Affiliations of authors: H. H. Nelson, K. T. Kelsey, Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA; D. C. Christiani, Occupational Health Program, Harvard School of Public Health, and Pulmonary and Critical Care Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston; E. J. Mark (Department of Pathology), J. C. Wain (Thoracic Surgery Unit, Department of Surgery), Massachusetts General Hospital, Harvard Medical School; J. K. Wiencke, Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, University of California at San Francisco.
Correspondence to: Karl T. Kelsey, M.D., M.O.H., Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Ave., Bldg. 1, Rm. 207, Boston, MA 02115-6021.
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ABSTRACT |
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INTRODUCTION |
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The K-ras gene encodes a protein that is known to be oncogenic when mutated or overexpressed [reviewed in (14)]. Mutation at codon 12 of K-ras is very specific for adenocarcinoma, occurring rarely in squamous cell or small-cell lung cancer. Ras proteins are known to act as signaling switches that relay growth signals from the cell surface to the MAP kinase cascade [reviewed in (15)]. As such, they are believed to contribute to hyperproliferative growth when they are mutated or overexpressed and may potentiate growth factor and hormone signaling. These proteins, however, are also involved in cell functions other than the MAP kinase cascade. Examples include interaction with the rho pathway (16,17) and integrin activation (18), suggesting that altered ras protein may affect the early metastatic potential of a neoplastic cell. Indeed, patients whose tumors have K-ras mutations have been observed to have poorer survival than patients whose tumors have no such mutations in some studies (19-28) but not in other studies (29-35). Differences in the demographic and clinical characteristics of these populations may also have an impact on these results. For example, some groups have reported that the occurrence of K-ras mutation in lung adenocarcinoma is associated with male sex (22,26-28,36), heavy smoking (26,27,37-39), and asbestos exposure (39-41).
Lung cancer is the leading cause of cancer mortality in the United States (42), and surgical resection for cure is often only applicable to early-stage NSCLC. Recent studies [reviewed in (43)] have suggested that combined modality treatment of NSCLC may improve survival in selected patients (43). One of the most critical questions in choosing the appropriate use of combined therapy is determining which patients might benefit from the more aggressive treatment. Several approaches have been suggested, including treatment based on markers of poor prognosis, such as the K-ras gene mutation in adenocarcinoma.
While the precise explanation for the lack of consensus among studies that have asked whether the K-ras mutation is associated with survival of patients with NSCLC is not known, the mutation-screening method and individual study design may be critical for observing an association between K-ras mutation status and patient outcome. In an effort to reduce misclassification attributable to tumor heterogeneity, we used a relatively nonsensitive mutation-screening method. This was done to bias the detection of mutations toward those tumors that were homogeneous for this alteration. It may be that ras mutations are important for prognosis only in tumors that are homogeneous for such alterations. Finally, we also used a prospective study design to assess the association of K-ras mutation with survival. Retrospective studies are problematic if selection criteria are biased by mutation status. That is, if K-ras mutation status is related to survival of disease, it is plausible that patient follow-up may be biased. Because surgery alone is often the treatment of choice for stage I NSCLC, patients without mutations might be significantly more likely to do well after surgery and, therefore, selectively not return for follow-up. These patients then may not appear in retrospectively designed studies, thus introducing significant bias toward a null result. Here, we report findings from a large, prospective study of patients with resected NSCLC in the New England area and examine the association of K-ras mutation status with sex, carcinogen exposure, and patient survival.
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SUBJECTS AND METHODS |
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Case subjects consisted of all newly diagnosed patients with resectable lung cancer who received treatment at the Massachusetts General Hospital (MGH) Thoracic Surgery, Oncology, and Pulmonary Services from November 1992 through December 1996. There were 461 case patients enrolled during that time period; this number represents approximately 90% of all eligible patients. Information regarding patient demographics and exposure histories was obtained with the use of an interviewer-administered questionnaire at the time of hospitalization. Written informed consent was obtained from all subjects.
