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WO2000028086A1 - Procedes et compositions pour dosages diagnostiques - Google Patents

Procedes et compositions pour dosages diagnostiques Download PDF

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WO2000028086A1
WO2000028086A1 PCT/US1999/025991 US9925991W WO0028086A1 WO 2000028086 A1 WO2000028086 A1 WO 2000028086A1 US 9925991 W US9925991 W US 9925991W WO 0028086 A1 WO0028086 A1 WO 0028086A1
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WO2000028086A9 (fr
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Jeffrey E. W. Miller
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IVS TECHNOLOGIES LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the present invention relates to the field of in vitro diagnostic assays, and, more particularly, to the diagnosis of hematologic cancers or infectious agents in biological samples.
  • a major impediment to identifying and stratifying hematologic malignancies in biological samples is the lack of a sensitive, fully integrated and cost-effective assay technology that simultaneously tests multiple genetic loci for the gene rearrangements often diagnostic for these malignancies.
  • PCR assays are routinely used to identify clonal lymphocyte populations by detecting clonal V-J rearrangements or chromosomal translocations within the immunoglobulin heavy chain and T-cell receptor genes. Additional primer sets are used to identify translocations that occur outside of these antigen receptor loci. Comprehensive clonality testing, therefore, requires multiple primer sets. Products from the individual genetic loci are analyzed separately using agarose or polyacrylamide gels to avoid misinterpretation, and small clonal populations remain difficult to identify. What is needed is an automated, sensitive and fully integrated PCR-based system which is capable of generating high-resolution and semi- quantitative data simultaneously from multiple genetic loci. Such an assay technology would have significant advantages over current testing methodologies.
  • Mantle cell lymphoma represents an example of a recently proposed diagnostic category within B-cell lineage non-Hodgkin's lymphomas (NHL) (Banks,
  • MCL carries a significantly worse prognosis compared to chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), or follicular lymphoma (Harris, 1994; Bosch, 1998).
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • follicular lymphoma Harris, 1994; Bosch, 1998.
  • discrimination between mantle cell lymphoma and CLL/SLL on the one hand, and follicular lymphoma on the other is sometimes uncertain on the basis of morphology and immunophenotype.
  • the translocation t(ll;14) involving bcl-l has been suggested to be a specific marker for this disease. However, this remains controversial (Brizard, 1997).
  • a sensitive PCR based assay may be of clinical utility in resolving these important differential diagnoses; one for which cutoff points may be changed may further allow flexibility to tailor test to greatest diagnostic specificity and sensitivity for the NHL's and expand the role of these tests beyond determination of clonality and have greater impact on monitoring disease post therapy.
  • Hematopoiesis refers to the processes responsible for production of the blood cells in the body. Erythrocytes, neutrophils, eosinophils, basophils, monocytes, platelets, and lymphocytes are examples of cells produced during hematopoietic cell proliferation and differentiation. These cells develop from hematopoietic pluripotent stem cells that respond to environmental and growth factor cues and differentiate down several defined pathways or cell lineages. There is strong circumstantial evidence for a common pluripotent precursor of the erythroid, granulocyte, monocyte, macrophage, megakaryocytic and lymphoid cell lines.
  • a small, well defined subset of undifferentiated donor stem cells can be used to reconstitute the entire blood system of a recipient following ablative therapy. Following reconstitution all members of terminally differentiated progeny blood cells can be genetically identified as arising from the donor cell population.
  • CML chronic myelogenous leukemia
  • Philadelphia chromosome, Ph+ Philadelphia chromosome, Ph+ (Rowley, JD, 1973).
  • This chromosome harbors a t(9;22) translocation and expresses a chimeric bcr-abl protein with elevated tyrosine kinase activity. Expression of this fusion protein is implicated in the loss of growth control evident in CML (Daley, GQ, 1990).
  • the Ph-f- chromosome is found in diverse and multi-lineage groups of cells from patients with CML. Therefore the translocation is likely to occur early in differentiation of these blood cells, evidence for a common multipotent precursor.
  • t(9;22) translocations are also present in a significant number of patients with acute lymphocytic leukemia (ALL), as well as acute myeloid leukemia (AML), lymphoma, multiple myeloma, and chronic neutrophilic leukemia.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • lymphoma multiple myeloma
  • chronic neutrophilic leukemia t(9;22) translocations give rise to transcription products that may encode different exon regions.
  • the vertebrate immune system consists of a number of distinct and specialized cell populations. Predominate among these are the B-and T-lymphocyte cell populations. These B-cell and T-cell lymphocytes are derived from stem cells of the lymphocyte cell lineage and can be identified by cell surface markers or their antigen receptor molecules. B-cells express membrane-bound or soluble forms of the B-cell receptors, composed of heavy and light protein chains. These receptors are also known as immunoglobulin (Ig) molecules. T-cells express heteroduplex ⁇ or ⁇ T-cell receptor (TcR) molecules.
  • B-and T-lymphocyte cell populations Predominate among these are the B-and T-lymphocyte cell populations. These B-cell and T-cell lymphocytes are derived from stem cells of the lymphocyte cell lineage and can be identified by cell surface markers or their antigen receptor molecules. B-cells express membrane-bound or soluble forms of the B-cell receptors, composed of heavy and light protein chains. These receptors are also known as immunoglobulin
  • antigen receptor molecules are assembled from multiple polymorphic gene segments (Tonegawa, 1983).
  • the variable (V) protion of antigen receptors is a domain of approximately 110 amino acids, containing three antigen binding or complementary determining regions (CDR) of 5-10 ammo acids, and more conserved framework regions (FR). Interaction between an antigenic determinant and the CDR regions of these receptor molecules constitute the phenomenon of antigen recognition at molecular level.
  • the immunoglobulin heavy chain gene is composed of multiple ( ⁇ 200) variable exon segments; a series of diversity (D) segments; joining (J) segments ( ⁇ 6) and a series of constant region (C ⁇ , ⁇ , ⁇ 3, ⁇ l, ⁇ 2 ⁇ , ⁇ 2 , e, ⁇ ) segments.
  • the constant region segments are the conserved protein coding regions that determine the localization and effector function of these antigen-binding receptor molecules.
  • variable exon regions have been assigned to different gene families based upon regions of similarity, termed framework regions (FR). These framework regions provide a coherent structural framework to the variable regions of these antigen receptor molecules. These framework regions have been exploited for diagnostic applications.
  • FR framework regions
  • Oligonucleotide primer molecules have been designed to anneal only to nucleic acid regions that are identical or nearly identical to these regions. There are three conserved framework regions (FRI, FRII, and FRIII) in the variable regions of the immunoglobulin heavy chain receptor. Approximately 33 % of rearrangements are likely to be in-frame; a term that signifies that the protein producing machinery can read the nucleic acid-encoded information in a manner consistent with the triplicate code of the translation machinery. If the rearrangement is nonfunctional (an unproductive rearrangement) the cell may rearrange the other allele (the comparable gene on the homologous chromosome).
  • FRI conserved framework regions
  • FRII FRII
  • FRIII conserved framework regions
  • genomic DNA of a B-or T-cell is examined these mechanisms predict that a single cell might harbor one or two specific rearrangements. However, only the productive in-frame rearrangement are likely to transcribe mRNA that will produce a receptor molecule. Therefore, analysis of genomic DNA might indicate the presence of more than one rearrangement whereas analysis of mRNA might suggest that there exists a single rearrangement.
  • Restriction fragment Southern blot hybridization (RF-SBH) analysis is DNA based testing that relies upon the presence of conserved restriction enzyme sites within the genomic region encoding the antigen receptors.
  • the method involves digestion of the genomic DNA with a several different enzymes that cleave the DNA at specific sequences. The fragments of DNA are separated on the basis of size on an electrophoretic agarose gel. This results in the alignment of fragments of the same length.
  • the digested and aligned DNA is transferred to a nylon or other membrane, and the location of specific regions within the digested DNA fragments is then determined by hybridization of specific DNA probes that are radiolabeled or produce a chemiluminescent signal.
  • RF-SBH analysis of normal patient DNA displays bands only at unrearranged germline positions. Although there are generally thousands of different rearrangements present in the normal patient sample, each rearrangement contributes a single undetectable signal at a specific location on the filter. The contribution to a specific location on the filter is much greater by fragments running at the size such as unrearranged genomic DNA and genomic DNA derived from an expanded clonal population of cells representing a single rearrangement.
