EGFR mutations underlie the sensitivity of lung cancers to erlotinib and gefitinib and can occur in any patient with this illness. Here we examine the frequency of EGFR mutations in smokers and men.
We determined the frequency of EGFR mutations and characterized their association with cigarette smoking status and male sex.
We tested 2,142 lung adenocarcinoma specimens for the presence of EGFR exon 19 deletions and L858R. EGFR mutations were found in 15% of tumors from former smokers (181 of 1,218; 95% CI, 13% to 17%), 6% from current smokers (20 of 344; 95% CI, 4% to 9%), and 52% from never smokers (302 of 580; 95% CI, 48% to 56%; P < .001 for ever v never smokers). EGFR mutations in former or current smokers represented 40% of all those detected (201 of 503; 95% CI, 36% to 44%). EGFR mutations were found in 19% (157 of 827; 95% CI, 16% to 22%) of tumors from men and 26% (346 of 1,315; 95% CI, 24% to 29%) of tumors from women (P < .001). EGFR mutations in men represented 31% (157 of 503; 95% CI, 27% to 35%) of all those detected.
A large number of EGFR mutations are found in adenocarcinoma tumor specimens from men and people who smoked cigarettes. If only women who were never smokers were tested, 57% of all EGFR mutations would be missed. Testing for EGFR mutations should be considered for all patients with adenocarcinoma of the lung at diagnosis, regardless of clinical characteristics. This strategy can extend the use of EGFR tyrosine kinase inhibitors to the greatest number individuals with the potential for substantial benefit.
A subset of lung adenocarcinomas appears preferentially sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). EGFR-activating mutations and never smoking are associated with response to TKIs.
To describe the morphology of adenocarcinomas responsive to TKIs, compare it to tumors in nonresponding patients, and correlate findings with EGFR mutations, gene copy number, and protein expression.
Material from 52 EGFR TKI-treated patients was studied: 29 responders and 23 nonresponders. Adenocarcinoma subtypes and morphologic features were defined in histologic and cytologic material. EGFR mutations were detected by sequencing, copy number by chromogenic in situ hybridization, and expression by immunohistochemistry.
Tumors from TKI responders tended to be better-differentiated adenocarcinomas with bronchioloalveolar carcinoma components. Nonresponders showed more heterogeneous morphology, higher grade, and more subtypes, and were more likely to show solid growth. In nonresponders, the only pure bronchioloalveolar carcinoma was mucinous, a subtype known to be negative for EGFR mutations. Using World Health Organization criteria, all tumors in both groups other than pure bronchioloalveolar carcinomas would be classified as adenocarcinomas, mixed subtype, thereby obscuring some of these distinctions. EGFR mutations were significantly more common in responders (22/29 vs 0/23; P < .001). Immunohistochemistry and chromogenic in situ hybridization results were not significantly correlated with EGFR mutations or response to TKIs in this study.
Overall, histologic differences exist between tumors that respond to TKIs and those that do not, although sampling affects classification, and there is significant histologic overlap between the 2 groups. Response is strongly associated with EGFR mutations.
Malignant pleural mesotheliomas (MPMs) often show CDKN2A and NF2 inactivation, but other highly recurrent mutations have not been described. To identify additional driver genes, we used an integrated genomic analysis of 53 MPM tumor samples to guide a focused sequencing effort that uncovered somatic inactivating mutations in BAP1 in 23% of MPMs. The BAP1 nuclear deubiquitinase is known to target histones (together with ASXL1 as a Polycomb repressor subunit) and the HCF1 transcriptional co-factor, and we show that BAP1 knockdown in MPM cell lines affects E2F and Polycomb target genes. These findings implicate transcriptional deregulation in the pathogenesis of MPM.
To compare preoperative size of Stage I and Stage II lung adenocarcinoma as measured by Computed Tomography (CT) and as assessed on gross pathology specimens.
Materials and Methods
47 patients diagnosed with Stage I or II lung adenocarcinoma were evaluated. Institutional Review Board permission was obtained. Tumor contours were delineated using a semi-automated segmentation algorithm and adjusted based on a radiologist’s input. Based on the tumor perimeter, maximal in-plane tumor diameter was calculated automatically. The largest single diameter from the pathology gross report was utilized. A paired t-test was used to examine the measurement difference between CT and pathology.