The protocol for staging and surgical intervention has been described elsewhere (44). Briefly, all patients underwent mediastinoscopy in addition to radiographic evaluation for preoperative staging. Patients who were judged to be candidates for complete extirpation of primary and nodal disease underwent thoracotomy. Postoperative staging was then done, and these data were added to the database maintained by the MGH Cancer Registry.
Tumor tissue was obtained from archived pathology specimens, and information describing the tumors (i.e., histology, size, and degree of differentiation) was available from clinical pathologic evaluation. The MGH Cancer Registry was used to obtain information on staging (clinical and pathologic, with the stage variable being designated as the higher of the two), as well as patient follow-up.
Of the 461 case patients enrolled, 21 were excluded because the resected tumor was
determined
to be either a recurrence of lung cancer or a metastatic lesion. For 69 case patients, a tissue
specimen
was not retrieved (i.e., an appropriate specimen was not available, or there was insufficient
material for
DNA extraction). Determination of K-ras mutation status was not possible for six case patients.
Thus, a
total of 365 case patients were included in the study of K-ras mutation (Table 1, A). There were 355 case patients within the cohort with available follow-up
information to
examine patient survival as a function of K-ras mutation.
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DNA was extracted with the use of a method modified from Banerjee et al. (45). From five to 10 adjacent 10-µm sections were cut from paraffin-embedded tumor blocks. Excess wax and normal tissue were removed, and the remaining material was placed in a 1.5-mL Eppendorf tube. Then 500 µL of digestion buffer (50 mM Tris-HCl, 1 mM EDTA, 0.5% Tween, and distilled deionized water) was added to the tissue. Samples were microwaved at high power for 30-60 seconds in 10-second intervals, after which they were centrifuged at 9000g for 10 minutes at room temperature, and the paraffin layer was removed manually. The remaining material was resuspended in digestion buffer and digested with 5 µL (10 mg/mL) of Proteinase K (PKA) for 3 hours at 65 °C (or overnight at 56 °C). Samples were then centrifuged at 5000g at room temperature for 5 minutes, and the supernatant was removed and PKA inactivated by heat treatment (100 °C for 10 minutes).
Mutation screening was done with the use of a modified method of Mitsudomi et al. (46). PCR amplification of the region surrounding codon 12 was performed under standard PCR conditions for 15 cycles with the use of primers 5'-CATGTTCTAATATAGTCACA and 5'-AACAAGATTTACCTCTATTG. The PCR product was then diluted 1 : 100 for a second round of PCR that used a mismatched upstream primer that introduces a BstNI restriction site. Again, standard PCR reaction conditions were used, and primers were 5'-ACTGAATATAAACTTGTGGTAGTTGGACCT and 5'-TCAAAGAATGGTCCTGCACC for 40 rounds. The PCR product was then digested with BstNI restriction endonuclease enzyme, and the products were analyzed on 4% MetaPhor® agarose gels (FMC BioProducts, Rockland, ME). Positive controls for the K-ras mutation genotype were NCI-H23 (mutated) and TK6 (wild-type). When mutations were detected, further PCR-RFLP protocols were used to determine the specific codon 12 mutation, as outlined by Mitsudomi et al. (46).
Statistical Analyses
Statistical analyses of mutation status and tumor traits included 2,
Wilcoxon
rank-sum of means, and unconditional logistic regression. Survival time was defined as the time
from
surgery to the patient's death, known recurrence, or the last time that the patient was
known to
be alive. Survival probability curves were constructed for various groupings of patients with the
use of
the Kaplan-Meier method, and differences between groups were tested with the use of the logrank
method. Cox's proportional hazards modeling was used to examine the simultaneous
effects of
several variables on patient's outcome. The data were consistent with the assumptions of
Cox's proportional modeling. All P values represent two-sided statistical tests and
are
considered to be statistically significant for P<.05.
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RESULTS |
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Given the very strong association between mutation and adenocarcinoma histology, all
subsequent
analyses were restricted to the patients with adenocarcinoma. A comparison of adenocarcinoma
case
patients with and without K-ras mutations in their tumors revealed a tendency for mutations to
occur in
former smokers and for those with mutations to have smoked for more pack-years and to have
quit
smoking for less time, although none of these associations were statistically significant (Table 1, B). The
distribution of K-ras mutations by measures of smoking intensity and duration is shown in Fig. 1
and Table 1, B.
