Mutational Analysis in Cytological Specimens of Advanced Lung Adenocarcinoma: A Sensitive Method for Molecular Diagnosis

ORIGINAL ARTICLE

Mutational Analysis in Cytological Specimens of Advanced Lung Adenocarcinoma: A Sensitive Method for Molecular Diagnosis Laura Boldrini, PhD,* Silvia Gisfredi, PhD,* Silvia Ursino, PhD,* Tiziano Camacci,* Editta Baldini, MD,† Franca Melfi, MD,‡ and Gabriella Fontanini, MD*

Introduction: The discovery that somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with sensitivity to the EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinomas, whereas Kras mutations are associated with resistance, has generated excitement among both clinicians and researchers studying non-small cell lung cancer (NSCLC). Mutational analysis may soon be very useful in choosing among a wide range of targeted therapies to individualize treatment to tumor characteristics. This analysis would be even more useful in patients with advanced NSCLC, in whom cytological specimens are often the only material available. Methods: We analyzed 23 archived cytologic specimens of advanced/metastatic lung adenocarcinomas for mutations in EGFR exons 18 to 21, and Kras exon 2. Results: Our data show that our cytological specimens were perfectly adequate for the molecular analysis of EGFR and Kras mutations. EGFR TK domain mutations were found in three cases (13.04%) and were associated with both female gender (p ⫽ 0.02) and a nonsmoking history (p ⫽ 0.008). Moreover, we explored the relationship between EGFR mutation status and the presence of Kras mutations. Kras mutations involving codon 12 in exon 2 were found in 5 (21.73%) of the 23 adenocarcinomas and were associated, where known, with smoking habits. We never found EGFR alterations in tumors with Kras mutations. Conclusions: Our results provide oncologists with a highly accurate laboratory method to identify biological predictors of the efficacy of different therapies, and they may have an important impact on clinical practice. This method may be particularly useful in patients with advanced/metastatic NSCLC. Key Words: Cytology, Non-small cell lung cancer, Epidermal growth factor receptor, Kras. (J Thorac Oncol. 2007;2: 1086–1090) Departments of *Surgery and †Medical Oncology, Oncology Department, Santa Chiara University Hospital, Pisa, Italy; and ‡Department of Cardiothoracic Surgery, University of Pisa, Pisa, Italy. Disclosure: The authors declare no conflict of interest. Address for correspondence: Laura. Boldrini, Department of Surgery, via Roma, 57 56126 Pisa, Italy. E-mail: [email protected] Copyright © 2007 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/07/0212-1086

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he discovery that somatic mutations in the epidermal growth factor receptor (EGFR) gene in lung adenocarcinomas are associated with sensitivity to the EGFR tyrosine kinase inhibitors (TKI) gefitinib1,2 and erlotinib,3 whereas Kras mutations are associated with resistance to TKIs,4 – 6 has generated excitement among clinicians and researchers studying non-small cell lung cancer (NSCLC). Mutational analysis should soon be very useful to clinicians in choosing among the wide range of targeted therapies that can be implemented for individual patients. For patients with advanced/metastatic lung cancer in whom cytological specimens, such as fineneedle aspirations, sputum, and bronchial washing or brushing are often the only materials available, mutational analysis should be particularly useful. In our study, we explored the feasibility of performing mutational analysis on diagnostic cytological specimens of lung adenocarcinoma, and we demonstrated that all of our samples were perfectly adequate for molecular analysis of both EGFR and Kras mutations by an automated sequencing method.

MATERIALS AND METHODS Patient and Specimen Characteristics We analyzed 23 archived cytological specimens, which were collected at our institute from NSCLC patients from April 2005 to April 2006. Samples were obtained as follows: 17 out of 23 by fine-needle aspiration, 1 by sputum, 4 by bronchial washing, and 1 by bronchial brushing. Specimens from sputum and bronchial brushing were processed by the liquid-based, thinlayer cytology Thin Prep 2000 method (Cytyc Co., Marlborough, MA). The material was fixed with the hemolytic and preservative solution Cytolit (Cytyc Co.). The cells were spun at 1500 rpm, and then the sediment was transferred to the Preservcyt (Cytyc Co.) solution to be processed with the T2000 automated processor, according to the manufacturer’s recommendations. The resulting slide was fixed in 95% ethanol and was stained with Papanicolaou. The slides of all of the specimens were incubated in xylene overnight to remove the coverslip, and then they were washed in 100% and 70% ethanol. The cells were microdissected, using a 25-gauge needle on a syringe as a microdissecting tool. While viewing the tissue through the microscope, the cell population of interest was gently scraped with

