Note: Descriptions are shown in the official language in which they were submitted.
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.
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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
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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.
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TABLE 1
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.
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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
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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
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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.
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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).
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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
21 51056
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
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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.
