detection of c-k-ras mutations in fine needle aspirates ...€¦12 in the c-k-ras gene have been...

[CANCER RESEARCH 50, 1279-1283, February 15. 1990]

Detection of c-K-ras Mutations in Fine Needle Aspirates from Human PancreaticAdenocarcinomas1

Darryl Shibata,2 ConcepciÃ³nAlmoguera, Kathleen Forrester,3 Jordan Dunitz, Sue Ellen Martin,Michele M. Cosgrove, Manuel Perucho,3 and Norman Arnheim

Department of Pathology, Los Angeles County-USC Medical Center. Los Angeles, California 90033 [D. S., S. E. M., M. M. C.J; Department of Biochemistry, StateUniversity of New York at Stony Brook, Stony Brook, New York 11794 [C. A., K. F., J. D., M. P.]; Department of Biological Sciences, University of Southern California,Los Angeles, California 90089-03 71 [N. A.J

ABSTRACT

Formalin-fixed paraffin-embedded tissue specimens obtained by fineneedle aspiration of pancreatic masses from 47 patients were examinedretrospectively for cytology and the presence of mutant c-K-ras onco-genes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification ofthe cellular c-K-ras sequences by the polymerase chain reaction. Of the36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogeneswere detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%)with atypical cytologies, and 0 of 3 with benign aspiration cytologies.The remaining 11 patients without pancreatic adenocarcinomas did nothave mutant c-K-ras genes detectable by the assay. The diagnosis ofpancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival inthe patients studied. Mutant c-K-ras genes were found at the time ofinitial clinical presentation in the majority of pancreatic adenocarcinomas,suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancerdiagnosis at the molecular genetic level.

INTRODUCTION

Genetic alterations are frequently associated with neoplasia.These genetic changes (point mutations, translocations, amplification, and deletions) alter cellular proto-oncogenes and/ortheir "suppressor" genes and contribute to the manifestations

of the malignant phenotype (1). The activated proto-oncogenes(oncogenes) most often associated with human neoplasia arethe members of the ras gene family (2). These highly conservedgenes (c-K-ras, c-H-ras, and N-ras) encode similar membrane-bound proteins possessing guanine nucleotide-binding and hy-drolytic activities. It is thought that ras proteins are involved inthe process of signal transduction across the cell membrane (3).Single point mutations in ras proto-oncogenes activate theironcogenic potential. Indeed, somatic point mutations at codon12 in the c-K-ras gene have been recently found in the majorityof human pancreatic carcinomas (4-6).

Pancreatic carcinoma accounts for 5% of all cancer deaths inthe United States (7). Despite advances in oncology, pancreaticcarcinoma is invariably fatal with a median survival of 2.8months (8) and a 5-year survival of 3-5% (7). FNA4 of pan

creatic masses has been utilized for cytological diagnosis tominimize patient morbidity in this essentially terminal disease.As reported in various studies (9, 10), aspiration cytology cangive an accurate diagnosis of carcinoma in 55-100% of pancreatic carcinomas. The cells obtained by FNA can represent

Received 6/6/89; revised 8/31/89; accepted 11/7/89The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by NIH Grants GM36745 (N. A.) and

CA38579 (M. P.).2To whom requests for reprints should be addressed.3 Present address: California Institute of Biological Research, 11099 N. Torrey

Pines Road, La Jolla, CA 92037.4The abbreviations used are: FNA, Fine needle aspiration; PCR. polymerase

chain reaction.

the earliest tissue samples of pancreatic tumors and are oftenthe only tissue available for analysis.

Using the RNAse A mismatch cleavage method (11, 12), wehave successfully analyzed pancreatic aspirates in paraffin-embedded tissue blocks from 47 patients for the presence of singlepoint mutations in the first coding exon of the c-K-ras gene.Since c-K-ras mutations are not found in nonneoplastic tissue(1-5), we reasoned that detection of such mutations could aidin diagnosis independent of cytological criteria. We also wantedto assess the effects of such mutations on clinical outcome.Since the number of cells obtained by FNA is often small, invitro DNA amplification by the PCR (13) was utilized toincrease the number of c-K-ras sequences present in the 5- toI0-/j.m sections of formalin-fixed paraffin-embedded aspirates(14) prior to RNA-DNA RNAse A mismatch cleavage analysis(4).

