141141
`
`K-Ras Point Mutations in the Blood.
`
`Plasma DNA of Patients with
`
`Colorectal Tumors
`
`Valeri Vasyukhinl, Maurice Strounl, Pierre Maurice2
`Jacqueline Lyauteyl, Christine Lederreyl
`and Philippe Ankerl-3
`
`[Departmenl of Plant Physiology. Pavilion des Isotopes, Faculty of Science
`University of Geneva, 20, hd d'Yvny 12] 1 Geneva, S witzerlnmi
`20ncahemaloiogy Division. University Hospital of Geneva. 24 rue Micheli-du Crest
`1205 Geneva. Switzerland
`
`INTRODUCTION
`
`Oncogene mutations are found with varying frequency among several
`tumor types and sometimes play an important role in their development
`(5,2). The identification of these mutations could be very important for the
`early detection of several cancers (4, 21). Normally, this is done by inves-
`tigating biopsy specimens which sometimes entails surgery or at least an in-
`vasive test. An easily accessible human material is blood plasma in which in-
`creased levels of DNA have been found in patients suffering from various
`malignant diseases (12, 16). The suggestion that an increased amount of
`DNA might originate from tumor cells was supported by the finding that
`some biophysical characteristics of cancer cell DNA (3) were also detected
`in the DNA extracted from the plasma of cancer patients (17). We have
`therefore investigated the possibility of finding activated oncogenes in the
`plasma of cancer patients which could be useful for the diagnosis and
`monitoring of various kind of malignancies. Among the different modifica-
`tions observed in oncogenes. point mutations of the ras genes are particular-
`ly significant. For instance. up to 50% of the colon adenocarcinoma tumors
`harbor a mutation in the ras genes, most of them taking place in codon l2 of
`K-ras (6, 9). These mutations usually occur during the transition from
`
`Foundation Exhibit 1053
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`I42
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`K—ras MUTATIONS IN COLON CANCER
`
`adenoma I to adenoma 11, before loss or mutation of the p53 gene (11). that
`is. relatively ‘early in the evolution of the tumor. For this reason we have
`screened the blood plasma DNA in search of mutations in codon 12 of the K-
`ras gene among 15 patients with intestinal cancer (13 undergoing ablative
`surgery shortly after blood collection).
`
`MATERIALS AND METHODS
`
`Materials. Concanavalin A-Sepharose (Pharmacia Biotech AG, Dilben-
`dorf, Switzerland). PCR Thermal Cycler 480 and Taq I polymerase (Perkin
`Elmer Cetus, Kuesnacht, Switzerland). Oligonucleotide primers and probes
`(Oncogene Science, Uniondale, NY.) Mutation-specific oligonucleotides
`(Microsynth, Windisch, Switzerland). Polyacrylamide (Fluka. Buchs. Swit-
`zerland). Zeta probe membranes (Bio-Rad, Hercules, CA). 3’end labeling
`kit and 32P-ddATP(Amersham, England).NuSieve 3:1 agarose (FMC, Rock-
`land ME)
`
`Patients and sources of DNA. Blood samples (20 to 30 ml) were collected
`on heparin from 15 informed and consenting patients with different stages of
`colorectal adenocarcinoma. The patients were not receiving any kind of can-
`cer drug during this period. The DNA was extracted from the blood cells
`and plasma, and for 13 of the patients, also from paraffin-embedded tumor
`samples. Blood was also taken from 10 healthy controls where an amount of
`400 ml had to be collected for plasma DNA isolation.
`
`DNA extraction. The tumor (10) and blood cell DNA (1) were extracted
`as previously described. The plasma DNA proved to be more delicate to ex-
`tract (16, 17). In brief, the plasma was subjected to phenol, ether and
`chloroform treatments. After dialysis against
`1 x SSC (0.15 M Sodium
`chloride, 0.015 M trisodium citrate), the material was passed through a Con-
`canavalin A-Sepharose column to remove the polysaccharides and then
`centrifuged in a CszSO4 gradient. After this centrifugation the DNA often
`formed two bands, one band in a normal position, the other higher. the
`DNA of this lighter fraction being still strongly attached to proteins. In these
`cases the DNA from each band was investigated separately.
