Doo Hyun Chung

Predictive and Prognostic Impact of Epidermal Growth Factor Receptor Mutation in Non–Small-Cell Lung Cancer Patients Treated With Gefitinib

PURPOSE This study was undertaken to investigate the effects of epidermal growth factor receptor (EGFR) mutation and its downstream signaling on response and survival in non-small-cell lung cancer (NSCLC) patients treated with gefitinib. PATIENTS AND METHODS For 90 consecutive NSCLC patients who had received gefitinib, EGFR mutation was analyzed by DNA sequencing of exons 18, 19, 21, and 23 in the EGFR tyrosine kinase domain. Expressions of phosphorylated (p) -Akt and p-Erk were determined via immunohistochemistry. Response rate, time to progression (TTP), and overall survival were compared between each group according to EGFR mutation, as well as p-Akt and p-Erk expression. RESULTS Seventeen patients (18.9%; 95% CI, 10.8 to 27.0) harbored EGFR mutations. These mutations include deletions in exon 19 in seven patients, L858R in six patients, G719A in three patients, and a novel A859T in one patient. Response rate in patients with EGFR mutation was 64.7% (11 of 17 patients; 95% CI, 42.0 to 87.4), in contrast to 13.7% (10 of 73 patients; 95% CI, 5.8 to 21.6) in patients without mutation (P < .001). Moreover, these 17 patients with EGFR mutation had significantly prolonged TTP (21.7 v 1.8 months; P < .001) and overall survival (30.5 v 6.6 months; P < .001) compared with the remaining 73 patients without mutation. Although no significant correlation was detected between EGFR mutation and expressions of p-Akt or p-Erk, p-Akt overexpression was associated with prolonged TTP in patients with EGFR mutation. CONCLUSION Our data further support the importance of EGFR mutation with regard to gefitinib sensitivity. In addition to its predictive role, EGFR mutation confers significant survival benefits on NSCLC patients treated with gefitinib.

Optimization of Patient Selection for Gefitinib in Non–Small Cell Lung Cancer by Combined Analysis of Epidermal Growth Factor Receptor Mutation, K-ras Mutation, and Akt Phosphorylation

Purpose: Mutations in epidermal growth factor receptor (EGFR) are strongly predictive of gefitinib efficacy in non–small-cell lung cancer. However, the presence of EGFR mutant nonresponses and nonmutant responses points out the need for more comprehensive analysis. Patients and Methods: For 69 non–small-cell lung cancer patients treated with gefitinib, we have extended our analysis to EGFR gene copy number by fluorescence in situ hybridization, mutations in K-ras, HER2, and exon 20 of EGFR by direct sequencing, and phosphatase and tensin homologue expression by immunohistochemistry, in addition to EGFR exons 18, 19, and 21, and phosphorylations of Akt and extracellular signal–regulated kinase reported previously. Results: EGFR mutation and high gene copy number were associated with better objective response in univariate analysis. However, only gefitinib-sensitive EGFR mutation was independently predictive of both response (P = 0.011) and survival (P = 0.002) in multivariate analysis. No patients with K-ras mutation, including two EGFR mutants, showed response. In EGFR nonmutants, patients with either K-ras mutation or p-Akt overexpression exhibited poor response and time-to-progression whereas patients with high gene copy number tended to have better outcomes in univariate analysis. In multivariate analysis of time-to-progression in EGFR nonmutants, K-ras mutation or p-Akt overexpression was associated with shorter time-to-progression (P = 0.017). No patient with HER2 mutation showed response to gefitinib. Reduced phosphatase and tensin homologue expression was not associated with gefitinib sensitivity. Conclusion: Gefitinib-sensitive EGFR mutation is the single most important predictor of gefitinib sensitivity. In addition to EGFR mutation, K-ras mutation and Akt phosphorylation aid in better prediction of gefitinib responsiveness in non–small-cell lung cancer.

Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Iressa, ZD1839) in chemotherapy-resistant non-small cell lung cancer.

