(CANCER RESEARCH 48, 5163-5166, September 15, 1988]

myc Family DNA Amplification in Small Cell Lung Cancer Patients' Tumors and

Corresponding Cell Lines1

Bruce E. Johnson,2 Robert W. Makuch,3 Alfreda D. Simmons, Adi F. Gazdar, David Burch, and Alan W. Cashell

NCI-Navy Medical Oncology Branch, National Cancer Institute and Naval Hospital [B. E. J., A. D. S., A. F. G.I, Pathology Department, Naval HospitalID. B., A. W. C.¡,Bethesda, Maryland 20814, and the Division of Biostatistics, Yale University School of Medicine [R. W. M.], New Haven, Connecticut 06510

ABSTRACT

Tumor specimens procured from 38 different small cell lung cancerpatients were studied for DNA amplification of the myc family of pro-tooncogenes (c-myc, \-myc, and 1.-myc). Six of the 38 specimens (16%)had 4-fold or greater myc family DNA amplification (N-myc in 4 and L-myc in 2). All 6 tumors with amplification came from patients who hadreceived combination chemotherapy. The myc family gene copy numberof the DNA prepared from 9 tumor cell lines established from these 38patients was similar to the myc family gene copy number in the DNAprepared from fresh tumor specimens from these same patients, mycfamily DNA amplification is present in 16% of small cell lung cancerpatients' tumors and the amplification pattern in the tumor cell lines is

representative of the fresh tumors obtained from the same patients.

INTRODUCTION

DNA amplification and mRNA expression of the myc familyof protooncogenes (c-myc, N-myc, and L-myc) has been identified in tumor cell lines and in tissues from small cell lungcancer patients by numerous investigators (1-9). DNA amplification of the myc family of protooncogenes has been associated with an altered clinical outcome in small cell lung cancerand other human cancers, c-myc DNA amplification in tumorcell lines established from chemotherapy treated small cell lungcancer patients has been associated with a shortened survival(6). In addition, N-myc DNA amplification in tumors fromuntreated childhood neuroblastoma patients has been linkedwith advanced stage disease and rapid tumor progression (10,11). Yakota et al. (12) examined a wide variety of epithelialcancers and associated c-myc DNA amplification with advancedaggressive disease. In the present study, molecular genetic characterization <ifmyc family DNA amplification in small cell lungcancer tumor specimens was undertaken in patients who havehad systematic initial evaluations and complete clinical follow-up. Thus we are able to examine the relationship between theclinical outcomes and myc family DNA amplification in thisadult malignancy.

Previous observations on the role of myc family DNA amplification in small cell lung cancer have been restricted primarilyto analysis of tumor cell lines (1-9) and paraffin fixed smallcell lung cancer tissue (13). There is a paucity of data aboutmyc family protooncogene amplification in a series of freshtumor specimens from small cell lung cancer patients or anyother single type of adult malignancy. In addition, the degreeof correlation between small cell lung cancer cell line amplification and fresh tumors has not been defined. We studied freshtumors from small cell lung cancer patients who also had tumor

Received 6/26/87; revised 3/15/88; accepted 6/16/88.The 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.

' The opinions and assertions contained herein are the private views of the

authors and are not to be construed as official or reflecting the views of theDepartment of the Navy or the Department of Defense.

2To whom requests for reprints should be addressed, at NCI-Navy Medical

Oncology Branch, Building 8, Room 5101, Naval Hospital, Bethesda, MD 20814.'Supported in part by Grant 5P30-CA-16359 from the National Cancer

Institute.

cell lines established and their myc family amplification statusstudied (6). Therefore, we can define the relationship betweenprotooncogene amplification in tumor cell lines and tumorsfrom the same small cell lung cancer patients.

MATERIALS AND METHODS

The tumor specimens adequate for molecular genetic analysis wereobtained from 38 different small cell lung cancer patients treated at theNCI-Navy Medical Oncology Branch from 1982 to 1987, 34 duringnecropsy examination and 4 during surgical biopsy. Normal tissue fromthe same patient was available for analysis from 34 of the 38 patients.The tumor and normal specimens were frozen at —70°Cuntil analysis.

The normal and tumor tissues were finely minced and disrupted inisotonic buffer plus 0.4% sodium dodecyl sulfate detergent, and DNAwas prepared by the method of Heiter et al. (14). Tumor cell linesestablished from 9 of the 38 patients were harvested during log phasegrowth, and DNA was prepared as previously described (6). The DNAwas digested with the restriction endonucleases EcoRl, BamHl, or Si/I,electrophoresed in 0.8% agarose gels, and transferred to nitrocelluloseby the method of Southern (15). The filters were hybridized to a 32Pradiolabeled Clal-EcoRl third exon c-myc fragment (c-myc probe) (16),an EcoRl-BamHl second exon N-myc fragment (N-myc probe) (17), ora Smal-EcoR\ third exon L-myc fragment (L-myc probe) (2). Smallcell lung cancer cell lines previously shown to be amplified for c-myc,N-myc, and L-myc (N417, H720, and H378, respectively) were used ascontrols for amplified tumor cell lines (1,2, 5). The variations in theamount of DNA loaded per lane were adjusted by standardizing themyc family gene signal to the signal made by either a 'I' radiolabeledsingle copy /V//I1-/'»'«!!nastriti releasing peptide fragment (nastritireleasing peptide probe) (18) or a Pst\-Pvull oxytocin fragment (oxy-tocin probe) (19).