The mean largest diameter of the tumors at CT and pathology was 29.53 mm and 24.04 mm, respectively. There was a statistically significant difference between the mean CT measurement and mean pathology measurement of 5.49 mm (standard deviation 9.08 mm, p < 0.001). The percent relative difference between the two measurements was 18.3% (standard deviation 28.2%).
There is a statistically significant difference between the tumor diameter as measured by CT and on pathology gross specimen. These differences could have implications in the treatment and prognosis of patients with early stage lung adenocarcinoma.
lung cancer; computed tomography; non-small cell lung cancer; staging; T descriptor; pathology measurement
Primary lung adenocarcinoma is extremely rare in the pediatric age group. There have been anecdotal reports of lesions that are histologically indistinguishable from adult-type pulmonary adenocarcinoma in young patients after treatment for nonpulmonary cancers. Herein, we present clinical, histopathologic, and molecular data on eight such cases.
Histopathologic evaluation of the tumors was performed according to the World Health Organization classification. Molecular studies for EGFR and KRAS mutations were performed on six patients with sufficient material.
All eight patients were never smokers, four males and four females. Median age at nonpulmonary cancer diagnosis was 14 years (range, 3–23 years). Pulmonary adenocarcinomas were diagnosed at a median age of 15 years (range, 10–24 years); tumors were 0.1 to 2.0 cm in size and in some cases coexisted with metastases from the original cancer. Retrospective review showed that in at least three patients, the nodules were radiographically present before chemotherapy. Of six patients whose tumors were tested for common EGFR and KRAS mutations, two were positive for the former and one for the latter. At a median follow-up of 11 months (range, 2–29 months), six patients remained well without lung nodules and two had additional small, peripheral lung nodules that have not been biopsied.
Pulmonary lesions found in young patients with pediatric cancers can be histologically indistinguishable from lung adenocarcinoma seen in adults, may display typical adenocarcinoma-associated mutations of EGFR and KRAS, and may precede the administration of cytotoxic chemotherapy.
Bronchioloalveolar carcinoma; Lung cancer; Adenocarcinoma; Secondary malignancies; Osteosarcoma; EGFR; KRAS
The mutually exclusive pattern of the major driver oncogenes in lung cancer suggests that other mutually exclusive oncogenes exist. We performed a systematic search for tyrosine kinase (TK) fusions by screening all TKs for aberrantly high RNA expression levels of the 3′ kinase domain (KD) exons relative to more 5′ exons.
We studied 69 patients (including 5 never smokers and 64 current or former smokers) with lung adenocarcinoma negative for all major mutations in KRAS, EGFR, BRAF, MEK1, and HER2, and for ALK fusions (termed “pan-negative”). A NanoString-based assay was designed to query the transcripts of 90 TKs at two points: 5′ to the KD and within the KD or 3′ to it. Tumor RNAs were hybridized to the NanoString probes and analyzed for outlier 3′ to 5′ expression ratios. Presumed novel fusion events were studied by rapid amplification of cDNA ends (RACE) and confirmatory RT-PCR and FISH.
We identified 1 case each of aberrant 3′ to 5′ ratios in ROS1 and RET. RACE isolated a GOPC-ROS1 (FIG-ROS1) fusion in the former and a KIF5B-RET fusion in the latter, both confirmed by RT-PCR. The RET rearrangement was also confirmed by FISH. The KIF5B-RET patient was one of only 5 never smokers in this cohort.
The KIF5B-RET fusion defines an additional subset of lung cancer with a potentially targetable driver oncogene enriched in never smokers with “pan-negative” lung adenocarcinomas. We also report for the first time in lung cancer the GOPC-ROS1 fusion previously characterized in glioma.
lung cancer; kinase; gene fusion; RET; ROS1; ALK
The discovery of RET fusions in lung cancers has uncovered a new therapeutic target for patients whose tumors harbor these changes. In an unselected population of non–small cell lung carcinomas (NSCLCs), RET fusions are present in 1% to 2% of cases. This incidence increases substantially, however, in never-smokers with lung adenocarcinomas that lack other known driver oncogenes. Although preclinical data provide experimental support for the use of RET inhibitors in the treatment of RET fusion-positive tumors, clinical data on response are lacking. We report preliminary data for the first three patients treated with the RET inhibitor cabozantinib on a prospective phase II trial for patients with RET fusion-positive NSCLCs (NCT01639508). Confirmed partial responses were observed in 2 patients, including one harboring a novel TRIM33–RET fusion. A third patient with a KIF5B–RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remain progression-free on treatment.
