Tissue Heterogeneity of EGFR Mutation in Lung Adenocarcinoma

Tissue Heterogeneity of EGFR Mutation in Lung Adenocarcinoma

BRIEF REPORT Tissue Heterogeneity of EGFR Mutation in Lung Adenocarcinoma Akira Sakurada, MD,* Humberto Lara-Guerra, MD,* Ni Liu, MSc,* Frances A. Sh...

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BRIEF REPORT

Tissue Heterogeneity of EGFR Mutation in Lung Adenocarcinoma Akira Sakurada, MD,* Humberto Lara-Guerra, MD,* Ni Liu, MSc,* Frances A. Shepherd, MD,† and Ming-Sound Tsao, MD‡

Tissue heterogeneity of EGFR gene mutation was studied in 10 formalin-fixed paraffin-embedded (FFPE) samples from four cases that demonstrated EGFR mutations in snap-frozen samples. EGFR mutations identical to those in frozen sample were demonstrated in 8 of 10 FFPE samples by direct sequencing and in 9 of 10 by fragment length analysis, but an exon-19 deletion mutation could not be identified in one FFPE sample analyzed by both techniques, despite multiple repeated assays. This suggests that some tumors may demonstrate intratumoral heterogeneity for the occurrence of EGFR mutation. Key Words: Epidermal growth factor receptor, Non-small cell lung cancer, Mutation, Heterogeneity. (J Thorac Oncol. 2008;3: 527–529)

M

utation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene is reported to occur frequently in Asian patients with non-small cell lung carcinoma, but less frequently in non-Asian patients.1 The presence of mutation is highly correlated with response to EGFR tyrosine kinase inhibitor (TKI) therapy,2,3 but available samples for mutational analysis are often limited to small, formalin-fixed paraffin-embedded (FFPE) biopsy tissue. In the recent studies performed on specimens from large randomized clinical trials of EGFR TKIs, specimens that contained sufficient tumor cells for study were available in only 24 to 28% of trial patients, and only 59 to 90% of the specimens demonstrated informative results for sequencing.4 –7 Thus, the efficiency of mutation detection in FFPE is an important limiting factor in these studies. The possibility of tissue heterogeneity for the occurrence of EGFR mutation within tumors is potentially an important consideration, as it might influence the results of mutation screening. With small biopsy samples, tissue heter*Princess Margaret Hospital and Ontario Cancer Institute, University Health Network; Departments of †Medicine and ‡Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Ming-Sound Tsao, MD, FRCPC, Department of Pathology, University Health Network, 200 Elizabeth Street, Toronto, Ontario, Canada M5C 2C5. E-mail: [email protected] Copyright © 2008 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/08/0305-0527

Journal of Thoracic Oncology • Volume 3, Number 5, May 2008

ogeneity may result in the analyzed sample being nonrepresentative of the tumor, leading to misinterpretation of the mutation results. Tissue heterogeneity for the occurrence of p53 or KRAS mutations has been reported in many types of tumors8 –10; this issue for EGFR mutation in lung cancer has not been addressed. To check the genetic heterogeneity and detectability of EGFR mutation on FFPE, we analyzed EGFR mutation on different paraffin block samples from separate areas of the same tumor that previously showed EGFR mutation by sequencing on fresh frozen specimens.

MATERIALS AND METHODS The University Health Network Research Ethics Board approved this study, which was conducted on four surgically resected adenocarcinoma tumors that had been found previously to harbor EGFR tyrosine kinase domain mutations upon analysis of their fresh frozen tumor samples (Table 1). Two cases (case P10 and P114) had L858R mutations, whereas the other two (case P163 and P229) had 15-nucleotide deletions (del_E746A750) in exon-19. All patients were never smokers. Paraffin blocks from these tumors were available for study. When multiple blocks were available, they were taken from different areas of the same tumor (Table 1). The histology of the same tumor in different blocks was not significantly different. Standard mutation analysis on exon-19 and exon-21 of EGFR were performed as described previously.6 Briefly, paraffin sections were scraped off the slides for DNA extraction, and DNA was amplified by the nested polymerase chain reaction (PCR). The PCR products were sequenced with the ABI3100 sequence analyzer (Applied Biosystems, Foster City, CA). For blocks with less than 50% estimated tumor cell content, enrichment by microdissection was performed. For each block, three independent PCR-sequencing reactions were performed. The fragment length analysis (FLA) method of EGFR mutation analysis was performed as previously reported by Pan et al.11 The genomic sequences of exons-19 and -21 were amplified using their respective forward primers and fluorescent-labeled reverse primers, and the PCR products were analyzed by ABI 3100 (Applied Biosystems). The length of the PCR products from exon-19 deletion mutants was evaluated with Genotypes Plot software (Applied Biosystems). The 222-base pairs (bp) of PCR products on exon-21 were treated with Sau39I, which digests wild-type sequence to produce a 173-bp fluorescent-labeled fragment, whereas the

