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Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer
ORIGINAL ARTICLE
Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer Marta Salido, MSc,*†‡ Lara Pijuan, MD, PhD,*‡ Luz Martínez-Avile´s, MSc,*‡ Ana B. Galva´n, MSc,* Israel Can˜adas, MSc,‡ Ana Rovira, PhD,‡§ Montserrat Zanui, MD,§ Alejandro Martínez, MD,‡§ Raquel Longaro´n,* Francisco Sole, PhD,*‡ Sergio Serrano, MD, PhD,*‡㛳 Beatriz Bellosillo, PhD,*‡ Murry W. Wynes, PhD,¶ Joan Albanell, MD, PhD,‡§㛳 Fred R. Hirsch, MD, PhD,¶ and Edurne Arriola, MD, PhD‡§
Introduction: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. Methods: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. Results: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p ⬍ 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. Conclusion: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK
*Pathology Department, Hospital del Mar, Parc de Salut Mar; †Animal and Vegetal Biology and Ecology Department, Anthropology Unit, BioScience Faculty, Universitat Auto`noma de Barcelona, Bellaterra; ‡Cancer Research Program, IMIM-Hospital del Mar, Parc de Salut Mar; §Oncology Department, Hospital del Mar, Parc de Salut Mar; 㛳Autonomous Barcelona University, Barcelona, Spain; and ¶University of Colorado Cancer Center, Aurora, Colorado. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Edurne Arriola, MD, PhD, Medical Oncology Department, Hospital del Mar, Passeig Maritim 25-29, Barcelona 08003, Spain. E-mail: [email protected] The first two authors contributed equally to this work. Copyright © 2010 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/11/0601-0021
amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies. Key Words: ALK, Non-small cell lung cancer, Amplification, Translocation, Polysomy. (J Thorac Oncol. 2011;6: 21–27)
L
ung cancer is the leading cause in the cancer-related deaths in developed countries.1 Although the prognosis of this disease remains poor, some clinically relevant advances have occurred in recent years. The identification of epidermal growth factor receptor (EGFR) mutations and amplification in a subset of patients with non-small cell lung cancer (NSCLC), coupled with the development of EGFR tyrosine kinase inhibitors, has opened new ways to treat this disease.2,3 Nevertheless, there is a need to find additional molecular targets to further improve patients’ outcome. Recently, the novel fusion transcript with transforming activity, formed by the translocation of echinoderm microtubule-associated protein-like 4 (EML4) (2p21) and anaplastic lymphoma kinase (ALK) (2p23), has been described in a subset of NSCLCs.4 ALK was originally involved in carcinogenesis in anaplastic large cell lymphoma as part of a chromosomal rearrangement as a fusion partner of nucleophosmin.5 Other fusion partners to ALK have been described since then and have been associated with other tumor types such as myofibroblastic tumors, B-cell lymphomas, and squamous cell carcinomas of the esophagus.6,7 The products of these translocations are fusion proteins with constitutively activated ALK tyrosine kinase, which plays a role in carcinogenesis by the aberrant phosphorylation of multiple intracellular substrates downstream of ALK-chimerical oncoproteins.8 In preclinical models of activated ALK, specific ALK targeting drugs inhibit cell proliferation and induce apoptosis in models “addicted” to this genetic aberration.9 Moreover, the analysis of clinical specimens in a number of studies suggests that this fusion gene may define a novel subclass of tumors within NSCLC characterized by distinct clinicopathological features in both Western and Asian populations.10 –13 Nevertheless, other reports have described cases that represent exceptions to this rule.14 There-
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fore, there are still many issues about the role of ALK in lung cancer that remain to be determined. Most importantly, the identification of EML4-ALK has provided clinicians with a novel potential therapeutic target as demonstrated by the favorable results of a phase I trial with a dual MET and ALK small molecule inhibitor that has showed promising clinical responses in patients harboring ALK translocations.15 Additional mechanisms of aberrant activation of ALK have been described in neuroblastomas. In these tumors, activating mutations and ALK amplification are the underlying mechanisms for ALK-dependent tumorigenesis16 –19 and are associated with a specific clinical phenotype in this disease. Nevertheless, copy number changes of ALK and their significance are poorly characterized in NSCLC and may represent an additional mechanism of activation of ALK and, therefore, play a role in the carcinogenesis of a subset of lung tumors. In this work, we sought to study the status of the ALK gene by fluorescence in situ hybridization (FISH) on a population of white patients and correlate it with the status of EGFR (mutations and copy number variations) in adenocarcinomas. We also correlated the genetic findings with clinical outcome.
PATIENTS AND METHODS Patient Population The samples included in this study were obtained from patients attending the Lung Cancer Unit in Hospital del Mar for diagnosis and treatment. The only inclusion criterion was the availability of tissue for biomarker studies. Clinical details of these patients were included in a database. This project was approved by the local ethics committee (CEIC-IMAS2009/3619/I).
Fluorescence In Situ Hybridization Four-micrometer paraffin-embedded histologic sections were used for FISH analysis. To assess the genetic status of EGFR and ALK, we used LSI EGFR/CEP7 and break-apart ALK (2p23) probes (Abbott Molecular Inc., Des Plaines, IL). To determine the fusion partner for ALK for cases with ALK translocation, we examined previously identified fusion partners EML4 (2p23), KIF5B (10p11.22), and TFG (3q12.2).20,21 Break-apart probes were designed using bacterial artificial chromosome (BAC) clones selected from the CHORI BAC/PAC resource (http://bacpac.chori.org): centromeric EML4: pooled RP11-804P20 and RP11-413N9, telomeric EML4: pooled RP11-798D22 and RP11-34L01; centromeric KIF5B: pooled RP11-633K11 and RP11-281A19 in spectrum Red, telomeric KIF5B pooled RP11-460H18 and RP11-166N17; and centromeric TFG: pooled RP11-320C17 and RP11-423M9, telomeric TFG: pooled RP11-49H3 and RP11-168G7. FISH was performed as described previously.22 Results were analyzed in a fluorescent microscope (Olympus, BX51) using the Cytovision software (Applied Imaging, Santa Clara, CA). A minimum of 50 nuclei was scored. Patients were classified as EGFR positive/negative according to criteria described in detail elsewhere.23 As criteria for copy number aberrations of ALK has not been established,
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we arbitrarily used the following cutoffs adapted from the criteria established for EGFR and HER2 in lung cancer specimens.23,24 Briefly, gain (including both low and high genomic gain) was defined as a mean copy number of 3 to 5 fusion signals in ⱖ10% of cells and amplification as the presence of ⱖ6 copies of ALK per cell in ⱖ10% of analyzed cells. In cases where clusters were observed, we reported the percentage of cells with clusters and considered amplified, cases with ⱖ10% of ALK clusters. On cases with ALK amplification, FISH with CEP2 (a centromeric alpha-satellite specific for chromosome 2) was performed (Abbott Molecular Inc.) to exclude polysomy. Normal fusion ALK, EML4, KIF5B, and TFG signals presented as an overlapping orange/ red and green (yellowish) signals. These probes were considered typically rearranged when separated green and orange/ red signals (at least by three times the signal diameter) were identified and atypically rearranged when a single orange or green signal was seen.