All patients with EGFR mutant lung cancers eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Smaller series have identified various mechanisms of resistance, but systematic evaluation of a large number of patients to definitively establish the frequency of various mechanisms has not been performed.
Patients with lung adenocarcinomas and acquired resistance to erlotinib or gefitinib enrolled onto a prospective biopsy protocol and underwent a re-biopsy after the development of acquired resistance. Histology was reviewed. Samples underwent genotyping for mutations in EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS and PIK3CA, and FISH for MET and HER2.
Adequate tumor samples for molecular analysis were obtained in 155 patients. Ninety-eight had second-site EGFR T790M mutations (63%, 95% CI 55-70%) and four had small cell transformation (3%, 95% CI 0-6%). MET amplification was seen in 4/75 (5%, 95% CI 1-13%). HER2 amplification was seen in 3/24 (13%, 95% CI 3-32%). We did not detect any acquired mutations in PIK3CA, AKT1, BRAF, ERBB2, KRAS, MEK1, or NRAS. (0/88, 0%, 95% CI 0-4%). Overlap among mechanisms of acquired resistance was seen in 4%.
This is the largest series reporting mechanisms of acquired resistance to EGFR TKI therapy. We identified EGFR T790M as the most common mechanism of acquired resistance, while MET amplification, HER2 amplification, and small cell histologic transformation occur less frequently. More comprehensive methods to characterize molecular alterations in this setting are needed to improve our understanding of acquired resistance to EGFR TKIs.
EGFR mutant lung cancer; lung adenocarcinoma; targeted therapy; acquired resistance; tyrosine kinase inhibitor therapy
In contrast to other primary EGFR mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1 and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases (2.2%, 95% CI 1.6 to 3.1%), all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (p<0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations, but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12bp, resulting in 13 different insertions which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors.
EGFR exon 20; EGFR; epidermal growth factor receptor; lung adenocarcinoma; driver oncogenes
Activating mutations in the tyrosine kinase domain of HER2 (ERBB2) have been described in a subset of lung adenocarcinomas (ADCs) and are mutually exclusive with EGFR and KRAS mutations. The prevalence, clinicopathologic characteristics, prognostic implications, and molecular heterogeneity of HER2-mutated lung ADCs are not well established in US patients.
Lung ADC samples (n=1478) were first screened for mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and negative cases were then assessed for HER2 mutations (exons 19–20) using a sizing assay and mass spectrometry. Testing for additional recurrent point mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1 and AKT was performed by mass spectrometry. ALK rearrangements and HER2 amplification were assessed by FISH.
We identified 25 cases with HER2 mutations, representing 6% of EGFR/KRAS/ALK-negative specimens. Small insertions in exon 20 accounted for 96% (24/25) of the cases. Compared to insertions in EGFR exon 20, there was less variability, with 83% (20/24) being a 12bp insertion causing duplication of amino acids YVMA at codon 775. Morphologically, 92% (23/25) were moderately or poorly differentiated ADC. HER2 mutation was not associated with concurrent HER2 amplification in 11 cases tested for both. HER2 mutations were more frequent among never-smokers (p<0.0001) but there were no associations with sex, race, or stage.
HER2 mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer worldwide and the availability of standard and investigational therapies targeting HER2, routine clinical genotyping of lung ADC should include HER2.
HER2; ERBB2; lung adenocarcinoma; EGFR; driver oncogenes
The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear.
We genotyped 3026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical and smoking history data.
EGFR mutations were found in 43% of never smokers (NS) and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of NS. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. NS were more likely than smokers to have KRAS G>A transition mutation (mostly G12D) (58% vs. 20%, p=0.0001). KRAS G12C, the most common G>T transversion mutation in smokers, was more frequent in women (p=0.007) and these women were younger than men with the same mutation (median 65 vs. 69, p=0.0008) and had smoked less.
The distinct types of KRAS mutations in smokers vs. NS suggest that most KRAS-mutant lung cancers in NS are not due to secondhand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens.
lung cancer; tobacco; EGFR; KRAS; molecular epidemiology
Pulmonary large cell carcinoma - a diagnostically and clinically controversial entity - is defined as a non-small cell carcinoma lacking morphologic differentiation as either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end-stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically-relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with 9 therapeutically-relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%), or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.
large cell carcinoma; TTF-1; ΔNp63/p40; EGFR; KRAS; ALK
Patients with stage IV lung adenocarcinoma and EGFR mutation derive clinical benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). Whether treatment with TKI improves outcomes in patients with resected lung adenocarcinoma and EGFR mutation is unknown.