`
`Ras gene amplification. Purified DNA (10—100 ng) was subjected to PCR
`amplification of the first exon of the K-ras gene in a volume of 100 U]. The
`primers were 5'-GACTGAATATAAACITGTGGTAGT—3' and
`5’-CTAT'I‘GTI‘GGATCATA'ITCGTCC-3’. The amplifications were per-
`formed in a buffer containing 50 mM KCl, 10 mM Tris-HCl at pH 8.3, 200
`
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`K-rus MUTATIONS IN COLON CANCER
`
`[43
`
`mM of each nucleotide. 1.8 mM MgC12. 0.2 uM of each primer and 2.5 units
`of AmpliTaq DNA polymerase. Thirty five cycles were performed for tumor
`and blood cell DNA and 45 cycles for plasma DNA (94° C for 1 min. 59°C
`for 1,5 min. 72°C for 1 min. The last cycle was followed by a 7 min extension
`at 72°C). The amplification products were analysed by electrophoresis in 8%
`polyacrylamide gel.
`
`Mutation detection. Two different methods were used for each sample :
`Hybridization of the PCR products with mutation specific oligonucleotide
`probes (20) and a more sensitive technique based on PCR amplification with
`point mutation specific primers also called PCR amplification for specific a1-
`leles (PASA)(14).
`According to the first method the PCR products, in equal quantities, were
`spotted on Zeta-probe membranes and hybridized with oligunucleotides
`specific for wild-type or mutant K-ras. (20). The oligonucleotide probes
`were end labeled with 32F ddATP. To discriminate between perfect and mis-
`matched hybrids, the final washing of the membrane was done in a solution
`containing 3 M tetramethylammonium chloride, 50 mM Tris-HCl at pH 8.0,
`0.2 mM EDTA. 0.1 % SDS at 58°C for l h.
`
`the DNA was subjected to PCR
`In the more sensitive technique (14),
`amplification with primers complementary to the normal GLY or to the
`mutated ALA, VAL, SER, ASP or CYS sequences. The mutation-specific
`primers have 3'-ends complementary to specific point mutations. Taq I
`polymerase enzyme lacks a 3’-exonuclease activity and is therefore unable to
`amplify DNA if the single base mismatch is located at the 3‘-end of the primer .
`Each PCR was done in a volume of 40 ul of a solution containing 50 mM KC].
`10 mM Tris-HCl at pH 8.3, 2 mM of each nucleotide, 0.7 mM MgC12, 0.2 mM of
`each primer and 1 unit of AmpliTaq DNA polymerase. Thirty five cycles were
`performed (94°C forl min, annealing at 550 to 62°C for 2 min, extension 72°C
`for l min). The last cycle was followed by 7 min extension at 72°C. Every reac-
`tion began with the hot-start technique. The primers were
`5’-ACITGTGGTAG'ITGGAGCI‘GG-3‘ for the wild type K-ras (anneal-
`ing 55°C), S’-ACITGTGGTAGTTGGAGCTGC~3’ for the ALA 12 mutant
`(annealing 62°C), 5'~ACITGTGGTAGTI‘GGAGCI‘GT—3‘ for the VAL 12
`mutant (annealing 61°C). 5’-ACITGTGGTAG'ITGGAGC1‘A-3’ for the SER
`12 mutant (annealing 59°C), S’-AC1'FGTGGTAGTFGGAGCTGA-3‘ for the
`ASP 12 mutant (annealing 60°C), 5’-ACITGTGGTAG'ITGGAGC'IT~3‘ for
`the CYS 12 mutant (annealing 59°C), and in each case the antisense primer
`5’-CI‘ATI‘GTTGGATCATA'ITCGTCC-3’. After amplification the reaction
`products were analysed by electrophoresis in 8% polyacrylamide gel. In
`some cases,
`the amplification products were run on a 4% NuSieve 3:1
`agarose (FMC). blotted on a Zeta probe membrane and hybridized with
`oligonucleotides specific for wild type or mutant K-ms (20).
