Purpose: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non–small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers.

Experimental Design: We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib.

Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5′ green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing.

Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib. Clin Cancer Res; 19(15); 4273–81. ©2013 AACR.

Translational Relevance

Lung cancers harboring chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene are associated with unique clinicopathologic features, including sensitivity to the tyrosine kinase inhibitor (TKI) crizotinib. Recently, several studies have suggested that ALK rearrangements co-occur with mutations in EGFR or KRAS at clinically relevant frequencies. If confirmed, this would have important consequences for therapeutic decision-making and may alter clinical laboratory workflow, as many centers reserve ALK testing for tumor specimens that are negative for mutations in EGFR and KRAS. Here, we present clinical genotyping data from 1,683 patients with NSCLC, focusing on the prevalence of EGFR and KRAS mutations in ALK-positive patients. We also report molecular studies on a cohort of 34 ALK-positive patients who underwent biopsies after the development of resistance to crizotinib to determine the prevalence of acquired EGFR and KRAS mutations in this population.

Treatment paradigms for non–small cell lung cancer (NSCLC) have recently shifted away from stratification of patients based upon histology alone and toward molecular classifications based on genetic alterations within “driver” oncogenes. NSCLCs harboring such alterations are often dependent on a single oncogenic pathway for cell survival, a concept known as oncogene addiction (1). In recent genotyping efforts, approximately 50% of pulmonary adenocarcinomas were identified as having at least one genetic alteration in an oncogenic driver, with higher rates observed among never-smokers (2–4). Notably, mutations within driver oncogenes are largely mutually exclusive with one another, overlapping in only 3% to 5% of cases (2, 3).

Identified in NSCLC in 2007, chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene define a new molecular subset of lung cancer (5). With an estimated frequency of 3% to 5% of NSCLC, ALK rearrangements are associated with unique clinical and pathologic features, including younger age, never or light smoking history, and adenocarcinoma histology (6–9). ALK rearrangements are also regarded as essentially mutually exclusive with mutations in other driver oncogenes (7, 10). However, several recent reports suggest an unexpectedly high degree of overlap between ALK rearrangements and mutations in KRAS or EGFR (3, 11–19). For example, in a recent analysis of 95 EGFR-mutant patients who participated in the phase III EURTAC trial, 15.8% were reported to have concomitant ALK rearrangements (20). Additional studies have identified the coexistence of EGFR mutations in up to 6% of ALK-positive patients within Western populations and in up to 11.8% among ALK-positive Asian populations (16, 18).

Recognition of ALK-positive patients is clinically important as ALK rearrangements are associated with marked sensitivity to the tyrosine kinase inhibitor (TKI) crizotinib. In early clinical studies of crizotinib in ALK-positive NSCLC, the objective response rate was 60% with median progression-free survival of 8 to 10 months (21–23). Despite the initial sensitivity of ALK-positive lung cancer to crizotinib, patients eventually develop resistance to therapy. Mechanisms of acquired crizotinib resistance have been identified in vitro and through molecular analysis of repeat biopsy specimens taken at the time of disease progression (16, 24–28). These include secondary mutations in the ALK tyrosine kinase (TK) domain, ALK fusion gene amplification, and upregulation of bypass signaling tracts, such as c-KIT and EGFR (16, 24, 26–28). In addition, one study reported loss of the ALK fusion gene and emergence of EGFR and KRAS mutations in crizotinib-resistant cancers (27).

The possibility of coexistence of ALK fusions and either EGFR or KRAS mutations would have profound effects on therapeutic choices and would also impact clinical laboratory workflow and resource allocation as many centers now reserve ALK FISH testing, a relatively labor-intensive assay, for cases that test negative for EGFR and KRAS mutations. In this multi-institutional study, we identified patients who underwent clinical genotyping for alterations in EGFR, KRAS, and ALK, examining the prevalence of coexisting EGFR or KRAS mutations among ALK-positive patients. We also present an updated analysis of a series of repeat biopsies from crizotinib-resistant, ALK-positive patients (26) to determine the frequency of EGFR and KRAS mutations in TKI-resistant disease.