Data were analyzed from a surgical database of patients with resected lung adenocarcinoma harboring EGFR exon 19 or 21 mutations. In a multivariate analysis, we evaluated the impact of treatment with adjuvant TKI.
The cohort consists of 167 patients with completely resected stage I–III lung adenocarcinoma. 93 patients (56%) had exon 19 del, 74 patients (44%) had exon 21 mutations, 56 patients (33%) received perioperative TKI. In a multivariate analysis controlling for sex, stage, type of surgery and adjuvant platinum chemotherapy, the 2-year DFS was 89% for patients treated with adjuvant TKI compared with 72% in control group (hazard ratio [HR] = 0.53; 95% confidence interval [CI] 0.28 to 1.03; p = 0.06). The 2-year OS was 96% with adjuvant EGFR TKI and 90% in the group that did not receive TKI (HR 0.62; 95% CI 0.26 to 1.51; p = 0.296).
Compared to patients who did not receive adjuvant TKI, we observed a trend toward improvement in disease free survival among individuals with resected stages I–III lung adenocarcinomas harboring mutations in EGFR exons 19 or 21 who received these agents as adjuvant therapy. Based on these data, 320 patients are needed for a randomized trial to prospectively validate this DFS benefit.
The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung.
SUBJECTS AND METHODS
The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens.
Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy.
Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients.
biopsy; lung cancer; molecular typing; personalized medicine; targeted therapy
PIK3CA encodes the p110α subunit of the mitogenic signaling protein phosphatidylinositol 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported.
We examined EGFR, KRAS, BRAF, HER2, PIK3CA, AKT1, NRAS, MEK1, and ALK in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA.
Twenty-three of 1125 (2%, 95% confidence interval (CI) 1–3%) patients had a mutation in PIK3CA, 12 in Exon 9 (10 E545K, 2 E542K) and 11 in Exon 20 (3 H1047L, 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range 34–78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI 49 – 86%) had coexisting mutations in other oncogenes - 10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions.
We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be performed on tumor specimens from patients enrolling on clinical trials of PI3K and other targeted therapies.
lung adenocarcinoma; oncogene; PIK3CA
There is persistent controversy as to whether EGFR/KRAS mutations occur in pulmonary squamous cell carcinoma (SQCC). We hypothesized that the reported variability may reflect difficulties in the pathologic distinction of true SQCC from adenosquamous carcinoma (AD-SQC) and poorly-differentiated adenocarcinoma (ADC) due to incomplete sampling or morphologic overlap. The recent development of a robust immunohistochemical approach for distinguishing squamous vs glandular differentiation provides an opportunity to reassess EGFR/KRAS and other targetable kinase mutation frequencies in a pathologically homogeneous series of SQCC.
Ninety-five resected SQCC, verified by immunohistochemistry as ΔNp63+/TTF-1−, were tested for activating mutations in EGFR, KRAS, BRAF, PIK3CA, NRAS, AKT1, ERBB2/HER2, and MAP2K1/MEK1. Additionally, all tissue samples from rare patients with the diagnosis of EGFR/KRAS-mutant “SQCC” encountered during5 years of routine clinical genotyping were reassessed pathologically.
The screen of 95biomarker-verified SQCC revealed no EGFR/KRAS (0%; 95%CI 0–3.8%), 4 PIK3CA (4%; 95% CI 1–10%) and 1 AKT1 (1%; 95% CI 0–5.7%) mutations. Detailed morphologic and immunohistochemical reevaluation of EGFR/KRAS-mutant SQCC” identified during clinical genotyping (n=16) resulted in reclassification of 10 (63%)cases as AD-SQC and 5 (31%) cases as poorly-differentiated ADC morphologically mimicking SQCC (i.e. ADC with “squamoid” morphology). One (6%) case had no follow-up.
Our findings suggest that EGFR/KRAS mutations do not occur in pure pulmonary SQCC, and occasional detection of these mutations in samples diagnosed as “SQCC” is due to challenges with the diagnosis of AD-SQC and ADC, which can be largely resolved by comprehensive pathologic assessment incorporating immunohistochemical biomarkers.