Immunohistochemistry for EGFR and ALK Four micrometers serial sections of paraffin blocks were used for the evaluation of EGFR and ALK done with the DAKO Cytomation EGFR PharmDxTM and ALK (D5F3) antibody (Cell Signaling, Danvers, MA), respectively. This antibody for ALK is able to detect the wild type and truncated proteins. Diaminobenzidine was used as the cromogen to identify positive results. The scoring for EGFR IHC was done considering staining intensity, as recommended by the manufacturer, from negative: 0, no detectable staining; to 3⫹, strong and continuous membranous staining. For ALK expression scoring, the percent of cells was determined within each staining intensity category 0 to 4⫹, and a hybrid score (H score) was calculated by the formula: % cells of 0 intensity⫹(% cells of 1 intensity ⫻ 1) ⫹(% cells of 2 intensity ⫻ 2)⫹ ⫹(% cells of 3 intensity ⫻ 3) ⫹ (% cells of 4 intensity ⫻ 4).25 We used the NCI-H2228 ALK translocated cell line as a positive control for ALK expression purchased from the American Type Culture Collection (http://www.ATCC.org).26
EGFR Mutation Detection DNA was extracted from macrodissected tumoral paraffin-embedded tissue using the QIAamp Tissue Kit (QIAGEN GMBH, Hilden, Germany) according to the manufacturer’s protocol. Mutational analysis of exons 18 to 21 of the EGFR gene was performed by direct sequencing using tumoral DNA with BigDye v3.1 following the manufacturer’s instructions and analyzed on an ABI3730XLSequencer (Applied Biosystems, Foster City, CA). Primers were designed using the Primer Express software (Applied Biosystems) and are available on request.
Statistical Analysis Statistical analysis was carried out with SPSS version 13.0 (SPSS, Inc., Chicago, IL). To analyze correlations between ALK status and clinical-pathologic variables and EGFR status, we used the 2 test or Fisher’s exact test. Survival analysis was performed by the Kaplan-Meier method. Curves were compared by the log-rank test. All the statistical tests were conducted at the two-sided 0.05 level of significance.
Copyright © 2010 by the International Association for the Study of Lung Cancer
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TABLE 1. Patients’ Characteristics (N: 107) Median age, yr (range) Gender Females Males Smoking status Never Former Current Performance status (ECOG) 0–1 2–3 Stage I II III IV Histology Adenocarcinomaa Squamous cell carcinoma NSCLC NOS LCC and BAC
66 (40–85) 25 (23%) 82 (77%) 16 (15%) 39 (36%) 52 (49%) 98 (92%) 9 (8%) 49 (46%) 6 (6%) 25 (23%) 27 (25%) 69 (65%) 30 (28%) 2 (2%) 6 (5%)
a Includes two cases of combined carcinomas (adenocarcinoma with large cell neuroendocrine carcinoma). NSCLC NOS, non-small cell lung cancer not otherwise specified; BAC, bronchioalveolar carcinoma (noninvasive tumor with lepidic spread); LCC, large cell carcinoma (exclusion diagnosis for a poorly differentiated tumor with absence of adenocarcinoma, squamous, or small cell component).
TABLE 2. ALK FISH Results Histology ALK FISH (N: 107) Normal Gain Amplification or ⬍10% clusters Translocation
ADC
SCC
NSCLC NOS
LCC and BAC
All
10 45 11
6 18 6
1 1 0
1 4 1
18 (17%) 68 (63%) 18 (17%)
0
0
0
3 (3%)
3a
One case was a combined carcinoma: ADC ⫹ large cell neuroendocrine carcinoma. ADC, adenocarcinoma; SCC, squamous cell carcinoma; NOS, not otherwise specified; LCC, large cell carcinoma; NSCLC, non-small cell lung carcinoma; BAC, bronchioaveolar carcinoma; FISH, fluorescence in situ hybridization; ALK, anaplastic lymphoma kinase. a
RESULTS Patients’ Characteristics One hundred seven patients were included in this study. Clinical-pathologic characteristics are detailed in Table 1. Stage I patients are overrepresented because tissue availability is higher in these surgical cases.
ALK Translocations The results of the FISH analysis of ALK are listed in Table 2. We found three cases with ALK gene translocation (Figure 1). Table 3 lists the characteristics of the translocated cases. Two cases showed a typical gene breakage signal of
ALK Gene Copy Number and Amplification
ALK, and for these, the FISH signal for EML4 was also aberrant, consistent with the translocation involving both genes. The third translocated case showed an atypical rearrangement with a deletion of the 5⬘ region of ALK in 70% of cells. In this case, the EML4 signal was normal, suggesting the existence of another fusion partner to ALK. We investigated the presence of TFG and KIF5B and found no translocation of these genes. Of note, this case was a combined carcinoma with an area of adenocarcinoma and an area of large cell neuroendocrine carcinoma (LCNEC), and the ALK aberration was restricted to the LCNEC area. Both patients with EML4-ALK translocation were elderly at diagnosis (⬎70 years), one of them was a never smoker female with a mucinous adenocarcinoma and the other was a heavy exsmoker male with a solid adenocarcinoma.
ALK Copy Number Gains and Amplification In our series, 11 cases (10%) showed amplification of ALK (Figure 2). Table 3 lists the characteristics of these patients along with seven cases (7%) with a small percentage of cells (⬍10%) showing clusters. Within the samples with amplification, five cases were adenocarcinomas, one was a bronchioalveolar carcinoma, and five had squamous cell histology. There was no statistical association between amplification and gender, age, smoking history, or histology. We observed a significant association between the presence of amplification and early stage (I–II) (p ⫽ 0.04). Notably, 9 of 11 cases with amplification were surgical cases and two cases with squamous histology showed locoregional and exclusively lung dissemination, respectively. The greater tissue availability in the surgical cases might perhaps allow detection of amplification in those specimens with a low percentage of positive cells. We found ALK copy number gains in 63% of cases. In the majority of these cases, gains were seen in a high proportion of cells (50 –95%). No statistically significant association was found between ALK copy number gain and any of the clinical or pathologic parameters analyzed.
ALK Protein Expression by Immunohistochemistry We analyzed the ALK protein expression by immunohistochemistry in 90 cases where tissue was available (including all translocated and amplified cases). We observed ALK expression in the two cases with a typical ALK translocation pattern that also showed a rearrangement of EML4 (Figure 3). The case with a loss of the centromeric probe was negative by IHC. In the remaining specimens, ALK protein expression was undetected with the assay used.