MATERIALS AND METHODS

Fine Needle Aspirates. Primary care patients at the Los AngelesCounty-USC Medical Center with pancreatic masses are routinelystudied by fine needle aspiration. Pancreatic masses were aspiratedintraoperatively or percutaneously with the guidance of computerizedaxial tomography or ultrasonography with thin needles (20 or 22gauge). Cell blocks were made in addition to stained smears. Fifty-fourcell blocks of FNAs obtained from 50 patients between the years 1981and 1987 were available for study. The DNA present in 51 cell blocksfrom 47 patients could be amplified by the polymerase chain reactionand analyzed for c-K-ras mutations. In three cases no amplificationsoccurred, despite having cells present. It is unclear why these threespecimens were inadequate substrates for the PCR. The aspirates withand without c-K-ras mutations were similar in cellular composition.

The consensus cytological diagnosis of pancreatic adenocarcinomawas made using standard criteria (9, 10) after review by three patholo-gists (D. S., M. M. C., and S. E. M.). AH of the pancreatic tumors werethought to be of ductal cell origin, although exact subclassification (15)was not possible. Clinical follow-up was obtained through review ofmedical records, death certificates, and the California Tumor Registry.Patients were considered to have pancreatic adenocarcinoma if theirclinical outcomes were consistent with terminal carcinoma. Except forone individual, all patients considered to have pancreatic carcinomadied within 1 year after fine needle aspiration. The one patient stillalive after 16 months had a pancreatic mass and an aspirate withmalignant cells. There were 36 pancreatic adenocarcinomas, 4 tumorsin the pancreatic region other than pancreatic adenocarcinoma, and 7benign lesions among the 47 patients successfully analyzed. Survivalwas the period between FNA and death. Staging (8) of the patients wasnot uniform, although disseminated mÃ©tastases(Stage IV) could bedocumented by laparotomy or radiological studies. Therefore, extentof disease at the time of aspiration was categorized as Stage IV or lessthan Stage IV (without disseminated mÃ©tastases).None of the tumorswere considered resectable (Stage I). Treatment of the pancreatic tumors was nonuniform and consisted of chemotherapy, palliative surgery, radiotherapy, or supportive care. The patients with or withoutmutated c-K-ras genes did not receive treatments which differed significantly (P > 0.05, x2)- The log-rank test (16) was used to compare

differences in survival.1279

on April 15, 2017. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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ras MUTATIONS IN PANCREATIC ADENOCARCÃ•NOMA

Polymerase Chain Reaction. The PCR was performed as previouslydescribed (4. 14). although modified for the heat-stable DNA polym-erase from Thermus aquaticus (17). One or. in cases where only a smallnumber of cells were present in the aspirate, three 5- to 10-^m paraffinslices from the FNA cell blocks were deparaffinized with one wash ofxylene and two washes with ethanol. An additional section was stainedto confirm the cytology. The desiccated tissue was then heated at 100Â°C

in 50 p\ of water for 7 min. The PCR was performed with an automatedthermal cycler (Perkin Elmer-Cetus, Norwalk, CT) for 50 cycles in thepresence of two primers ( I n\\ each) specific for the c-K-ras first codingexon (4). Four units of the heat-stable polymerase (TAQ, Perkin ElmerCetus) were used for each reaction. Denaturation was at 95Â°Cfor 30sec, annealing at 42Â°Cfor 15 sec, and polymerization at 72Â°Cfor 1

min. Temperature transitions occurred as fast as possible.Detection of Mutant c-K-ras Genes. Single point mutations within

the first coding exon of the c-K-ras gene were analyzed by the RNAseA mismatch cleavage assay (11), as previously described (4). Uniformlylabeled RNA probes corresponding to the first coding exon of the c-K-ras gene were synthesized with SP6 polymerase in vitro transcriptionsystem (18) using pAKlNGly (11), as previously described (4), exceptthat 100 ^M unlabeled CTP were used. The extent of amplification byPCR of c-K-ras sequences from the aspirates was estimated by nonde-naturing polyacrylamide gel electrophoresis and staining with ethidiumbromide, as well as hybridization to the c-K-ras probe. Aliquots of theamplified PCR reaction were accordingly adjusted and hybridized tothe RNA probe. The conditions for hybridization, RNAse A digestion,and analysis by denaturing polyacrylamide gels and autoradiographywere reported previously (4).