DNA amplification was quantitated by serial dilution of DNA toobtain a hybridization signal equivalent to a single-copy signal fromDNA prepared from normal tissue or a nonamplified small cell lungcancer cell line (e.g., a 20-fold amplification is indicated if a 1:20dilution is necessary to achieve single-copy intensity). The presence ofan amplified myc family signal was also confirmed by densitometriccomparison of the signal from the tumor DNA versus the signal fromthe normal DNA standardized by the signal from the single copy genecontrol (gastrin releasing peptide or oxytocin probe). The myc familygene was considered to be amplified if the signal from the tumor DNAwas at least 4-fold greater than the normal tissue DNA signal.

The clinical characteristics and course of the small cell lung cancerpatients who had their tumor studied for myc family DNA amplificationwere reviewed. The initial stage, date of initial chemotherapy, responseto therapy, date tumor was obtained for study, and the date of deathwere noted. If a tumor cell line was established at any time during thelife of the 38 small cell lung cancer patients whose fresh tumor specimenhad been studied, the date the tumor tissue was obtained for cell culturestudies was identified.

Statistical methods for the analysis of the data included Fisher's

exact test for the analysis of the 2x2 contingency tables and use ofthe nonparametric Wilcoxon rank sum test for the analysis of continuous variables (20). For the analysis of survival data, the Kaplan-Meiermethod was used to examine the data graphically (21). The generalizedWilcoxon test of Gehan (22) was used to compare the survival timesbetween patients with and without myc family DNA amplification.Survival time was measured from the data on study to the date of death.All P values are of the two-sided type. Finally, to calculate the proba-

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myc FAMILY DNA AMPLIFICATION IN SCLC TUMORS

bility that by chance alone myc family DNA amplification of a tumorcell line and a tumor from the same small cell lung cancer were thesame, we assumed: un the observations were independent; and (/>)thea priori probability of a myc match (c-myc, N-myc, L-myc, or no myc)between a tumor and the corresponding cell line was 0.25. The binomialdistribution was then used to calculate the probability that all the pairsare matched by chance alone.

RESULTS

The tumors from small cell lung cancer patients analyzed formyc family DNA amplification were obtained at different timesduring the patients' treatment. Of the 38 tumor specimens, 32

were harvested during necropsy examinations of patients whodied after administration of combination chemotherapy. Sixother tumor samples were harvested prior to the patient's

receiving combination chemotherapy (4 from surgical biopsiesand 2 from postmortem examinations).

The DNA from 4 patients' tumors (patients 1, 2, 3, and 4)was amplified for the N-myc protooncogene while the normaltissue obtained from the same patients had a normal diploidcopy number (Fig. 1). Dilutional studies of the N-myc signalfrom the tumor DNA prepared from patients 1-4 showedapproximately 4-, 20-, 20-, and 10-fold greater intensity, respectively, compared to the signal from DNA prepared fromnormal tissue from the same patient. The tumor DNA of

Bam H1

3.1 kb^

1.9kb—

patients 5 and 6 had L-myc amplification while a diploid copywas present in the normal tissue from these patients (Fig. 2).The L-myc signal from the tumor DNA from patients 5 and 6showed approximately 10-fold greater intensity compared tothe signal from the DNA prepared from normal tissues fromthe same patients. The DNA from all 38 tumors had a signalintensity equivalent to that of normal tissue when hybridizedto a radiolabeled c-myc fragment. All 6 tumor specimens withmyc family DNA amplification came from patients who hadbeen treated with combination chemotherapy.