EGFR and ALK in Adenocarcinomas EGFR protein expression (1⫹ to 3⫹) was seen in 45 of 64 patients (70%). Fifty-six patients were evaluated for EGFR by FISH. Of these, 28 (50%) were FISH positive (15% amplifications) and 28 (50%) negative. EGFR mutation status was studied in 68 patients, and mutation on exons 18, 19, and 21 were found in one (2%), six (9%), and four (6%) patients, respectively. These cases were characterized by the clinical
Copyright © 2010 by the International Association for the Study of Lung Cancer
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FIGURE 1. A, Left (arrows): atypical anaplastic lymphoma kinase (ALK) rearrangement with deletion of 5⬘ region of ALK (loss of green-labeled probe centromeric to ALK). Right: normal echinoderm microtubule-associated protein-like 4 (EML4) fusion pattern. B, Left (arrows): typical gene breakage signal of ALK (yellow wild-type allele, and single red and green probe signals for the rearranged allele). Right (arrows): EML4 typical probe breakage.
features typical of these patients subgroup (female, nonsmokers). Regarding the association between EGFR status and ALK, we found a statistically significant association between ALK amplification and FISH positivity for EGFR (p value ⬍0.001). No statistically significant association with EGFR mutations or protein overexpression was observed (Supplementary Table 1, http://links.lww.com/JTO/A38). Two of the cases with ALK clusters in a low percentage of cells harbored an EGFR mutation on exon 21. No EGFR mutations were found in the patients with ALK translocations.
Survival Analysis We sought to explore the potential impact on survival of ALK aberrations. Median follow-up for the whole series was 10.6 months (0.1– 61.2). As the majority of patients with ALK amplification or ⬍10% of clusters were stage I patients, we stratified all stage I patients according to ALK status in two manners. First, we analyzed amplified (six patients with ALK amplification) versus nonamplified (33 patients with copy number gains, four with disomic tumors, and five patients with ⬍10% clusters), excluding the patient with ALK translocation. Second, as the definition of ALK amplification
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we used is not validated and we had observed clusters in a low proportion of cells, we grouped these patients with amplification cases (N: 11) versus nonamplified patients (N: 37). Because of the low number of patients in each group, we continued the analysis with the second approach (grouping amplification with clusters). Median follow-up for stage I patients was 11.5 months (1.2–37.2). Five patients within the stage I subgroup received adjuvant chemotherapy, three of them within the ALK amplified group and two of them in the nonamplified group. Mean overall survival was 32.4 months and 28.6 months for patients with ALK amplification(⫹clusters) and for those without ALK amplification, respectively (median OS was not reached for either group). This difference was not statistically significant (p ⫽ 0.4) (survival curves as Supplementary Figure 1, http://links.lww.com/JTO/A39). All three cases with translocation were alive without disease progression at the timing of writing this work. Interestingly, one of the patients with a pathologically confirmed stage IV adenocarcinoma (pleural metastases) harboring an EML4-ALK translocation is alive after 5 years of follow-up.
Copyright © 2010 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 6, Number 1, January 2011
ALK Gene Copy Number and Amplification
TABLE 3. Characteristics of Patients with ALK Aberrations Patient
ALK FISH
Percentage Cells
ALK IHC
Gender
Age (yr)
Smoking
Histology
Stage
106 997 215 1259 495 692 1123 956 806 278 180 1432 1493 524 1370 1328 1119 1062 1024 839 246
EML4-ALK EML4-ALK ?-ALK Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Clusters Clusters Clusters Clusters Clusters Clusters Clusters
64 66 70 15 70 86 20 10 40 90 80 90 22 52 3 5 8 8 5 7 6
POS POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
M F M M F M M M M M M M M F M F M M M F M
74 72 74 59 73 55 50 53 71 82 79 48 65 53 66 70 54 77 59 64 51
Former Never Current Current Never Current Current Current Former Former Former Current Current Current Former Never Current Former Current Never Current
ADC ADC ADC SCC ADC ADC SCC ADC SCC SCC SCC ADC ADC BAC ADC ADC ADC SCC ADC ADC ADC
pIA IV(Pl) pIIIB IV(L) pIIIA pIA pIIIB pIIB pIA IIIB pIA pIA pIB pIA pIA pIB pIIIA pIA pIA pIA pIV(B)
M, male; F, female; ADC, adenocarcinoma; SCC, squamous cell carcinoma; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; ALK, anaplastic lymphoma kinase.
FIGURE 2. A, Anaplastic lymphoma kinase (ALK) amplification (orange/green fusion signals) in a cell with three copies of CEP2 (red signals). B, Polysomic cells from the same amplified case showing 3 to 4 copies of ALK and 3 to 4 copies of the chromosome 2 centromere.
FIGURE 3. Immunohistochemical staining analysis of anaplastic lymphoma kinase (ALK) protein expression. Pictures are at ⫻40 magnification. A, Case with ALK overexpression H score: 260. B, Case with ALK overexpression H score: 80. Both cases correspond to the tumors from patients with echinoderm microtubule-associated proteinlike 4 (EML4)-ALK translocations by fluorescence in situ hybridization (FISH). Copyright © 2010 by the International Association for the Study of Lung Cancer
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DISCUSSION Lung cancer is a devastating disease, and the need of developing better treatments is an urgent need for our patients. Targeted therapy strategies have recently demonstrated the importance of selecting patients according to the biologic characteristics of the tumors.27 The discovery of a new potentially relevant oncogenic event in lung cancer, the EML4-ALK translocation, and the development of ALK inhibitors with promising results in preclinical models and early clinical trials provides the rationale for the comprehensive characterization of this new genetic aberration in patients with lung cancer. Our work confirms the low incidence (3%) of ALK translocation in an unselected population of patients diagnosed with NSCLC. Both typical translocations involving EML4 and ALK showed a good correlation to FISH and IHC. Nevertheless, the atypically rearranged case was not detectable by IHC, probably resulting from a lack of protein expression derived from this translocation. This stresses the need of further characterizing these atypical translocations and their predictive value for sensitivity to ALK inhibitors. Moreover, the ability of IHC to detect the protein expression in tumors harboring the typical EML4-ALK rearrangement supports the use of IHC with this sensitive antibody as an easy and inexpensive clinical tool for mass screening of patients with advanced NSCLC for this particular rearrangement. Regarding the clinical-pathologic characteristics of patients with ALK translocations, our findings are in contrast with previously reported ones, which associated ALK translocations with nonsmokers and young age patients.11–13 The patients in our series were elderly, two of them with a smoking history and one had LCNEC histology, a distinct subtype of lung tumor which has not previously been reported harboring ALK aberrations. Globally, although the number of patients with translocations is low and we lack the statistical power to draw definitive conclusions, we believe that screening of this genetic aberration at this moment should include the general population of patients with NSCLC, maybe except for those with EGFR mutations that seem to be mutually exclusive with ALK translocations. Interestingly, we found an unexpectedly high prevalence of ALK gene amplifications and copy number gains in NSCLC. This is the first study reporting such high frequency of this genetic aberration. Other reports that have evaluated large populations of patients with NSCLC by FISH have been published. One of them screened more than 600 cases28 and reported a prevalence of 0.5% of ALK amplification. More recent works evaluating ALK gene status by FISH10,11 do not report amplification in their series. These differences with our findings may be explained, at least in part, by the fact that the previous studies used tissue microarrays (TMAs) or small biopsies from patients with advanced disease for FISH evaluation. Supporting this hypothesis, we observed that the percentage of cells within the tumor containing the amplification or clusters is frequently low, which may explain the possibility of this genetic aberration being missed in a TMA sample or in a small biopsy. Accordingly, we detected ALK amplification or clusters in surgical specimens (except for