  • One advantage of RF-SBH analysis is that the probes that are used to detect the location of the cut and aligned fragments can be large and relatively comprehensive.
  • Ten micrograms of genomic DNA is generally required for each of the restriction enzyme patterns generated for RF-SBH analysis.
  • Nine digestion patterns are usually required for a comprehensive B-cell and T-cell clonal analysis.
  • Three enzymes are generally used for each probe.
  • the digestion patterns are analyzed using probes specific for the immunoglobulin heavy chain gene; three additional digestion patterns are analyzed with probes directed to the ⁇ -light chain gene; and three digestion patterns are analyzed using probes to the T-cell receptor ⁇ -gene loci.
  • the amount of DNA required for RF-SBH analysis (approximately 90 ⁇ g) precludes its use for the many small clinical samples that are more routinely available.
  • RF-SBH analysis is very labor-intensive, requires approximately a week to complete, requires the use of radioactive or chemiluminescent probes, and clonal bands are reliably detected only when the clonal population represents 5 % or more of the cells present in the sample.
  • restriction fragment Southern blot hybridization analysis requires large amounts of DNA and is successful only if the clonal population is present at high frequency in the sample.
  • PCR requires less DNA, is more rapid, less costly and more sensitive than RF-SBH clonality assays. For these reasons, PCR-based assays are becoming more widely used in the clinical laboratory as the initial molecular assay used to identify clonal B-cell and T-cell populations (Bourguin, 1990; Trainor, 1991; Slack, 1993; Benhattar, 1995; Lombardo, 1996).
  • PCR polymerase chain reaction
  • CDR3 region located at the junction of rearranged V and J segments in both IgH and TcR receptor genes, provides a direct means for identifying clonal rearrangements.
  • CDR3 regions are flanked by relatively conserved framework (FR) and joining regions.
  • the sequences of the CDR3 regions comprises the entirety of rearrangements that occur during the generation of antibody and T-cell receptor diversity. Therefore expansion or representation of a specific-sized rearrangement is indicative and diagnostic for clonal B-cell (or T-cell) populations.
  • the immunoglobulin heavy chain (IgH) gene is a target for identification of clonal IgH gene rearrangements in B-cell leukemias and non- Hodgkin's lymphomas.
  • Each somatic cell is diploid; therefore rearrangement of the antigen receptor genes in B-cells and T-cells result in one or two size- and sequence-specific VDJ junctions at each rearranged antigen receptor locus. Since the PCR amplifies the population of DNA segments that lie between two DNA primer sequences, DNA from cell populations that arise from a single cell (clonal cell populations) produce unique clonal products. The presence of one or several single-sized amplicon products is diagnostic for clonality (i.e., cells arising from a single cell).
  • the template population contains one or two specific over- represented rearrangements and one or two unique amplified products are apparent superimposed on the normal distribution of polyclonal products.
  • This over representation may reflect the expansion of a specific clone during a normal immunologic response, or, more likely, may be indicative of a transformed clone of neoplastic B-cells or T-cells.
  • Oligoclonal patterns are sometimes present in the absence of disease if the number of cells in the sample is small, if there is an expansion of one or several cells in the sample, or if specific template molecules present in the sample have been preferentially amplified by a biological process (e.g. , transcription).
  • PCR assays rely on multiple primer sets that recognize the relatively conserved framework regions of the IgH and TcR genes. These regions flank the diverse V-J regions which serve as markers of clonality (Trainor, 1991; Lombardo, 1996). Additional primers are used to target genes, such as Bcl2 and bcr-abl, that can be translocated into the IgH gene in a large proportion of defined leukemias and lymphomas.
  • follicular lymphomas have reciprocal t(14;18) chromosomal translocations that juxtapose the Bcl-2 gene on chromosome 18 with the IgH gene on chromosome 14 (Ladanyi, 1992), and the leukemic cells from more than 95% of patients with chronic myelogenous leukemia have bcr-abl t(9;22) translocations (Melo, 1996).
  • PCR primers that target sequences that are present on separate chromosomes generate translocation products only if there is a chromosomal translocation.
  • clonality can be determined only after complementary DNA is transcribed from mRNA populations.
  • PCR-based methods over RF-SBH analysis is that very small amounts of genomic DNA are required. The amount of DNA recovered from single 4 ⁇ m glass slides is often sufficient for the identification of clonality (Crisan, 1990).
  • PCR-based analyses compatible with the many small clinical samples which are routinely available.
  • PCR analysis is relatively rapid.
  • clonal bands are generally apparent only when the clonal population of cells which contribute to the genomic DNA represent 1-5 % or more of the cells used in the analysis.
  • primers that are chosen to target the receptor genes are consensus primers. Due to the extreme diversity of the receptor gene families, these "consensus" primers are not sufficiently homologous to identify all clonal receptor rearrangements. Therefore, a negative result as determined by a PCR assay is not always indicative of a normal cell population. In fact, testing centers usually retest patient samples with RF-SBH methods following a negative PCR result.
  • PCR based assays are more sensitive than RF-SBH analysis when the PCR primers-are homologous to the target and there are no inhibitors of PCR present in the sample.
  • PCR-based analysis require small amounts of DNA, the assays are successful only if representation of the clonal population is approximately 1 percent or greater of the total population analyzed, and only if both PCR primers are complementary to the receptor gene sequence in the clonal population.
  • One advantage of PCR-based methods is that several primer pairs can be used for the analysis. The use of an algorithm or a series of primer sets generally increases the ability to identify clonal B-cell tumors (Lombardo, 1996). PCR assays used for detection of clonal B-and T-cells have gained popularity over RF-SBH assays in recent years.
  • PCR-based analysis must be reexamined using RF-SBH analysis to assure comprehensive clonality testing.
  • RF-SBH analysis as well suffers from decreased sensitivity in some situations; there are cases of lymphoid malignancies identified using PCR that were determined to be normal by RF-SBH analysis (Lehman, 1995).
  • the time and difficulties associated with RF-SBH testing, and the more stringent specimen requirements provide incentive to maximize the sensitivity, accuracy and specificity of PCR based analyses.
  • thermocycler programs are generally needed to circumvent differences in the length and guanosine:cytosine content of multiple primer set combinations. This complication has given rise to testing algorithms with as many as 11 primer sets and 6 different thermocycler programs (Lombardo, 1996). Amplified products from each of these reactions are then fractionated and analyzed separately to avoid misinterpretation of the data (Slack, 1993). Agarose and polyacrylamide gel electrophoresis are most commonly used for analysis. In addition to the difficulties with target heterogeneity, there are two practical limitations with current PCR testing: first, multiple PCR primer sets are required to identify a majority of B-cell and T-cell neoplasms. These multiple individual reactions require individual firactionation and analysis, and often separate thermocycler programs.
  • thermocy tiers In addition to contributing an inordinate burden on the thermocy tiers, this strategy more importantly increases labor costs to clinical laboratories.
  • a second limitation is the subjective visual interpretation of these data following amplification and fractionation (Slack, 1993). Heteroduplex analysis (Bottaro, 1994), denaturing gradient gel electrophoresis (Bourguin, 1990; Delfau-Larue, 1998; Greiner, 1995), temperature gradient gel electrophoresis (Linke, 1995; Menke, 1995), single-strand conformational polymorphism analysis (Signoretti, 1999; Thunberg, 1998) and EtBr staining of agarose and polyacrylamide gels (Benhattar, 1995) are among the methods used to identify clonal bands.
  • Secondary targets must include primers directed to the MBR of the bcl2 gene to identify the high frequency of t(14;18) translocations that occur in approximately 70% of follicular lymphomas.
  • Consensus and family-specific framework 1 (FRI) primers are also useful for detection of clonal CDR3 rearrangements.
  • TcR ⁇ gene The most specific target for PCR detection of T-cell clonality is the TcR ⁇ gene (Benhattar, 1995). However this locus is not useful for RF-SBH analysis. The paucity of V gene segments result in restriction patterns consistent with clonality even from polyclonal populations (Uppenkamp, 1988). Therefore the TcR ⁇ gene is typically examined by RF-SBH. However, the limited number of V region gamma genes allowed identification of a very useful variable region consensus primer that is used with a consensus J region primer. The identification of clonality is difficult using agarose gel electrophoresis. Polyclonal and clonal products are indistinguishable. Polyacrylamide gel electrophoresis is recommended to obtain the resolution necessary to detect clonal products within the background of a polyclonal population (Benhattar, 1995).