Journal of Thoracic Oncology • Volume 2, Number 12, December 2007

Journal of Thoracic Oncology • Volume 2, Number 12, December 2007

the needle. The tip of the needle with the procured tissue fragments was carefully placed into a small PCR tube containing the appropriate buffer. Gentle shaking of the tube ensured that the tissue detached from the tip of the needle.

PCR for EGFR Exons 18 to 21 Total DNA was extracted from the specimens, using a standard acid– guanidium–phenolchloroform method. DNA extraction was then performed, using a spin column procedure (QIAamp Tissue Kit, Qiagen). The eluted DNA was used as template in a standard 20-␮l PCR reaction mixture consisting of 20 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM deoxynucleoside triphosphate, 8 pmol each of sense and antisense primer, and 2.5 units of AmpliTaq Gold (Applied Biosystems). Primers used for the amplification were as follows: for exon 18, 5=-ACCCTTGTCTCTGTGTTCTTGTCC-3= 5=-AGACCATGAGAGGCCCTGC-3=; for exon 19, 5=-GCACCATCTCACAATTGCCAGTTA-3= 5=-GAGGTTCAGAGCCATGGACCC-3=; for exon 20, 5=-CACACTGACGTGCCTCTCCCTCCC-3= 5=-CTCCCCTCCCCGTATCTCCCTTCC-3=; and for exon 21, 5=-CCATGATGATCTGTCCCTCACA-3= 5=-AGGAAAATGCTGGCTGACCTAAAG-3=. PCR product sizes for EGFR exons 18, 19, 20, and 21 were 207, 194, 247, and 235 bp, respectively. Because all of the primers had similar melting temperatures, the same PCR conditions were used to simultaneously amplify all four exons (in separated reaction tubes): after initial denaturation at 94°C (7 minutes), there were 35 cycles of denaturation at 94°C for 60 seconds, annealing at 58°C for 60 seconds, and synthesis at 72°C for 60 seconds, followed by a final extension at 72°C for 7 minutes. As a negative control, the DNA template was omitted from the reaction. The amplification products were separated on 1.5% agarose gels and visualized by ethidium– bromide staining. For the detection of mutations, PCR products were purified with ExoSAP-IT (Amersham Biosciences) and sequenced using a cyclic sequencing kit (ALFexpress II, Amersham Biosciences), according to the manufacturer’s recommendations.

Mutagenic PCR for Kras Exon 2 DNA previously extracted was amplified by a mutagenic PCR assay. We used a mismatched upstream primer for codon 12 amplification and a mismatched downstream primer for codon 13 amplification, which introduced a BstNI and a HaeIII restriction site, respectively, in the wild-type allele. The primers used were as follows: Kras/12 (sense) 5=-ACTGAATATAAACTTGTGGTAGTTGGACCT-3= (nt 99 –128) and (antisense) 5=-CTGTATCAAAGAATGGTCCTGCACCAGTA-3= (nt 232–260),

Mutational Analysis in NSCLC

Kras/13 (sense) 5=-GTACTGGTGGAGTATTTGATAGTGTATTAA-3= (nt 1–30) and (antisense) 5=-GTATCGTCAAGGCACTCTTGCCTAGG-3= (nt 134 –159). The underlined bases represent mismatches.

RFLP Analysis (Restriction Fragment Polymorphism Analysis) BstNI digestion of the wild-type codon 12 allele yielded two bands of 133 and 29 bp, whereas the mutant remained intact (162 bp). HaeIII digestion of the wild-type codon 13 allele yielded fragments of 85, 48, and 26 bp, whereas the mutant yielded only two fragments of 85 and 74 bp. (A constant HaeIII site at nucleotide 85 yielded an 85-bp fragment in all samples).

Statistical Analysis All statistical analyses were carried out using Statistica software (Stat-soft). A chi-square test was used to analyze the associations between the different variables. The a priori level of significance was set at a p value of less than 0.05.

