Abstract:
Changes in tumor cell RNA and DNA are utilized to detect the progression and the temporal changes in resistance to chemotherapy in human tumors.

Description:
This application is a continuation-in-part of copending application Ser. No. 234,096 filed Aug. 19, 1988 as a continuation-in-part of application Ser. No. 046,127 filed May 5, 1987 and abandoned Dec. 5, 1988. 
    
    
     BACKGROUND OF THE INVENTION 
     The efficiency of cancer chemotherapy protocols tends progressively to decrease in inverse proportion to the target tumor&#39;s progressive increase in drug resistance. Accordingly, early detection of drug resistance would significantly benefit the development, choice and timing of alternative treatment strategies. Currently, the multidrug resistant (MDR) gene offers a potential for monitoring tumor resistance to some natural agents such as the vinca alkaloids, Vincristine and Vinblastine; antibiotics such as Daunorubicin, Actinomycin D, Doxorubicin, Mitomycin C, Etoposide (VP-16), Teniposide (VM-26) and Mithramycin. 
     Amplification of genes associated with drug resistance has been monitored by a modified polymerase chain reaction (PCR) assay, as described in Kashani-Sabet, et al., &#34;Detection of Drug Resistance in Human Tumors by in Vitro Enzymatic Amplification,&#34; Cancer Res. 48:5775-5778 (1988). Acquired drug resistance has been monitored by the detection of cytogenetic abnormalities, such as homogeneous chromosome staining regions and double minute chromosomes. 
     Several shortcomings attend these procedures. Gene amplification techniques other than PCR are applicable only to DNA, require at least 10 6  tumor cells and cannot discriminate less than two to four fold changes, whereas drug resistant tumors may be indicated by lower gene amplification levels. Drug resistance has been manifested by tumors in the absence of gene amplification or cytogenetic abnormalities. The detection of tumor progression by imaging lacks reliability and precision. 
     No efficient, generally applicable non-invasive procedure for the early detection of or for monitoring the changes in drug resistance over time is presently known. 
     SUMMARY OF THE INVENTION 
     This invention utilizes changes in tumor cell RNA and DNA to detect the progression and the temporal changes in resistance to chemotherapy of human tumors. Such changes are evidenced, for example, by qualitative and quantitative differences in RNA and DNA and by the differences and degree of differences between the Southern analysis patterns of DNA from specific cancer cell genes. 
     The invention also includes the identification of human cancer marker genes characterized by unique gene transcript DNA patterns and pattern changes revealed, for example, by Southern analysis as cells pass progressively from a normal to a cancerous or drug resistant state. Procedures for the clinical monitoring of tumor progression and of the beginning and progression of drug resistance by comparison of DNA patterns of sequential tumor gene transcripts are described. 
     DESCRIPTION OF THE PCR ASSAY 
     FIG. 1 is a schematic diagram outlining the steps of a modified PCR assay useful in the invention. Two converging, preferably about 15 to 25 base, oligoprimers oriented in opposite directions, are provided for the 5&#39; and 3&#39; ends of the gene sequence to be analyzed. See Kashani-Sabet, et al., supra. 
     Tumor cells for the PCR assay are obtained from patients&#39; tissue or peritoneal fluid, and total RNA for use as a template is isolated. 
     To replicate a specific sequence which preferably includes a restriction site, the oppositely oriented primers are annealed to the RNA template. Addition of reverse transcriptase yields first strand polymerization. Cycles of denaturation, annealing, and polymerization ensue upon addition of heat-stable DNA Polymerase. This process is continued for a plurality of rounds. Inclusion of ribonuclease A after the completion of round one tends to eliminate RNA which may compete for primer binding. 
     In general, the amplified sequence, or a restriction fragment thereof, is detected in the reaction product by hybridization with a complementary probe. The amplified DNA is cut with a restriction enzyme. The resulting fragments are separated by gel electrophoresis. The gel is then laid on a piece of nitrocellulose, and a flow of an appropriate buffer is set up through the gel, perpendicular to the direction of electrophoresis, toward the nitrocellulose filter. The flow causes the DNA fragments to be carried out of the gel onto the filter, where they bind, so that the distribution of the DNA fragments in the gel is replicated on the nitrocellulose. The DNA is then denatured and fixed onto the filter. A complementary radioactively labeled probe is then hybridized to the DNA sequence on the filter. Autoradiography of the filter identifies which fragment or fragments contain the sequence under study, each fragment being identified according to its molecular weight. A variation on this technique is to hybridize and do autoradiography directly in the gel, rather than on a nitrocellulose filter. 