`For each sample all the specified probes and primers were used.
`
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`I44
`
`K-ras MUTATIONS IN COLON CANCER
`
`RESULTS
`
`Using the selective dot-blot hybridization with oligonucleotide probes, we
`detected a Valine mutation (Figure l) in one of the two plasma specimens for
`which we had neither tumor nor blood cell sample (patient 15). Out of the [3
`tumors analysed, six (46%) presented mutations (GLY to VAL, CYS 01'
`ALA) while with the same technique these mutations could not be revealed
`in the corresponding plasma DNA. Indeed, a PCR amplification followed by
`dot—blot hybridization allows a point mutation to he usually clearly iden-
`tified only if 10 % (8). at most in our hands 2 °/o (Figure l). of the total
`amplified DNA carries a point mutation.
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`I)
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`ease e e To 0 e
`9 ‘19 99 99 .9 ’
`
`GLY
`
`‘ .
`
`.
`
`'
`
`VAL
`
`
`
`FIG. 1. Detection of point mutations in codon l2 of the K-ras gene by dot-blot hybridization.
`The DNA was amplified. spotted in equal quantities onto Zeta probe membranes and
`hybridized with oligonucleotides specific for wild type or mutant K-ras as described in
`“Material and Methods". Well No.l. Negative control (no DNA in PCR): No. 2. DNA trom
`human placenta; No.3.,4.,5. DNA from three different samples of normal blood plasma; No. 6.
`DNA from the tumor of patient 4; No.7.8. DNA from the blood plasma of patient 4, L and H
`respectively (plasma DNA was often found in two bands after (352304 centrifugation : L. low
`density DNA; H, high density DNA);No. 9. DNA from the blood plasma of patient I5: No. IO to
`I8. Mixture of normal human placenta DNA and mutant DNA trorn cell line SW 480 (which har-
`bors a Valine mutation of codon I2 of the K-ras gene on both alleles) in varying proportions
`which were used as a template for PCR amplification with 50%. 25%. I0%. 5%. 2%. |%. 0.5%.
`0.25%. O.I% ot mutant DNA. respectively.
`
`To improve the sensitivity we used the technique of PCR amplification
`using mutation-specific primers with 3’-ends complementary to the point
`
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`K-rus MUTATIONS IN COLON CANCER
`
`I45
`
`mutation. This method enables identification of mutations in a DNA sample
`mixed with a l04 to 105 fold excess of normal non-mutated DNA (18). With
`this assay the blood plasma of live out of the six mutation positive patients
`presented in a reproducible way the same mutation as identified in the
`tumor (Table I) while the other possible point mutations of codon 12 did not
`appear.
`
`Table 1. Patients with ooiorecta/ adenocarcinomas studied
`for ras gene mutations in plasma DNA
`E
`
`Patients Age/Sex
`Tumor
`Tumor
`Tumor size
`Tumor Mutant ras gene
`location
`stage
`(cma)
`mutation
`in plasma
`
`
`1
`64/M
`Rectum
`B
`4 x 4 x 0.8
`ALA
`ALA
`
`
`peritoneal nodules CYS
`
`CYS
`
`6 x 6 x 2.5
`
`VAL
`
`VAL
`
`x 4
`
`2
`
`59/M Colon (relapse)
`
`59/F
`
`Colon
`
`D
`
`C
`
`x 7
`
`59/M
`Rectum
`A
`3.5 x 3.5 x 1.2
`VAL
`VAL
`
`
`not detected
`perirectal iniiltrations VAL
`D
`65/M Rectum (relapse)
`12
`‘—
`
`13
`
`74/F
`
`Colon (relapse)
`
`D
`
`peritoneal nodules
`
`VAL
`
`VAL
`
`5 S
`
`even negative tumors are not shown on the table. nor the 10 controls oi healthy donors.