Study populations

Patients (n = 1,683) with NSCLC who underwent nonsequential testing for mutations in EGFR, KRAS, and ALK between March 2009 and June 2012 were identified. Patients were seen at Massachusetts General Hospital (MGH; n = 1,619), Memorial Sloan-Kettering Cancer Center (n = 33), and the University of California at Irvine (n = 31). All patients had biopsy-proven NSCLC. Medical records were reviewed to extract data on clinical and pathologic features. This study was approved by the Institutional Review Board at each of the participating institutions.

Patients with ALK-positive NSCLC (n = 34) with acquired resistance to crizotinib underwent biopsies of their resistant tumors at one institution (MGH) between January, 2009 and October, 2012. Specimens were reviewed for histologic confirmation of malignancy. Total nucleic acid was extracted in all specimens for ALK TK domain sequencing as outlined below. Repeat ALK FISH and the multiplexed SNaPshot assay were conducted in all specimens with sufficient tissue for analysis.

Tumor pathology and mutation analysis

Tumor histology was classified according to World Health Organization criteria. EGFR and KRAS testing were conducted using SNaPshot (29). In cases where SNaPshot was unavailable, mutation analysis was conducted by a combination of a multiplexed PCR-based sizing assay, allele-specific PCR, direct sequencing and mass spectrometry genotyping.

ALK molecular analysis

ALK FISH was conducted on formalin-fixed and paraffin-embedded (FFPE) tissue using a dual-color break-apart probe specific to the ALK locus (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular). Samples were considered positive if more than 15% of cells showed split ALK 5′ and 3′ probe signals or isolated 3′ signals (7).

In ALK-positive, crizotinib-resistant biopsy specimens, total nucleic acid was extracted and the ALK TK domain (exons 20–28) was sequenced as previously described (26).

Immunohistochemistry

Immunohistochemical staining was conducted on representative tissue sections from FFPE tissue blocks. Immunohistochemistry (IHC) was conducted with an anti-ALK monoclonal antibody (clone 5A4, Novacastra) at 1:50 dilution (30).

Statistical analysis

Fisher exact test and Wilcoxon rank-sum test were used to assess the association of genotype status with clinicopathologic features. An exact calculation of the binomial distribution is used to obtain the upper bound of a one-sided 95% CI for the frequency of ALK-positive cases with a second driver mutation. Data analysis was computed by SAS 9.2 (SAS Institute), and all P values were two-sided.

Screening patient characteristics

We identified 1,687 NSCLC specimens that had undergone clinical genotyping for abnormalities in EGFR, KRAS, and ALK. Testing for each genetic alteration was conducted in 100% of specimens. A total of 1,683 patients were included, with 4 patients having 2 separate primary NSCLCs analyzed. Genotyping for EGFR and KRAS mutations was conducted using the SNaPshot assay in 1,603 (95.1%) cases. Mutations in EGFR and KRAS were identified in 301 (17.8%) and 465 (27.6%) specimens, respectively. ALK FISH was conducted on all samples. ALK rearrangements were found in 75 (4.4%) cases. The clinical and pathologic features of these patients are summarized in Table 1.

Table 1.