  • the t(9;22) translocation is diagnostic for CML and ALL, and to a lesser extent other hematologic malignancies.
  • Bcr-abl translocation testing is generally done by fluorescence in situ hybridization (FISH) analysis or reverse transcriptase-Polymerase chain reaction (RT-PCR).
  • RT-PCR testing is done using a series of primer pairs to amplify the various breakpoint regions encompassing the most common breakpoint cluster regions, m-bcr and M-bcr (Melo, 1996).
  • the products are generally transferred to membranes and detected using a radioactive or chemiluminescent probe. These assays generally take approximately one week to perform and to generate a result.
  • IgH, Bell and Bcl2 testing (the latter two for mantle cell and follicular lymphomas, respectively) is generally done using PCR or RF-SBH assays.
  • the products are often detected using a radioactive or chenriluminescent probe following transfer of the amplified product to a membrane (Crescenzi, 1988).
  • the products can also be detected within polyacrylamide gels (Segal, 1994).
  • the present invention provides a method for detecting clonal cell populations in a biological sample.
  • the invention comprises providing a biological sample containing DNA sequences that are diagnostic for cell clonality, mixing a portion of said sample with separate primer compositions, each containing at least one differentially-labeled primer that recognizes at least one DNA sequence region diagnostic for cell clonality, incubating said mixtures under conditions that result in amplification of intervening DNA sequence region(s), combining a portion of the amplification products from individual amplification mixtures, and analyzing the amplification products in the mixture to detect amplification product(s) diagnostic for cell clonality.
  • portions of the sample are mixed with a primer composition comprising at least three primers consisting of at least two differentially-labeled primer which recognize and bind to the DNA sequence region that is diagnostic for the diagnostic marker.
  • the mixture is incubated under amplification conditions, and the products analyzed.
  • the biological sample comprises mRNA encoding sequences diagnostic for the diagnostic marker, and the sample is contacted with a reverse transcriptase enzyme and oligonucleotide primer complex and incubated under conditions sufficient to produce cDNA sequences from the mRNA.
  • the biological sample comprises cells or tissue suspected of harboring the diagnostic marker, and the sample is contacted with a modified tRNA primer molecule and reverse transcriptase enzyme complex, and incubated to produce cDNA sequences to the mRNA.
  • Figure 1A presents a diagram depicting regions targeted by B-cell panel primers in accordance with the invention, together with the size range of the corresponding amplicon products.
  • the FR3, FR1 and Mbr-bc/2 sense primers are 5'-labeled with 6-FAM, NED and ROX, respectively.
  • the downstream ' antisense consensus JH primer is unlabeled;
  • Figure IB presents a diagram depicting regions targeted by B + T cell panel primers in accordance with the invention, together with the size range of the corresponding amplicon products.
  • the FR3, FR1 and TcRV ⁇ sense primers are 5'-labeled with 6-FAM, NED and ROX, respectively.
  • the downstream antisense JH and TcRJ ⁇ primers are unlabeled.
  • Figure 2 presents data generated in analyzing placental DNA (panel a, IG), PBL genomic DNA (panel b, 3G) and cDNA (panel c, 7G) using the B-cell primer panel in accordance with the invention.
  • Products are Framework 3 (Green), Framework 1 (Blue) and mbr-bc/2 (black) primer combinations. Red peaks are size standards. Size in basepairs is displayed across the top of the figure. Relative fluorescence units are listed on the Y-axis.
  • the normal Gaussian distribution of Framework 3 amplicons are bounded by two prominent peaks at 69 and 129 base pairs (arrows) in human placental DNA, and to a lesser extent in PBL genomic DNA.
  • the table (bottom of the figure) are data on prominent 69 and 129 basepair peaks from the runs in panels a, b, and c.
  • Size in basepairs, peak height and area, and data point are indicated;
  • Figure 3 presents data generated in analyzing placental DNA (panel a, 2G),
  • Panels a, b, c and d represent dilutions of 30% , 20% , 10% and 5% , respectively.
  • the mean size dete ⁇ nined for the " highlighted clonal FR3 band (green) was 103.91bp with a standard deviation of 0.012bp for these separate assays;
  • Figure 6 presents data generated using the B + T cell analysis of samples containing SUP-Tl DNA.
  • Panels a, b, c and d represent data from 30% (15G.Y), 20% (12G.Y), 10% (lOG.Y) and 5% (8G,Y) dilutions of SUP-Tl DNA into tonsil DNA.
  • the FR3 (green) and TcR ⁇ (black) products are shown. Arrows denote the ubiquitous FR3 peak used for quantifying data.
  • the table (bottom of the figure) lists data from the FR3 peak (15G, 12G, 10G, and 8G) and prominent clonal (182 and 187bp) peaks (15Y,
  • Figure 7 presents data generated using the clinical samples 1, 2 and 4 respectively.
  • PCR amplification products are generated using the B-cell primer panel.
  • Mbr-bc/2 products black are apparent at 194, 147 and 237bp in samples 1, 2 and 4, respectively.
  • Figure 8 presents data generated using the clinical samples 13, 14, 15 and 16 respectively. Products were generated using B + T cell primer panel. FR3 (green) and TcR ⁇ (black) products are shown. Size standards are red. Note: clonal rearrangements of the TcR ⁇ gene detected using the TcR ⁇ primer set in samples 13, 15 and 16 (black), and the clonal IgH rearrangement detected in sample 14 using the FR3 primer set (green).
  • NED-labeled Bcr b2-2 primer Black peaks were generated using the 6-FAM-labeled Bcr b3-2 primer. Red peaks are size standards. Peaks include a cDNA control amplicon (green peak) of approximately 93 basepairs (panels b-d) generated using the Abl 1-1 + Abl 2-2 primer set (arrows). Data generated testing the K562 cDNA sample resulted in production of nine peaks representing various transcription products from the t(9:22) translocation.
  • the present invention provides a method for detecting a diagnostic marker in a biological sample.
  • the invention comprises providing a biological sample comprising DNA encoding sequences diagnostic for the diagnostic marker, mixing the sample with at least three primers comprising at least two differentially- labeled primers which recognize and bind to at least one mterveriing DNA sequence region in the DNA that is diagnostic for the diagnostic marker, " incubating the mixture under conditions sufficient to result in detectable amplification of the intervening DNA sequence region, and analyzing the products of the amplification step to detect the individual amplification product diagnostic for the diagnostic marker.
  • portions of the sample are mixed separately with at least two primer compositions, each comprising at least one differentially- labeled primer which recognize and bind to the DNA sequence region that is diagnostic for the diagnostic marker.
  • the mixtures are incubated separately under amplification conditions, and combined prior to analysis.
  • biological sample is defined as a sample derived from a biological source, and includes samples previously subjected to any chemical techniques, such as, for example, extractions, conversions, amplifications and the like;
  • cell monoclonality or clonality is defined as a population of cells or nucleic acid sequences arising from a single cell;
  • neoplasm or neoplasia is defined as a new and abnormal growth of tissue, which may be benign or cancerous; a tumor or malignancy; a new growth of tissue serving no physiological function; or the process of tumor formation;
  • geneetic locus or locus is defined as the site in a linkage map or on a chromosome where the gene for a particular trait is located. Any one of the alleles of a gene may be present at this site; "alleles” are defined as mutually exclusive forms of the same gene, occupying the same locus on homologous chromosomes, and governing the same biochemical or developmental process;
  • detection platform is defined as an instrument used to detect the constituent products of an assay or reaction mixture
  • differentiated is defined as a having a label that can be identified or distinguished form other labels by any means, including the use of an analytical instrument or detection platform;
  • primer is defined as a specific nucleic acid polymer used by an enzyme to initiate new template-specific DNA or RNA synthesis
  • “recognize and bind” is defined as the annealing of, for example, a primer molecule to a complementary nucleic acid sequence, ordinarily under defined conditions of stringency to recognize the possibility of one or more specific base mismatches.
  • one embodiment of the present invention employs two panels of differentially-labeled primers.
  • each panel targets and amplifies three separate sequence regions diagnostic for B-cell or B-cell and T-cell clonality.
  • the differentially-labeled amplified products are loaded and fractionated on a high-throughput, sensitive, automated capillary electrophoresis platform capable of single base resolution, automated sizing and relative quantification, and differential amplicon detection.
  • the data are automatically archived for subsequent analysis.