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two cases) where more tissue is available for analysis. Another reason for the difficulty of detection of these alterations on a TMA section is that the cells harboring amplification were scattered within the specimen. Nevertheless, ALK amplification could also represent an early genetic event in a subset of lung carcinomas. In neuroblastoma, focal high-level amplification has been described as an oncogenic event and selects cells sensitive to ALK inhibitors16,19,29. The clinical significance of ALK amplifications and increased copy number is not known in lung cancer. The lack of ALK protein expression by our immunohistochemical assay, recently validated as a highly sensitive antibody for detection of ALK translocated cases30 together with the low percentage of cells with amplification in most cases, suggests that amplification might not be a biologically relevant event or predict response to ALK targeting molecules. Furthermore, ALK is contained in regions already reported as copy number polymorphisms (http://projects.tcag.ca/variation/). This could explain the large percentage of cases with copy number gain in our series. In addition, the association of EGFR FISH positivity with ALK amplification may define a subset of cases with more frequent aneuploidy as a result of genomic instability. Nevertheless, lack of protein detection with this novel antibody in the cases with copy number gain and amplification does not definitely rule out the potential benefit of ALK inhibitors in this population, as demonstrated in colorectal patients without EGFR protein expression that do respond to therapeutic monoclonal antibodies targeting EGFR.31 Moreover, genetic alterations present initially in a small percentage of the tumor cells have shown to have a relevant biologic role in subsequent phases of disease (i.e., resistant to initial targeted therapy).32 Future studies will be needed to see whether high ALK gene copy number and/or amplification represent important association to sensitivity to ALK fusiontargeted therapies. In summary, we confirm that ALK translocations are infrequent in patients with NSCLC. Immunohistochemical analysis optimally detects EML4-ALK translocations and may represent a clinically easy way to screen patients with NSCLC for this alteration. Copy number gains and amplifications are a frequent event in NSCLC and ALK amplification typically coexists with EGFR amplification. Although these findings lack prognostic significance in our series, our results pave the way to study the predictive role of ALK amplification in NSCLC to potentially expand the spectrum of patients that benefit from ALK inhibitors, alone or combined with EGFR inhibitors.
ACKNOWLEDGMENTS Supported partially by Plan Nacional de Investigacio´n Científica, Desarrollo e Innovacio´n Tecnolo´gica (I⫹D⫹I), iniciativa Ingenio 2010, programa Consolider and Instituto de Salud Carlos III (ISCIII)/FEDER (RD06/0020/0109, RD07/0020/2004) and PN de I⫹D⫹I 2008-20011 and ISCIII/FEDER-Subdireccio´n General de Evaluacio´n y Fomento de la Investigacio´n (PS09/01594, PS09/01285, and PS09/01296). This work was also supported by a grant from DIUE de la Generalitat de Catalunya (2009 SGR 321).
Copyright © 2010 by the International Association for the Study of Lung Cancer
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The authors thank Fundacio´ Cellex (Barcelona) for a generous donation to the Group of Molecular Therapeutics and Biomarkers (Cancer Research Program, IMIM-Hospital del Mar); Carme Melero and María Rodríguez for technical support and Sergi Mojal for collaboration in the statistical analysis; and the Tumor Bank of the Pathology Department of Hospital del Mar and Xarxa de Bancs de Tumors de Catalunya for providing tissue samples. REFERENCES 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71–96. 2. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304: 1497–1500. 3. Uramoto H, Mitsudomi T. Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? Br J Cancer 2007; 96:857– 863. 4. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007; 448:561–566. 5. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science 1994;263:1281–1284. 6. Gascoyne RD, Lamant L, Martin-Subero JI, et al. ALK-positive diffuse large B-cell lymphoma is associated with Clathrin-ALK rearrangements: report of 6 cases. Blood 2003;102:2568 –2573. 7. Griffin CA, Hawkins AL, Dvorak C, et al. Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res 1999;59: 2776 –2780. 8. Ma Z, Cools J, Marynen P, et al. Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis. Blood 2000;95:2144 –2149. 9. Settleman J. Cell culture modeling of genotype-directed sensitivity to selective kinase inhibitors: targeting the anaplastic lymphoma kinase (ALK). Semin Oncol 2009;36:S36 –S41. 10. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009;15:5216 –5223. 11. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4ALK. J Clin Oncol 2009;27:4247– 4253. 12. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol 2009;22:508 –515. 13. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 2008;3:13–17. 14. Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009;174:661– 670.
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15. Kwak EL, Camidge DR, Clark J, et al. Clinical activity observed in a phase I dose escalation trial of an oral c-met and ALK inhibitor, PF-02341066. In ASCO 2009, Orlando, 2009. 16. Janoueix-Lerosey I, Lequin D, Brugieres L, et al. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 2008;455:967–970. 17. Mosse YP, Laudenslager M, Longo L, et al. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 2008;455: 930 –935. 18. Caren H, Abel F, Kogner P, et al. High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. Biochem J 2008;416:153–159. 19. Chen Y, Takita J, Choi YL, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature 2008;455:971–974. 20. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131: 1190 –1203. 21. Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009;15:3143– 3149. 22. Salido M, Tusquets I, Corominas JM, et al. Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry. Breast Cancer Res 2005;7:R267–R273. 23. Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005;97:643– 655. 24. Cappuzzo F, Varella-Garcia M, Shigematsu H, et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J Clin Oncol 2005;23:5007–5018. 25. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003;21:3798 –3807. 26. NCI-Navy Medical Oncology Branch cell line supplement. J Cell Biochem Suppl 1996;24:1–291. 27. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947– 957. 28. Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia 2008;10:298 –302. 29. George RE, Sanda T, Hanna M, et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 2008;455:975– 978. 30. Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clin Cancer Res 2010;16:1561–1571. 31. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol 2005; 23:1803–1810. 32. Turke AB, Zejnullahu K, Wu YL, et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 2010;17: 77– 88.