Clinicopathologic features of EGFR, KRAS, and ALK-positive patients

Clinical characteristicEGFR (n = 301)KRAS (n = 465)ALK (n = 75)P-value for ALK vs. EGFRP-value for ALK vs. KRAS
Age at diagnosis    <0.001 <0.001 
 Median 64 66 56   
 Range 26–92 26–92 29–87   
Sex    <0.001 0.022 
 Male 86 (29%) 170 (37%) 38 (51%)   
 Female 215 (71%) 295 (63%) 37 (49%)   
Ethnicitya    0.192 <0.001 
 Caucasian 227 (76%) 436 (96%) 64 (85%)   
 Asian 47 (16%) 8 (2%) 9 (12%)   
 Other 23 (8%) 11 (2%) 2 (3%)   
Smoking Historyb    0.239 <0.001 
 Never 173 (57%) 18 (4%) 49 (65%)   
 Smoker 128 (43%) 445 (96%) 26 (35%)   
Pathology    0.104 0.072 
 Adenocarcinoma 287 (95%) 432 (93%) 71 (95%)   
 Adenosquamous 6 (2%) 9 (2%) 0 (0%)   
 Squamous 2 (1%) 4 (1%) 3 (4%)   
 Other NSCLC 6 (2%) 20 (4%) 1 (1%)   
Stagec    <0.001 <0.001 
 Stage I 78 (26%) 175 (38%) 4 (5%)   
 Stage II 16 (5%) 55 (12%) 8 (11%)   
 Stage III 43 (14%) 69 (15%) 18 (24%)   
 Stage IV 163 (55%) 165 (35%) 45 (60%)   
Stage at Testingd    0.002 <0.001 
 Stage I 68 (23%) 158 (34%) 4 (5%)   
 Stage II 14 (5%) 47 (10%) 4 (5%)   
 Stage III 33 (11%) 56 (12%) 12 (16%)   
 Stage IV 185 (62%) 204 (44%) 55 (74%)   
Clinical characteristicEGFR (n = 301)KRAS (n = 465)ALK (n = 75)P-value for ALK vs. EGFRP-value for ALK vs. KRAS
Age at diagnosis    <0.001 <0.001 
 Median 64 66 56   
 Range 26–92 26–92 29–87   
Sex    <0.001 0.022 
 Male 86 (29%) 170 (37%) 38 (51%)   
 Female 215 (71%) 295 (63%) 37 (49%)   
Ethnicitya    0.192 <0.001 
 Caucasian 227 (76%) 436 (96%) 64 (85%)   
 Asian 47 (16%) 8 (2%) 9 (12%)   
 Other 23 (8%) 11 (2%) 2 (3%)   
Smoking Historyb    0.239 <0.001 
 Never 173 (57%) 18 (4%) 49 (65%)   
 Smoker 128 (43%) 445 (96%) 26 (35%)   
Pathology    0.104 0.072 
 Adenocarcinoma 287 (95%) 432 (93%) 71 (95%)   
 Adenosquamous 6 (2%) 9 (2%) 0 (0%)   
 Squamous 2 (1%) 4 (1%) 3 (4%)   
 Other NSCLC 6 (2%) 20 (4%) 1 (1%)   
Stagec    <0.001 <0.001 
 Stage I 78 (26%) 175 (38%) 4 (5%)   
 Stage II 16 (5%) 55 (12%) 8 (11%)   
 Stage III 43 (14%) 69 (15%) 18 (24%)   
 Stage IV 163 (55%) 165 (35%) 45 (60%)   
Stage at Testingd    0.002 <0.001 
 Stage I 68 (23%) 158 (34%) 4 (5%)   
 Stage II 14 (5%) 47 (10%) 4 (5%)   
 Stage III 33 (11%) 56 (12%) 12 (16%)   
 Stage IV 185 (62%) 204 (44%) 55 (74%)   

aOther includes “Hispanic,” “African-American,” and “Native American” ethnicities. Ethnicity was unavailable in 14 patients.

bNever smokers have smoked less than100 cigarettes per lifetime. Smokers have smoked more than100 cigarettes (current or former). Smoking status was unavailable in 2 patients.

cClinical stage represents stage at initial diagnosis. Stage was determined according to current American Joint Commission on Cancer Guidelines. Stage was not available for 2 patients.

dRepresents stage at time of molecular testing. Stage was not available for 1 patient.

Lack of overlapping mutations in driver oncogenes

The overall frequency of coalterations among any of the 3 tested oncogenes was 0.06% (1/1,687). Only 1 case of overlap, involving concomitant mutations in EGFR and KRAS, was observed by allele-specific PCR. Insufficient tumor tissue was available for confirmatory SNaPshot testing of this specimen. Among 376 patients found to have EGFR mutations (n = 301) or ALK rearrangements (n = 75), there were no cases of coexisting EGFR and ALK alterations identified.

