EGFR-mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKIs). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Since afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. Fluorescent in situ hybridization analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR T790M were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR mutant tumors with acquired resistance to EGFR TKIs.
EGFR mutations; lung cancer; EGFR tyrosine kinase inhibitors; erlotinib; afatinib; cetuximab; HER2 amplification; EGFR T790M; acquired resistance
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.
Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for over 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole genome sequence analysis revealed frequent structural re-arrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40–50% of cases are positive. To uncover other potential targetable mutations, we performed whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, D816) wildtype melanoma. Surprisingly, we found a somatic BRAF L597R mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF V600 mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that 2 (4%) harbored L597 mutations and another 2 involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by MEK inhibition. A patient with BRAF L597S mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data demonstrate clinical significance to BRAF L597 mutations in melanoma.
melanoma; BRAF L597; whole genome sequencing; BRAF inhibitor; MEK inhibitor; TAK-733
Erlotinib (Tarceva) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, which effectively targets EGFR-mutant driven non–small-cell lung cancer. However, the evolution of acquired resistance because of a second-site mutation (T790M) within EGFR remains an obstacle to successful treatment.
We used mathematical modeling and available clinical trial data to predict how different pharmacokinetic parameters (fast versus slow metabolism) and dosing schedules (low dose versus high dose; missed doses with and without make-up doses) might affect the evolution of T790M-mediated resistance in mixed populations of tumor cells.
We found that high-dose pulses with low-dose continuous therapy impede the development of resistance to the maximum extent, both pre- and post-emergence of resistance. The probability of resistance is greater in fast versus slow drug metabolizers, suggesting a potential mechanism, unappreciated to date, influencing acquired resistance in patients. In case of required dose modifications because of toxicity, little difference is observed in terms of efficacy and resistance dynamics between the standard daily dose (150 mg/d) and 150 mg/d alternating with 100 mg/d. Missed doses are expected to lead to resistance faster, even if make-up doses are attempted.
For existing and new kinase inhibitors, this novel framework can be used to rationally and rapidly design optimal dosing strategies to minimize the development of acquired resistance.
EGFR-mutant lung cancer; Erlotinib; Evolutionary cancer modeling; Pharmacokinetic modeling; Acquired resistance; EGFR T790M mutation
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
The study of patients carrying germline EGFR mutations, which have been found in cases of familial lung adenocarcinoma, could provide unique insight into lung cancer risk and carcinogenesis in non-smokers. However, investigations into the biology of germline EGFR mutations have been hampered by the lack of an effective strategy for screening for carriers. We hypothesized that patients with lung cancers found to harbor the EGFR T790M resistance mutation prior to treatment, an uncommon occurrence, would be likely to carry underlying germline T790M mutations.
Eleven unrelated patients were identified with lung cancer harboring an EGFR T790M mutation from a 7-year institutional experience with tumor genotyping. Ten patients had benign tissue available, which was anonymously tested for presence of germline EGFR mutations.
Five of 10 cases carried a germline T790M mutation (50%, CI 27%–73%). One patient’s cancer exhibited a distinctive indolent growth which has also been described in preclinical studies of T790M-mutant cancers. A second patient underwent resection of 6 separate primary lung adenocarcinomas, each carrying different sensitizing EGFR mutations as well as T790M.
Genotyping of lung cancers, now commonly performed to predict benefit from treatment with EGFR tyrosine kinase inhibitors, can also be used as a screening tool to identify patients at risk of carrying germline EGFR mutations. Once identified, these patients and their families can be studied prospectively in order to explore appropriate screening strategies. Further studies using existing oncogenomic data to provide insight into underlying germline genetics are warranted.
Familial lung cancer; EGFR mutations; genetic susceptibility; T790M
This article reviews important complications of targeted drug therapies for solid malignancies that can be identified on diagnostic imaging. Wherever possible, known or proposed mechanistic explanations for drug complications are emphasized.