Copyright © 2010 by the International Association for the Study of Lung Cancer
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Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer Marta Salido, MSc,*†‡ Lara Pijuan, MD, PhD,*‡ Luz Martínez-Avile´s, MSc,*‡ Ana B. Galva´n, MSc,* Israel Can˜adas, MSc,‡ Ana Rovira, PhD,‡§ Montserrat Zanui, MD,§ Alejandro Martínez, MD,‡§ Raquel Longaro´n,* Francisco Sole, PhD,*‡ Sergio Serrano, MD, PhD,*‡㛳 Beatriz Bellosillo, PhD,*‡ Murry W. Wynes, PhD,¶ Joan Albanell, MD, PhD,‡§㛳 Fred R. Hirsch, MD, PhD,¶ and Edurne Arriola, MD, PhD‡§
Introduction: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. Methods: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. Results: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p ⬍ 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. Conclusion: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK
*Pathology Department, Hospital del Mar, Parc de Salut Mar; †Animal and Vegetal Biology and Ecology Department, Anthropology Unit, BioScience Faculty, Universitat Auto`noma de Barcelona, Bellaterra; ‡Cancer Research Program, IMIM-Hospital del Mar, Parc de Salut Mar; §Oncology Department, Hospital del Mar, Parc de Salut Mar; 㛳Autonomous Barcelona University, Barcelona, Spain; and ¶University of Colorado Cancer Center, Aurora, Colorado. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Edurne Arriola, MD, PhD, Medical Oncology Department, Hospital del Mar, Passeig Maritim 25-29, Barcelona 08003, Spain. E-mail: [email protected] The first two authors contributed equally to this work. Copyright © 2010 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/11/0601-0021
amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies. Key Words: ALK, Non-small cell lung cancer, Amplification, Translocation, Polysomy. (J Thorac Oncol. 2011;6: 21–27)
L
ung cancer is the leading cause in the cancer-related deaths in developed countries.1 Although the prognosis of this disease remains poor, some clinically relevant advances have occurred in recent years. The identification of epidermal growth factor receptor (EGFR) mutations and amplification in a subset of patients with non-small cell lung cancer (NSCLC), coupled with the development of EGFR tyrosine kinase inhibitors, has opened new ways to treat this disease.2,3 Nevertheless, there is a need to find additional molecular targets to further improve patients’ outcome. Recently, the novel fusion transcript with transforming activity, formed by the translocation of echinoderm microtubule-associated protein-like 4 (EML4) (2p21) and anaplastic lymphoma kinase (ALK) (2p23), has been described in a subset of NSCLCs.4 ALK was originally involved in carcinogenesis in anaplastic large cell lymphoma as part of a chromosomal rearrangement as a fusion partner of nucleophosmin.5 Other fusion partners to ALK have been described since then and have been associated with other tumor types such as myofibroblastic tumors, B-cell lymphomas, and squamous cell carcinomas of the esophagus.6,7 The products of these translocations are fusion proteins with constitutively activated ALK tyrosine kinase, which plays a role in carcinogenesis by the aberrant phosphorylation of multiple intracellular substrates downstream of ALK-chimerical oncoproteins.8 In preclinical models of activated ALK, specific ALK targeting drugs inhibit cell proliferation and induce apoptosis in models “addicted” to this genetic aberration.9 Moreover, the analysis of clinical specimens in a number of studies suggests that this fusion gene may define a novel subclass of tumors within NSCLC characterized by distinct clinicopathological features in both Western and Asian populations.10 –13 Nevertheless, other reports have described cases that represent exceptions to this rule.14 There-
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fore, there are still many issues about the role of ALK in lung cancer that remain to be determined. Most importantly, the identification of EML4-ALK has provided clinicians with a novel potential therapeutic target as demonstrated by the favorable results of a phase I trial with a dual MET and ALK small molecule inhibitor that has showed promising clinical responses in patients harboring ALK translocations.15 Additional mechanisms of aberrant activation of ALK have been described in neuroblastomas. In these tumors, activating mutations and ALK amplification are the underlying mechanisms for ALK-dependent tumorigenesis16 –19 and are associated with a specific clinical phenotype in this disease. Nevertheless, copy number changes of ALK and their significance are poorly characterized in NSCLC and may represent an additional mechanism of activation of ALK and, therefore, play a role in the carcinogenesis of a subset of lung tumors. In this work, we sought to study the status of the ALK gene by fluorescence in situ hybridization (FISH) on a population of white patients and correlate it with the status of EGFR (mutations and copy number variations) in adenocarcinomas. We also correlated the genetic findings with clinical outcome.
PATIENTS AND METHODS Patient Population The samples included in this study were obtained from patients attending the Lung Cancer Unit in Hospital del Mar for diagnosis and treatment. The only inclusion criterion was the availability of tissue for biomarker studies. Clinical details of these patients were included in a database. This project was approved by the local ethics committee (CEIC-IMAS2009/3619/I).
Fluorescence In Situ Hybridization Four-micrometer paraffin-embedded histologic sections were used for FISH analysis. To assess the genetic status of EGFR and ALK, we used LSI EGFR/CEP7 and break-apart ALK (2p23) probes (Abbott Molecular Inc., Des Plaines, IL). To determine the fusion partner for ALK for cases with ALK translocation, we examined previously identified fusion partners EML4 (2p23), KIF5B (10p11.22), and TFG (3q12.2).20,21 Break-apart probes were designed using bacterial artificial chromosome (BAC) clones selected from the CHORI BAC/PAC resource (http://bacpac.chori.org): centromeric EML4: pooled RP11-804P20 and RP11-413N9, telomeric EML4: pooled RP11-798D22 and RP11-34L01; centromeric KIF5B: pooled RP11-633K11 and RP11-281A19 in spectrum Red, telomeric KIF5B pooled RP11-460H18 and RP11-166N17; and centromeric TFG: pooled RP11-320C17 and RP11-423M9, telomeric TFG: pooled RP11-49H3 and RP11-168G7. FISH was performed as described previously.22 Results were analyzed in a fluorescent microscope (Olympus, BX51) using the Cytovision software (Applied Imaging, Santa Clara, CA). A minimum of 50 nuclei was scored. Patients were classified as EGFR positive/negative according to criteria described in detail elsewhere.23 As criteria for copy number aberrations of ALK has not been established,
22
we arbitrarily used the following cutoffs adapted from the criteria established for EGFR and HER2 in lung cancer specimens.23,24 Briefly, gain (including both low and high genomic gain) was defined as a mean copy number of 3 to 5 fusion signals in ⱖ10% of cells and amplification as the presence of ⱖ6 copies of ALK per cell in ⱖ10% of analyzed cells. In cases where clusters were observed, we reported the percentage of cells with clusters and considered amplified, cases with ⱖ10% of ALK clusters. On cases with ALK amplification, FISH with CEP2 (a centromeric alpha-satellite specific for chromosome 2) was performed (Abbott Molecular Inc.) to exclude polysomy. Normal fusion ALK, EML4, KIF5B, and TFG signals presented as an overlapping orange/ red and green (yellowish) signals. These probes were considered typically rearranged when separated green and orange/ red signals (at least by three times the signal diameter) were identified and atypically rearranged when a single orange or green signal was seen.