Four patients with KRAS mutations were noted to have abnormal ALK FISH patterns that did not meet criteria for the presence of an ALK rearrangement (Table 2). Such abnormal ALK FISH patterns involved single isolated green 5′ probes in 3 (75%) cases. The last case involved an ALK FISH pattern with a single isolated red 3′ probe that was considered unusually small. To determine whether these cases harbored functional ALK rearrangements leading to expression of an ALK fusion protein, ALK immunohistochemistry (clone 5A4) was conducted. Previous immunohistochemical studies using the ALK 5A4 antibody have shown a sensitivity and specificity of 100% among 29 ALK FISH-positive adenocarcinoma specimens and 110 ALK FISH-negative samples (30). Sufficient tissue was available for ALK immunohistochemical testing in 3 of the KRAS-positive, abnormal ALK FISH cases. Immunohistochemistry was negative for ALK expression in 3/3 (100%) specimens, including the case with an isolated, unusually small red 3′ probe on ALK FISH (Fig. 1). Thus, these abnormal ALK FISH patterns were not consistent with functional ALK gene rearrangements. Therefore, altogether 0 of 75 (0%; upper 95% CI: 4%) patients with ALK rearrangements were found to have concomitant mutations in EGFR or KRAS (Table 3).

Figure 1.

Immunohistochemical staining for ALK. Specimens (A–C) show negative ALK immunohistochemical staining for 3 patients with KRAS mutations and abnormal ALK FISH. Specimens (D–F) depict positive ALK immunohistochemical staining from 3 representative patients with positive ALK FISH.

Figure 1.

Immunohistochemical staining for ALK. Specimens (A–C) show negative ALK immunohistochemical staining for 3 patients with KRAS mutations and abnormal ALK FISH. Specimens (D–F) depict positive ALK immunohistochemical staining from 3 representative patients with positive ALK FISH.

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Table 2.

Molecular analysis of patients with KRAS mutations and abnormal ALK FISH

Patient no.EGFR mutationKRAS mutationALK FISH patternALK IHC
WT Gly12Cys Isolated green 5′ probe Negative 
WT Gly12Ser Isolated green 5′ probe Negative 
WT Gly13Cys Isolated green 5′ probe N/A 
WT Gly12Val Isolated red 3′ probea Negative 
Patient no.EGFR mutationKRAS mutationALK FISH patternALK IHC
WT Gly12Cys Isolated green 5′ probe Negative 
WT Gly12Ser Isolated green 5′ probe Negative 
WT Gly13Cys Isolated green 5′ probe N/A 
WT Gly12Val Isolated red 3′ probea Negative 

aALK FISH—isolated red 3′ probe considered “unusually small.”

Table 3.

Summary of overlapping mutations in EGFR, KRAS, and ALK

# Single mutationsEGFRKRASALK
EGFR (n = 301) – 
KRAS (n = 465) – 
ALK (n = 75) – 
# Single mutationsEGFRKRASALK
EGFR (n = 301) – 
KRAS (n = 465) – 
ALK (n = 75) – 

Repeat biopsies in ALK-positive, crizotinib-refractory NSCLC

We previously reported an analysis of crizotinib resistance in 18 ALK-positive patients who underwent repeat biopsies at the time of progression (26). Here, we present an updated and expanded analysis of 34 repeat biopsy specimens from ALK-positive, crizotinib-refractory patients. Specimens were examined for mechanisms of acquired resistance. In all cases, patients had initially responded to crizotinib but subsequently developed progressive disease after a median of 9 months (range 3–34 months). A majority of repeat biopsies were obtained before or within 1 month of crizotinib discontinuation (Table 4). If sufficient tissue was available, specimens underwent repeat ALK FISH, direct sequencing of the ALK TK domain, and SNaPshot testing.

Table 4.