EGFR; KRAS; TTF-1; p63; squamous cell carcinoma
The detection of mutations in the epidermal growth factor receptor (EGFR) gene, which predict sensitivity to treatment with EGFR tyrosine kinase inhibitors (TKIs), represents a major advance in the treatment of lung adenocarcinoma. KRAS mutations confer resistance to EGFR -TKIs. The prevalence of these mutations in African-American patients has not been thoroughly investigated.
We collected formalin-fixed, paraffin-embedded material from resected lung adenocarcinomas from African-American patients at three institutions for DNA extraction. The frequencies of EGFR exon 19 deletions, exon 21 L858R substitutions and KRAS mutations in tumor specimens from African-American patients were compared to data in Caucasian patients (n=476).
EGFR mutations were detected in 23 of the 121 specimens from African-American patients (19%, 95% CI 13–27%), while KRAS mutations were found in 21 (17%, 95% CI 12−25%). There was no significant difference between frequencies of EGFR mutations comparing African-American and Caucasian patients, 19% vs. 13% (61/476, 95% CI 10–16%) (p=0.11). KRAS mutations were more likely among Caucasians, 26% (125/476, 95% CI 23−30%) (p=0.04).
This is the largest study to date examining the frequency of mutations in lung adenocarcinomas in African-Americans. Although KRAS mutations were somewhat less likely, there was no difference between the frequencies of EGFR mutations in African-American patients as compared to Caucasians. These results suggest that all patients with advanced lung adenocarcinomas should undergo mutational analysis prior to initiation of therapy.
EGFR mutation; KRAS; African-Americans; racial differences
The EGFR mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/sequencing assay. MET amplification is also analyzed.
Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFR mutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH.
Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37).
The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients.
Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown.
All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry (IHC), and analyzed by array-based comparative genomic hybridization (aCGH). Previously untreated tumors were subjected to global gene expression arrays.
We analyzed 45 thymic tumors (thymoma n=38 (type A: n=8, type B2: n=22, type B3: n=8), and thymic carcinoma n=7). One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, while the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A-B2 thymomas vs. type B3 and thymic carcinomas. Moreover, aCGH could readily distinguish squamous cell carcinomas of the thymus vs. the lung, which can often present a diagnostic challenge.
Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathological, and therapeutic implications for the treatment of thymic malignancies.
Thymoma; Thymic Carcinoma; EGFR, RAS mutations; KIT mutations; mutational profiling; genomic analysis
To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This revealed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with under-expression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.
The IGF receptor type 1 (IGF-1R) pathway is frequently deregulated in human tumors and has become a target of interest for anti-cancer therapy.
We used a panel of 22 non-small cell lung cancer (NSCLC) cell lines to investigate predictive biomarkers of response to R1507, a fully-humanized anti-IGF-1R monoclonal antibody (Ab; Roche). 5 lines were moderately sensitive (25–50% growth inhibition) to R1507 alone. While levels of phospho-IGF-1R did not correlate with drug sensitivity, 4 out of 5 sensitive lines displayed high levels of total IGF-1R versus 1 out of 17 resistant lines (p = 0.003, Fisher's Exact). Sensitive lines also harbored higher copy numbers of IGF-1R as assessed by independent SNP array analysis. Addition of erlotinib or paclitaxel to R1507 led to further growth inhibition in sensitive but not resistant lines. In one EGFR mutant lung adenocarcinoma cell line (11–18), R1507 and erlotinib co-treatment induced apoptosis, whereas treatment with either drug alone induced only cell cycle arrest. Apoptosis was mediated, in part, by the survival-related AKT pathway. Additionally, immunohistochemical (IHC) staining of total IGF-1R with an anti-total IGF-1R Ab (G11;Ventana) was performed on tissue microarrays (TMAs) containing 270 independent NSCLC tumor samples. Staining intensity was scored on a scale of 0 to 3+. 39.3% of tumors showed medium to high IGF-1R IHC staining (scores of 2+ or 3+, respectively), while 16.7% had scores of 3+.
In NSCLC cell lines, high levels of total IGF-1R are associated with moderate sensitivity to R1507. These results suggest a possible enrichment strategy for clinical trials with anti-IGF-1R therapy.
EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.
Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.
The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.
To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFRT790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFRL858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFRT790M-expressing animals develop tumors with longer latency than EGFRL858R+T790M-bearing mice and in the absence of additional kinase domain mutations.
These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFRT790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.