     Table I identifies target and primer sequences and restriction sites for eleven gene transcripts. 
     
                                           TABLE I__________________________________________________________________________Oligonucleotide Primers of RNAExpression in Drug Resistant Tumor Cells                Location of the Oligonucleotide                in the Nucleotide Sequence*     Amplified Fragment                Primers     Predicted          Restriction                5&#39;oligo                      3&#39;oligoTranscript     Size (bp)          Site  nucleotide                      nucleotide                            Probe__________________________________________________________________________DHFR      136  Ava II                1301-1321                      1406-1386                            1364-1340dTMP synthase     171  Pst I -3-21 168-146                            122-101T kinase  184  Hinf I                58-83 242-219                            141-119DNA pol α     202  Hae III                138-158                      340-318                            240-215DNA pol β     108  Kpn I 21-46 129-103                            98-73c-fos     121  Pst I 908-927                      1029-1010                            985-961c-myc     300  Alu I  1-24 300-277                            216-193H-ras     273  Msp I 1661-1680                      2183-2202                            1782-1763Multidrug 332  Hph I 16-39 342-321                            201-180Resistant (MDR) Iβ Actin     240  Bgl II                25-44 269-245                            155-132Phosphglycerate     166  Alu I 1364-1386                      1529-1507                            1405-1427Kinase (PGK)__________________________________________________________________________ *See Journal of Clinical Laboratory Analysis, Vol. 3, No. 5 (August 1989) (In Press). 
    
     FIGS. 2-7 are schematic maps which identify the target, primer and probe sequences and the position of the primers for use in PCR assays of the DHFR, dTMP, DNA polymerase β, c-fos, c-myc, and H-ras genes. Optimum amplification requires selection of appropriate primers for each selected gene sequence. 
     As shown in FIG. 2, DHFR-3 (#3) is the 3&#39;-5&#39; oligoprimer complementary to DNA (bases 1301-1321) having the sequence CGG AGG TCC TCC CGC TGC TGT. #2 is the 5&#39;-3&#39; oligoprimer complementary to mRNA (bases 1386-1406) having the sequence GAG CGG TGG CCA GGG CAG GTC. The target sequence bases 1301-1406 includes an Ava 2 restriction site. The probe for identifying the target sequence has the sequence GTT CTG GGA CAC AGC GAC GAT GCA. 
     Oligoprimers and probes for the dTMP synthase gene are shown in FIG. 3. The target sequence includes bases -3 to 168. #2 is the 3&#39;-5&#39; oligoprimer complementary to DNA (bases -3 to 21) having the sequence GCC ATG CCT GTG GGC CGG CCT TCC CCG GAG. #3 is the 5&#39;-3&#39; primer complementary to mRNA (bases 146-168) having the sequence AGG GTG CCG GGG TTG CCC GTG CGGT. #4 (bases 101 to 122) is the probe for identifying the target sequence. The probe has the sequence AGG ATG TGT TGT GGA TCT GCC CA. The target sequence includes a PstI restriction site. 
     Oligoprimers and probes for the DNA polymerase β gene are shown in FIG. 4. #1 is the 5&#39;-3&#39; oligoprimer complementary to DNA (exon 1, bases 21-46) having a sequence of GGA GCT GGG TTG CTC CTG CTC CCG T. #2 is the 5&#39;-340  oligoprimer complementary to m-RNA (exon 1 bases 103-129) having a sequence GCC TTC CGT TTG CTC ATG GCG GCC T. #3 is the probe (bases 73-98) for identifying the target sequence bases 21 to 129. The probe sequence is ACC AGG GAC TAG AGC CCT CTC CCA G. The target sequence includes a KpnI restriction site. 
     Oligoprimers and probes for the c-fos gene are shown in FIG. 5 #1 is the 5&#39;-3&#39; oligoprimer complementary to m-RNA (exon 1, bases 908-927) having a sequence ACG CAG ACT ACG AGG CGT CA. #2 is the 5&#39;-3&#39; oligoprimer complementary to DNA (exon 1, bases 1010-1029) having a sequence CTG CGC GTT GAC AGG CGA GC. The target sequence includes bases 908 to 1029. #4 is the probe for identifying the target sequence (bases 961-985) has the sequence TGA GTG GTA GTA AGA GAG GCT ATC. The target sequence includes a Pst I restriction site. 