ALK-positive patients with acquired crizotinib resistance

PatientTiming (months)aDuration (months)bALK fusionALK amplificationALK secondary mutationEGFR mutationKRAS mutation
MGH0NZ 20 Positive No No WT WT 
MGH001 4.5 Positive No No WT WT 
MGH010 Positive No No WT WT 
MGH011 34 Positive No S1206Y WT WT 
MGH013 Positive No No WT WT 
MGH016 Positive No No WT WT 
MGH017 23+ Positive No No WT WT 
MGH018 0.5 10 Positive No G1202R N/A N/A 
MGH019 <0.5 Positive No No WT WT 
MGH020 13 N/A N/A L1196M N/A N/A 
MGH021 12 Positive No 1151Tins, G1269A WT WT 
MGH022 Positive No No WT WT 
MGH023 12 Positive No No WT WT 
MGH024 15 Positive No No N/A N/A 
MGH025 11 N/A N/A No WT WT 
MGH027 N/A N/A No N/A N/A 
MGH028 14 N/A N/A No N/A N/A 
MGH029 Positive Yes No N/A N/A 
MGH030 Positive No No WT WT 
MGH031 N/A N/A Positive No No WT WT 
MGH032 21 Positive No No N/A N/A 
MGH033 Positive No No WT WT 
MGH034 Positive Yes No WT WT 
MGH035 N/A 14 Positive No No WT WT 
MGH036 0.5 13 Positive No No WT WT 
MGH037 11 Positive No G1269A WT WT 
MGH038 Positive No L1196M WT WT 
MGH040 Positive No No WT WT 
MGH044 Positive Yesc No WT WT 
MGH045 13 Positive No L1196M N/A N/A 
MGH051 <0.5 Positive No No WT WT 
MGH054 8.5 Positive No No WT WT 
MGH055 N/A N/A No N/A N/A 
MGH058 14 Positive No No WT WT 
PatientTiming (months)aDuration (months)bALK fusionALK amplificationALK secondary mutationEGFR mutationKRAS mutation
MGH0NZ 20 Positive No No WT WT 
MGH001 4.5 Positive No No WT WT 
MGH010 Positive No No WT WT 
MGH011 34 Positive No S1206Y WT WT 
MGH013 Positive No No WT WT 
MGH016 Positive No No WT WT 
MGH017 23+ Positive No No WT WT 
MGH018 0.5 10 Positive No G1202R N/A N/A 
MGH019 <0.5 Positive No No WT WT 
MGH020 13 N/A N/A L1196M N/A N/A 
MGH021 12 Positive No 1151Tins, G1269A WT WT 
MGH022 Positive No No WT WT 
MGH023 12 Positive No No WT WT 
MGH024 15 Positive No No N/A N/A 
MGH025 11 N/A N/A No WT WT 
MGH027 N/A N/A No N/A N/A 
MGH028 14 N/A N/A No N/A N/A 
MGH029 Positive Yes No N/A N/A 
MGH030 Positive No No WT WT 
MGH031 N/A N/A Positive No No WT WT 
MGH032 21 Positive No No N/A N/A 
MGH033 Positive No No WT WT 
MGH034 Positive Yes No WT WT 
MGH035 N/A 14 Positive No No WT WT 
MGH036 0.5 13 Positive No No WT WT 
MGH037 11 Positive No G1269A WT WT 
MGH038 Positive No L1196M WT WT 
MGH040 Positive No No WT WT 
MGH044 Positive Yesc No WT WT 
MGH045 13 Positive No L1196M N/A N/A 
MGH051 <0.5 Positive No No WT WT 
MGH054 8.5 Positive No No WT WT 
MGH055 N/A N/A No N/A N/A 
MGH058 14 Positive No No WT WT 

aIndicates the time interval between last crizotinib dose and repeat biopsy. Repeat biopsies conducted while the patient is still receiving crizotinib are depicted with a “0”.

bIndicates the approximate duration the patient was treated with crizotinib.

cLow level ALK amplification.

Persistence of the ALK fusion gene at the time of acquired crizotinib resistance

Our earlier report of acquired resistance to crizotinib in patients with ALK rearrangements showed the continued presence of the ALK fusion transcript upon rebiopsy (26). However, it has recently been reported that patients may lose the ALK gene rearrangement upon treatment with crizotinib (27). We therefore conducted repeat ALK FISH testing on the additional crizotinib-resistant specimens. Among 34 specimens, a total of 29 were evaluable. Tumor material in the remaining 5 samples was exhausted due to sequencing efforts. Notably, in 29 of 29 (100%) cases, repeat ALK FISH confirmed the continued presence of an ALK fusion gene despite acquired resistance to crizotinib (Tables 4 and 5).

Table 5.