There was no marked difference in clinical characteristicsand course between patients whose tumors had myc familyDNA amplification and those whose tumors did not. Comparedto patients whose tumors did not have myc family DNA amplification, patients whose tumors had myc family DNA amplification presented with a similar proportion of women (2 of 6

BamH1

7.0kb

3.1 kb

i-

Fig. I. Auloradiograms of DNA from small cell lung cancer tumors, normaltissue, and tumor cell lines hybridized to N-m>'c and a gastrin releasing peptideprobe. Ten pg of DNA prepared from tumors, normal tissue, and tumor cell lineswas digested with //«mill restriction endonuclease. Southern blot prepared, andhybridized to NB-1 and a gastrin releasing peptide probe. Small cell lung cancercell lines which have previously been shown to be amplified for L-myc. N-myc,and c-m>r (H378, H720, and N417, respectively) were used as controls foramplified tumor cell lines, (2, 12, 13). The tumor for each patient is designatedas / and the normal tissue as v. Small cell lung cancer cell line HS26 wasestablished from patient 2's tumor tissue and cell line H689 was established frompatient 3's tumor tissue. The "s wir signal [1.9 kilobases (A/»|of the tumor is

more intense in the tumor and tumor cell line than the normal tissue. The singlecopy gene control (gastrin releasing peptide) signal appears at 3.1 kilobases andthe similar intensity of the signals in all lanes show similar amounts of DNAhave been added to each lane.

Fig. 2. Autoradiograms of DNA from small cell lung cancer tumors andnormal tissue hybridized to L-myc and gastrin releasing peptide probes. Southernblots were prepared as in Fig. 1 and hybridized to MP labeled L-myc and gastrinreleasing peptide probes. The L-myc signal [7 kilobases (kb)] is more intense inthe tumor than the normal tissue in patients 5 and 6. The diploid copy control,gastrin releasing peptide signal (3.1 kilobases) are equivalent in all lanes showingsimilar amounts of DNA were loaded in all lanes.

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myc FAMILY DNA AMPLIFICATION IN SCLC TUMORS

versus 9 of 23; P = 1.0), limited stage patients (1 of 6 versus 11of 32; P = 0.64) and a similar proportion achieved complete orpartial responses to combination chemotherapy (5 of 6 versus22 of 30; P = 0.72). The ages of the two groups was also similar(P=0.41).

The survival from the initiation of chemotherapy of the 4patients whose tumors had N-nyc amplifications was 4, 11, 12,and 13 months and both patients with L-myc amplification hada survival period of 9 months. Survival of this subset of patientswhose tumors had myc family DNA amplification was notmarkedly different from that seen in the other 32 patients whosetumors were not amplified (median, 11 months; range, 0-92; P= 0.63) or from that observed in large series of chemotherapytreated small cell lung cancer patients (23-25).

Nine of these 38 patients (27%) had tumor cell lines established and have had their myc gene copy number previouslyreported (6). Six of the cell lines were established at the sametime in the patient's treatment course as the tumor tissue was

obtained; 3 at diagnosis prior to initiation of chemotherapy and3 at the time of tumor progression following chemotherapyadministration. The remaining 3 tumor cell lines were established prior to initiation of chemotherapy and the tumor specimens adequate for DNA analysis were obtained at relapsefollowing chemotherapy administration, myc family DNA amplification was present in both the tumor cell lines and tumorsfrom two patients. The tumor cell line H526 was establishedfrom the bone marrow of patient 2 prior to the initiation ofcombination chemotherapy while the tumor specimens wereharvested at postmortem examination 4 months later aftercombination chemotherapy and chest irradiation. The cell lineH689 was established from the pleural effusion of patient 3after the patient had relapsed following administration of combination chemotherapy and the tumor was obtained 4 weekslater at his postmortem examination. N-»yc DNA amplification was present in both the tumor cell lines and tumor specimens from these patients and to a similar degree (H526, 40-fold, patient 2, 20-fold; and H689, 40-fold, patient 3, 20-fold;Fig. 1). The other 7 cell lines established from patients whosetumors were studied in this series had diploid copy numbers forthe myc family genes, in agreement with the findings in theirrespective tumors (Table 1). Therefore, the copy number for allthree myc family genes was similar in all nine matched tumorcell lines and tumors from the same small cell lung cancerpatients. The probability of this event occurring by chance aloneis extraordinarily small (P < 0.001).

DISCUSSION

Small cell lung cancer cell lines are commonly amplified andexpress abundant amounts of one of the myc family of protoon-cogenes, c-myc, N-wyc, and L-myc (1-9). This study of DNAfrom 38 small cell lung cancer patients' tumors showed 6 (16%)

were amplified for one of the myc family of genes (4 N-mycand 2 L-myc). The incidence of myc family DNA amplification