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Sakurada et al.

mutant sequence produced an 87-bp fluorescently labeled fragment due to the creation of a Sau39I restriction site by the L858R mutation. TABLE 1. Patients Characteristics Case No. Gender Age Stage P10

Female

62

IB

P114 Female

42

IIIA

P163 Female

66

IB

P229 Female

66

IB

Paraffin Blocks Available

EGFR Status

Histology Well-differentiated adenocarcinoma Moderately differentiated adenocarcinoma Well-differentiated adenocarcinoma Well-differentiated adenocarcinoma

L858R

2

L858R

1

Del_E746_A750

3

Del_E746_A750

2

TABLE 2. Results of Direct Sequencing and Fragment Length Analysis on the FFPE Samples Case No.

Mutation In Frozen Tumor Tissue

P10

L858R

P114 P163

L858R Del_E746_A750

P229

Del_E746_A750

Paraffin Block No.

Direct Sequencing Analysis

Fragment Length Analysis

1 2 1 1 2 3 1 2

L858R L858R Wild type Wild type Del_E746_A750 Del_E746_A750 Del_E746_A750 Del_E746_A750

L858R L858R L858R Wild-type 15nt deletion 15nt deletion 15nt deletion 15nt deletion

RESULTS Mutations identical to those found in the frozen tumor specimens were confirmed in 9 of 10 paraffin blocks. Three independent repeats of PCR and sequencing assays failed to detect the L858R mutation in paraffin block P114#1 or the 15-nucleotide deletion of exon-19 in the P163#1 block (Table 2). Using the FLA method, L858R mutation was confirmed in all five paraffin blocks including P114#1 that was negative by direct sequencing assay. In 4 of 5 paraffin blocks from tumors P163 and P229, the exon-19 15nucleotide deletion was readily found. However, this deletion could not be identified in DNA isolated from the P163#1 paraffin block, either by repeated direct sequencings or using FLA on DNA isolated independently from serial paraffin sections (Figure 1).

DISCUSSION We have provided evidence for the existence of intratumoral heterogeneity for EGFR tyrosine kinase domain mutations in lung adenocarcinoma. With the limited number of cases studied, we only found such heterogeneity with the exon-19 deletion. In the current study, P163#1 was negative for deletion not only by direct sequencing but also by FLA. Possible reasons why we did not detect the deletion from this paraffin block include: low concentration of mutant sequences caused by high normal cell contamination, or intratumoral heterogeneity for mutation occurrence. The first possibility is unlikely because the H&E section showed more than 50% population of tumor cells with a histologic appearance identical to the other blocks positive for this deletion.

FIGURE 1. The histopathology, direct sequencing, and fragment length analysis results on three different paraffin blocks of case P163. Left pictures show histology (H&E staining) of paraffin sections from blocks P163#1(A), #2(D), and #3(G). All sections showed similar morphology. Deletion was demonstrated in P163#2(E) and P163#3(H) as indicated by black arrows but not in P163#1(B). The 15-nt deletion was also demonstrated by LFA in P163#2(F) and P163#3(I) as indicated by red arrows, but not in P163#1(C).