  • the invention thus provides an efficient and cost-effective method for initial diagnosis of a diagnostic marker, such as for cancer, and for the subsequent monitoring of residual disease.
  • information on the identity, size, and relative quantity of each clonal product can be compared with data generated during subsequent analyses.
  • These feature of the invention provide a coherent approach for monitoring patients and for assessing treatment regimens.
  • the invention tests cell clonality simultaneously and specifically at multiple genetic loci. This novel approach improves the speed, the sensitivity and the specificity of the initial identification and provides additional information useful in subsequent monitoring of monoclonal cell populations.
  • the invention increases the throughput and lowers the cost of diagnostic clonal analyses.
  • the biological sample comprises mRNA encoding sequences diagnostic for the diagnostic marker, and the sample is contacted with a reverse transcriptase enzyme and oligonucleotide primer complex and incubated under conditions sufficient to produce cDNA sequences from the mRNA.
  • the biological sample comprises cells or tissue suspected of harboring the diagnostic marker, and the sample is contacted with a modified tRNA primer molecule and reverse transcriptase enzyme complex, and incubated to produce cDNA sequences to the mRNA.
  • RNA specific for the clonal or translocated targets will increase the sensitivity of the present invention for certain specimens.
  • the mRNA population is first converted to complementary DNA (cDNA), which can be generated using in vivo or in vitro cDNA synthesis methods.
  • cDNA complementary DNA
  • In vivo cDNA synthesis methods can produce cDNA from smaller numbers of cells and tissue biopsy samples and can stabilize samples in the field.
  • Targeting cDNA enhances the signal from lymphoproliferative populations that express the clonal receptor or translocation product.
  • cDNA synthesis capitalizes on the amplification of the single copy gene rearrangements that occur during transcription. Hundreds or even thousands of RNA copies are produced from a single genomic DNA template during transcription.
  • each of the primer sets designed for use with the invention directs the amplification of specific DNA sequences diagnostic for, e.g. , lympho-proliferative disease.
  • the amplicon products are specific rearrangements or translocations diagnostic for B-cell and T-cell clonality. These primer combinations will be seen to be very efficient for identifying B-cell and T-cell lymphoproliferative disease.
  • Each primer combination produces characteristic amplified products within a defined size range, which is designed to minimize the overlap in size distribution between the various amplicon products.
  • the FRI + JH primer combination produces amplicons from genomic DNA that are primarily 339-399 base pairs in size.
  • DNA is analyzed, amplified product are also detected in the 69-129 base pair range. Similarly, the FR3 + JH primer combination produces amplicons that are primarily in the 69-129 base pairs in size.
  • Primers detecting t(14;18) translocations target the major breakpoint region (Mbr) of bcl-2 and the JH region of the IgH. This primer combination produces amplicon products that are 80-300 base pairs in size.
  • the TV ⁇ 4- TJ ⁇ primers amplify products in two Poisson distribution patterns that range from 120-160 and 162-201 base pairs in size.
  • a second TV ⁇ 4- TJ ⁇ primer set produces amplicon peaks in the 235-270 basepair range.
  • Differential labeling of the amplification products in each primer set allows the simultaneous amplification and fractionation of diagnostic product with single base resolution and verification of the primer-specific and size-related specificity of the amplified products.
  • the use of multiple fluorescent primers allows multiplex PCR amplification, or singlicate amplifications coupled with multiplex detection. This feature of the invention lowers the costs, increases the throughput, and allows for the simultaneous identification of clonal rearrangements of the Ig heavy chain gene, TcR ⁇ alleles and t(14;18) translocations.
  • primers useful for the practice of the present invention include, but are not limited to, the following: B-cell Clonality Panel: Detects: clonal IgH rearrangements and bcl2 translocations
  • T-cell Clonality Panel Detects: both clonal TcR ⁇ chain and TcR ⁇ chain gene rearrangements.
  • B-cell + T-cell Clonality Panel Detects: clonal IgH gene rearrangements and TcR ⁇ chain gene rearrangements
  • Bcr-abl Panel Simultaneously confirms control abl transcript and identifies bcr-abl t(9;22) translocations.
  • An evaluation is conducted to detect the presence of clonal B-cells or T-cells using genomic DNA extracted from normal patients and patients demonstrated to have clonal B-cell or T-cell cancers.
  • Sources of the DNA include peripheral blood, lymph nodes, bone marrow, spleen, salivary gland and an abdominal mass.
  • Samples consisting of peripheral blood or bone marrow are separated on Ficoll- Hypaque prior to extraction of DNA.
  • the DNA is extracted using routine and well- established extraction protocols available from the manufacturers (e.g., DNAzol,
  • the DNA is quantified by spectrophotometric readings at 260nm and diluted to 20-500ng/ ⁇ L prior to analysis.
  • a sample comprising 0.1-2.5 ⁇ g of DNA is amplified in multiplex or singlicate reactions with a total volume of 33 ⁇ L or 55 ⁇ L using the primers in the B-cell, or one of the B-cell + T cell panels.
  • the master mix contains 2mM MgCl 2 , 0.2mM dNTPs, 25pM of each primer and 100-lOOOng of sample genomic DNA. To this mixture is added approximately 1.5U of AmpliTaq DNA Polymerase (PE Applied Biosystems) per reaction, in a total volume of 33 or 55 ⁇ L. The amplification is performed for 35 cycles in a model 9600
  • PE Thermocycler PE Applied Biosystems using the following amplification program: 3 minutes at 95 °C; 35 cycles of: 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 1 minute; followed by 10 minutes at 72°C; then 4°C until examined.
  • a l ⁇ L aliquot of each of the amplification reactions from a single clinical sample are combined and denatured for 3 minutes at 95 °C in 15 ⁇ L of distilled formamide containing 0.5 ⁇ L of
  • GSROX50-400HD size standards PE Applied Biosystems. Immediately following the heating, the samples are placed on ice for 5 minutes, then centrifuged briefly to collect the condensate prior to loading on the PE 310 instrument (PE Applied Biosystems).
  • the master mix contains 2mM MgCl 2 , 0.2mM dNTPs, 25pM of each primer except 50pM of the JH primer. To this mixture is added approximately 1.5U of AmpliTaq DNA Polymerase (PE Applied Biosystems) per reaction, in a total volume of 33 or 55 ⁇ L. The amplification is performed for 35 cycles in a model 9600 PE Thermocycler (PE Applied Biosystems) using the following amplification program: 3 minutes at 95°C; 35 cycles of: 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute; followed by 10 minutes at 72°C; then 4°C until examined.
  • PE Applied Biosystems PE Thermocycler
  • a 2 ⁇ L aliquot of the amplification reaction from a clinical sample is denatured for 3 minutes at 95 °C in 15 ⁇ L of distilled formamide containing 0.5 ⁇ L of GSROX50-400HD size standards (PE Applied Biosystems). Immediately following the heating, the samples are placed on ice for 5 minutes, then centrifuged briefly to collect the condensate prior to loading on the
  • Oligonucleotides HPLC purified; Operon Technologies or PE Applied Biosystems
  • Deoxynucleotide triphosphate stocks are diluted from lOOmM stocks (Pharmacia or Gibco/BRL Cat# 10297-018).
  • Human placental DNA Sigma# D-4642
  • whole blood samples from normal persons and human genomic DNA (Boehringer Mannheim # 1 691 112) are used as negative controls.
  • Injection of the sample is for six seconds at 15kV with the capillary run at 15kV at a temperature at 60 °C, using the GS_STR_POP4_lmL module, and the POP4 filter D matrix file.
  • the samples are run for 26 minutes at a current value of approximately 8-9 ⁇ A.
  • the data collection is performed using ABI 310 collection software, and the analysis is performed with GENESCAN 2.1 PE Applied Biosystems software.
  • HEX FR3 A consensus primer that targets the framework 3 region of the IgH variable region. This primer is 5' end labeled with HEX.
  • NED Mbr A primer that targets the major breakpoint region of the Bcl2 proto- oncogene on chromosome 18. This primer is 5' end labeled with NED.
  • JH A consensus antisense primer that targets the joining region of the IgH gene on chromosome 14.
  • Example 1 demonstrates that RNA can provide a preferred target for clonal analysis. Previous studies have confirmed this for both B-cell (Voena, 1997) and T-cell clonal disease (Veelken, 1996).
  • 35mL of fresh peripheral blood is collected into 5 vacutainer tubes with Li-heparin used as an anticoagulant.
