CA2157056A1 - Detection of human tumor progression and drug resistance - Google Patents

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

21 S70~

DETECTION OF HUMAN TUMOR
PROGRESSION AND DRUG RESISTANCE

BACRGROUND OF THE lNV~NLlON

The efficiency of cancer chemotherapy protocols tends progressively to decrease in inverse proportion to the target tumor'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., "Detection of Drug Resistance in Human Tumors by ln Vitro Enzymatic Amplification,: 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 Io6 tumor cells and cannot discriminate less than two to four fold changes, whereas drug resistant tumors may be indicated 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.

'- 21 57056 SU~ARY 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 quantative 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 Figure 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' and 3' ends of the gene sequence to be analyzed. See Kashani-Sabet, et al., supra.
Tumor cells for the PCR assay are obtained from patients's tissue or peritoneal fluid, and total RNA for use as a template is isolated as described.
To replicate a specific sequence which preferably includes a restriction site, the oppositely oriented primers are annealed to the RNA template. Addition of reverse transciptase 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 1 identifies target and primer sequences and restriction sites for eleven gene transcripts.

Oligonucleotide Primers of RNA
Expression in Drug Resistant Tumor Cells Transcript A-,.pliried Franment Location of the Oliqonucleotide in the Nucleotide Sequence*
Fre:dicled Re~lli.,lion Primers Probe Size (bp) Site 5'oligo 3'oligo nucleo- nucleo-tide tide DHFR 136 Ava ll 1301-1321 1406-1386 1364-1340 dTMP synthase 171 Pst I -3-21 168-146 122-101 T kinase 184 Hinf 1 58-83 242-219 141-119 DNA pol ~ 202 Hae lll 138-158 340-318 240-215 DNA pol,~ 108 Kpn 1 21-46 129-103 98-73 c-fos 121 Pst 1 908-927 1029-1010 985-961 c-myc 300 Alu 1 1-24 300-277 216-193 H-ras 273 Msp 1 1661-1680 2183-2202 1782-1763 Multidrug 332 Hph 1 16-39 342-321 201-180 Resistant (MDR) I

,~Actin 240 Bgl ll 25-44 269-245 155-132 Phosphglycerdle 166 Alu 1 1364-1386 1529-1507 1405-1427 Kinase (PGK) * See Journal of Clinical Laboratory Analysis, Vol. 3, No. 5 (August 1989) (In Press).

Figures 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 ~, 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'-5' oligoprimer complementary to DNA (bases 1301-1321) having the sequence CGG AGG TCC TCC CGC TGC TGT. #2 is the 5'-3' 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'-5' oligoprimer complementary to DNA
(bases -3 to 21) having the sequence GCC ATG CCT GTG GCC
GGC TCG GAG. #2 is the 5'-3' primer complementary to mRNA
(bases 146-168) having the sequence AGG GTG CCG GTG CCC GTG
CGG T. #4 (bases 101 to 122) is the probe for identifying the target sequence. The probe has the sequence AGG ATG
TGT GTT GGA TCT GCC CCA. The target sequence includes a PstI restriction site.
Oligoprimers and probes for the DNA polymerase ~ gene are shown in Fig. 4. #1 is the 5'-3' 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'-3' 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'-3' oligoprimer complementary to m-RNA (exon 1, bases 908-927) having the sequence ACG CAG ACT
ACG AGG CGT CA. #2 is the 5,-3, 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'-3' 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'-3' oligoprimer complementary to DNA or a probe for RNA (exon 2, bases 193-216). It has a sequence AGG AGC CTG CCT TTC
CAC AGA. #3 is a 5'-3' oligoprimer (exon 2, 277-300) having the probe sequence CGG TGT CTC CTC ATG GAG CAC CAG.
There is a base 87 AluI restriction site between 1 and 300.
The target sequence, bases 1-300 includes an Alu restriction site at base 87.
Oligoprimers and probes for the H-ras gene are shown in Figure 7. The amplified fragment stretches from base 1661 to base 2202 (541 DNA bases, 273 RNA bases). #1, a sense oligonucleotide, span bases 16610-1680 and contains the sequence: 5'-TGAGGAGCGATGACGGAATA-3'. #2 is an antisense oligonucleotide, encodes nucleotides 2183 to 2202 and has the sequence: 5'-GACTTGGTGTTGTTGATGGC-3'. #3 is the probe oligonucleotide spanning bases 1763-1782 and encodes the sequence: 5'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.
Oligoprimers and probes for the DNA polymerase ~ gene are:
5' GCT A~A GCT GGT GAG AAG TAT A

3' CTC ATC AGC ATC AAG GGC ATC AT
Probe TCC TGG CGT GCC TGA ACC AGC TTC GA
Oligoprimers and probes for the MDR1 gene are:
5' AGC AGC TGA CAG TCC AAG AAC A

3' GTT GCT GCT TAC ATT CAG GTT TC
Probe AGA GAC ATC ATC TGT AAG TCG G
Table II relates some of the several genes useful in this invention to chemotherapeutic agents.