Among the patients with
adenocarcinoma, 53.8%
(95% CI = 46.8%-61.0%) were female. The prevalence of K-ras
mutation
was higher among women (26.2% [95% CI =
18.1%-35.6%]) than among men (17.4% [95% CI
=
10.3%-26.7%]). The spectrum of mutations in codon 12 of K-ras, including
the
prevalence of specific amino acid changes, is presented in Table 2.
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When the spectrum of mutations in the K-ras gene at codon 12 was examined by sex, we
observed that the majority of mutations were GT and occurred at the second guanine,
giving rise
to a cysteine. When this change was compared with all other amino acid substitutions combined,
there
was an excess of cysteine mutations among women, which did not reach statistical significance
(OR
= 2.6; 95% CI = 0.73-9.1).
To examine K-ras mutation as a marker of patient outcome, we calculated Kaplan-Meier
survival
probability curves for groups of patients and examined the differences by using the logrank test.
Considering all adenocarcinoma case patients for whom we had survival data (n = 195),
there
was a statistically significant association between K-ras mutation and decreased survival time (Fig.
2, A; P = .009, logrank test). When the ras-survival
association
was examined in a stage-specific manner, it was observed that the only stratum where there was a
statistically significant association between K-ras mutation and worse outcome was among stage I
cases (Fig. 2,
B; P = .002, logrank test).
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DISCUSSION |
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We also observed that women were statistically significantly more likely to have mutations in
codon
12 of the K-ras oncogene. There have been reports of a preponderance of K-ras mutations in
males,
but these were small studies, which primarily included only nonsmoking women, and none of the
studies
were prospective in design (as is the current study). Hence, the results of these other
investigations
either are not comparable or were subject to various selection biases (22,26,27,36). At the same time, the increase in lung cancer incidence among women that has been
consistently reported in recent years is clearly associated with an increase in adenocarcinoma
incidence
(1-13). Our data suggest that one mechanism for this increase might
involve
hormonally mediated selection of K-ras mutant clones. The second event that is needed to negate
the
ras-induced senescence experimentally observed by Serrano et al. (47)
might
be related to hormonal effects on cells of an adenocarcinoma histology. It is known that the
adenocarcinoma precursor cell can exhibit estrogen binding and express estrogen receptors (48). Furthermore, Kato et al. (49) have shown
that
K-ras mutation can enhance the expression of estrogen receptors and induce cell transformation in
NIH3T3 mouse embryo fibroblast cells. Finally, Caracta et al. (50) have
observed that adenocarcinoma cell lines, unlike other NSCLC cell lines, express both estrogen
receptor and estrogen receptor ß. Consequently, our data are consistent with the
following
sequence: Cigarette smoking induces K-ras mutation. The resultant clones are further expanded
by a
second event that may involve the growth-promoting effects of hormones (like estrogen) that may
be
specific for the adenocarcinoma histology. This model would account for the increased
occurrence of
K-ras mutations that we observed in women.
We also investigated the outcome of surgical therapy in the patients studied and found a statistically significant association between the presence of a K-ras mutation and aggressive disease. The proportion of tumors with K-ras mutation was slightly higher with increasing stage, and those tumors with mutation tended to be larger and to have associated lymph node metastasis. Associations of K-ras mutation with worsened patient survival are consistent with many previous studies (19-28). In addition, this study demonstrates that K-ras mutation has potential predictive value in a U.S. population that includes both men and women.
Of particular import is the very strong association between K-ras mutation and poorer survival among patients with stage I tumors, supported by results from other groups (19,20,22,25,27). In addition, our data suggest that the K-ras-survival association may be specific to stage I disease. One hypothesis consistent with these data is that K-ras mutation confers a metastatic advantage. That is, stage I K-ras mutant tumors may be more likely to have micrometastases undetected at diagnosis. Once clinically evident metastasis has occurred, there is no difference in survival based on ras mutation status. Consistent with this hypothesis is the expectation that K-ras mutations are overrepresented among patients with metastasis at the time of diagnosis, as is true in this study. It must be noted, however, that the design of this study is insufficient for determining if K-ras mutation status is associated with patient outcome in advanced disease because the cohort was restricted to patients treated with surgical resection.