  • the blood is pooled and diluted with approximately an equal volume of RPMI 1640.
  • a 35mL aliquot of blood is then layered onto 15mL of Ficoll in a 50mL tube, and centrifuged at 2,500 rpm for 10 minutes. The interface is removed and resuspended in RPMI (using an excess of media so that cells will pellet through the remaining Ficoll).
  • This suspension is centrifuged for 10 minutes at 2,000 rpm, and the pellet is washed with PBS, then again centrifuged and resuspended in RPMI 1640 with 10% FCS.
  • the Ficoll-separated cells are removed from the refrigerator and 30mL of the cells are transferred to a 50mL tube, with PBS added to a total volume of 50mL.
  • the suspension is centrifuged at 2,000 RPM for 5 minutes, and the pellet is resuspended in
  • RNA Preparation The cell pellet is resuspended in lmL of RNA Stat 60 (Tel-Test, Inc) and incubated for 5 minutes at room temperature. 200 ⁇ L of chloroform is added and the suspension is agitated, then allowed to settle for 5 minutes. The tube is centrifuged at high speed and the supernatant is transferred to a fresh tube. 500 ⁇ L of 2-propanol is added, the suspension is mixed and allowed to settle for 5 minutes. The RNA is then centrifuged, the pellet is rinsed with RNase-free 70% EtOH and Savant-dried. The RNA pellet is then resuspended in 400 ⁇ L of RNase-free dH 2 O.
  • RNA prepared as described above is then subjected to cDNA synthesis according to known techniques, generally as follows: To two microfuge tubes are added: 20 ⁇ L 5x first strand buffer
  • Both tubes are incubated at 42 °C for 90 minutes, then heated to 100 °C for two minutes and quick chilled on ice. The material is then collected by a brief centrifugation.
  • the master mix contains 2mM MgCl 2 , 0.2mM dNTPs, 25pM of each primer and 5 ⁇ L of sample cDNA, and to this mixture is added approximately 1.5U of
  • AmpliTaq DNA Polymerase (PE Applied Biosystems) per reaction, in a total volume of 33 or 55 ⁇ L.
  • the amplification is performed for 35 cycles in a model 9600 PE Thermocycler (PE Applied Biosystems) using the following amplification program: Three minutes at 95 °C; 35 cycles of: 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for one minute; followed by 10 minutes at 72°C; then 4°C until examined.
  • a l ⁇ L aliquot of each of the amplification reactions is combined and denatured for 3 minutes at 95°C in 15 ⁇ L of distilled formamide containing 0.5mL of GSROX50-400HD size standards (PE Applied Biosystems).
  • the samples are placed on ice for five minutes, then centrifuged briefly to collect the condensate prior to loading on the PE 310 instrument.
  • Analytical Parameters Injection of the sample is for six seconds at 15kV with the capillary run at 15kV at a temperature, at 60°C, using the GS_STR_POP4_lmL module, and the POP4 filter D matrix file.
  • the samples are run for 26 minutes at a current value of approximately 8-9 ⁇ A.
  • the data collection is performed using ABI 310 collection software, and the analysis is performed with GENESCAN 2.1 PE Applied Biosystems software.
  • the data gathered from clinical samples reveals an additional advantage to the present invention: Two amplified clonal products produced with the framework 3 primers differed in size to the base with two amplified products produced from the same clinical sample using the framework 1 primer set. Therefore, independent verification of clonality can be achieved within a single multiplex run. The presence of more than two bands is not unusual and may be indicative of early neoplasms which later go through somatic rearrangements (Davi, 1996).
  • the high resolution obtainable with the present method also allows diagnostic follow-up of minimum residual disease. Clonal products can be identified to the base due to the resolution possible with this approach. Proofreading of DNA polymerases for detection of amplicon products generated from B-cell populations has be recommended by some investigators (Linke, 1995). Generic AmpliTaq polymerase (PE Applied Biosystems) is found to be very effective in generating amplicon product from positive samples, although single base additions are seen in the majority of the peaks. Data from these examples will also demonstrate that the multiplex reaction produced amplified products identical in size to products produced using separate PCR reactions.
  • cells are added to a mixture comprising at least one differentially labeled and modified reverse transcriptase cognate primer tRNA molecule wherein the tRNA molecule is modified to anneal to the 3 '-region of an RNA molecule whose detection is diagnostic for monoclonality.
  • the complementary DNA synthesis reaction proceeds in a mixture such that the amplification product can be fractionated and differentially detected.
  • a nucleic acid sample is added to a mixture comprising at least one modified reverse transcriptase cognate primer tRNA molecule wherein the tRNA molecule is modified to anneal to the 3 ' region of a nucleic acid molecule whose detection is diagnostic for monoclonality.
  • the complementary DNA synthesis reaction proceeds in a mixture comprising at least one modified deoxynucleotide triphosphate or analog thereof such that the amplification product can be fractionated and differentially detected.
  • Example 3 In addition to the Examples above, clinical examples are provided demonstrating the efficacy and efficiency of the present invention.
  • Samples are obtained from the specimen archives of the Molecular Diagnostics Laboratory, Emory University Hospital, Atlanta, GA, representing material discarded after appropriate diagnostic studies had been performed. Samples are obtained from healthy individuals, patients diagnosed with Hodgkin's disease, clonal B-cell or T-cell lymphoproliferative disorders and human control cell lines.
  • Peripheral blood from healthy volunteers is anticoagulated with EDTA, and the peripheral blood lymphocytes (PBLs) are isolated by diluting the whole blood with RPMI 1640 and banding on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells collected at the interface are washed 3x with RPMI prior to DNA or RNA extraction.
  • PBLs peripheral blood lymphocytes
  • DNA is extracted from clinical samples, including: peripheral blood, lymph nodes (fresh and frozen), bone marrow, salivary gland, an abdominal mass, tissue and spleen.
  • the extracted DNA is resuspended in TE buffer at a final concentration of lO ⁇ g-lOO ⁇ g per mL.
  • Coded dilutions of positive control DNAs are utilized to assess assay sensitivity and are obtained from the Armed Forces Institute of Pathology (AFIP; Washington, DC). Positive control DNA is diluted into tonsil DNA extracted from healthy volunteers to final dilutions of 30% , 20% , 10% and 5% (w/w%). The placental DNA is obtained from Sigma Chemical Co. (St. Louis, MO).
  • RNA is extracted from these dilutions using standard procedures. Extracted DNA is resuspended in TE buffer (10 mM TrisHCl, 1 mM EDTA, Ph 8.0) to 50 ⁇ g-200 ⁇ g per mL. Total RNA is used for cDNA synthesis.
  • FBS heat inactivated fetal bovine serum
  • L-glutamine L-glutamine
  • Penicillin and Streptomycin The cells are harvested and counted. Serial ten-fold dilutions are made into peripheral blood lymphocytes (PBLs) collected from healthy volunteers. DNA and RNA are extracted from these dilutions using standard procedures. Extracted DNA is resuspended in TE buffer (10 mM TrisHCl, 1 mM EDTA, Ph 8.0) to 50 ⁇ g-200 ⁇ g per mL. Total RNA is used for cDNA synthesis.
  • RNA is extracted from PBLs and human cell lines using RNA STAT-60 (Tel-Test, Inc., Friends wood, TX) according to the manufacturer's instructions.
  • Complementary DNA is synthesized in vitro using MMLV Superscript II reverse transcriptase enzyme (Gibco/BRL, Gaithersburg, MD) with random hexamer primers according to the manufacturer's instructions.
  • Template Amplification is amplification of MMLV Superscript II reverse transcriptase enzyme (Gibco/BRL, Gaithersburg, MD) with random hexamer primers according to the manufacturer's instructions.
  • DNA or cDNA samples are tested using the B 4- T Cell Clonality Assay Kit (Cat# 102-24; IVS Technologies, Carlsbad, CA).
  • the assay kit contains protocols, reagents and controls for complete B-cell and T-cell clonality testing on 24 samples. Briefly, four (4) master mixes are used in the Example.
  • the PCR primers, as identified in Example 2 are synthesized and purified by HPLC (Perkin Elmer; Operon Technologies).
  • Upstream sense primers '-end-labeled with 6-FAM, HEX or NED dyes targeted either the IgH framework 1 (FRI) or framework 3 (FR3) regions, the major breakpoint region (mbr) of the bcl2 gene, or the variable region of the TcR ⁇ chain gene.