     Oligoprimers and probes for the c-myc gene are shown in FIG. 6. #1 is a 5&#39;-3&#39;0 oligoprimer complementary to either DNA or RNA (exon 1, bases 1-24) having a sequence of TCC AGC TTG TAC CTG CAG GAT CTG. #2 is the 5&#39;-3&#39; oligoprimer complementary to DNA or a probe for RNA (exon 2, bases 193-216). It has a sequence GAC CAC CGA GGG GTC GAT GCA CTC T. #3 is a 5&#39;-3&#39; oligoprimer complementary to only RNA (exon 2, 277-300) having the sequence AGG AGC CTG CCT CTT TTC CAC AGA. There is a base 87 AluI restriction site between 1 and 300. The target sequence, bases 1-300 includes an Alu I restriction site at base 87. 
     Oligoprimers and probes for the H-ras gene are shown in FIG. 7. The amplified fragment stretches from base 1661 to base 2202 (541 DNA bases, 273 RNA bases). #1, a sense oligonucleotide, spans bases 1661-1680 and contains the sequence: 5&#39;-TGAGGAGCGATGACGGAATA-3&#39;. #2 is an antisense oligonculeotide, encodes nucleotides 2183 to 2202 and has the sequence: 5&#39;-GACTTGGTGTTGTTGATGGC-3. #3 is the probe oligonucleotide spanning bases 1763-1782 and encodes the sequence: 5&#39;-ACCTCTATAGTAGGGTCGTA-3. #1 and #2 are used as primers for the polymerization assay. #3 is used as the probe to detect the amplified target sequence. The 273 base RNA sequence contains a cleavage site for MspI at position 1786 which yields two fragments of 136 and 137 base pairs in length upon digestion. Only the 171 base pair cleavage fragment contains the sequence complementary to #3. Hybridization of the digested PCR product with the end labeled probe should yield only one band. 
     Table II relates some of the several genes useful in this invention to chemotherapeutic agents. 
     
                       TABLE II______________________________________Gene             Cancer Chemotherapeutic Agents______________________________________TS CycleDHFR             Methotrexate (MTX)dTMP Synthase    Cisplatin, 5FUra, FdUrdThymidine Kinase Cisplatin, MTX, 5FUra, FdUrdDNA Repair EnzymesDNA polymerase α            CisplatinDNA polymerase β            Cisplatin, araC, alkylating            agents, some natural products,            and X-ray RadiationOncogenesc-fos            Cisplatinc-myc            Cisplatin, MTX, araC, VP-16H-ras            CisplatinMultidrug Resistance GenesMDR I            Adriamycin, Actinomycin DTopoisomerase II colchicine, Vinblastine,            Vincristine, daunorubicin,            VP-16, VM-26 and mithramycinGlutathione-S    Alkylating agentsTransferase (GST)______________________________________ 
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     The preferred embodiments of the invention utilize the DNA polymerase α and β genes, the dTMP gene, the DHFR gene, the MDR gene and the c-fos, c-myc and H-ras oncogenes. 
     The DNA polymerase β gene has been shown to be elevated in drug resistant tumor cells treated with antimetabolites, e.g., ara-C, alkylating agents, some natural products, e.g., VP-16, and cisplatin. Changes in the DNA of DNA polymerase β evidence the progression of tumor formation and temporal changes in drug resistance. 
     Most chemotherapeutic agents damage DNA directly or indirectly. The dTMP synthase cycle is the sole de novo source of thymidine, the availability of which is rate limiting in DNA synthesis and the repair of DNA damage. The dTMP cycle accordingly has been a selected target for several cancer therapeutic agents, such as methotrexate (MTX), 5-fluorouracil (5-FUra) and fluorodeoxyuridine (FdUrd). 1  Tumor cells resistant to cisplatin display increased levels of dTMP synthase by elevated gene expression in vitro and by gene amplification in vivo. 2   
    
     Pattern Difference Between The DNA of DNA Polymerase β From Normal and Cancer Tissues 
     FIGS. 8-11 depict EcoRI digestion for Southern analyses of DNA polymerase β DNA from four types of human cancer. 
     FIG. 8 is a Southern analysis comparison of the DNA of DNA polymerase β DNA from a human colon carcinoma HCT8 cell lines sensitive (S), and resistant (D) to cisplatin, normal colon tissue (N) and colon carcinoma tissue from a patient (PK) that failed cisplatin and 5 fluorouracil chemotherapy. The lane PK pattern from the carcinoma cells includes a band at a 5.5 Kb not present in the normal tissue pattern. 
     FIG. 9 is a Southern analysis of the DNA of the DNA polymerase β from the cancer tissue of six human ovarian carcinoma patients. Patients DM, MD, TS, BD and DL) were treated with cisplatin in combination with 5 fluorouracil. Patient HS was treated with cisplatin in combination with cytoxane. The polymerase β DNA from all patients except DM lost a high molecular weight band (20Kb) upon development of resistance to chemotherapy. A low molecular weight band (5.5 Kb) was lost in 3 of the 6 drug resistant patients, i.e., patients DL, BD, and D. In FIG. 9, lane D pertains to a drug resistant ovarian cell line and lane S pertains to drug sensitive ovarian cell line. 