Data from in vitro studies are congruent with the hypothesis that K-ras mutant tumors have increased metastatic potential. Ras has repeatedly been shown to transform fibroblasts. In addition, bronchial epithelial cells transformed with the Ki-ras virus no longer arrest cell growth in response to transforming growth factor ß (51). Other work has shown that mutant ras transfected into epithelial cells weakens their adhesion to the substratum (52). More recently, it has been demonstrated that the ras protein is involved in integrin activation (18). The implication from this work is that ras mutation might alter integrin functioning and, thus, change cell morphology and behavior. Since the ras protein is also linked to the rho-signaling pathway (involved in membrane ruffling), it is possible that mutation might affect the ability of the cell to crawl, which also might make ras mutants more likely to metastasize.
The strong association of K-ras mutation with patient outcome, particularly in patients with stage I adenocarcinoma, has important clinical implications that should be investigated further.
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NOTES |
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We thank Kathryn Springer, Dr. J. Brain, David Miller, Linda Lineback, and the Massachusetts General Hospital Cancer Registry for their assistance in data collection and management.
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REFERENCES |
---|
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---|
1
Thun MJ, Lally CA, Flannery JT, Calle EE, Flanders WD, Heath
CW
Jr. Cigarette smoking and changes in the histopathology of lung cancer. J Natl Cancer Inst1997
;89:1580-6.
2 Lubin JH, Blot WJ. Assessment of lung cancer risk factors by histologic category. J Natl Cancer Inst 1984;73:383-9.[Medline]
3 Brownson RC, Chang JC, Davis JR. Gender and histologic type variations in smoking-related risk of lung cancer. Epidemiology 1992;3:61-4.[Medline]
4 Travis WD, Travis LB, Devesa SS. Lung Cancer [published erratum appears in Cancer 1995;75:2979]. Cancer 1995;75(1 Suppl):191-202.[Medline]
5 Wynder EL, Hoffmann D. Smoking and lung cancer: scientific challenges and opportunities. Cancer Res 1994;54:5284-95.[Medline]
6 Parkin DM, Sasco AJ. Lung cancer: worldwide variation in occurrence and proportion attributable to tobacco use. Lung Cancer 1993;9:1-16.
7 Peto R, Lopez AD, Boreham J, Thun M, Heath C Jr, Doll R. Mortality from smoking worldwide. Br Med Bull 1996;52:12-21.[Abstract]
8
Zang EA, Wynder EL. Differences in lung cancer risk between
men
and women: examination of the evidence. J Natl Cancer Inst 1996;88:183-92.
9 McDuffie HH, Klaassen DJ, Dosman JA. Female-male differences in patients with primary lung cancer. Cancer 1987;59:1825-30.[Medline]
10 McDuffie HH, Klaassen DJ, Dosman JA. Men, women and primary lung cancera Saskatchewan personal interview study. J Clin Epidemiol 1991;44:537-44.[Medline]
11 Zang EA, Wynder EL. Cumulative tar exposure. A new index for estimating lung cancer risk among cigarrette smokers. Cancer 1992;70:69-76.[Medline]
12 Risch HA, Howe GR, Jain M, Burch JD, Holowaty EJ, Miller AB. Are female smokers at higher risk for lung cancer than male smokers? A case-control analysis by histologic type. Am J Epidemiol 1993;138:218-93.
13 Osann KE, Anton-Culver H, Kurosaki T, Taylor T. Sex differences in lung-cancer risk associated with cigarette smoking. Int J Cancer 1993;54:44-8.[Medline]
14 Barbacid M. Ras genes. Annu Rev Biochem 1987;56:779-827.[Medline]
15 Bos, JL. p21ras: an oncoprotein functioning in growth factor-induced signal transduction. Eur J Cancer 1995;31A:1051-4.