`Adenocarcinomas were classified according to Duke (Cohen et al..1989) : A. confined to mus~
`cularis propria; B, extension to muscularis proprla. but confined to colon; C, metastatic to
`regional lymph nodes; D. metastatic tumor outside regional lymph nodes.
`
`Interestingly enough. when we investigated with the same sensitive tech-
`nique the DNA present in the peripheral nucleated blood cells, the mutation
`observed in the plasma DNA could not be detected (Figure 2. patients 1 and
`2). As controls we used DNA from human placenta, normal lymphocyte
`DNA and ten different samples of plasma DNA from healthy donors which
`were all found to be negative.
`
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`I40
`
`K405 MUT/i TIONS IN COLON CANCER
`
`12 34 5678 9101112131415161718
`
`12 3 4 5 6 7 89101112131415161718
`
`12 3 4 5 6 7 a 9101112131415161718
`
`GLY
`
`ALA
`
`
`
`CYS
`
`FIG. 2. Detection of point mutations in codon l2 oi the K-ras gene by PCR with mutation-
`specific primers. DNA was subjected to PCFI amplification with primers complementary to nor-
`mal and to mutated sequences. The mutation-specific primers have 3'-ends complementary to
`specific point mutations. Lanes l and I8. Markers (pBR 322 DNA digested with Hae III); Lane
`3. DNA from human placenta; Lane 4. DNA from normal human blood cells; lanes 5 to IO.
`DNA from six different samples of normal blood plasma; lane II. DNA from blood plasma of
`patient 2; lane l2. DNA from blood cells of patients 2; lane l3. DNA from the tumor of patient 2:
`lanes I4 to I5. DNA from blood plasma of patient I, H and L, respectively (Plasma DNA were
`often found in two bands after CsZSO4 centrifugation, L.
`low density DNA; H. high density
`DNA); lane I6. DNA from bood cells of patient I; lane I7. DNA from the tumor of patient I.
`
`The specificity of the technique using point mutation specific primers
`(PASA) was checked by hybridizing the amplification product with labelled
`mutation specific probes (Figure 3).
`
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`K—ras MU 'l'/l I'IUNS [N CUL UN CANCER
`
`147
`
`123456
`
`
`
`VAL- *
`
`123456
`
`FIG. 3. Southern blot hybridization of amplification products obtained by PCR mutation
`primers. DNA was subjected to PCR amplification with primers complementary to a Valineo
`mutated sequence. The mutation specific primers have 3’-ends complementary to specific
`point mutations. A) The DNA was amplified. run on a 4% NuSieve agarose electrophoresis. B)
`blotted on a Zetaprobe membrane and hybridized with an oligonucleotide probe specific for a
`mutant K-ras as described in "Material and Methods". Lane 1 to 2. DNA from blood plasma oi
`patient 7, H and L, respectively; Lane3. DNA from the tumor of patient 7; Lane 4 to 5. DNA
`from blood plasma of patient 1, H and L respectively; lane 6. DNA lrom the tumor of patient I.
`
`DISCUSSION
`
`Out of 15 patients six showed a point mutation at codon 12 of the K—ras
`gene in the DNA extracted from the blood plasma. In one case the mutation
`was detected by dot-blot hybridization with oligonucleotides specific for the
`mutant K-ras. In five out of the six cases where a mutation had been found
`
`in the tumor sample with the hybridization technique, the same mutation
`could be found in the plasma by using polymerase Chain reaction with muta-
`
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`I48
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`K-ms MUTATIONS IN COLON CANCER
`
`tion specific primers. This very sensitive technique which allowed recently
`the detection of ras gene mutations in pancreatic juice and peripheral blood
`of patients with pancreatic adenocarcinoma (18) has been proven to be reli-
`able. Indeed. the specificity of our findings is demonstrated by the following
`facts: a) all samples presented a wild type allele except the DNA of the
`negative control SW 480 cell line which has a mutation on both alleles. b)
`the control DNA from healthy donors did not present any of the mutations
`tested. c) the mutations appearing in the plasma DNA correlated specifically
`with the mutation found in the tumor while the other possible K-ras muta-
`tion of codon 12 could not be detected. d)
`the amplification product
`hybridizes with a labelled oligonuclcotide specific to the mutation.