Table 1 myc family oncogene amplification of tumor cell lines and tumors fromthe same small cell lung cancer patients

myc familygenec-mycN-mycL-mycNo

mycfamilyDNAamplificationTotalCell

line02079Tumor02079

identified in small cell lung cancer patients' tumors in this study

is similar to the incidence of myc family DNA amplificationidentified in other recent investigations of adult solid tumors.Wong et al. (13) studied DNA from paraffin embedded smallcell lung cancer tissue and showed that 5 of 45 (11%) hadgreater than 3-fold amplification of c-myc (2 examples) or N-myc (3 examples). Yakota (12) demonstrated c-myc DNA amplification in 8 of 71 (11%) tumors from patients with a widevariety of epithelial neoplasms which did not include any smallcell lung cancer tumors. In this study we have examined DNAprepared directly from small cell lung cancer tumors and haveextended the analysis of myc family genes to include L-myc.DNA amplification of the myc family is still detected in a smallminority (16%) of these patients' tumors. Although we observed

an incidence (4 of 38) of N-myc DNA amplification in smallcell lung cancer tumors similar to that of Wong et al. (3 of 45),we did not find any examples of c-myc amplification in thefresh small cell lung cancer tissue. It is possible that we couldnot detect the lower levels of c-myc amplification (5- and 7-fold) Wong et al. (13) detected using the slot blot technique.Alternatively, our patient cohort may not have had any c-mycDNA amplification in their tumors, or it may not have beenlarge enough to rule out this low probability of occurrence withhigh statistical power (i.e., in excess of 80%).

The copy number of the myc family genes in the amplifiedtumors is similar to that reported by other investigators studying N'-wvf and L-myc in small cell lung cancer cell lines and

tumors. The 4-, 10-, 20-, and 20-fold DNA amplification of N-myc described in the 4 small cell lung cancer tumors in thisstudy is similar to the 14-, 22-, and 33-fold amplification of N-myc in the small cell lung cancer tissue studied using slot blottechniques of DNA extracted from paraffin blocks. Althoughthe N-myc copy number of the tumor cell lines H526 and H689(40- and 40-fold, respectively) reported here is less than thatpreviously reported by our group (115-fold for H526 and 170-fold for H689), it is consistent with the N-wyc copy numberfor H689 depicted in the dilutional studies (5). The higherestimates previously reported are potentially explained by thealternative method of quantitation used by cutting out theappropriate 32P-labeled N-myc bands from the Southern hy

bridization blot and counting the bands in a liquid scintillationcounter (5). The degree of L-myc DNA amplification reportedhere (patient 5, 10-fold; and patient 6, 10-fold) is similar to thesingle case (patient 5) previously reported by our group (2).Although we can compare only 4 N-myc DNA amplified tumorsfrom these small cell lung cancer patients, the N-myc DNAamplification pattern was not obviously similar to the bimodalpattern described in childhood neuroblastoma where tumorDNA appears to be amplified either 3-10-fold or 100-300-fold(10,11).

myc family DNA amplification had been shown previouslyto be associated with a poor clinical outcome. Studies of c-mycDNA amplification in tumor cell lines established from treatedsmall cell lung cancer patients and N-wir DNA amplificationin tumors obtained from untreated patients with childhoodneuroblastoma have associated shortened survival with mycfamily DNA amplification (6, 11). In addition Yakota et al.(12) associated c-myc DNA amplification with aggressive disseminated primary tumors. This study of myc family DNAamplification in 38 tumor specimens obtained from small celllung cancer patients showed that the 6 patients whose tumorshad N-myc or L-myc DNA amplification did not have a remarkably different clinical course than patients whose tumorsdid not. One must be careful in interpreting the significance of

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myc FAMILY DNA AMPLIFICATION IN SCLC TUMORS

DNA amplification of the myc family genes in small cell lungcancer because the survival of these patients is short (less than12 months). This shortened survival makes it difficult to identify a biological variable which may actually be important inthe genesis and progression of small cell lung cancer because itdoes not obviously change the observed short survival. In agreement with the previous observation that myc family DNAamplification is more prevalent in tumor cell lines establishedfrom chemotherapy treated small cell lung cancer patients'

tumors (6), the present study showed myc family DNA amplification was observed solely in tumor specimens obtained fromchemotherapy treated small cell lung cancer patients althoughwe studied only six tumors from untreated patients.

In addition, we have demonstrated the myc family DNA copynumber was similar in 9 tumor cell lines and tumor specimensfrom the same patients. Because of the potential growth advantage demonstrated by higher cloning efficiencies and shorterdoubling times of c-myc amplified small lung cancer cell linesin cell culture (4, 26), some degree of selection for amplificationin vitro may occur. However, we have not encountered this inthe tumor cell lines and tumors studied thus far.

Thus, our ability to study DNA samples obtained from smallcell lung cancer patients' cell lines and tumor specimens for

myc family amplification allows certain observations to bemade, myc family DNA amplification is present in 16% of thepatients studied and was observed only in patients who hadreceived combination chemotherapy. The myc family DNAamplification pattern is identical in 9 tumor specimens and 9tumor cell lines obtained from the same small cell lung cancerpatients suggesting that the DNA amplification of the mycfamily of protooncogenes is not exclusively an artifact of cellculture.

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1988;48:5163-5166. Cancer Res   Bruce E. Johnson, Robert W. Makuch, Alfreda D. Simmons, et al.   Patients' Tumors and Corresponding Cell Lines

Family DNA Amplification in Small Cell Lung Cancermyc

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