Immunohistochemistry for EGFR and ALK Four micrometers serial sections of paraffin blocks were used for the evaluation of EGFR and ALK done with the DAKO Cytomation EGFR PharmDxTM and ALK (D5F3) antibody (Cell Signaling, Danvers, MA), respectively. This antibody for ALK is able to detect the wild type and truncated proteins. Diaminobenzidine was used as the cromogen to identify positive results. The scoring for EGFR IHC was done considering staining intensity, as recommended by the manufacturer, from negative: 0, no detectable staining; to 3⫹, strong and continuous membranous staining. For ALK expression scoring, the percent of cells was determined within each staining intensity category 0 to 4⫹, and a hybrid score (H score) was calculated by the formula: % cells of 0 intensity⫹(% cells of 1 intensity ⫻ 1) ⫹(% cells of 2 intensity ⫻ 2)⫹ ⫹(% cells of 3 intensity ⫻ 3) ⫹ (% cells of 4 intensity ⫻ 4).25 We used the NCI-H2228 ALK translocated cell line as a positive control for ALK expression purchased from the American Type Culture Collection (http://www.ATCC.org).26
EGFR Mutation Detection DNA was extracted from macrodissected tumoral paraffin-embedded tissue using the QIAamp Tissue Kit (QIAGEN GMBH, Hilden, Germany) according to the manufacturer’s protocol. Mutational analysis of exons 18 to 21 of the EGFR gene was performed by direct sequencing using tumoral DNA with BigDye v3.1 following the manufacturer’s instructions and analyzed on an ABI3730XLSequencer (Applied Biosystems, Foster City, CA). Primers were designed using the Primer Express software (Applied Biosystems) and are available on request.
Statistical Analysis Statistical analysis was carried out with SPSS version 13.0 (SPSS, Inc., Chicago, IL). To analyze correlations between ALK status and clinical-pathologic variables and EGFR status, we used the 2 test or Fisher’s exact test. Survival analysis was performed by the Kaplan-Meier method. Curves were compared by the log-rank test. All the statistical tests were conducted at the two-sided 0.05 level of significance.
Copyright © 2010 by the International Association for the Study of Lung Cancer
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TABLE 1. Patients’ Characteristics (N: 107) Median age, yr (range) Gender Females Males Smoking status Never Former Current Performance status (ECOG) 0–1 2–3 Stage I II III IV Histology Adenocarcinomaa Squamous cell carcinoma NSCLC NOS LCC and BAC
66 (40–85) 25 (23%) 82 (77%) 16 (15%) 39 (36%) 52 (49%) 98 (92%) 9 (8%) 49 (46%) 6 (6%) 25 (23%) 27 (25%) 69 (65%) 30 (28%) 2 (2%) 6 (5%)
a Includes two cases of combined carcinomas (adenocarcinoma with large cell neuroendocrine carcinoma). NSCLC NOS, non-small cell lung cancer not otherwise specified; BAC, bronchioalveolar carcinoma (noninvasive tumor with lepidic spread); LCC, large cell carcinoma (exclusion diagnosis for a poorly differentiated tumor with absence of adenocarcinoma, squamous, or small cell component).
TABLE 2. ALK FISH Results Histology ALK FISH (N: 107) Normal Gain Amplification or ⬍10% clusters Translocation
ADC
SCC
NSCLC NOS
LCC and BAC
All
10 45 11
6 18 6
1 1 0
1 4 1
18 (17%) 68 (63%) 18 (17%)
0
0
0
3 (3%)
3a
One case was a combined carcinoma: ADC ⫹ large cell neuroendocrine carcinoma. ADC, adenocarcinoma; SCC, squamous cell carcinoma; NOS, not otherwise specified; LCC, large cell carcinoma; NSCLC, non-small cell lung carcinoma; BAC, bronchioaveolar carcinoma; FISH, fluorescence in situ hybridization; ALK, anaplastic lymphoma kinase. a
RESULTS Patients’ Characteristics One hundred seven patients were included in this study. Clinical-pathologic characteristics are detailed in Table 1. Stage I patients are overrepresented because tissue availability is higher in these surgical cases.
ALK Translocations The results of the FISH analysis of ALK are listed in Table 2. We found three cases with ALK gene translocation (Figure 1). Table 3 lists the characteristics of the translocated cases. Two cases showed a typical gene breakage signal of
ALK Gene Copy Number and Amplification
ALK, and for these, the FISH signal for EML4 was also aberrant, consistent with the translocation involving both genes. The third translocated case showed an atypical rearrangement with a deletion of the 5⬘ region of ALK in 70% of cells. In this case, the EML4 signal was normal, suggesting the existence of another fusion partner to ALK. We investigated the presence of TFG and KIF5B and found no translocation of these genes. Of note, this case was a combined carcinoma with an area of adenocarcinoma and an area of large cell neuroendocrine carcinoma (LCNEC), and the ALK aberration was restricted to the LCNEC area. Both patients with EML4-ALK translocation were elderly at diagnosis (⬎70 years), one of them was a never smoker female with a mucinous adenocarcinoma and the other was a heavy exsmoker male with a solid adenocarcinoma.
ALK Copy Number Gains and Amplification In our series, 11 cases (10%) showed amplification of ALK (Figure 2). Table 3 lists the characteristics of these patients along with seven cases (7%) with a small percentage of cells (⬍10%) showing clusters. Within the samples with amplification, five cases were adenocarcinomas, one was a bronchioalveolar carcinoma, and five had squamous cell histology. There was no statistical association between amplification and gender, age, smoking history, or histology. We observed a significant association between the presence of amplification and early stage (I–II) (p ⫽ 0.04). Notably, 9 of 11 cases with amplification were surgical cases and two cases with squamous histology showed locoregional and exclusively lung dissemination, respectively. The greater tissue availability in the surgical cases might perhaps allow detection of amplification in those specimens with a low percentage of positive cells. We found ALK copy number gains in 63% of cases. In the majority of these cases, gains were seen in a high proportion of cells (50 –95%). No statistically significant association was found between ALK copy number gain and any of the clinical or pathologic parameters analyzed.
ALK Protein Expression by Immunohistochemistry We analyzed the ALK protein expression by immunohistochemistry in 90 cases where tissue was available (including all translocated and amplified cases). We observed ALK expression in the two cases with a typical ALK translocation pattern that also showed a rearrangement of EML4 (Figure 3). The case with a loss of the centromeric probe was negative by IHC. In the remaining specimens, ALK protein expression was undetected with the assay used.