`The presence of double bands sometimes observed might be due to the
`fact that an incompletely extended primer/template complex can migrate
`faster than the completed duplex. .
`The presence of point mutations in the DNA of the blood plasma. while
`absent in the nucleated cells of the circulating blood, indicates that ap-
`parently there are no or very few circulating tumor cells in the blood.
`However. Tada el al (18) using the same sensitive technique. did find muta-
`tions in peripheral blood cells of patients with pancreatic adenocarcinoma in
`two out of eight patients. This discrepancy can be explained by the particular
`characteristics of the intestinal tissue which sheds its cells after the last
`division directly into the tract and thus not readily in the blood stream. The
`finding of point mutations of the K-ras gene in the stool of patients with
`colorectal adenocarcinoma (13) demonstrates the exfoliation of the intes-
`tinal cells. In another study. we have also found mutations in the plasma
`DNA of patients presenting myeloid disorders (19). Even in this case. the
`mutations are more readily detectable in the plasma DNA than in the blood
`cell DNA or even in a bone marrow biopsy. Taking into account these ob-
`servations, it would be interesting to look for ras mutations in the plasma
`DNA of patients with pancreatic adenocarcinoma to see if the rate of muta-
`tions detected would not be higher than in the blood cells.
`In any case. these findings suggest that part of the DNA found in the plas-
`ma of cancer patients may be released from intact tumors and not necessari-
`ly from tumor cells lysed, either by necrosis or apoptosis. This DNA release
`could be due to an active mechanism as found in several cell types in culture
`(1, 15). As shown by previous unpublished observations from our laboratory,
`this DNA release is more pronounced in tumor cells than in normal cells.
`Our study provides an extension of the interesting approach proposed by
`Sidransky er a1 (13) who have studied mutated oncogenes in the DNA from
`exfoliated tumor cells present in stool. Identification of mutations in the
`plasma offers the possibility to detect the appearance of a tumor which
`would not be in direct contact with extracorporeal media. Ras gene muta-
`tions are frequent in many cancers (5, 2)). Other oncogenes or tumor supres-
`sor genes could also be studied in the plasma DNA. After tumor removal
`this approach could be useful in the follow up of the disease by testing the
`
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`K-ms MUTATIONS IN COLON CANCER
`
`[49
`
`plasma periodically for the presence or absence of the specific oncogene
`found in several types of cancer cells.
`
`ACKNOWLEDGEMENTS
`
`We thank Dr. P. Meyer for the blood samples, Dr. J. Weintraub for provid-
`ing us with paraffin embedded tumors and Dr. A.P. Sappino for the SW 480
`cell line. We thank Dr. H. Turler for critical reading of the manuscript.This
`work was supported by grants from the Swiss National Fund for Scientific
`Research No 31-37382-93 . from the “Ligue Genevoise contre le Cancer”
`and from the OJ. lsvet Fund No 747.
`
`SUMMARY
`
`Increased levels of plasma DNA have been observed in patients with
`various malignancies. Ras gene mutations are frequently found in several
`tumor types and seem particularly significant.We now show that the blood
`plasma DNA of five out of six patients with colorectal adenocarcinoma.
`whose tumors presented mutations in codon l2 of the K-ras, harboured the
`same mutations. These mutations were absent in the cells isolated from the
`
`peripheral blood collected shortly before surgery. Ten healthy controls were
`negative. The finding of activated oncogenes in the plasma of cancer
`patients could be useful for the diagnosis and monitoring of various cancers.
`
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