RESULTS

Single nucleotide substitutions within the first coding exonof the c-K-ras gene were detected by RNAse A cleavage at singlemismatches in RNA-DNA hybrids and polyacrylamide gel electrophoresis of the RNAse A-resistant products (4). Fig. 1 showsthe results from two of these experiments. The mismatch-specific RNA bands of 66 and 43 nucleotides (indicated byarrows) are diagnostic of the presence of c-K-ras genes mutantat codon 12 in the SK-CO-1 tumor cell line, used as a positivecontrol (4, 19), as well as in some of the samples (Fig. IA, lanes2, 3, 4, 7, 8, and 10; Fig. IB, lanes 12 through 17 and 19).These bands diagnostic of a mismatch were undetectable inDNA amplified from normal human fibroblasts, used as anegative control, as well as in other FNA samples (Fig. IA,lanes 1, 5, 6, 9, and 11; Fig. IB, lanes 18 and 20). Althoughthe exact nature of each mutation has not been characterized,based on the mobility of the mismatch bands,5 it can be deduced

that samples 2 and 7 (Fig. 1/1) and sample 15 (Fig. 1A) containa mutation at the first position of codon 12, while the rest ofthe positive samples are mutant at the second position of codon12. The RNA-DNA mismatch cleavage assays were interpretedwithout prior knowledge of the cytological or clinical data. Toavoid false positive diagnosis, we have been conservative in theinterpretation of the results and only those cases with a clearmismatch pattern have been considered positive. The results ofthese experiments are summarized in Table 1. Thirty-six of the47 patients with pancreatic masses in this study had clinicalfollow-up and outcome consistent with pancreatic carcinoma.c-K-ras mutations were detected in 20 of the 36 (56%) patients.By cytological criteria alone, 25 of these 36 patients had anaspirate consistent with a diagnosis of carcinoma. Among these25 patients, 18 (72%) had a mutated c-K-ras gene. Eleven ofthe 36 patients with pancreatic carcinoma had FNA specimensthat were not characteristic of pancreatic carcinoma. Eight ofthese aspirates were judged atypical and three benign. Interest-

oo

I 2345 6 7 89 IO160-

120-

90-

67-

t-tflâ€¢¿�fffrtf--

B 8*:

CL to

f234-194-

118-

72- â€¢¿� f â€¢¿�*

Fig. 1. RNA-DNA mismatch analysis of c-K-raj sequences from paraffin-embedded tissue sections of pancreatic FNAs. A. M, molecular weight marker: P,RNA probe pAKINGly; N, negative control using DNA amplified from normalhuman fibroblasts; SK-CO-1, positive control using DNA amplified from a coloncarcinoma cell line which contains a valine mutation (G I 1) at the second positionof codon 12. B. Same as A except that the molecular weight marker M was a//Â«clll digest of 0 X 174 DNA. The arrows at the righi indicate the positions ofthe codon 12-specific mismatch bands. Positive specimens are numbers 2-4, 7,8. 10, 12-17. and 19.

Table 1 AspÃralesfrom 47 patients: cytology and K-ras mutations

PancreaticadenocarcinomaCytologymalignantCytologyatypicalCytology

benignBenign

pancreaticdiseaseCytologyatypicalCytology

benignNumber36258371ftrai-mutated20

(55%)18(72%)200(1(1

Pancreatic region tumors not pancreaticadenocarcinoma0

5 Unpublished observations.

" Lymphoma. sarcoma, and gastric and bile duct carcinoma.

ingly, c-K-ras mutations were detected in FNAs from two ofthe eight patients with atypical cytology. An example of thecytology not diagnostic of carcinoma but containing mutated c-K-ras genes is seen in Fig. 2. Aspirates of four tumors from thepancreatic region which were not pancreatic adenocarcinomas(lymphoma, sarcoma, and gastric and bile duct carcinoma) andseven benign lesions did not contain mutated c-K-ras sequencesdetectable by our assay.

FNAs of pancreatic masses may score negative in our assay1280



ras MUTATIONS IN PANCREATIC ADENOCARCINOMA

The factors affecting survival are presented in Fig. 3. Patientswith Stage IV disease at the time of aspiration lived significantlyshorter (P < 0.001), with a median survival of 2.5 monthsversus 7 months for patients with less than Stage IV disease.This effect of stage on survival has been previously documented(8). The effects of c-K-ras mutations on survival were notstatistically significant (P > 0.05). The median survival afteraspiration was 3.5 months for patients with c-K-ras mutationsand 5 months for patients without c-K-ras mutations. Mediansurvival for patients with Stage IV disease was 2.5 months inthe presence of c-K-ras mutations and 3.2 months in the absenceof c-K-ras mutations. Patients with less than Stage IV diseasehad median survivals of 7.5 months with c-K-ras mutations and7 months without c-K-ras mutations.