EGFR and ALK in Adenocarcinomas EGFR protein expression (1⫹ to 3⫹) was seen in 45 of 64 patients (70%). Fifty-six patients were evaluated for EGFR by FISH. Of these, 28 (50%) were FISH positive (15% amplifications) and 28 (50%) negative. EGFR mutation status was studied in 68 patients, and mutation on exons 18, 19, and 21 were found in one (2%), six (9%), and four (6%) patients, respectively. These cases were characterized by the clinical
Copyright © 2010 by the International Association for the Study of Lung Cancer
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FIGURE 1. A, Left (arrows): atypical anaplastic lymphoma kinase (ALK) rearrangement with deletion of 5⬘ region of ALK (loss of green-labeled probe centromeric to ALK). Right: normal echinoderm microtubule-associated protein-like 4 (EML4) fusion pattern. B, Left (arrows): typical gene breakage signal of ALK (yellow wild-type allele, and single red and green probe signals for the rearranged allele). Right (arrows): EML4 typical probe breakage.
features typical of these patients subgroup (female, nonsmokers). Regarding the association between EGFR status and ALK, we found a statistically significant association between ALK amplification and FISH positivity for EGFR (p value ⬍0.001). No statistically significant association with EGFR mutations or protein overexpression was observed (Supplementary Table 1, http://links.lww.com/JTO/A38). Two of the cases with ALK clusters in a low percentage of cells harbored an EGFR mutation on exon 21. No EGFR mutations were found in the patients with ALK translocations.
Survival Analysis We sought to explore the potential impact on survival of ALK aberrations. Median follow-up for the whole series was 10.6 months (0.1– 61.2). As the majority of patients with ALK amplification or ⬍10% of clusters were stage I patients, we stratified all stage I patients according to ALK status in two manners. First, we analyzed amplified (six patients with ALK amplification) versus nonamplified (33 patients with copy number gains, four with disomic tumors, and five patients with ⬍10% clusters), excluding the patient with ALK translocation. Second, as the definition of ALK amplification
24
we used is not validated and we had observed clusters in a low proportion of cells, we grouped these patients with amplification cases (N: 11) versus nonamplified patients (N: 37). Because of the low number of patients in each group, we continued the analysis with the second approach (grouping amplification with clusters). Median follow-up for stage I patients was 11.5 months (1.2–37.2). Five patients within the stage I subgroup received adjuvant chemotherapy, three of them within the ALK amplified group and two of them in the nonamplified group. Mean overall survival was 32.4 months and 28.6 months for patients with ALK amplification(⫹clusters) and for those without ALK amplification, respectively (median OS was not reached for either group). This difference was not statistically significant (p ⫽ 0.4) (survival curves as Supplementary Figure 1, http://links.lww.com/JTO/A39). All three cases with translocation were alive without disease progression at the timing of writing this work. Interestingly, one of the patients with a pathologically confirmed stage IV adenocarcinoma (pleural metastases) harboring an EML4-ALK translocation is alive after 5 years of follow-up.
Copyright © 2010 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 6, Number 1, January 2011
ALK Gene Copy Number and Amplification
TABLE 3. Characteristics of Patients with ALK Aberrations Patient
ALK FISH
Percentage Cells
ALK IHC
Gender
Age (yr)
Smoking
Histology
Stage
106 997 215 1259 495 692 1123 956 806 278 180 1432 1493 524 1370 1328 1119 1062 1024 839 246
EML4-ALK EML4-ALK ?-ALK Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Amplified Clusters Clusters Clusters Clusters Clusters Clusters Clusters
64 66 70 15 70 86 20 10 40 90 80 90 22 52 3 5 8 8 5 7 6
POS POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
M F M M F M M M M M M M M F M F M M M F M
74 72 74 59 73 55 50 53 71 82 79 48 65 53 66 70 54 77 59 64 51
Former Never Current Current Never Current Current Current Former Former Former Current Current Current Former Never Current Former Current Never Current
ADC ADC ADC SCC ADC ADC SCC ADC SCC SCC SCC ADC ADC BAC ADC ADC ADC SCC ADC ADC ADC
pIA IV(Pl) pIIIB IV(L) pIIIA pIA pIIIB pIIB pIA IIIB pIA pIA pIB pIA pIA pIB pIIIA pIA pIA pIA pIV(B)
M, male; F, female; ADC, adenocarcinoma; SCC, squamous cell carcinoma; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; ALK, anaplastic lymphoma kinase.
FIGURE 2. A, Anaplastic lymphoma kinase (ALK) amplification (orange/green fusion signals) in a cell with three copies of CEP2 (red signals). B, Polysomic cells from the same amplified case showing 3 to 4 copies of ALK and 3 to 4 copies of the chromosome 2 centromere.
FIGURE 3. Immunohistochemical staining analysis of anaplastic lymphoma kinase (ALK) protein expression. Pictures are at ⫻40 magnification. A, Case with ALK overexpression H score: 260. B, Case with ALK overexpression H score: 80. Both cases correspond to the tumors from patients with echinoderm microtubule-associated proteinlike 4 (EML4)-ALK translocations by fluorescence in situ hybridization (FISH). Copyright © 2010 by the International Association for the Study of Lung Cancer
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DISCUSSION Lung cancer is a devastating disease, and the need of developing better treatments is an urgent need for our patients. Targeted therapy strategies have recently demonstrated the importance of selecting patients according to the biologic characteristics of the tumors.27 The discovery of a new potentially relevant oncogenic event in lung cancer, the EML4-ALK translocation, and the development of ALK inhibitors with promising results in preclinical models and early clinical trials provides the rationale for the comprehensive characterization of this new genetic aberration in patients with lung cancer. Our work confirms the low incidence (3%) of ALK translocation in an unselected population of patients diagnosed with NSCLC. Both typical translocations involving EML4 and ALK showed a good correlation to FISH and IHC. Nevertheless, the atypically rearranged case was not detectable by IHC, probably resulting from a lack of protein expression derived from this translocation. This stresses the need of further characterizing these atypical translocations and their predictive value for sensitivity to ALK inhibitors. Moreover, the ability of IHC to detect the protein expression in tumors harboring the typical EML4-ALK rearrangement supports the use of IHC with this sensitive antibody as an easy and inexpensive clinical tool for mass screening of patients with advanced NSCLC for this particular rearrangement. Regarding the clinical-pathologic characteristics of patients with ALK translocations, our findings are in contrast with previously reported ones, which associated ALK translocations with nonsmokers and young age patients.11–13 The patients in our series were elderly, two of them with a smoking history and one had LCNEC histology, a distinct subtype of lung tumor which has not previously been reported harboring ALK aberrations. Globally, although the number of patients with translocations is low and we lack the statistical power to draw definitive conclusions, we believe that screening of this genetic aberration at this moment should include the general population of patients with NSCLC, maybe except for those with EGFR mutations that seem to be mutually exclusive with ALK translocations. Interestingly, we found an unexpectedly high prevalence of ALK gene amplifications and copy number gains in NSCLC. This is the first study reporting such high frequency of this genetic aberration. Other reports that have evaluated large populations of patients with NSCLC by FISH have been published. One of them screened more than 600 cases28 and reported a prevalence of 0.5% of ALK amplification. More recent works evaluating ALK gene status by FISH10,11 do not report amplification in their series. These differences with our findings may be explained, at least in part, by the fact that the previous studies used tissue microarrays (TMAs) or small biopsies from patients with advanced disease for FISH evaluation. Supporting this hypothesis, we observed that the percentage of cells within the tumor containing the amplification or clusters is frequently low, which may explain the possibility of this genetic aberration being missed in a TMA sample or in a small biopsy. Accordingly, we detected ALK amplification or clusters in surgical specimens (except for