Summary of ALK-positive, crizotinib-refractory cases

Resistant casesALK fusionALK amplificationALK secondary mutationEGFR mutationKRAS mutation
34 29/29 (100%) 3/29 (10.3%) 7/34 (20.6%) 0/25 (0%) 0/25 (0%) 
Resistant casesALK fusionALK amplificationALK secondary mutationEGFR mutationKRAS mutation
34 29/29 (100%) 3/29 (10.3%) 7/34 (20.6%) 0/25 (0%) 0/25 (0%) 

Gene amplification has been reported to mediate resistance in a number of oncogene-driven malignancies (31, 32), including ALK-rearranged NSCLC (26–28). In our crizotinib-resistant cohort, repeat ALK FISH also permitted detection of gene amplification. In 2 patients (MGH029 and MGH034), ALK FISH was notable for high-level amplification, and a third specimen (MGH044) showed low-level gene amplification. In total, 3 of 29 (10.3%) patients exhibited amplification of the ALK gene at the time of crizotinib resistance.

Secondary mutations in ALK TK domain as mediators of crizotinib resistance

We next evaluated crizotinib-resistant specimens for secondary mutations in the ALK TK domain. Total nucleic acid was extracted and direct sequencing of the ALK TK domain was carried out as described previously (26). Among 34 tested specimens, a total of 8 secondary mutations were identified among 7 (20.6%) patients (Tables 4 and 5). The resistant specimen from MGH021 contained 2 separate secondary mutations: an amino acid substitution (G1269A) and an insertion mutation (1151Tins). A majority of secondary mutations involved missense mutations (S1206Y, G1202R, L1196M, and G1269A), all of which have been previously reported as mediators of crizotinib resistance (24, 26, 27). The most frequently identified secondary mutation was the gatekeeper L1196M mutation. However, this substitution was present in only 3 (9.3%) specimens, underscoring that the ALK gatekeeper mutation is not the dominant mechanism of crizotinib resistance.

Absence of EGFR and KRAS mutations in crizotinib-resistant cancers

Aberrant coactivation of EGFR signaling, in the absence of EGFR mutations, has been observed as a mechanism of crizotinib resistance in vitro and within patient-derived specimens (16, 26). One recent report also identified the emergence of EGFR and KRAS mutations among 11 ALK-positive patients following development of resistance to crizotinib (27). In a separate study of 7 ALK-positive patients with acquired crizotinib resistance, 1 patient was found to have ALK gene amplification as well as EGFR high polysomy and an acquired EGFR L858R mutation (28). We therefore tested repeat biopsy specimens from crizotinib-resistant, ALK-positive patients for mutations in EGFR and KRAS using the SNaPshot assay (Table 4). In 25 patients with available tissue, EGFR mutations were identified in 0 of 25 (0%; upper 95% CI: 11%) cases. Similarly, KRAS mutations were found in 0 of 25 (0%; upper 95% CI: 11%) specimens.

In NSCLC, genetic alterations in EGFR, KRAS, and ALK are considered to be largely mutually exclusive (7, 10, 33). However, recent series have suggested that EGFR mutations and ALK rearrangements coexist within patients at clinically meaningful frequencies (3, 15–19). The question of whether overlap exists between these driver oncogenes is clinically relevant and would impact therapeutic choices. In vitro studies suggest that concomitant alterations in EGFR and ALK lead to mutual resistance to EGFR or ALK TKI monotherapy (16). In limited clinical series of patients reported to have both alterations, isolated responses to erlotinib, gefitinib, and crizotinib have been described (11, 12, 16, 18, 19). However, to the best of our knowledge, there have been no reports in which individual patients with co-existing EGFR mutations and ALK rearrangements responded to both EGFR and ALK TKIs.

In this study, we examined a large Western population of patients with NSCLC who underwent clinical genotyping. In contrast to the reports mentioned above, we found no cases of coexisting EGFR mutations and ALK rearrangements across 1,687 screened specimens, supporting the mutual exclusivity of these 2 genetic alterations. We did identify a few cases of concomitant KRAS mutations and abnormal ALK FISH. While abnormal, however, these FISH patterns did not meet criteria for an ALK rearrangement. Specifically, most cases (3/4) involved single isolated green 5′ probes, rather than the typical split probe or isolated 3′ probe pattern. To confirm the absence of a functional ALK rearrangement in these specimens, ALK immunohistochemistry was conducted and found to be negative in 3 of 3 cases tested. In a prior series using this antibody, ALK immunohistochemistry had a negative predictive value of 100% (30). Thus, these findings are unlikely to be due to false-negative staining, as the probability of obtaining 3 false-negative immunohistochemical results among these cases would be less than 0.000001. We therefore conclude that these abnormal ALK FISH patterns are unlikely to represent functional ALK gene rearrangements. Altogether, these findings support the lack of overlap between ALK rearrangements and either EGFR or KRAS mutations.