Familiarity with the toxicity profiles of different targeted cancer therapies is important for identifying drug-related complications and for differentiating drug effects from disease progression. A mechanistic understanding may be useful for associating individual drugs with their complications and for predicting the complications of emerging agents.
bevacizumab; drug toxicities; imatinib; ipilimumab; targeted agents; temsirolimus; vemurafenib
This brief report summarizes Dr. Pao's talk at the 54th Annual Meeting of the Thomas L. Petty Aspen Lung Conference, in Aspen, Colorado, on June 11, 2011. In this talk, Dr. Pao discussed three main topics: (1) DETECT (DNA Evaluation of Tumors for Enhanced Cancer Treatment), (2) MyCancerGenome.org (web-based decision support), and (3) DIRECT (DNA-mutation Inventory to Refine and Enhance Cancer Treatment).
lung cancer; driver mutations; targeted therapy; MyCancerGenome
Our previous study revealed that 90% (47 of 52; 95% CI: 0.79–0.96) of Chinese never-smokers with lung adenocarcinoma harbor known oncogenic driver mutations in just four genes: EGFR, ALK, HER2, and KRAS. Here, we examined the status of known driver mutations specifically in female never-smokers with lung adenocarcinoma.
Tumors were genotyped for mutations in EGFR, KRAS, ALK, HER2, and BRAF. Data on age, stage, tumor differentiation, histological subtypes, and molecular alterations were recorded from 349 resected lung adenocarcinomas from female never-smokers. We further compared the clinicopathological parameters according to mutational status of these genes.
Two hundred and sixty-six (76.2%) tumors harbored EGFR mutations, 16 (4.6%) HER2 mutations, 15 (4.3%) EML4-ALK fusions, seven (2.0%) KRAS mutations, and two (0.6%) BRAF mutations. In univariate analysis, patients harboring EGFR mutations were significantly older (p<0.001), whereas patients harboring HER2 mutations were significantly younger (p=0.036). Higher prevalence of KRAS (p=0.028) and HER2 (p=0.021) mutations was found in invasive mucinous adenocarcinoma (IMA). The frequency of EGFR mutations was positively correlated with acinar predominant tumors (p=0.002). Multivariate analysis revealed that older age at diagnosis (p=0.013) and acinar predominant subtype (p=0.005) were independent predictors of EGFR mutations. Independent predictors of HER2 mutations included younger age (p=0.030) and IMA (p=0.017). IMA (p=0.006) and poor differentiation (p=0.028) were independently associated with KRAS mutations.
The frequency of driver mutations in never-smoking female lung adenocarcinoma varies with histological subtypes and age at diagnosis. These data have implications for both clinical trial design and therapeutic strategies.
Lung adenocarcinoma; Female; Never smoker; EGFR mutation; HER2 mutation; Acinar; Mucinous; Age
Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non–small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement.
Patients and Methods
Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.
Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response.
ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.
EGFR mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, they develop resistance, often due to acquisition of a second-site mutation (T790M). Current EGFR TKIs select for T790M in preclinical models of acquired resistance. We explored whether all EGFR TKIs similarly select for the T790M mutation using data from early clinical trials and established in vitro models of acquired resistance.
We analyzed the clinical characteristics of 8 patients with metastatic EGFR mutant lung adenocarcinoma who were treated first-line with XL647 and then progressed. XL647 is an ATP-competitive inhibitor of EGFR, HER2, KDR, and EPHB4. Additional molecular preclinical studies were performed to characterize resistance.
Four patients displayed confirmed partial responses (PRs), three patients had unconfirmed PRs, and one patient displayed stable disease. Only one of five patients’ tumor samples available for analysis after disease progression harbored the T790M mutation. Eight patients subsequently received erlotinib, with (n=3) or without (n=5) chemotherapy. Three of five patients treated with single agent erlotinib derived additional benefit, staying on drug up to 9 months. EGFR mutant PC-9 cells with acquired resistance to XL647 did not harbor the T790M mutation, displayed a distinct mRNA profile from PC-9 cells with T790M-mediated resistance, and were moderately sensitive to erlotinib in growth inhibition assays. Crystal structure analyses of XL647/EGFR T790M did not reveal a different binding mode from that of erlotinib.