16 Khosravi-Far RS, Solski PA, Clark GJ, Kinch MS, Der CJ. Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol Cell Biol 1995;15:6443-53.[Abstract]
17 Qiu RG, Chen J, Kirn D, McCormick F, Symons M. An essential role for Rac in Ras transformation. Nature 1995;374:457-9.[Medline]
18 Hughes PE, Renshaw MW, Pfaff M, Forsyth J, Keivens VM, Schwartz MA, et al. Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 1997;88:521-30.[Medline]
19 Slebos RJ, Kibbelaar RE, Dalesio O, Kooistra A, Stam J, Meijer CJ, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 1990;323:561-5.[Abstract]
20 Rosell R, Li S, Skacel Z, Mate JL, Maestre J, Canela M, et al. Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients. Oncogene 1993;8:2407-12.[Medline]
21 Rosell R, Monzo M, Molina F, Martinez E, Pifarre A, Moreno I, et al. K-ras genotypes and prognosis in non-small-cell lung cancer. Ann Oncol 1995;6 Suppl 3:S15-20.[Medline]
22 Silini EM, Bosi F, Pellegata NS, Volpato G, Romano A, Nazari S, et al. K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma. Virchows Arch 1994;424:367-73.[Medline]
23 Fukuyama Y, Mitsudomi T, Sugio K, Ishida T, Akazawa K, Sugimachi K. K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer. Br J Cancer 1997;75:1125-30.[Medline]
24 Cho JY, Kim JH, Lee YH, Chung KY, Kim SK, Gong SJ, et al. Correlation between K-ras gene mutation and prognosis of patients with nonsmall cell lung carcinoma. Cancer 1997;79:462-7.[Medline]
25 Kwiatkowski DJ, Harpole DH Jr, Godleski J, Herndon JE 2nd, Shieh DB, Richards W, et al. Molecular pathologic substaging in 244 stage I non-small-cell lung cancer patients: clinical implications. J Clin Oncol 1998;16:2468-77.[Abstract]
26 De Gregorio L, Manenti G, Incarbone M, Pilotti S, Pastorino U, Pierotti MA, et al. Prognostic value of loss of heterozygosity and KRAS2 mutations in lung adenocarcinoma. Int J Cancer 1998;79:269-72.[Medline]
27 Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki T. ras gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res 1992;52:2903-6.[Abstract]
28 Kern JA, Slebos RJ, Top B, Rodenhuis S, Lager D, Robinson RA, et al. C-erbB-2 expression and codon 12 K-ras mutations both predict shortened survival for patients with pulmonary adenocarcinomas. J Clin Invest 1994;93:516-20.[Medline]
29 Isobe T, Hiyama K, Yoshida Y, Fujiwara Y, Yamakido M. Prognostic significance of p53 and ras gene abnormalities in lung adenocarcinoma patients with stage I disease after curative resection. Jpn J Cancer Res 1994;85:1240-6.[Medline]
30 Keohavong P, DeMichele MA, Melacrinos AC, Landreneau RJ, Weyant RJ, Siegfried JM. Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 1996;2:411-8.[Abstract]
31 Keohavong P, Zhu D, Melacrinos AC, DeMichele MA, Weyant RJ, Luketich JD, et al. Detection of low-fraction K-ras mutations in primary lung tumor using a sensitive method. Int J Cancer 1997;74:162-70.[Medline]
32 Siegfried JM, Gillespie AT, Mera R, Casey TJ, Keohavong P, Testa JR, et al. Prognostic value of specific KRAS mutations in lung adenocarcinomas. Cancer Epidemiol Biomarkers Prev 1997;6:841-7.[Abstract]
33
Greatens TM, Niehans GA, Rubins JB, Jessurun J, Kratzke RA,
Maddaus MA, et al. Do molecular markers predict survival in non-small-cell lung cancer? Am J
Respir Crit Care Med 1998;157:1093-7.