We recognize that these findings are in contrast to some recent reports (14–19). One explanation for such discordant findings may involve ethnic differences between screening populations. Specifically, the higher prevalence of EGFR mutations within Asian NSCLC populations may increase the chance of detecting dual EGFR and ALK alterations (34). Indeed, the highest frequencies of concomitant EGFR mutations and ALK rearrangements have been reported in Asian populations (17–19). For instance, in one study describing concomitant EGFR mutations in 11.8% of patients with ALK rearrangements, the overall prevalence of EGFR mutations in the study population (n = 444) was 51.4% (18).

In addition to ethnic variations, our findings may differ from other reports due to the stage distribution of our patient population. A significant proportion of patients in this study had early-stage disease at the time of mutation testing. It is therefore possible that we did not capture additional genetic alterations that may have accumulated later during the disease course. Another potential explanation for reports of dual genetic alterations may be the presence of multiple separate primary tumors in patients, each harboring different driver mutations. When overlapping genetic alterations in ALK and EGFR are suspected, ALK and EGFR mutant-specific immunohistochemistry may help distinguish whether both abnormalities are colocalized in the same cells. Our findings may also differ from those of prior studies due to possible reporting bias and choice of screening techniques. ALK testing can be conducted using ALK immunohistochemistry, reverse transcription PCR (RT-PCR), and ALK FISH. While ALK FISH is the only clinically validated assay and current gold standard, this technique is technically challenging (35). In prior reports of coexisting EGFR mutations and ALK rearrangements, various methods of ALK testing were used, including ALK FISH, ALK immunohistochemistry, RT-PCR, and rapid amplification of cDNA ends (RACE)-coupled PCR (11–19). In contrast, all patients in our study were screened using ALK FISH. Of note, ALK FISH positivity in this study was defined as more than 15% of cells showing positive signals. This is consistent with the definition of ALK positivity used in clinical trials of crizotinib, and this threshold is above the technical background noise of the assay (21, 36).

As the molecular profile of malignancies may evolve over the course of treatment, we also examined the molecular characteristics of ALK-positive cancers after the development of resistance to crizotinib. Crizotinib-resistant specimens were examined for ALK gene amplification, secondary ALK TK domain mutations, and emergence of EGFR and KRAS mutations. Of particular note, we found that the presence of the ALK fusion gene as assessed by FISH was preserved across all tested specimens despite the presence of acquired crizotinib resistance. This finding is consistent with the oncogene addiction paradigm as well as results from repeat biopsy series in patients with EGFR mutations and acquired resistance to EGFR inhibitors (32, 37). Although we and others have previously identified ligand-dependent activation of EGFR as a potential resistance mechanism to crizotinib (16, 26, 38), we found no cases of EGFR or KRAS mutations among 25 examined specimens, including those cases with upregulation of activated EGFR by immunohistochemistry. Thus, our findings are different from a previous study reporting that ALK-positive patients develop ALK-negative tumors harboring new mutations in EGFR following treatment with crizotinib (27). It remains possible that the patients identified in the other study had 2 separate primary cancers, an EGFR-mutant lung cancer and an ALK-positive lung cancer, and the EGFR-mutant lung cancer emerged upon treatment with crizotinib. In such cases, more comprehensive genetic analyses may inform whether pretreatment and resistant cancers are derived from a common ancestor clone. Nevertheless, our findings on a larger cohort of patients, suggest that such occurrences will be rare in cancers with acquired crizotinib resistance.