The findings of this exploratory study suggest different EGFR TKIs may select for distinct mechanisms of resistance. These results raise the possibility that different EGFR TKIs could be sequentially used to improve outcomes in patients with EGFR mutant lung cancer. Further work investigating this hypothesis is warranted.
non-small cell lung cancer; EGFR mutations; XL647; EGFR tyrosine kinase inhibitors; acquired resistance; gefitinib; erlotinib; afatinib
Erlotinib is effective for epidermal growth factor receptor (EGFR) mutant lung cancer, but CNS penetration at standard daily dosing is limited. We previously reported that intermittent “pulsatile” administration of high-dose (1500 mg) erlotinib once weekly was tolerable and achieved concentrations in cerebrospinal fluid exceeding the half maximal inhibitory concentration for EGFR mutant lung cancer cells in a patient with leptomeningeal metastases; we now expand this paradigm to a series of 9 patients. We retrospectively identified patients with EGFR mutant lung cancer treated with pulsatile erlotinib for CNS metastases (brain and/or leptomeningeal) that occurred despite conventional daily erlotinib or other EGFR tyrosine kinase inhibitors. Mutations in available lung and CNS tissue were correlated with efficacy. Erlotinib was administered as monotherapy at a median dose of 1500 mg weekly. Best CNS radiographic response was partial in 67% (6/9, including 2 with isolated leptomeningeal metastases), stable disease in 11% (1/9), and progressive disease in 22% (2/9). Median time to CNS progression was 2.7 months (range, 0.8–14.5 months) and median overall survival was 12 months (range, 2.5 months–not reached). Treatment was well tolerated. No acquired resistance mutations in EGFR were identified in the CNS metastases of 4 patients, including 1 harboring T790M outside the CNS. Pulsatile erlotinib can control CNS metastases from EGFR mutant lung cancer after failure of standard daily dosing. CNS disease may not harbor acquired resistance mutations that develop systemically. A prospective trial is planned.
CNS metastases; EGFR; erlotinib; lung cancer; pulsatile dosing
Non–small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance.
Given the unprecedented efficacy of EGFR tyrosine kinase inhibitors (TKI) in advanced EGFR-mutant lung cancer, adjuvant TKI therapy is an appealing strategy. However, there are conflicting findings regarding the potential benefit of adjuvant EGFR-TKI in patients with lung cancer harboring EGFR mutations. To better understand these results, we studied the natural history of lung cancers which recurred despite adjuvant TKI.
Patients with recurrent EGFR-mutant lung cancer following adjuvant TKI were identified using an IRB approved mechanism. Recurrent cancer specimens were tested for resistance mutations. Sensitivity to re-treatment with EGFR-TKI was evaluated.
Twenty-two patients with cancers harboring an EGFR sensitizing mutation received adjuvant erlotinib or gefitinib for a median of 17 months (range 1–37 months). T790M was more common in cancers which recurred while receiving TKI than in those which recurred after stopping TKI (67% vs. 0%, p=0.011). Fourteen patients who developed recurrence after stopping EGFR-TKI were re-treated, with a median time to progression of 10 months and radiographic response seen in 8 of 11 patients with evaluable disease (73%).
Recurrence of EGFR-mutant lung cancer after stopping adjuvant TKI should not preclude a trial of TKI re-treatment; a phase II trial of erlotinib in this setting is underway. Studies of adjuvant EGFR-TKI will underestimate the potential survival benefit of adjuvant TKI for patients with EGFR-mutant lung cancers if re-treatment at recurrence is not given.