  • Unlabeled downstream antisense primers targeted the joining regions of the IgH and TcR ⁇ chain genes.
  • Fifty nanograms to one microgram (50ng-l ⁇ g) of human genomic DNA or complementary DNA are amplified in separate reactions (55 ⁇ l total volume) in a single plate using AmpliTaq DNA polymerase (PE Applied Biosystems) and the following thermocycler program: 95 °C for 3 minutes; 35 cycles of: 30 sec at 94°C, 30 sec at 55°C, 1 minute at 72°C; 5 minutes at 72°C.
  • thermocycler automatically maintained the amplicon products at 4°C.
  • the capillary runs at 60°C are initiated with 6 second injections and ran 24 minutes at 15 kV using the GS STR POP4 D lmL run module.
  • Data are collected using ABI 310 collection software (PE Applied Biosystems) and analyzed with TVS Technologies' dye matrices and sizing parameters using ABI GeneScan ® (v2.0) software.
  • the B-cell panel ( Figure 1A) included primers that targeted the IgH FRI and FR3 regions, and the major breakpoint region (mbr) of bcl2. This panel identified rearrangements within the V-J region of the IgH, and t(14;18) translocations common in follicular lymphomas.
  • the B 4- T cell panel ( Figure IB) identified the IgH rearrangements (IgH FRI and FR3), and rearrangements of the TcR ⁇ gene.
  • Amplification of either genomic DNA (Green line, Figures 2, panel b, and 3, panel b) or cDNA ( Figures 2, panel c, and 3, panel c) from human PBLs with the IgH FR3 primer set produced a Gaussian distribution of product amplicons that ranged in size from 69-129 basepairs. Adjacent peaks within the FR3 product profile are separated by exactly 3 basepairs. Placental DNA tested using the IgH FR3 primer set produced two prominent amplicon peaks that circumscribed this region ( Figures 2, panel a, and 3, panel a). These peaks are reproducibly determined to be 69 and 129 basepairs in size.
  • Analyzing cDNA from PBLs using the FRI primer set produces a Gaussian distribution of amplicons that ranged in size from 339-399 basepairs ( Figures 2, panel c, and 3, panel c). Consistent with the FR3 product data, adjacent peaks in the FRI product profile are separated by exactly 3 basepairs. As predicted, approximately two hundred and seventy (270) basepairs separated amplicon peaks from similar locations within the FRI and FR3 Gaussian distributions. Comparison of the sizes and relative heights of the IgH FRI and IgH FR3 Gaussian products from the B-cell and
  • Amplification reactions using the mbr-bc/2 primer set did not produce any specific bands using DNA from normal persons.
  • the product profiles for the TcR ⁇ primer set differed markedly depending on the DNA source used for testing. As expected, placental DNA produced no apparent amplicon products.
  • the amplicon profile generated with genomic DNA is a Gaussian distribution between 162-201 basepairs ( Figure 3, panel b).
  • the lack of a discrete three base pair spacing for ⁇ rearrangements probably reflects the fact that ⁇ cells are a small subpopulation, and that there is no selection for in-frame ⁇ rearrangements in ⁇ T-cells or B-cells.
  • Complementary DNA produced a product profile that appeared to be oligoclonal within the same 162-20 lbp size range ( Figure 3, panel c). This product profile is probably due to transcription amplification within expanded clones in the peripheral blood.
  • human clonal T-cell lines (CEM and Jurkat) are serially diluted ten-fold into peripheral blood lymphocytes from healthy volunteers. DNA is extracted from approximately 10 4 cells to make each of the extracts. The assay detected approximately 1 clonal cell in 10 2 cells by testing DNA extracted from these serial dilutions ( Figure 4).
  • a coded proficiency panel (from AFIP; Karen Bijwaard, MS, and Dr. Jeffrey Taubenberger) is utilized.
  • DNA is extracted from the human SUP-Tl, SUDHL5, Raji and MOLT-4 cell lines.
  • the SUDHL5 and Raji cell lines contain clonal IgH gene rearrangements.
  • the SUP-Tl and MOLT4 cell lines contain clonal TcR ⁇ rearrangements.
  • a 104bp clonal IgH FR3 amplicon can be identified in all four of the coded samples that contained diluted SUDHL5 DNA.
  • the relative intensity of the specific 104bp FR3 amplicon peak is proportional to the concentration of the SUDHL5 DNA in each of the coded samples ( Figure 5, Table 3).
  • 182bp and 188bp clonal TcR ⁇ amplicons are identified in four of the other coded samples. These samples contained diluted SUP-Tl DNA, and the relative intensities of the peaks in these samples are proportional to the dilutions of SUP-Tl DNA ( Figure 6, Table 3).
  • This assay also identified a HObp clonal IgH FR3 amplicon in two of the remaining samples. These samples represented the 30% and 20% dilutions of Raji DNA. The assay did not detect this amplicon in the 10% and 5% dilutions.
  • Intra-assay reproducibility of relative peak height quantification for several clonal amplicons is examined by comparing the peak heights of the clonal peak and the ubiquitous 69 basepair FR3 amplicon peak in the B-cell, and B 4- T cell panels. Since the B-cell and B 4- T cell panels are separate capillary electrophoretic runs and analyses of the same FR3 amplification reactions, they represent intra-assay tests.
  • the clonal 103.9bp FR3 peak detected in the 30% dilution of SUDHL5 DNA from the B-cell panel analysis had a peak height of 6652 units.
  • the peak height of the ubiquitous FR3 band at 69bp had a height of 870 units, giving a ratio of "7.6" for these two peaks.
  • These same amplicons had heights of 6121 and 838, respectively, for a ratio of "7.3" in data from the B 4- T cell panel. This is > 95% agreement for the ratio of heights, of these peaks.
  • the relative peak heights for the remaining dilutions of SUDHL5 DNA are listed in Table 3.
  • Amplicon peaks generated from monoclonal samples are examined by intra- assay and inter-assay reproducibly testing.
  • the mean value assigned to the IgH FR3 amplicon peaks detected in the coded dilution proficiency panel from AFIP is 103.91 basepairs with a standard deviation of 0.012 basepairs.
  • the mean value assigned to the two TcR ⁇ amplicon peaks detected in SUP-Tl samples are 182.5 and 187.87 basepairs, with standard deviations of 0.07 and 0.05 basepairs, respectively (bottom of Figure 6).
  • the assay identified bc ⁇ 1(14;18) translocations (mbr-b 2 rearrangements), as well as clonal IgH FR3 rearrangements, in several follicular lymphomas (Figure 7). Two discrepant results are encountered. First, the assay did not detect a clonal rearrangement in the TcR ⁇ locus (black line, sample 14, Figure 8) that is previously detected using PCR (Table 1). However this assay identified an additional clonal IgH rearrangement of 88bp from the same patient sample using the IgH FR3 primer set
  • the automated semi-quantitative B-cell and T-cell assay system evaluated in this study provided a simple and likely cost effective solution to these problems.
  • the testing panels included primers that identified the majority of B-cell and T-cell neoplasms.
  • the master mixes and primer sets were developed for use together in a single thermocycler program. Labor-intensive gel fractionation and subjective visual interpretation of the individual reaction products were replaced with automated, sensitive, high-resolution, differential fluorescence quantification that simultaneously identified and quantified amplified products from multiple genetic loci.
  • Amplicon products consistent with the expected size range were generated with each of the primer sets in the panel and were ⁇ eproducibly sized to within a single basepair.
  • the assay identified specific and reproducible amplicon size ranges for IgH FRI, FR3 and TcR ⁇ primer sets using both DNA and cDNA from polyclonal B-cell and T-cell populations. These profiles were consistent with the expected size and
  • Gaussian distribution patterns independently determined for these CDR3 regions (Rock, 1994), and were internally consistent as well.
  • the size range of amplicon products generated using the IgH FR3 primers (69-129bp) was 60 nucleotides, and mirrored the corresponding IgH FRI primer (340-400bp) profile.
  • the IgH FRI and IgH FR3 primers are targeting similar representative template populations, and confirmed that the length of rearrangements within the CDR3 region of the IgH genes are limited to approximately 20 amino acids.
  • the size range for Gaussian amplicon products generated using the TcR ⁇ primer set was 162-201 nucleotides.
  • the FR3 primer targets the conserved amino acid sequence, AspThrAlaValTyrTyrCys, located at the 3' end of the IgH FR3 region.