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Furthermore, we have independently confirmed that FLA is sensitive enough to detect mutant alleles even at 100-fold lower levels than concurrently present wild-type alleles, especially for deletion mutations (data not shown).11 Although we cannot exclude other less obvious mechanisms, heterogeneity is the likely reason for our finding. To the best of our knowledge, no similarly designed studies to evaluate tumor heterogeneity of EGFR mutations have been reported. Nagai et al.12 previously reported that among non-small cell lung cancer cell lines screened, 11 demonstrated EGFR mutations in 100% of cells whereas eight cell lines had mutation in only 10% or less of the tumor cells. The latter results are consistent with the presence of genetic heterogeneity or instability of the mutations in these cell lines. EGFR mutation status has been assessed in atypical adenomatous hyperplasia, BAC, and invasive adenocarcinoma in Japanese patients, and the frequencies of mutation were reported to be 3%, 11%, and 42%, respectively.13 This increasing frequency of EGFR mutation with histologic progression of malignancy has led to the hypothesis that atypical adenomatous hyperplasia lesion with EGFR mutation preferentially progress because of an mutation-induced growth advantage. Mutation screening on normal bronchial epithelium of patients with EGFR-mutant tumor has also revealed the presence of identical EGFR mutations in 9 of 21 (43%) of corresponding normal epithelial samples studied.14 The authors speculated that this high frequency of mutations in normal epithelium is caused by the “limited field effect.” These results suggest that tumors with EGFR mutation acquire the mutation early during carcinogenesis. The finding of multiple EGFR mutations in a single specimen has been reported in a number of studies.3,4,6 Multiple simultaneously detected EGFR mutations may suggest the possibility of intratumoral heterogeneity and metachronous generation of different mutations. Among those multiple mutations, concurrent T790M mutation associated with exon-19 deletions or L858R mutation appear to confer acquired resistance to TKIs.15 However, such mutations have been detected only at very low frequency, both in recurrent and untreated tumors.15 Although the impact of multiple mutations is not fully understood, it might indicate the instability of EGFR mutations. Nevertheless, based on the T790M result, it is possible that the existence of even a minor subpopulation of EGFR wild-type tumor cells in dominant population of EGFR mutant tumor cells could impact on response to TKIs. Therefore, although the frequency of intratumoral heterogeneity appears low, this might become an important issue when only very small biopsy specimens are available for mutation analysis to direct therapy. The effect of genetic heterogeneity on the response and prognosis with TKI therapy warrants further studies. Recently, there has been discussion on the sensitivity of the direct sequencing method to detect EGFR mutations in FFPE samples.16 Using this technique, we failed to detect the original mutation/deletion in 2 of 10 blocks after at least three independent PCR assays. However, one of these negative cases yielded a positive result by a single FLA study, which had been optimized to detect L858R or exon-19 deletion

Heterogeneity of EGFR mutations

mutations. This is consistent with the results of Pan et al.11 In conclusion, we have identified intratumoral heterogeneity in an adenocarcinoma with minor component of EGFR wildtype tumor cells and a majority of tumor cells having exon-19 deletion mutation. The clinical significance of intratumoral heterogeneity warrants future studies.

ACKNOWLEDGMENTS This work is supported by the Ontario Cancer Research Network grant 03-Apr-0324 and a grant from the Jacqueline Seroussi Memorial Foundation for Cancer Research. Dr Shepherd holds the Scott Taylor Chair in Lung Cancer Research. Dr Tsao holds the M. Qasim Choksi Chair in Lung Cancer Translational Research. REFERENCES 1. Sakurada A, Shepherd FA, Tsao MS. Epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer: impact of primary or secondary mutations. Clin Lung Cancer 2006;7(Suppl 4):S138 – 44. 2. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmall-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129 –2139. 3. Pao W, Miller VA. Epidermal growth factor receptor mutations, smallmolecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. J Clin Oncol 2005;23:2556 –2568. 4. Eberhard DA, Johnson BE, Amler LC, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 2005;23:5900 –5909. 5. Bell DW, Lynch TJ, Haserlat SM, et al. Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials. J Clin Oncol 2005;23:8081– 8092. 6. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer—molecular and clinical predictors of outcome. N Engl J Med 2005;353:133–144. 7. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005;23:5892–5899. 8. Iwamatsu H, Nishikura K, Watanabe H, et al. Heterogeneity of p53 mutational status in the superficial spreading type of early gastric carcinoma. Gastric Cancer 2001;4:20 –26. 9. Klein CA, Blankenstein TJ, Schmidt-Kittler O, et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683– 689. 10. Kitago M, Ueda M, Aiura K, et al. Comparison of K-ras point mutation distributions in intraductal papillary-mucinous tumors and ductal adenocarcinoma of the pancreas. Int J Cancer 2004;110:177–182. 11. Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn 2005;7:396 – 403. 12. Nagai Y, Miyazawa H, Huqun, et al. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 2005;65:7276 –7282. 13. Yoshida Y, Shibata T, Kokubu A, et al. Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung. Lung Cancer 2005;50:1– 8. 14. Tang X, Shigematsu H, Bekele BN, et al. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res 2005;65:7568 –7572. 15. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.[see comment]. PLoS Med 2005;2:e73. 16. Ja¨nne PA, Borras AM, Kuang YA, et al. A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res 2006;12:751–758

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