

Also disclosed by the present invention is a method for determining whether the Ig V genes of the B-CLL cells of a B-CLL patient are mutated, comprising comparing CD38 expression of B-CLL cells in a biological sample from the subject to a baseline level of CD38 expression of B-CLL cells, wherein a lower level of CD38 expression in relation to the baseline level of CD38 expression indicates IG V gene mutation. In one embodiment of the invention, CD38 expression of B-CLL cells in the biological sample is compared to the baseline level of CD38 expression by comparing the percentage of total B-CLL cells which are CD38.sup.+ to a baseline percentage of CD38.sup.+ B-CLL cells, wherein a lower percentage of CD38.sup.+ B-CLL cells in relation to the baseline percentage is indicative of mutated IgV genes of the B-CLL cells. Preferably, the percentage of CD38.sup.+ B-CLL cells indicating Ig V gene mutation is 15% or less, more preferably is 20% or less, even more preferably is 25% or less, and most preferably is 30% or less. In another embodiment of the invention, CD38 expression of the B-CLL cells in the biological sample is compared to the baseline level of CD38 expression by comparing the density of CD38 surface membrane expression of the B-CLL cells in relation to a baseline of CD38 surface membrane expression, wherein a lower density of CD38 surface membrane expression in relation to the baseline is indicative of mutated IgV genes of the B-CLL cells.
 

Claim 1 of 10 Claims

1. A method for determining indications of the prognosis of and aggressiveness of disease in a subject with B cell chronic lymphocytic leukemia ("B-CLL"), the method comprising: measuring CD38 expression by B-CLL cells in a blood sample from the subject; determining the percentage of B-CLL cells in the sample that are CD38.sup.+; and comparing said percentage to a cut-off value, wherein a percentage of CD38.sup.+ B-CLL cells from the subject that is greater than or equal to the cut-off value indicates a poor prognosis and an aggressive disease course and a percentage of CD38.sup.+ B-CLL cells from the subject that is less than the cut-off value indicates an indolent disease course, wherein the cut-off value is determined by: measuring CD38 expression by B-CLL cells in a blood sample from each of a cohort of B-CLL patients; determining the percentage of B-CLL cells that are CD38.sup.+ in each of the cohort patients: determining whether the B-CLL in each of the cohort patients is aggressive or indolent; and plotting the percentage of B-CLL cells that are CD38.sup.+ in the cohort patients vs. aggressiveness or indolence in the cohort patients to establish a cut-off value that distinguishes between patients with aggressive disease and patients with indolent disease.

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