Non-small cell lung cancer; adjuvant; EGFR; tyrosine kinase inhibitor; T790M
An American Society of Clinical Oncology (ASCO) focused update updates a single recommendation (or subset of recommendations) in advance of a regularly scheduled guideline update. This document updates one recommendation of the ASCO Guideline Update on Chemotherapy for Stage IV Non–Small-Cell Lung Cancer (NSCLC) regarding switch maintenance chemotherapy.
Recent results from phase III clinical trials have demonstrated that in patients with stage IV NSCLC who have received four cycles of first-line chemotherapy and whose disease has not progressed, an immediate switch to alternative, single-agent chemotherapy can extend progression-free survival and, in some cases, overall survival. Because of limitations in the data, delayed treatment with a second-line agent after disease progression is also acceptable.
Seven randomized controlled trials of carboxyaminoimidazole, docetaxel, erlotinib, gefitinib, gemcitabine, and pemetrexed have evaluated outcomes in patients who received an immediate, non–cross resistant alternative therapy (switch maintenance) after first-line therapy.
In patients with stage IV NSCLC, first-line cytotoxic chemotherapy should be stopped at disease progression or after four cycles in patients whose disease is stable but not responding to treatment. Two-drug cytotoxic combinations should be administered for no more than six cycles. For those with stable disease or response after four cycles, immediate treatment with an alternative, single-agent chemotherapy such as pemetrexed in patients with nonsquamous histology, docetaxel in unselected patients, or erlotinib in unselected patients may be considered. Limitations of this data are such that a break from cytotoxic chemotherapy after a fixed course is also acceptable, with initiation of second-line chemotherapy at disease progression.
The management of non-small cell lung cancer (NSCLC) has been transformed by the observation that lung adenocarcinomas harboring mutations in EGFR are uniquely sensitive to EGFR tyrosine kinase inhibitors (TKIs). In these patients, acquired resistance to EGFR-TKI develops after a median of 10-14 months, at which time the current standard practice is to switch to conventional cytotoxic chemotherapy. Several possible mechanisms for acquired resistance have been identified, the most common being the development of an EGFR T790M gate-keeper mutation in over 50% of cases. In this review, we discuss recent advances in the understanding of acquired TKI resistance in EGFR-mutant lung cancer and review therapeutic progress with second generation TKIs and combinations of targeted therapies.
Aberrant forms of the anaplastic lymphoma kinase (ALK) have been implicated in the pathogenesis of multiple human cancers, where ALK represents a rational therapeutic target in these settings. In this study, we report the identification and biological characterization of X-376 and X-396, two potent and highly specific ALK small molecule tyrosine kinase inhibitors (TKIs). In Ambit kinome screens, cell growth inhibition studies, and surrogate kinase assays, X-376 and X-396 were more potent inhibitors of ALK but less potent inhibitors of MET compared to PF-02341066 (PF-1066), an ALK/MET dual TKI currently in clinical trials. Both X-376 and X-396 displayed potent anti-tumor activity in vivo with favorable pharmacokinetic and toxicity profiles. Similar levels of drug sensitivity were displayed by the three most common ALK fusion proteins in lung cancer (EML4-ALK variants E13;A20, E20;A20, and E6b;A20) as well as a KIF5B-ALK fusion protein. Moreover, X-396 could potently inhibit ALK kinases engineered with two point mutations associated with acquired resistance to PF-1066, L1196M and C1156Y, when engineered into an E13;A20 fusion variant. Lastly, X-396 displayed synergistic growth inhibitory activity when combined with the mTOR inhibitor rapamycin. Our findings offer preclinical proof-of-concept for use of these novel agents to improve therapeutic outcomes of patients with mutant ALK-driven malignancies.
ALK; ALK fusions; tyrosine kinase inhibitor; cancer; non-small cell lung cancer; rapamycin; targeted therapeutics; acquired resistance; gatekeeper mutation; PF-1066; X-376; X-396
EML4–ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4–ALK-positive tumors.
By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684.
Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4–ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066.
Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4–ALK fusions and ALK mutations.