26
two cases) where more tissue is available for analysis. Another reason for the difficulty of detection of these alterations on a TMA section is that the cells harboring amplification were scattered within the specimen. Nevertheless, ALK amplification could also represent an early genetic event in a subset of lung carcinomas. In neuroblastoma, focal high-level amplification has been described as an oncogenic event and selects cells sensitive to ALK inhibitors16,19,29. The clinical significance of ALK amplifications and increased copy number is not known in lung cancer. The lack of ALK protein expression by our immunohistochemical assay, recently validated as a highly sensitive antibody for detection of ALK translocated cases30 together with the low percentage of cells with amplification in most cases, suggests that amplification might not be a biologically relevant event or predict response to ALK targeting molecules. Furthermore, ALK is contained in regions already reported as copy number polymorphisms (http://projects.tcag.ca/variation/). This could explain the large percentage of cases with copy number gain in our series. In addition, the association of EGFR FISH positivity with ALK amplification may define a subset of cases with more frequent aneuploidy as a result of genomic instability. Nevertheless, lack of protein detection with this novel antibody in the cases with copy number gain and amplification does not definitely rule out the potential benefit of ALK inhibitors in this population, as demonstrated in colorectal patients without EGFR protein expression that do respond to therapeutic monoclonal antibodies targeting EGFR.31 Moreover, genetic alterations present initially in a small percentage of the tumor cells have shown to have a relevant biologic role in subsequent phases of disease (i.e., resistant to initial targeted therapy).32 Future studies will be needed to see whether high ALK gene copy number and/or amplification represent important association to sensitivity to ALK fusiontargeted therapies. In summary, we confirm that ALK translocations are infrequent in patients with NSCLC. Immunohistochemical analysis optimally detects EML4-ALK translocations and may represent a clinically easy way to screen patients with NSCLC for this alteration. Copy number gains and amplifications are a frequent event in NSCLC and ALK amplification typically coexists with EGFR amplification. Although these findings lack prognostic significance in our series, our results pave the way to study the predictive role of ALK amplification in NSCLC to potentially expand the spectrum of patients that benefit from ALK inhibitors, alone or combined with EGFR inhibitors.
ACKNOWLEDGMENTS Supported partially by Plan Nacional de Investigacio´n Científica, Desarrollo e Innovacio´n Tecnolo´gica (I⫹D⫹I), iniciativa Ingenio 2010, programa Consolider and Instituto de Salud Carlos III (ISCIII)/FEDER (RD06/0020/0109, RD07/0020/2004) and PN de I⫹D⫹I 2008-20011 and ISCIII/FEDER-Subdireccio´n General de Evaluacio´n y Fomento de la Investigacio´n (PS09/01594, PS09/01285, and PS09/01296). This work was also supported by a grant from DIUE de la Generalitat de Catalunya (2009 SGR 321).
Copyright © 2010 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 6, Number 1, January 2011
The authors thank Fundacio´ Cellex (Barcelona) for a generous donation to the Group of Molecular Therapeutics and Biomarkers (Cancer Research Program, IMIM-Hospital del Mar); Carme Melero and María Rodríguez for technical support and Sergi Mojal for collaboration in the statistical analysis; and the Tumor Bank of the Pathology Department of Hospital del Mar and Xarxa de Bancs de Tumors de Catalunya for providing tissue samples. REFERENCES 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71–96. 2. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304: 1497–1500. 3. Uramoto H, Mitsudomi T. Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? Br J Cancer 2007; 96:857– 863. 4. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007; 448:561–566. 5. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science 1994;263:1281–1284. 6. Gascoyne RD, Lamant L, Martin-Subero JI, et al. ALK-positive diffuse large B-cell lymphoma is associated with Clathrin-ALK rearrangements: report of 6 cases. Blood 2003;102:2568 –2573. 7. Griffin CA, Hawkins AL, Dvorak C, et al. Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res 1999;59: 2776 –2780. 8. Ma Z, Cools J, Marynen P, et al. Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis. Blood 2000;95:2144 –2149. 9. Settleman J. Cell culture modeling of genotype-directed sensitivity to selective kinase inhibitors: targeting the anaplastic lymphoma kinase (ALK). Semin Oncol 2009;36:S36 –S41. 10. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009;15:5216 –5223. 11. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4ALK. J Clin Oncol 2009;27:4247– 4253. 12. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol 2009;22:508 –515. 13. Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 2008;3:13–17. 14. Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009;174:661– 670.
ALK Gene Copy Number and Amplification
15. Kwak EL, Camidge DR, Clark J, et al. Clinical activity observed in a phase I dose escalation trial of an oral c-met and ALK inhibitor, PF-02341066. In ASCO 2009, Orlando, 2009. 16. Janoueix-Lerosey I, Lequin D, Brugieres L, et al. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 2008;455:967–970. 17. Mosse YP, Laudenslager M, Longo L, et al. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 2008;455: 930 –935. 18. Caren H, Abel F, Kogner P, et al. High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. Biochem J 2008;416:153–159. 19. Chen Y, Takita J, Choi YL, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature 2008;455:971–974. 20. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131: 1190 –1203. 21. Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009;15:3143– 3149. 22. Salido M, Tusquets I, Corominas JM, et al. Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry. Breast Cancer Res 2005;7:R267–R273. 23. Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005;97:643– 655. 24. Cappuzzo F, Varella-Garcia M, Shigematsu H, et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J Clin Oncol 2005;23:5007–5018. 25. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003;21:3798 –3807. 26. NCI-Navy Medical Oncology Branch cell line supplement. J Cell Biochem Suppl 1996;24:1–291. 27. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947– 957. 28. Perner S, Wagner PL, Demichelis F, et al. EML4-ALK fusion lung cancer: a rare acquired event. Neoplasia 2008;10:298 –302. 29. George RE, Sanda T, Hanna M, et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 2008;455:975– 978. 30. Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clin Cancer Res 2010;16:1561–1571. 31. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol 2005; 23:1803–1810. 32. Turke AB, Zejnullahu K, Wu YL, et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 2010;17: 77– 88.
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