  • the IgH FRI primer targets a conserved GluValGlnLeuValGluSerGly sequence at the very 5' end of the FRI region.
  • the size of amplicons generated using these primer sequences are separate by approximately 90 amino acids, or 270 basepairs, consistent with the spacing of these regions in the heavy chain of the immunoglobulin receptor (Kabat, 1987). Further, neighboring amplicon peaks within the Gaussian distribution differed by 3 basepairs. This is consistent with single codon differences, which is the expected size difference between productively rearranged members of the polyclonal population.
  • the discrepant results would have been unlikely to affect diagnosis or therapy.
  • the first discrepant result (Tables 1 and 2; sample 9) was an additional amplicon peak (TcR ⁇ ) which was verified by subsequent testing.
  • This CLL patient had a B-lineage assigned by flow cytometric immunophenotyping of the same bone marrow specimen.
  • the second discrepancy (Tables 1 and 2; sample #14) concerns a mediastinal T-lineage lymphoblastic lymphoma in a 22 year old and reflects the new identification of a clonal IgH rearrangement but failure to identify a clonal rearrangement at TCR ⁇ . Again, immunophenotyping indicated T-lineage.
  • the present assay system simultaneously examines rearrangements at multiple genetic loci using matched sets of differentially labeled primers. This approach saves time, increased sample throughput and eliminated the separate thermocycler reactions and separate analyses common to other assay systems (Benhattar, 1995; Lombardo, 1996; Abdel-Reheim, 1996). Second, labor intensive and error-prone post-amplification detection steps were eliminated and replaced by automated fluorescence detection and quantitation of products, which saved analysis time, substantially improved the quality of the resulting data. Complete fractionation and analysis with this system was approximately 30 minutes per sample. Clonal peaks were quantified and sized to the exact base, and data were automatically archived for review and storage. Single primer B-cell only assays use proofreading Taq polymerase to generate high resolution data. However, detection takes 2-3 hours (Linke, 1997). Whereas this integrated B-cell and T-cell assay uses conventional Taq polymerase to generate precise and semi-quantitative data from multiple genetic loci, and detection and data analysis is complete in less than 30 minutes.
  • This integrated and semi-quantitative approach generated a body of precise information about each B-cell and T-cell tumor. These data may prove useful in further stratifying malignancies, by yielding prognostic information or specifying treatment protocols, or alternatively they may provide information useful for post therapeutic monitoring.
  • Peripheral blood from healthy volunteers is anticoagulated with EDTA, and the peripheral blood lymphocytes (PBLs) are isolated by diluting the whole blood with RPMI 1640 and banding on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells collected at the interface are washed 3x with RPMI prior to DNA or RNA extraction.
  • PBLs peripheral blood lymphocytes
  • DNA is extracted from control samples.
  • the extracted DNA is resuspended in TE buffer (10 mM TrisHCl, 1 mM EDTA, Ph 8.0) at a final concentration of lO ⁇ g-lOO ⁇ g per mL.
  • Positive control human cell lines used in this study are obtained from the American Type Culture Collection (ATCC) or from other investigators.
  • the CEM and Jurkat T-cell lines, and the K562 CML cell lines are grown in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (FBS), L-glutamine, Penicillin and Streptomycin.
  • FBS heat inactivated fetal bovine serum
  • the cells are harvested and DNA was extracted from these cells using standard procedures. Extracted DNA is resuspended in TE buffer (10 mM TrisHCl, 1 mM EDTA, Ph 8.0) to 50 ⁇ g-200 ⁇ g per mL. Total RNA is used for cDNA synthesis.
  • RNA is extracted from PBLs and human cell lines using RNA STAT-60 (Tel-Test, Inc. , Friendswood, TX) according to the manufacturer's instructions.
  • Complementary DNA is synthesized in vitro using MMLV Superscript II reverse transcriptase enzyme (Gibco/BRL, Gaithersburg, MD) with random hexamer primers according to the manufacturer's instructions.
  • Genomic DNA or cDNA(4-) and cDNA(-) samples are tested using the Bcr-abl Assay Kit (TVS Technologies, Carlsbad, CA).
  • the assay kit contains protocols, reagents and controls for complete Bcr-abl testing on 24 samples.
  • PCR primers are synthesized and purified by HPLC (Perkin Elmer; Operon Technologies).
  • HPLC Perkin Elmer; Operon Technologies.
  • the master mixes for both the first and second round reactions contain 2mM MgCl 2 , 0.2mM dNTPs, 25pM of each primer and 5 ⁇ L of sample cDNA, and to this mixture is added approximately 1.5U of AmpliTaq DNA Polymerase (PE Applied Biosystems) per reaction, in a total volume of 55 ⁇ L.
  • PE Applied Biosystems AmpliTaq DNA Polymerase
  • Upstream sense primers targeted the Abl gene on chromosome 9 (Abll-1, bases 152-171 , GeneBank Accession #X16416) and the Bcr gene on chromosome 22 (Bcr b2-l, bases 3128-3149, Genebank Accession #X02596; Bcr el-1, bases 1571-1589,
  • Downstream antisense primers targeted the Abl gene (AW3-1, bases 455-438, GeneBank Accession #X16416).
  • Master mixes consisted of the following sets of primer pairs: Abll-1 4- Abl3-1; Bcr el-1 4- Abl3-1; and Bcr b2-l 4- Abl3-1.
  • thermocycler program 95 °C for 3 minutes; 35 cycles of: 30 sec at 94° C, 30 sec at 55 °C, 1 minute at 72° C; 5 minutes at 72°C. Following this program the thermocycler automatically maintained the amplicon products at 4°C.
  • the upstream sense primer that targeted the Abl gene on chromosome 9 (Abll-1, bases 152-171, GeneBank Accession #X16416) and the upstream sense primer that targeted the Bcr gene on chromosome 22 (Bcr el -2, bases 1600-1619, Genebank Accession #X02596) are 5' end-labeled with HEX.
  • the upstream sense primer that targeted the Bcr gene on chromosome 22 (Bcr b3-2, bases 3234-3253, Genebank Accession #X02596) is 5' end-labeled with 6-FAM.
  • the upstream sense primer that targeted the Bcr gene on chromosome 22 (Bcr b2-2, bases 3165-3182, Genebank Accession #X02596) is 5' end-labeled with NED. Unlabeled downstream antisense primers targeted the Abl gene (Abl2-2, bases
  • thermocycler program 95 °C for 3 minutes; 35 cycles of: 30 sec at 94° C, 30 sec at 55 °C, 1 minute at 72°C; 5 minutes at 72°C. Following this program the thermocycler automatically maintained the amplicon products at 4°C.
  • One microliter aliquots from the amplification reactions are combined in 50 ⁇ L of deionized formamide containing 2 ⁇ L of the GS-400HD ROX labeled size standards (PE Applied Biosystems). Alternatively, 0.5 ⁇ L of each reaction product is combined with 25 ⁇ L of formamide and l ⁇ L of size standards. Bcr-abl analysis is done combining the amplified products from second round reactions 1-6. In addition, in preliminary experiments the second round reactions 1 + 4-6 are combined, fractionated and detected separately to allow differential interpretation of green peaks arising from the Abl 1-1 and the Bcr el-2 primers. Mixtures are heated to 95°C for 3 minutes, quick chilled and loaded on an ABI 310 capillary electrophoresis instrument.
  • the capillary runs at 60°C are initiated with 6 second injections and ran 24 minutes at 15 kV using the GS STR POP4 D lmL run module.
  • Data are collected using ABI 310 collection software (PE Applied Biosystems) and analyzed with IVS Technologies' dye matrices and IVS Technologies' sizing parameters using ABI GeneScan® (v2.0) software.
  • Data generated by testing cDNA from the t(9;22) positive control K562 cell line includes nine (9) prominent amplicon products between 50-400 basepairs (five blue peaks, three green peaks and a yellow peak; Figure 9).
  • This cell line has a characterized Bcr-abl translocation that generates a series of transcripts representing the
  • This assay generated a single minor yellow amplicon peak at 165bp from placental DNA samples that is specific for the Bcr b3-2 primer.
  • Genomic DNA and RNA ( " cDNA -minus) samples
  • Data generated from the genomic DNA samples, and the cDNA-minus K562 RNA sample, are a single prominent Abll-1 primer-specific peak of 154 basepairs.
