WO1991015600A1 - Detection of minimal residual disease in lymphoid malignancies - Google Patents

Abstract

A method for detection of minimal residual disease in lymphoid malignancies by the following steps: (i) amplifying DNA from an original tumor using universal J and V primers in a polymerase chain reaction; (ii) preparing a single stranded probe from a discrete fragment of the amplification product of step (i); (iii) amplifying DNA from a test specimen suspected of containing residual disease with the same J and V primers in a polymerase chain reaction; (iv) hybridizing the products of steps (ii) and (iii); (v) subjecting the hybridization product to S1 nuclease digestion; and (vi) determining the presence or absence in the digestion product of a sequence of substantially the same length as the probe prepared in step (ii).

Description

DETECTION OF MINIMAL RESIDUAL DISEASE IN LYMPHOID MALIGNANCIES

This application is a continuation of United States application Serial No. 501,496 filed 30 March 1990.

FIELD OF INVENTION This invention relates to a method for detecting minimum residual diseases (MRD) in lymphoid malignancies.

BACKGROUND OF INVENTION One of the major problems in treating lymphoproliferative disease is the diffuculty in detecting minimal residual disease (MRD) . The sensitivity of the human eye in examining a bone marrow aspirate smear is not great. To detect a malignant population immunologically, approximately 10% of the cells have to belong to the malignant clone. Enhancement of the immunologic sensitivity, such as by K-S (Kolmogorov-Smirnov) curves, is sometimes possible, but even with this technique, the sensitivity can be as low as 5%, and this method will only work with surface immunoglobulin positive tumors. Molecular biology techniques, such as gene rearrangement studies by Southern hybridization, also have only enough sensivitivy to detect a clone that comprises between 2-5% of a sample.

The polymerase chain reaction (PCR) has been used to detect MRD in lymphoproliferative lesions. (Stetler-Stevenson, et al., Blood 72;1822-1825 (1988) ; Crescenzi, et al. Proc. Nat. Acad. Sci. USA 85:4869-4873 (1988).) However, these studies have been most successful only in those lesions, such as fcliicuiar lymphoma or Bur iι_ 'Ξ j.yιπρhoma, where a well-defined chromosomal translocation exists, and it is possible to amplify across this translocation. In these cases, if the translocation is present, amplified material will be present; if the sought after translocation does not exist, no amplified genetic material will be generated.

Unfortunately, most lymphoid neoplasms do not have identifiable and will characterized chromosomal translocations. Therefore, attempts have been made to perform "generic" PCR, using universal primers. These studies utilize the fact that immunoglobulin and/or T-cell receptor genes undergo clonal somatic rearrangement in lymphoid neoplasms; if the DNA has not undergone rearrangement, the primers will be too far apart in the genome to form an amplifiable product by PCR. Several groups have attempted to detect MRD amplifying for either the T-cell receptor ₇-chain gene, or for the immunoglobulin heavy chain gene. These latter methods require sequencing and then synthesis of a clonospecific oligonucleotide probe, steps which are not performed in every laboratory and can take weeks to perform.

The method of Yamada, et al. Proc. Nat. Acad. Sci. USA 8j5:5123-5127 (1989) involves amplifying a rearranged immunoglobulin heavy chain gene, which is present in all B- and pre-B-cell neoplasms, diseases which comprise more than 90% of lymphoid neoplasms. In this method, a universal JJJ primer, one that will hybridize to all six JJJ sequences, and a universal Vg primer, which recognizes the conserved framework region 3 (FR3) of the variable region, are used to amplify the rearranged immunoglobulin heavy gene, in which the JJJ and FR3 regions are brought within 200 base pairs of each other. The amplified product is then sequenced, and a clonospecific oligonucleotide probe is synthesized that recognizes the unique CDR III region of the gene which is the portion of the rearranged DNA that represents the highly variable junction the of the DNA splicing. Different tumors, because of the different nucleotide insertions in the junctional region, produced different length products.

The invention provides a simple method for the detection of MRD. The method is specific for a particular tumor without the need for sequencing the junctional region and subsequent manufacture of clonospecific oligonucleotide probes.

SUMMARY OF THE INVENTION Pursuant to this invention both the original tumor specimen and the test specimen are amplified with identical PCR primers. The amplified product from the original tumor is used as a probe for the amplification product of the test specimen. Hybrids will form because both of the amplification products are produced with identical primers thus providing complementary end portions. However, internal sequences will hybridize only if the amplified test specimen includes MRD sequences since only such sequences will share the unique V-J-D rearrangement with the accompanying N-Segments that characterize the tumor of interest.

If this hybridization mixture is then subjected to Si nuclease digestion, only perfectly matched hybrids will remain intact, whereas those with mismatches will be cleaved into smaller pieces. When the digestion product is run on a gel and subjected to autoradiography, a band will appear at the position of the full length product only if residual tumor is present. This procedure is illustrated by Figure 1.

All of the methods used in the practice of this invention are well established techniques in molecular biology, and can be found in any molecular biology laboratory manual. See e.g. , Sambrook, J. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press (1989) . EXEMPLIFICATION OF THE INVENTION The invention will be exemplified in part by reference to Figure 1. In general, the invention may comprise the following five steps:

1. Isolate DNA from the original tumor sample and amplify by PCR using universal J and V primers. See, Yamada, et al., supra.

2. Run the amplification product on a gel, cut a discrete band, and extract the DNA.

3. Prepare by any known means a labelled single stranded probe from the DNA extracted in step 2. For example, a single stranded labelled probe can be made directly using the transcription initiators of the plasmid and ³²S labelled nucleotides. The labelled product should be phenol-extracted, ethanol precipitated and resuspended.

4. Process specimen to be tested for MRD by repeating the procedure of step 1. The PCR product should be phenol-extracted, ethanol precipitated, and resuspended.

5. As generally illustrated by Figure 1, denature and hybridize the products of steps 3 and 4, preferably at about 60^βC. Perform S^ nuclease digestion, precipitate product and run on a polyacrylamide gel. If a tumor is present in the specimen a band as indicated in the left column of the Figure 1 gel will appear at the length of the discrete band cut in step 2, usually about 120-200 bp, depending on the specific tumor. If no tumor is present no such band will appear. Shorter bands produced by Sj nuclease digestion elimination of a mismatch region may appear as illustrated b the right column of the Figure 1 gel.

Claims

CLAIMS :

1. A method for detection of residual disease in lymphoid malignancies which comprises

(i) amplifying DNA from an original tumor using universal J and V primers in a polymerase chain reaction;

(ii) preparing a single stranded probe from a discrete fragment of the amplification product of step (i) ;

(iii) amplifying DNA from a test specimen suspected of containing residual disease with the same J and V primers in a polymerase chain reaction;

(iv) hybridizing the products of steps (ii) and (iii) ;

(v) subjecting the hybridization product to Si nuclease digestion; and

(vi) determining the presence or absence in the digestion product of a sequence of substantially the same length as the probe prepared in step (ii) .

2. A method as defined by claim 1 in which the digestion product is subjected autoradiograph to determine the presence or absence in the digestion product of said sequence of substantially the same length as probe prepared in step (ii) .

3. A method as defined by claim 1 in which said probe prepared in step (ii) is prepared from a DNA fragment about 120 to about 200 base pairs in length.

4. A method as defined by claim 1 in which the amplification in steps (i) and (ii) is accomplished by the polymerase chain reaction.