RESULTS Patient and Specimen Characteristics There were 14 men (60.9%) and 9 women (39.1%), with ages at diagnosis ranging from 44 to 77 years (mean age 62.3 years, median 64 years). Smoking habits were known for 14 of the 23 patients: there were 5 nonsmokers (35.7%, all women) and 9 smokers (64.3%, 7 men and 2 women). Cytologic type of the adenocarcinoma was determined according to WHO criteria7; for three cases, BAC pattern was predominant. Advanced pathologic staging of lung cancers was determined according to the revised International System for Staging Lung Cancer8 for four cases; in the other patients, it was not possible to perform staging, because the cytological specimen represented the only diagnostic tool. All twenty-three cytological specimens (17 fine-needle aspirations, 1 sputum in thin-prep, 4 bronchial washings, and 1 bronchial brushing in thin-prep) were informative, containing enough cells to isolate a sufficient amount of DNA for mutational analysis. Also, liquid-base cytology seems to be a reliable cytologic method for this molecular approach.

EGFR Mutations EGFR TK domain mutations were found in three cases (13.04%). Of those three mutations, two were in-frame deletions in exon 19, and one was a point mutation in exon 20. The in-frame deletions in exon 19 involved five codons, E746 –A750, from nucleotide 2235 to 2249; the mutation in exon 20 resulted in a threonine-to-methionine amino acid change at position 790 (T790M) in the kinase domain. All three cases were from women who had never smoked (Table 1). Representative nucleotide sequences of the EGFR mutations are shown in Figure 1. We analyzed the relationship between EGFR mutations and clinicopathologic features of adenocarcinomas (Table 2).

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TABLE 1. Genetic Alterations in the Kinase Domain of the EGFR Gene (Exons 18 –21) Case No. 1 2 23

Histology

Sex

Age (yr)

Smoking Status

Exon

Aminoacidic Alteration

Adeno BAC Adeno

F F F

44 57 45

Never Never Never

19 19 20

E746 A750 del E746 A750 del T790M

EGFR mutations in adenocarcinomas were associated with both female gender (p ⫽ 0.02) and with smoking habits (p ⫽ 0.008).

Kras Mutations Examples of representative RFLP products for the Kras mutational analysis are shown in Figure 2.

A.

Case 1, 2

DISCUSSION Accumulating evidence suggests that gefitinib provides a survival benefit to patients with NSCLCs harboring EGFR mutations. Therefore, screening for EGFR mutations in all advanced NSCLC patients is expected to become a routine procedure. To examine a large number of samples, and to deal with the variety of clinical settings in which the samples are taken, an EGFR mutation test that is sensitive and integrated into the standard procedures for cancer diagnosis is needed. This report examines an approach to mutational analysis of a variety of cytological specimens (fine-needle aspirations, sputum in thin-prep, bronchial washing, and

Wild type

EGFR Exon 19

EGFR protein EGFR gene

Kras mutations were not found in adenocarcinomas with EGFR mutations (Table 2), suggesting a mutually exclusive relationship. Kras mutations were found in 5 (21.73%) of the 23 adenocarcinomas, involving codon 12 in exon 2 (Table 3).

744 I K E L R E A T S 752 2230 ATC AAG GAA TTA AGA GAA GCA ACA TCT 2256 ATC AA -------------------------------------A ACA TCT

Case 1, 2

Wild type

B.

EGFR Exon 20

EGFR protein EGFR gene Case 23

788 L I T Q L 792 2362 CTC ATC ACG CAG CTC 2376 CTC ATC ATG CAG CTC

Case 23

FIGURE 1. Mutations in the EGFR gene in non-small cell lung cancer. (A) One pattern of an in-frame deletion in EGFR exon 19, and its representative electropherogram. (B) T790M mutation in EGFR exon 20.

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TABLE 2. EGFR Mutations in 23 Cytological Specimens of Advanced Lung Adenocarcinoma

TABLE 3. Case No.