  • cDNA from normal persons Data include a control abl (green peak) amplicon of approximately 93 basepairs representing the amplicon product from the Abll-1 4- Abl2-2 primer set ( Figure 9).
  • a control green peak of approximately 93 basepairs represents amplicon product from the Abll-1 4- Abl2-2 primer set and is used as a quality control in all cDNA containing samples to assure the integrity of the RNA and cDNA.
  • This abl gene product is somewhat smaller than the size predicted from distances calculated using the Accession numbers listed in GenBank database (97 basepairs). However, this product is reproducibly sized at 93 basepairs using the present assay system. Genomic DNA and cDNA(-) RNA-only controls did not yield a band at 93b ⁇ .
  • a green peak is evident at 154 basepairs in data generated testing genomic DNA and RNA samples.
  • the origin and identity of this product is not yet known. However, it is amplified using the Abl 1-1 primer, it does not represent a product from a t(9;22) translocation, and is seen in genomic DNA and not in other cDNA samples.
  • Other major green peaks, which are generated using the Bcr el-2 primer, are indicative of ela2 or ela3 bcr-abl translocation products arising from transcripts generated from translocations within the minor (m-bcr) breakpoint region.
  • Data from t(9;22)-positive K562 cDNA includes two additional Bcr el-2 primer specific peaks: A peak of approximately 187 basepairs (that represents an 51 a2 product amplified using the Bcr el-2 4- Abl 2-2 primers), and a peak of approximately 371 basepairs (predicted from GenBank sequences to be 375bp) that represents an ela2 product amplified using the Bcr el-2 4- Abl 3-2 primer pairs. It is interesting that sizes of several amplicon peaks are less than predicted by exactly 4 basepairs using several different primer pairs.
  • the 126 and 311 basepair products represent amplicon products from the Bcr b2-2 + Abl2-2 and Bcr b2-2 4- Abl3-2 primer sets, respectively.
  • the 62, 171 and 356 basepair peaks are Bcr b2-2 primer-specific. Although the specific identity of these amplicons have not been dete ⁇ nined, the 171 basepair product is 45 bases bigger than the 126bp peak, and the 356bp product is 45bp bigger than the 311bp peak. Therefore it is likely that these peaks represent amplicon produced from a common splice variant. They are not seen in data from genomic DNA or RNA.
  • K562 cDNA-specific amplicon peaks are internally consistent and redundant using these multiple multi-loci primer sets. These products represent the approximate distances expected for the control b3a2 (exon 14/exon 2) bcr-abl transcription product. The presence of blue peaks is diagnostic for the presence of Bcr b2 (exon 13) in the translocation transcription product.
  • the blue peak at 31 lbp (the product amplified between Bcr b2-2 4- Abl 3-2; Bcr exon 13 4- Abl exon 3 primers) is predicted to be 69bp larger than the yellow peak of 242bp (Bcr b3-2 4- Abl 3-2; Bcr exon 14 4- Abl exon 3).
  • the present invention provides a useful approach to standardized testing for a variety of clonal hematolymphoid malignancies.
  • the majority of clinical PCR-based testing performed prior to the invention utilizes assays that are internally validated and rely on quality controls maintained by the individual institutions.
  • the present integrated assay kit includes specific validated primer sets, testing parameters and size standards, and provides a PCR assay to replace many assays, including the RF-SBH B-& T-cell assay previously available (Oncor).
  • the present integrated assay system for semi-quantitative B-cell, T-cell and hematology testing is able to reduce costs and turnaround time, and provide additional useful information, while providing comparable diagnostic sensitivity and accuracy.
  • An interesting example of the additional information is provided in Example 4, above. An additional amplicon product of approximately 45bp is identified. This product is likely a splice variant, unlikely to be identified using conventional bcr-abl assays.
  • Multiple differentially-labeled primer sets will be used together to test for multiple hematologic malignancies, simultaneously targeting either multiple genes within a single locus (e.g., FRI and FR3 IgH primer sets), or simultaneously testing multiple genetic loci (FR3 IgH 4- TcRg 4- bcl2; or as demonstrated using the Bcr-abl assay, Example 4); multiple primer sets in accordance with the invention will simultaneously test for a variety of related or unrelated infectious disease agents or pathogens.
  • the present invention can be used to produce specific differentially-labeled primer sets to produce amplicon product simultaneously from the nucleic acid present in infectious agents.
  • the number of different amplicon products detected will increase as technology improves in detection. It has been shown that the present invention will identify at least four different labels.
  • the number of amplicons detected may be increased by further modifying the primer design, for example, by adding size- specificity to the label specificity currently employed using the invention.
  • improvement in the number and efficiencies of labeling reagents and moieties will increase the number of targets that can be examined using the invention, and improved algorithms and instrumentation will allow better discrimination between the emission spectra of labeling reagents, further expanding the capabilities and uses of the invention.
  • the ABI 377 gene sequencer was used for fractionation and detection in parallel with the 310 capillary electrophoresis instrument. This embodiment required pouring acrylamide gels, there were modifications in amplicon preparation and the total detection time for a sample was 2-3 hours instead of approximately 30 minutes. Although the performance characteristics of the present method using the 377 platform have not been rigorously examined, amplicon profiles appeared to be similar. However, in limited studies the data from the 310 capillary instrument appeared to be superior to the 377 slab gel instrument.
  • the present invention also demonstrated that the amplicon signal from cDNA is greater than the signal generated using genomic DNA (compare Figures 2, panel b, and 3, panel b, with Figures 2, panel c, and 3, panel c). Therefore, it is considered advantageous to test expressed transcripts using this assay system.
  • In vivo cDNA synthesis technology which produces stable cDNA in viable samples, will also improve the sensitivity of the assay system of the invention and eliminate costs and difficulties associated with handling and transporting RNA samples.
  • the present invention contemplates additional primer panels, including additional primers for the
  • RT-PCR bcr-abl assay and a T-cell panel targeting several TcR gene loci, to expand the neoplasms identified with this integrated assay system.
  • the present invention can be coupled with preparative capillary electrophoresis for gene discovery or for rapid preparation and sequencing of specific amplicon products related to antigen receptors. These embodiments would be analogous to cell sorting using flow cytometry. Such an approach is expected to provide a rapid means to identify patient-specific or allele-specific primers and probes useful for quantifying and monitoring residual disease.
  • Panel refers to the products combined for capillary electrophoresis detection and analysis. Primer designation indicates the origin of the peaks examined. Peak height is automatically measured by the instruments and expressed in relative fluorescence units. Size is determined by the instrument and expressed in basepairs. The ratio is determined by dividing the peak height of the clonal amplicon peak by the reference peak.

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Abstract

L'invention concerne un procédé servant à détecter un marqueur diagnostique dans un échantillon biologique. Selon un mode de réalisation, l'échantillon biologique comprend des séquences de codage d'ADN efficaces pour le diagnostic du marqueur diagnostique. On mélange l'échantillon à au moins trois amorces comprenant au moins deux amorces différentiellement marquées qui reconnaissent et qui se lient à au moins une région de séquence d'ADN intermédiaire dans l'ADN qui est efficace pour diagnostiquer le marqueur diagnostique. On incube ensuite le mélange dans des conditions suffisantes pour obtenir une amplification que l'on peut détecter de la région de séquence d'ADN intermédiaire. On analyse les produits de la phase d'amplification pour détecter le produit d'amplification individuel efficace pour le diagnostic du marqueur diagnostique.
PCT/US1999/025991 1998-11-06 1999-11-04 Procedes et compositions pour dosages diagnostiques Ceased WO2000028086A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004033728A3 (fr) * 2002-10-11 2004-08-12 Univ Erasmus Amorces d'amplification d'acides nucleiques pour etudes de la clonalite basee sur la pcr
JP2006501842A (ja) * 2002-10-11 2006-01-19 エラスマス・ユニバーシティー・ロッテルダム Ｐｃｒベースのクローン性研究のための核酸増幅プライマー
EP2460889A3 (fr) * 2002-10-11 2012-07-04 Erasmus Universiteit Rotterdam Amorces d'acides nucléiques pour les'études de la clonalité des translocations BCL2-IGH basées sur PCR
US8859748B2 (en) 2002-10-11 2014-10-14 Jacobus Johannes Maria van Dongen Nucleic acid amplification primers for PCR-based clonality studies
US10280462B2 (en) 2002-10-11 2019-05-07 Jacobus Johannes Maria van Dongen Nucleic acid amplification primers for PCR-based clonality studies

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