Fig. 2. Cytology of a fine needle aspiration cell block containing mutant c-K-ras genes. The poorly cellular aspirate was considered cytologically atypical butnot diagnostic of pancreatic adenocarcinoma. The clinical outcome was consistentwith pancreatic adenocarcinoma. A. The entire cell block is shown and only a fewatypical cells were present (arrow) among other benign cells. H & E x 20. B. highpower magnification of the atypical cells in A. H & E x 1000.

Table 2 Median survival for pancreatic adenocarcinomaOnly the 25 patients with pancreatic adenocarcinoma and cytology diagnostic

of pancreatic adenocarcinoma were analyzed. Number of patients is given inparentheses.

Median survival (months)

All patientsra.v-mutatedNormal rasAll

tumors4(25)

3.5(18)5(7)<

StageIV7(9)

7.5 (6)7(3)Stage

IV2.5(16)

2.5(12)3.2 (4)

because of several reasons. First, they may not contain neoplas-tic tissue due to sampling errors. Second, some mutations inthe first coding c-K-ras exon may not be recognized by theRNAse enzyme or mutations may be present outside of the firstcoding exon. Finally the c-K-ras gene may not be mutated. Toavoid errors due to sampling, we analyzed only the 25 aspirateswith cells diagnostic of pancreatic adenocarcinoma for thefrequency of the codon 12 c-K-ras mutations and their clinicalsignificance (Table 2). These 25 patients consisted of 8 malesand 17 females, with an average age of 59.5 years. No significantage or sex differences were present between patients with orwithout mutated c-K-ras genes (/*> 0.05, x2)- Sixty-seven % ofpatients with c-K-ras mutations were Stage IV while 57% ofthe patients without c-K-ras mutations were Stage IV (Table2). This difference was not significant (P > 0.05, x2)-

DISCUSSION

The RNAse A mismatch cleavage analysis after gene amplification by the PCR is a powerful tool for detection of pointmutations in cellular genes. We have demonstrated that thismethod is useful for the detection of mutant c-K-ras oncogenes

present in routinely obtained clinical specimens of pancreaticadenocarcinoma. Because the c-K-ras mutations were specific

for malignant tissue, our approach represents a direct application for cancer diagnosis at the molecular genetic level. Thepolymerase chain reaction allows in vitro DNA amplificationof the scant amounts of DNA present in the small numbers ofcells obtained by fine needle aspiration, thereby yielding quantities suitable for analysis. Human papilloma viral sequenceshave also been detected in fine needle aspirations of metastatictumors using the PCR (20).

In this study, the majority (72%) of pancreatic adenocarci-nomas defined cytologically after FNA had detectable c-K-ras

mutations at the time of clinical presentation. In a previousstudy (4), 95% of pancreatic adenocarcinomas contained mutated K-ras genes. There are several possible explanations for

this difference, although the total number of tumors that havebeen examined in these studies is relatively small and thedifference between the two studies is not statistically significant(P> 0.05, x2)- One possibility is that the distribution of tumor

cells containing c-K-ras mutations may not be homogeneous

and the smaller number of cells sampled by FNA compared toa typical tissue section may have not contained sufficient cellswith the c-K-ras mutation to be detectable in the assay. In

addition, while the earlier work was a study of advanced disease(68% of pancreatic tumors were sampled at autopsy), the fineneedle aspirations were performed as the initial step for thedefinitive diagnosis of pancreatic carcinoma and the materialwe analyzed in this study represented the earliest possiblesampling of the tumor cells. Therefore, the differences in thefrequency of c-K-ras mutations between these two studies isalso consistent with mutations in c-K-ras being acquired during

progression of the tumors. Consistent with this hypothesis isthe presence of mutant c-K-ras genes in metastatic pancreaticcarcinoma (4, 21). In addition, a steady increase in mutated c-K-ras gene incidence has been detected during colorectal early

tumor development (22, 23) and progression, including themetastatic stage (23).

The clinical contribution of mutated c-K-ras genes to pancreatic carcinoma is uncertain. The effects of c-K-ras mutations

on survival were not statistically significant with the numbersof patients studied. Similar results have also been reported (6).Additional studies are needed to clarify the role of mutated c-K-ras genes in pancreatic carcinoma. The presence of mutated

1281




SURVIVAL VS. STAGE

Fig. 3. Survival curves of patients with pancreatic carcinoma. Only the 25 patients withpancreatic adenocarcinoma and cytology diagnostic of pancreatic adenocarcinoma arepresented (see text).