Our study has several implications. In rare cases of suspected overlapping genetic alterations in driver oncogenes, our findings underscore the importance of using confirmatory molecular testing, preferably using different genotyping techniques. Our findings may also affect clinical laboratory workflow and resource allocation since the lack of concomitant genetic alterations in EGFR, KRAS, and ALK may permit sequential genetic testing in centers where ALK FISH is not readily available. However, this must be balanced against the need for timely acquisition of genotyping data to guide rapid initiation of therapy, particularly at the time of diagnosis. For this reason, a simultaneous molecular testing strategy also remains a viable approach. Finally, this study has implications for our understanding of crizotinib resistance. Consistent with prior reports, we observed ALK fusion gene amplification and secondary mutations in the ALK TK domain in a subset of ALK-positive patients with acquired crizotinib resistance. Nonetheless, the number of crizotinib-resistant patients examined in this and other studies have been relatively small, and a significant proportion of these patients still have unknown mechanisms of resistance to date. Resistance is likely to be a molecularly complex process, and studies of repeat biopsies in larger cohorts of crizotinib-resistant patients are needed.

There are several potential limitations to our study. It is possible that we did not detect genetic alterations present at very low allelic frequencies within our study population. To minimize this likelihood, all ALK testing was conducted using the current gold-standard assay, ALK FISH. Moreover, EGFR and KRAS mutation testing was conducted using the SNaPshot assay, which has analytic sensitivity to detect mutations present at a frequency of approximately 5% (29). Another consideration is that mutations in EGFR or KRAS may have been present, but they were outside of the mutation hotspot regions designated in our multiplexed assays. This is less likely as more than 95% of mutations in both EGFR and KRAS are captured by these assays. One last consideration is the presence of intratumoral heterogeneity. In this study, molecular testing was conducted on a single biopsy or resection specimen and thus, may not be reflective of all sites of disease. This is mainly relevant to treatment resistant specimens, as studies have shown clonality of driver gene alterations in pretreatment samples (39, 40). However, if this were a common occurrence in resistance, one would have expected to observe an EGFR or KRAS mutation in at least one of the resistant biopsies examined.

Finally, while this manuscript was under review, 2 smaller screening studies were published, confirming similar findings in 208 and 99 patients, respectively (41, 42). Together, our analyses provide support for the mutual exclusivity of alterations in EGFR, KRAS, and ALK within the Western population. By examining a series of biopsy specimens from ALK-positive patients after development of resistance to crizotinib, we also show that this lack of overlap persists in the resistant cancers.

A.J. Iafrate is employed (other than primary affiliation; e.g., consulting) as a consultant in Pfizer and Bioreference Labs. J.A. Engelman has a commercial research grant from Novartis, Sanofi-Aventis, and AstraZeneca; has ownership interest (including patents) in Gatekeeper; and is a consultant/advisory board member of Novartis, Sanofi-Aventis, Chugai, and AstraZeneca. D. Dias-Santagata is a consultant/advisory board member of BioReference Laboratories, Inc. A.T. Shaw is a consultant/advisory board member of Pfizer, Novartis, Ariad, Chugai, and Daiichi sankyo. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.F. Gainor, A.M. Varghese, G.J. Riely, M.E. Arcila, M. Ladanyi, J.A. Engelman, A.T. Shaw

Development of methodology: J.F. Gainor, R. Katayama, G.J. Riely, M.E. Arcila

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.F. Gainor, A.M. Varghese, S.H. Ignatius Ou, S. Kabraji, M.M. Awad, R. Katayama, M. Mino-Kenudson, G.J. Riely, A.J. Iafrate, M.E. Arcila, D. Dias-Santagata, A.T. Shaw

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.F. Gainor, S.H. Ignatius Ou, M.M. Awad, B.Y. Yeap, A.J. Iafrate, M.E. Arcila, M. Ladanyi, J.A. Engelman, D. Dias-Santagata, A.T. Shaw

Writing, review, and/or revision of the manuscript: J.F. Gainor, S.H. Ignatius Ou, M. Mino-Kenudson, B.Y. Yeap, G.J. Riely, A.J. Iafrate, M.E. Arcila, M. Ladanyi, J.A. Engelman, A.T. Shaw

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.F. Gainor, S. Kabraji, M.M. Awad, A. Pawlak

Study supervision: G.J. Riely, M.E. Arcila, A.T. Shaw

The authors thank Emily Howe at Massachusetts General Hospital for help in coordinating clinical testing of crizotinib-resistant specimens. They also thank “Be a Piece of the Solution” and the “Evan Spirito Memorial Foundation” for support of lung cancer research.

This work was supported by grants from the U.S. NIH 5R01CA164273-02 and 5P50CA090578-10, and by a V Foundation Translational Research Grant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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