     The FIG. 10 Southern analysis shows that the DNA from the DNA polymerase β gene from tissue from four breast carcinoma patients BC1-BC4 is characterized by an additional band at 5.2 Kb and at 5.5 Kb as compared with normal tissue (NBT). Tissue from three of the four patients (BC 1-3 ) yielded an additional band at 5.5 Kb. The 5.2 Kb bands provide a marker to discriminate normal from neoplastic tissue. The D and S lanes relate to drug resistant and drug sensitive human breast tissues. 
     The FIG. 11 Southern analysis shows that the DNA of the DNA polymerase β gene from human leukemia cells resistant to cisplatin (DDP), VP-16 or MTX has additional bands at about 15 Kb as compared to the same gene from normal tissue(s) lane 5. These band changes provide markers for drug resistance in neoplastic cells, including human leukemia cells. A like band change is not observed in the case of cells resistant to ara-C. 
     The foregoing experiments utilized normal tissue and untreated tissue as standards representing drug sensitive cells. Cells obtained from a patient prior to treatment and stored provide an internal drug sensitive cell standard. 
     Normal and colon carcinoma tissues were obtained from five separate patients and analyzed by the methods previously described for their restriction enzyme fragment pattern for DNA polymerase α (FIG. 12a) and DNA polymerase β (FIG. 12b). 
     FIG. 12a shows by Southern analysis that the restriction enzyme pattern of the DNA from DNA polymerase α is similar for the normal (N 1-5) and colon carcinoma (T 1-5) samples. 
     FIG. 12b, shows a Southern analysis of the restriction enzyme patterns of the DNA of DNA polymerase β, the tumor samples T1-T5 lack bands at 12 Kb and 15 Kb, present in the normal tissue samples (N 1-5). Two bands at 5.2 Kb and 5.5 Kb, not present in the normal tissue samples, are present in the colon cancer samples. 
     This invention includes visualization of temporal changes in the restriction enzyme fragment patterns and fragment pattern differences between normal, sensitive, and drug resistant tissue to monitor all stages of the progression of human tumor growth and of drug resistance. 
     Labelled nucleotide sequences in Southern or Northern analysis bands are routinely quantified by comparison of signal intensity from such bands with a standard. When the amount of the target sequence is quite small, such quantification techniques may be inadequate. 
     Pursuant to this invention the quantification of small DNA samples from tissue or cells is readily and efficiently accomplished. 
     The intensity of the signal from a labelled target sequence in a given Southern analysis band is a function of the number of rounds of amplification required to yield a band of preselected or predetermined signal intensity. See, e.g., Kashani-Sabet, supra. 
     This invention entails the determination of a set of standards which identify quantatively the signal intensity from a unique or preselected Southern analysis band after a selected number of PCR amplification rounds. 
     Comparison of the signal intensity of like Southern analysis bands derived from patient cell or tissue samples similarly amplified for a like number of rounds provides a ready and efficient monitor of the progress of both tumor size and tumor drug resistance. 
     EXAMPLE 
     Tissue or cell samples are prepared with known, progressively increasing quantities of a gene transcript which yields a unique cancer marker band. For example, colon carcinoma tissue or cell samples containing progressively increasing specific amounts of the transcript of DNA polymerase β are prepared. The DNA polymerase β target DNA sequence in each sample is PCR amplified under like conditions for each of a plurality of predetermined rounds. The intensity of the signal from each amplified sample after each predetermined plurality of rounds is recorded to provide a set of standards. Each standard in the set is the quantified intensity of the signal after one of the predetermined pluralities of amplification rounds. 
     DNA from a patient tissue cell sample is subjected to Southern analysis by the method used to prepare the standards. The intensity of the signal from the unique marker band after amplification for one or more of the pluralities of rounds used to prepare the standards is measured. Comparison of these signal intensity measurements from DNA of the patient sample with the standards provides a monitor of the existence and progress of tumor size and of tumor drug resistance of the patient samples. 
     The absence and continuing absence of a signal from the patient samples indicates freedom at least from the type of tumor to which the analyses apply. The initial appearance of a signal from a patient sample at a given amplification round level is evidence of incipient or appearing drug resistance. Increase in the magnitude of the signal in subsequently taken samples provides a temporal monitor of tumor progression per se and of tumor resistance to chemotherapy.