34 Nemunaitis J, Klemow S, Tong A, Courtney A, Johnston W, Mack M, et al. Prognostic value of K-ras mutations, ras oncoprotein, and c-erb B-2 oncoprotein expression in adenocarcinoma of the lung. Am J Clin Oncol 1998;21:155-60.[Medline]
35
Graziano SL, Gamble GP, Newman NB, Abbott LZ, Rooney M,
Mookherjee S, et al. Prognostic significance of K-ras codon 12 mutations in patients with
resected
stage I and II non-small-cell lung cancer. J Clin Oncol 1999;17:668-75.
36 Lung ML, Wong M, Lam WK, Lau KS, Kwan S, Fu KH, et al. Incidence of ras oncogene activation in lung carcinomas in Hong Kong. Cancer 1992;70:760-3.[Medline]
37 Slebos RJ, Hruban RH, Dalesio O, Mooi WJ, Offerhaus GJ, Rodenhuis S. Relationship between K-ras oncogene activation and smoking in adenocarcinoma of the human lung. J Natl Cancer Inst. 1991;83:1024-7.[Abstract]
38 Westra WH, Slebos RJ, Offerhaus GJ, Goodman SN, Evers SG, Kensler TW, et al. K-ras oncogene activation in lung adenocarcinomas from former smokers. Evidence that K-ras mutations are an early and irreversible event in the development of adenocarcinoma of the lung. Cancer 1993;72:432-8.[Medline]
39 Husgafvel-Pursiainen K, Hackman P, Ridanpaa M, Anttila S, Karjalainen A, Partanen T, et al. K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int J Cancer 1998;53:250-6.
40
Husgafvel-Pursiainen K, Karjalainen A, Kannio A, Anttila S,
Partanen T, Ojajarvi A, et al. Lung cancer and past occupational exposure to asbestos. Role of
p53
and K-ras mutations. Am J Respir Cell Mol Biol 1999;20:667-74.
41
Nelson HH, Christiani DC, Wiencke JK, Mark EJ, Wain JC,
Kelsey
KT. K-ras mutations and occupational asbestos exposure in lung adenocarcinoma:
asbestos-related
cancer without asbestosis. Cancer Res 1999;59:4570-3.
42
Cancer statistics, 1999. CA Cancer J Clin 1999;49:8-31, 1.
43
Belani CP. Ramanathan RK. Combined-modality treatment of
locally advanced non-small cell lung cancer: incorporation of novel chemotherapeutic agents.Chest 1998;113(1 Suppl):53S-60S.
44 McLoud TC, Bourgouin PM, Greenberg RW, Kosiuk JP, Templeton PA, Shepard JA, et al. Bronchogenic carcinoma: analysis of staging in the mediastinum with CT by correlative lymph node mapping and sampling. Radiology 1992;182:319-23.[Abstract]
45 Banerjee SK, Makdisis WF, Weston AP, Mitchell SM, Campbell DR. Microwave-based DNA extraction from paraffin-embedded tissue for PCR amplification. Biotechniques 1995;18:768-70, 772-3.[Medline]
46 Mitsudomi T, Viallet J, Mulshine JL, Linnoila RI, Minna JD, Gazdar AF. Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 1991;6:1353-62.[Medline]
47 Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p15INK4a. Cell 1997;88:593-602.[Medline]
48 Saunders PT, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 1997;154:R13-6.[Abstract]
49 Kato K, Ueoka Y, Kato K, Hachiya T, Nishida J, Wake N. Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation. Oncogene 1997;15:3037-46.[Medline]
50 Caracta CF, Powell C, Brody JS. Estrogen receptor status of lung cancer cell lines [abstract]. Am J Respir Crit Care Med 1999;159:A204.
51 Reddel RR, Ke Y, Kaighn ME, Malan-Shibley L, Lechner JF, Rhim JS, et al. Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation. Oncogene Res 1988;3:401-8.[Medline]
52 Kinch, MS, Burridge K. Altered adhesions in ras-transformed breast epithelial cells. Biochem Soc Trans 1995;23:446-50.[Medline]
Manuscript received July 7, 1999; revised September 24, 1999; accepted October 4, 1999.
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