Gefitinib has shown meaningful antitumor activity with tolerable toxicity in chemotherapy‐refractory NSCLC in previous studies. Moreover, EGFR expression failed to show a correlation with response. In an attempt to identify predictive markers of response, we have investigated the tumoral expression of key signaling molecules of EGFR (EGFR, p‐EGFR, p‐Akt, p‐Erk, p‐STAT3) by immunohistochemistry and analyzed their correlations with response. Of 65 patients who received gefitinib (250 mg/day) for chemotherapy‐refractory NSCLC, there were 14 partial responses (21.5%), 21 stable diseases (32.3%) and 21 progressive diseases (32.3%). Median durations of overall survival and time to progression were 6.7 months and 2.8 months, respectively. Immunohistochemistry was performed in 34 patients with evaluable tissue specimens. EGFR was overexpressed (2+ or 3+) in 32.4% and p‐EGFR was positive in 26.5%. The expressions of p‐Akt, p‐Erk and p‐STAT3 were positive (1+ or 2+) in 50%, 38.2% and 79.4%, respectively. The EGFR expression was not correlated with p‐EGFR or the downstream molecules. EGFR or p‐EGFR status did not correlate with response. Positive expression of p‐Erk was significantly associated with poor response (38.1% in −, 14.3% in 1+, 0% in 2+; p = 0.046). Furthermore, tumors with positive p‐Akt and negative p‐Erk nuclear expression exhibited the best response (60%), whereas there was no response in the opposite [p‐Akt (−), p‐Erk (+)] cases. Intense nuclear staining of p‐Akt (2+) was associated with prolonged TTP (HR 0.25, 95% confidence interval [CI] 0.08–0.79, p = 0.018) and OS (HR 0.16, 95% CI 0.04–0.62, p = 0.008). These results support the assumption that gefitinib responsiveness might be predicted by activated EGFR downstream molecules such as p‐Akt and p‐Erk.

Anaplastic lymphoma kinase translocation: a predictive biomarker of pemetrexed in patients with non-small cell lung cancer.

Introduction: This study compared the efficacy of pemetrexed in patients with anaplastic lymphoma kinase (ALK)-positive versus ALK-negative (epidermal growth factor receptor [EGFR] mutant or wild type [WT] for both ALK and EGFR) non-small cell lung cancer (NSCLC). Methods: Patients with advanced NSCLC who received second-line pemetrexed and beyond between March 2007 and April 2010 were screened for EGFR mutations and ALK rearrangements at Seoul National University Hospital. The clinical and in vitro efficacy of pemetrexed was evaluated for each genotypic group. Results: Ninety-five NSCLC patients were genotyped as follows: 43 (45%) EGFR mutation, 15 (16%) ALK translocation, and 37 (39%) WT. The overall response rate was superior in ALK-translocated patients compared with EGFR mutant or WT patients (46.7 versus 4.7 versus 16.2%, p = 0.001). ALK-positive patients showed longer time to progression than EGFR mutant or WT patients (9.2 versus 1.4 versus 2.9 months, p = 0.001). ALK positivity alone was a significant predictor for overall response rate (hazard ratio [HR] = 0.07, 95% confidence interval [CI]: 0.01–0.32; p = 0.001) and time to progression (HR = 0.44, 95% CI: 0.24–0.80; p = 0.007). ALK positivity remained independently significant regardless of treatment line (HR = 0.43, 95% CI: 0.24–0.77; p = 0.005). Thymidylate synthase mRNA levels in ALK-positive cells were significantly lower compared with control cells (p < 0.05). Conclusion: Pemetrexed is an effective treatment in patients with ALK-positive NSCLC. ALK positivity was independently predictive of pemetrexed efficacy in NSCLC patients.

High MET Gene Copy Number Leads to Shorter Survival in Patients with Non-small Cell Lung Cancer

Introduction: Activation of MET, either by increased gene copy number (GCN) or mutation, has been detected in various cancers. We investigate the clinicopathologic features of MET gene copy in nonsmall cell lung cancer (NSCLC). Methods: Tumor tissues were obtained from 180 resected NSCLCs, including 97 squamous cell carcinomas (SCCs) and 72 adenocarcinomas. No patient received epidermal growth factor receptor (EGFR)-targeted therapy. EGFR and MET GCNs were studied using fluorescence in situ hybridization (FISH) and were estimated according to the University of Colorado Cancer Center (UCCC) criteria. For MET, we also assessed GCNs using the Cappuzzo system. Results: FISH-positive MET was observed in 16.7% using the UCCC criteria; specifically, amplification was seen in 3.9% and high polysomy in 12.8%. FISH-positive MET status was significantly correlated with FISH-positive EGFR (p = 0.003). In the Cappuzzo system, high MET GCN (mean, ≥5 copies/cell) was found in 6.7% and also associated with FISH-positive EGFR (p = 0.031). MET gene copy status was not associated with gender, smoking history, histology, or stage. However, true MET amplification was more frequent in patients with SCC than in those with adenocarcinoma. FISH-positive MET status predicted worse survival in patients with NSCLC at advanced stages (p = 0.034) and in patients with SCC (p = 0.028). In multivariate analyses, increased MET GCN was significantly associated with shorter survival in patients with SCC, as analyzed using both the UCCC and Cappuzzo criteria (p = 0.019 and 0.008). Conclusions: Our results suggest that increased MET GCN would be an independent poor prognostic factor in SCC of the lung.

NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor β1 production

Although NKT cells has been known to exert protective roles in the development of autoimmune diseases, the functional roles of NKT cells in the downstream events of antibody-induced joint inflammation remain unknown. Thus, we explored the functional roles of NKT cells in antibody-induced arthritis using the K/BxN serum transfer model. NKT cell–deficient mice were resistant to the development of arthritis, and wild-type mice administrated with α-galactosyl ceramide, a potent NKT cell activator, aggravated arthritis. In CD1d−/− mice, transforming growth factor (TGF)-β1 was found to be elevated in joint tissues, and the blockade of TGF-β1 using neutralizing monoclonal antibodies restored arthritis. The administration of recombinant TGF-β1 into C57BL/6 mice reduced joint inflammation. Moreover, the adoptive transfer of NKT cells into CD1d−/− mice restored arthritis and reduced TGF-β1 production. In vitro assay demonstrated that interleukin (IL)-4 and interferon (IFN)-γ were involved in suppressing TGF-β1 production in joint cells. The adoptive transfer of NKT cells from IL-4−/− or IFN-γ−/− mice did not reverse arthritis and TGF-β1 production in CD1d−/− mice. In conclusion, NKT cells producing IL-4 and IFN-γ play a role in immune complex–induced joint inflammation by regulating TGF-β1.

Heterogeneity of Genetic Changes Associated with Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancer

Background: Anaplastic lymphoma kinase (ALK)-rearranged non–small-cell lung cancer (NSCLC) is markedly sensitive to the ALK inhibitor crizotinib. However, acquired resistance to crizotinib is inevitable through several mechanisms. Therefore, this study was conducted to identify genetic alterations associated with crizotinib resistance. Methods: Tumor samples were derived from seven ALK-positive NSCLC patients who showed acquired resistance to crizotinib, and these patients were analyzed for ALK, EGFR, and KRAS mutations and ALK and EGFR gene amplifications. In vitro cytotoxicity of crizotinib and ALK downstream signals were compared between crizotinib-naive and -resistant NSCLC cells. Results: After a median duration of 6 months (range, 4–12 months), seven ALK-positive NSCLC patients developed acquired resistance to crizotinib. Three patients harbored secondary ALK mutations, including one patient with both mutations: L1196M (n = 2) and G1269A (n = 2). Of note, one patient displayed ALK gene copy number gain (4.1-fold increase compared with the pre-crizotinib specimen) and EGFR L858R mutation with high polysomy. The amphiregulin concentration was high in the supernatant fluid from five patients with malignant pleural effusion (116.4–18934.0 pg/ml). SNU-2535 cells derived from a patient who harbored the G1269 mutation were resistant to crizotinib treatment similar to H3122 CR1 cells. L1196M and G1269A mutant clones were less sensitive to crizotinib and ALK downstream signals were ineffectively suppressed in these clones. Conclusions: Genetic changes associated with crizotinib resistance are heterogeneous in ALK-rearranged NSCLC patients who respond to crizotinib and subsequently develop resistance.

Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non-small cell lung cancer patients

Fluorescence in situ hybridization (FISH) is currently used to detect non-small cell lung cancer (NSCLC) patients with anaplastic lymphoma kinase (ALK) gene rearrangement, who are candidates for ALK inhibitor therapy. However, FISH may not be a practical method for screening for ALK-positive patients in a large population due to its cost and difficulty in interpretation. We investigated the role of immunohistochemistry (IHC) to screen for ALK rearrangement in advanced NSCLC. We identified 1,166 stage IIIB or IV NSCLC patients with non-squamous histology from the Seoul National University Hospital NSCLC database. To enrich ALK-positive cases, we selected 262 patients who were either EGFR wild-type or non-responders to previous EGFR tyrosine kinase inhibitors (TKI). ALK IHC and ALK FISH were performed on formalin-fixed, paraffin-embedded tissue. ALK protein was expressed in 28 (10.7%) tumors in 262 patients. ALK FISH was positive in 25 (9.5%) cases. All patients with IHC score of 3 (n=9) were FISH-positive and all patients with score of 0 (n=234) were FISH-negative. Among patients with IHC scores of 1 and 2, five (83.3%, 5/6) and eleven (84.6%, 11/13) were FISH-positive, respectively. The sensitivity and specificity of ALK IHC with intensity score of 1 or more were 100% and 98.7%, respectively. IHC can be a useful test for screening ALK FISH-positive cases in advanced NSCLC. FISH testing should be considered for advanced NSCLC patients with tumors showing mild to moderate staining for ALK by IHC to confirm ALK translocation.

Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: Comparison with tumor-infiltrating T cells and the status of oncogenic drivers.

PURPOSE Programmed cell death-1 (PD-1)/programmed cell death-ligand-1 (PD-L1) pathway-targeted immunotherapy has beneficial therapeutic effects in pulmonary squamous cell carcinoma (SqCC) patients. However, the expression patterns of PD-1 and PD-1 ligands (PD-Ls) in pulmonary SqCC remain unclear. Moreover, the association between the PD-1/PD-Ls pathway and the status of oncogenic drivers in pulmonary SqCC is unknown. METHODS PD-L1 and PD-L2 expression in tumor cells and the numbers of PD-1(+) and CD8(+) tumor-infiltrating lymphocytes (TILs) were examined in 331 resected SqCC tumors along with matched lymph node metastases from 77 cases using immunohistochemistry. EGFR and FGFR1 and MET expression and genetic status were also examined. RESULTS PD-L1 and PD-L2 expression was detected in 26.9% and 23.9% of the pulmonary SqCC samples, respectively. PD-L1 and PD-L2 expression was maintained or increased in the metastatic lymph node tumors in 81.1% and 93.5% of the 77 cases, respectively. The numbers of PD-1(+) and CD8(+) TILs were significantly positively correlated (P<0.001). Cases displaying high PD-L1 expression exhibited consistently high CD8(+) T cell infiltration (P<0.001), even in subgroup analyses according to age, smoking status, tumor size, lymph node metastasis, stage, and the EGFR, MET and FGFR1 status. Moreover, MET expression in the tumors was significantly correlated with high PD-L2 expression and increased PD-1(+) TILs (P=0.001 for both). Increased numbers of CD8(+) or PD-1(+) TILs were significantly associated with prolonged disease-free survival of these patients, whereas PD-L1 and PD-L2 expression had no significant prognostic implications. CONCLUSION PD-L1 and PD-L2 expression in pulmonary SqCC is associated with an increased number of CD8(+) TILs and increased MET expression, which might provide therapeutic insight into targeting the PD-1/PD-Ls pathway in pulmonary SqCC.

Epidermal Growth Factor Receptor Mutation in Lung Adenocarcinomas: Relationship with CT Characteristics and Histologic Subtypes

PURPOSE To retrospectively identify quantitative computed tomographic (CT) features that correlate with epidermal growth factor receptor (EGFR) mutation in surgically resected lung adenocarcinomas stratified by the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS), and European Respiratory Society (ERS) classification in an East Asian cohort of patients known to have a high prevalence of EGFR mutations. MATERIALS AND METHODS An institutional review board approved this study and waived informed consent. In 153 surgically resected lung adenocarcinomas, EGFR mutation was determined by direct DNA sequencing. Histologic subtype was classified according to IASLC/ATS/ERS classification of lung adenocarcinoma. At preoperative chest CT, the percentage of ground-glass opacity (GGO) volume and total tumor volume of each tumor were measured by using a semiautomated algorithm. Distribution of EGFR mutation according to histologic subtype, percentage of GGO volume, and total tumor volume was evaluated by using the Fisher exact test, the Student t test, trend analysis, and multiple logistic regression analysis. RESULTS Exon 21 missense mutation was more frequent in lepidic predominant adenocarcinomas than in other histologic subtypes (odds ratio, 3.44; 95% confidence interval: 1.53, 7.74; P = .003). GGO volume percentage in tumors with exon 21 missense mutation (61.7% ± 31.9 [standard deviation]) was significantly higher than that in EGFR wild-type tumors (30.0% ± 38.5) (P = .0001) and exon 19-mutated tumors (28.9% ± 37.7) (P = .0006). A significant trend of prevalence of exon 21 missense mutation increasing along with increasing GGO volume (P = .0008) was found. CONCLUSION GGO volume percentage in tumors with exon 21 missense mutation was significantly higher than that in tumors with other EGFR mutation status. This can be related to the fact that exon 21 missense mutation was significantly more frequent in lepidic predominant adenocarcinomas, including adenocarcinoma in situ, minimally invasive adenocarcinoma, and lepidic predominant invasive adenocarcinoma, according to IASLE/ATS/ERS classification.