CO

IV)

o2Oo.

SURVIVAL VS. RAS MUTATIONS100

3 i</5

O) 40-

â€”¿�<STAGE4(N=9)â€”¿�STAGE 4(N-16)

â€”¿�ras t (N. 18)â€”¿�ras - (N-7)

012345

SURVIVAL VS. RAS MUTATIONS: <STAGE 4100-

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â€”¿�rast (N,6)â€”¿�ras-(N=3)

SURVIVAL VS. RAS MUTATIONS: STAGE 4

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01234567 9 10 11 12

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c-K-ras genes in the majority of pancreatic adenocarcinomas at

the time of clinical presentation may reflect the aggressiveclinical behavior of a disease which is largely resistant to chemotherapy and radiotherapy and seldom resectable. For comparison, using similar highly sensitive DNA probe analysis (19, 22-24), only approximately 40-50% of all colonie carcinomas [5year survival rate (1), 49-54%] were found to have mutated rasgenes. The analysis of pancreatic dysplasia (25, 26) or smallresectable pancreatic adenocarcinomas, which were not presently studied and are potentially curable, may yield furtherinsight into the role of ras oncogene mutations in early pancreatic oncogenesis. As multiple genetic alterations may bepresent in tumors (22), the investigation of multiple oncogenesor anti-oncogenes may yield better understanding of malignancy. The methods used in this study allow selective geneticanalysis of human tissues routinely obtained and stored. Furtherinsight into the role of c-K-ras mutations and pancreatic carcinoma may be possible with prospective as well as previouslyconducted well defined clinical studies if paraffin-embeddedtissue samples are available.

The cytomorphological diagnosis of pancreatic carcinomawas more sensitive than diagnosis based on the presence of c-K-ras mutations. The two methods were complementary, however, as more accurate diagnosis was possible if both techniqueswere utilized. In two cases in which c-K-ras mutations weredetected, the cytology was considered atypical but not diagnostic of carcinoma (Fig. 2). The cytological criteria for malignancymust be conservative to avoid a false positive diagnosis, sinceinflammatory atypia is difficult to distinguish from better differentiated carcinomas (9). Moreover, the cytological diagnosisof malignancy is difficult if only a few atypical cells are present.By using the presence of mutated c-K-ras genes as a criterionfor malignancy, two of the eight cytologically atypical aspiratesfrom patients with pancreatic adenocarcinoma could be correctly classified as malignant. Review of the cytology clearly

demonstrated cells which had features of malignancy but weretoo few in number for unequivocal diagnosis. The PCR withsubsequent DNA-RNA mismatch assay allows analysis independent of morphology and would be most useful when thecytology is atypical or when the suspicious cells are few innumber.

In conclusion, we have detected c-K-ras mutations in cellsobtained by fine needle aspiration in the majority of pancreaticadenocarcinomas, suggesting an important and early role of thec-K-ras oncogene in tumorigenesis. However, since there arenegative cases, mutations at codon 12 in the c-K-ras gene arenot absolutely required for development and/or progression ofpancreatic adenocarcinoma. In addition, although none of thepancreatic tumors analyzed other than pancreatic carcinomashas c-K-ras mutations, tumors of a different primary site butmetastatic to the pancreas and containing c-K-ras oncogenescould also be detected by our method. Therefore, the detectionof c-K-ras oncogenes with mutations at codon 12 in pancreaticaspirations is not completely reliable for diagnosis becauseneither is their presence diagnostic of pancreatic carcinoma noris their absence a criterion for benign disease. However, sincethe mutations appear specific for malignant disease, our oncogene detection approach, in combination with cytomorphological evaluation, allows a more definitive diagnosis of malignancyon aspirates which by cytomorphology alone are not diagnosticof malignancy. Distinguishing between normal and mutant ras-containing tumors and/or between relative oncogene dosagemay be useful for classification at the molecular level and mayalso aid in the evaluation of new therapeutic modalities forpancreatic carcinoma.

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1990;50:1279-1283. Cancer Res Darryl Shibata, Concepcion Almoguera, Kathleen Forrester, et al. Human Pancreatic Adenocarcinomas

Mutations in Fine Needle Aspirates fromrasDetection of c-K-

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