Mutational Analysis in NSCLC

Kras Alterations Histology

Sex

Age (yr)

Smoking Status

Codon

Adeno Adeno Adeno Adeno Adeno

F F M M M

52 77 76 68 68

Unknown Current Current Unknown Current

12 12 12 12 12

EGFR Status Variables Age (yr) ⱕ65.5 ⬎65.5 Sex Male Female Smoking status Nonsmokers Longtime smokers Kras status Wild type Mutated

n

wt

mut

p

12 11

9 11

3 0

0.07

14 9

14 6

0 3

0.02

5 9

2 9

3 0

0.008

18 5

15 5

3 0

0.32

1 A.

2

3

4 Cn M

Mutagenic PCR Kras codon 12 162 bp

1 B.

RFLP Analysis Kras codon 12

2

3

4

M

162 bp 133 bp

FIGURE 2. Mutations in the Kras gene in non-small cell lung cancer. (A) Gel electrophoresis of mutagenic PCR products for codon 12 of Kras. Lanes 1 through 3, NSCLC samples; lane 4, control sample; Cn, negative control (with no DNA added); M, molecular weight marker (100 bp). (B) Gel electrophoresis of PCR products digested with restriction enzyme BstNI. Lane 1, homozygous sample for codon 12 mutation (uncut PCR product of 162bp); lane 2, heterozygous sample (bands at 162 and 133bp); lane 3, homozygous normal sample (band at 133bp); lane 4, homozygous normal control; M, molecular weight marker (50 bp).

bronchial brushing in thin-prep) by scraping cells off of the diagnostic glass slide without destaining processes, giving faster results. Information about EGFR mutations is, therefore, available at the time when the oncologists determine the treatment regimen for each patient, and we have found this information to be very useful. Moreover, our test uses diagnostic samples, and, therefore, patients do not need to undergo additional procedures. Our method is also based on a simple, widely available technique of automated sequencing; therefore, our analysis is easily integrated into clinical practice and is expected to help medical specialists use the latest research results to select the optimal treatment strategy for lung cancer patients. Because EGFR mutations in lung can-

3 7 12 14 19

cers were limited to the first four exons (exons 18 –21) of the TK domain in previous studies,1,2,9,10 –12 we searched for mutations in these four exons in our 23 cytological specimens of advanced lung adenocarcinoma. EGFR TK domain mutations were found in three cases (13.04%); the frequency of EGFR mutations was within the range already reported in other studies.13 Deletions in exon 19, targeting a five-codon region (codons 746 –750), as well as a mutation in exon 20 (T790M), have both been reported previously.10 –12,14 Most studies have shown a significant association of EGFR mutations, particularly exon 19 deletions, and response to TKIs.15–24 On the other hand, the T790M substitution has been reported in progressive lesions after gefitinib or erlotinib therapy.25,26 Our patient (number 23) was a nonsmoking female with a stage IV adenocarcinoma of the lung, who showed progression of the disease after 14 months of therapy with gefitinib (250 mg/day) as the first-line therapy; the T790M mutation was found in the biopsy performed in this patient after the treatment with the EGFR-TKI. Recent studies also showed a differential impact on survival of different EGFR mutations.27–29 Consistent with previous studies,1,2,9 –12,14,30 EGFR mutations were associated with both female gender and a nonsmoking history. Moreover, we explored the relationship between EGFR mutational status and the presence of Kras mutations. Kras is a critical downstream effector of the EGFR pathway, which has been found to be mutated in about 15% to 30% of lung adenocarcinomas.12,31 Activating mutations of Kras usually occur in codons 12 and 13 of exon 232,33 and have been reported to be associated with intrinsic TKI resistance.5 Mutations in Kras are commonly associated with a history of tobacco smoke exposure,31 and several studies have shown that EGFR-sensitizing mutations and Kras mutations are mutually exclusive.4,5,10 –12,28,32,33 Consistent with all previous studies, in our series of lung adenocarcinomas, Kras mutations involving codon 12 in exon 2 were found in 5 (21.73%) of the 23 adenocarcinomas and were associated, where known, with smoking habits; we never found EGFR alterations in tumors with Kras mutations. In conclusion, we have demonstrated the feasibility of performing mutational analysis on archived cytological specimens. This may be especially important for metastatic NSCLC patients, where cytologic material frequently is all that is available. Testing of EGFR and Kras mutations is likely to become indispensable in selecting the best treatment options for individual patients with lung cancer.

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ACKNOWLEDGMENTS CP

This study was supported by AIDA (Italian Association Women Against Lung Cancer) and by the Grant COFIN 2004 no. 2004061208 of the Italian Minister of University and Scientific Research. REFERENCES

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