A critical step in detecting variants from next-generation sequencing data is post hoc filtering of putative variants called or predicted by computational tools. Here, we highlight four critical parameters that could enhance the accuracy of called single nucleotide variants and insertions/deletions: quality and deepness, refinement and improvement of initial mapping, allele/strand balance, and examination of spurious genes. Use of these sequence features appropriately in variant filtering could greatly improve validation rates, thereby saving time and costs in next-generation sequencing projects.
Genetically engineered mouse models (GEMMs) of human cancer were first created nearly 30 years ago. These early transgenic models demonstrated that mouse cells could be transformed in vivo by expression of an oncogene. A new field emerged, dedicated to generating and using mouse models of human cancer to address a wide variety of questions in cancer biology. The aim of this review is to highlight the contributions of mouse models to the diagnosis and treatment of human cancers. Because of the breadth of the topic, we have selected representative examples of how GEMMs are clinically relevant rather than provided an exhaustive list of experiments. Today, as detailed here, sophisticated mouse models are being created to study many aspects of cancer biology, including but not limited to mechanisms of sensitivity and resistance to drug treatment, oncogene cooperation, early detection, and metastasis. Alternatives to GEMMs, such as chemically induced or spontaneous tumor models, are not discussed in this review.
To determine if tumor regression following treatment with gefitinib correlates with the presence of sensitizing mutations in EGFR.
Patients and Methods
Patients with resectable stage I and II non-small cell lung cancer (NSCLC) enriched for the likelihood of EGFR mutation (≤ 15 pack year cigarette smoking history and/or a component of bronchioloalveolar carcinoma) received preoperative gefitinib for 21 days. Tumor specimens were analyzed for EGFR and KRAS mutations and EGFR protein expression and amplification. Patients with ≥ 25% reduction in tumor size measured bidimensionally at 3 weeks and/or patients with an EGFR mutation received adjuvant gefitinib for 2 years post-operatively.
50 patients with stage I/II NSCLC were treated. After 21 days of preoperative gefitinib a response of ≥ 25% was observed in 21/50 (42%) patients. 17/21 patients with a response had an EGFR mutation and 4/21 patients with a response did not (p=0.0001). 25/50 patients were eligible to receive adjuvant gefitinib. With a median follow-up of 44.1 months, 2-year disease free survival for EGFR mutant patients and for those who received adjuvant gefitinib was not statistically different than those who were EGFR wild-type and those who did not receive adjuvant gefitinib. The median disease free and overall survivals have not been reached.
The presence of sensitizing EGFR mutations correlates with radiographic response. A short course of preoperative treatment serves a platform for evaluating activity of new agents and assures sufficient tumor availability for correlative analyses.
Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.
The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.
Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.
We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.
Patients with EGFR-mutant lung adenocarcinoma develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) after a median of 10–16 months. In half of these cases a second EGFR mutation, T790M, underlies acquired resistance. We undertook this study to examine the clinical course of patients harboring the T790M mutation following progression on TKI.
EGFR-mutant lung cancer patients with acquired resistance to EGFR TKIs were identified as part of a prospective re-biopsy protocol where post-progression tumor specimens were collected for molecular analysis. Post-progression survival and characteristics of disease progression were compared in patients with and without T790M.
We identified T790M in the initial re-biopsy specimens from 58/93 patients (62%, 95% confidence interval 52%–72%). T790M was more common in biopsies of lung/pleura tissue and lymph nodes than in more distant sites (p=0.014). Median post-progression survival was 16 months (interquartile range 9–29 months); patients with T790M had a significantly longer post-progression survival (p=0.036). Patients without T790M more often progressed in a previously uninvolved organ system (p=0.014) and exhibited a poorer performance status at time of progression (p=0.007).
Among patients with acquired resistance to EGFR TKIs, the presence of T790M defines a clinical subset with a relatively favorable prognosis and more indolent progression. Knowledge of T790M status is therefore important for the clinical care of these patients as well as for the optimal design and interpretation of clinical trials in this setting.
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.