TABLE II

Gene Cancer Chemotherapeutic Aqents TS Cycle DHFR Methotrexate (MTX) dTMP Synthase Cisplatin, 5FUra, FdUrd Thymidine Kinase Cisplatin, MTX, 5FUra, FdUrd DNA Repair Enzymes DNA polymerase ~ Cisplatin DNA polymerase ~ Cisplatin, araC, alkylating agents, some natural products, and X-ray Radiation Oncoqenes c-fos Cisplatin c-myc Cisplatin, MTX, araC,VP-16 H-ras Cisplatin Multidruq Resistance Genes MDR I Adriamycin, Actinomycin D
Topoisomerase II colchicine, Vinblastine, Vincristine, daunorubicin, VP-16, VM-26 and mithryamcin Gluthathione-S Transferase Alkyalting agents (GST) DESCRIPTION OF PREFERRED EMBODIMENTS
The preferred embodiments of the invention utilize the DNA polymerase ~ and ~ genes, the dTMP gene, the DHFR gene, the MDR gene and the c-fos, c-myc and H-ras oncogenes.

The DNA polymerase ~ 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 ~ 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 ln vitro and by gene amplification ln vivo. 2 Pattern Difference Between The DNA of DNA Polymerase ~ From Normal and Cancer Tissues Figs. 8-11 depict EcoRI digestion for Southern analyses of DNA polymerase ~ DNA from four types of human cancer.
Fig. 8 is a Southern analysis comparison of the DNA of DNA polymerase ~ DNA from 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.

1 Bertino, J.R., "Toward Selectivity in Cancer Chemotherapy: The Richard and Hinda Rosenthal Foundation Award Lecture," Cancer Res. 39:293-304 (1979).
2 Scanlon, K.J., et al., supra; Lu, Y., et al., "Biochemical and Molecular Properties of cisplatin-Resistant A2780 Cells Grown in Folinic Acid," J.Biol.Chem. 283:4891-4894 (1988).

Fig. 9 is a Southern analysis of the DNA of the DNA
polymerase ~ 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.
Patients HS was treated with cisplatin in combination with cytoxane. The polymerase ~ 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 ~ 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 (BC13) 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 lane relate to drug resistant and drug sensitive human breast tissues.
The Fig. 11 Southern analysis shows that the DNA of the DNA polymerase ~ 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) lanes 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 ~ (Fig. 12a) and DNA polymerase (Fig. 12b).
Figure 12a shows by Southern analysis that the restriction enzyme pattern of the DNA from DNA polymerse is similar for the normal (N 1-5) and colon carcinoma (T 1-5) samples.
Figure 12b, shows a Southern analysis of the restriction enzyme patterns of the DNA of DNA polymerase ~, the tumor samples Tl-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 - 2~1 57G56 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 ~ are prepared. The DNA polymerase ~
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 signals 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 E~ se and of tumor resistance to chemotherapy.

Claims (7)

1. A process which comprises:
(a) providing a transcript of a gene, said gene having an intron flanked by first and second exons, said transcript including mRNA and DNA from which said mRNA was transcribed, said mRNA having a first portion complementary only to a sequence of said first exon and an abutting second portion complementary only to a sequence of said second exon;
(b) providing first and second PCR primers, said first PCR primer being complementary to each of said first portion of mRNA and to said sequence of said first exon complementary thereto, said second PCR primer being complementary to each of said abutting portio of said mRNA and to said sequence of said second exon complementary thereto;
(c) annealing said primers to said MRNA and thereafter adding reverse transcriptase to provide a double stranded DNA molecule, one strand of which has the sequence of said mRNA and the other strand of which is complementary to said sequence of said MRNA;
(d) subjecting said double stranded DNA molecule to polymerase chain reaction amplification utilizing said first and second primers to simultaneously amplify (i) said double stranded DNA molecule and (ii) said template DNA;
wherein said amplified double stranded DNA which has no sequence complementary to said DNA of said intron and is of substantially lower molecular weight than said amplified double stranded DNA which includes DNA of said intron;
(e) utilizing the difference in molecular weight to separate said amplified double standard DNA molecule substantially free of said amplified intron DNA, and (f) denaturing said separated double stranded DNA
molecule to provide an amplification product of said mRNA
free of DNA of said intron.

2. A method as defined by claim 1 in which said transcript of a gene is a transcript of a human oncogene.

3. A method as defined by claim 1 in which said transcript of a gene is a transcript of a human H-ras ocogene or a human c-myc ocogene.

4. A method as defined by claim 1 in which said transcript of a gene is a transcript of a human c-myc gene.

5. A method as defined by claim 1 in which said gene is a transcript of a human c-myce gene, said first primer has the sequence of SEQ ID NO: 1 and said second primer has the sequence of SEQ ID NO: 2.

6. A method as defined by claim 1 in which said transcript of a gene is a transcript of a human H-ras ocogene.

7. A method as defined by claim 1 in which said gene is a transcript of a human H-ras gene, said first primer has the sequence of SEQ ID NO: 4 and said second primer has the sequence of SEQ ID NO: 5.