Kazumi Nishino

Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas.

Purpose: Examination of somatic epidermal growth factor receptor (EGFR) mutations is now a diagnostic routine for treatment of cancer using EGFR tyrosine kinase inhibitors (EGFR-TKI). Circulating tumor DNA is a promising target for noninvasive diagnostics. We evaluated its utility by quantitatively detecting activating and resistant mutations, which were measured with BEAMing (beads, emulsion, amplification, and magnetics). Experimental Design: Twenty-three patients with lung cancer with progressive disease after EGFR-TKI treatment and 21 patients who had never been treated with EGFR-TKIs were studied. Their primary tumors were confirmed to have activating mutations. In the plasma DNA of each patient, the activating mutation found in the corresponding primary tumor and the T790M resistance mutation were quantified by BEAMing. Results: In 32 of 44 patients, activating mutations were detected in the plasma DNA [72.7%; 95% confidence interval (CI), 58.0%–83.6%]. The T790M mutation was detected in 10 of 23 patients in the first group (43.5%; 95% CI, 25.6%–53.4%). The ratio of T790M to activating mutations ranged from 13.3% to 94.0%. The peak of the distribution of the mutation allele fraction in the plasma DNA was in the 0.1% to 1% range. Conclusions: The major advantage of BEAMing is its ability to calculate the fraction of T790M-positive alleles from the alleles with activating mutations. This feature enables the detection of increases and decreases in the number of T790M mutations in cancer cells, regardless of normal cell DNA contamination, which may be useful for monitoring disease progression. Circulating tumor DNA could potentially be used as an alternative method for EGFR mutation detection. Clin Cancer Res; 17(24); 7808–15. ©2011 AACR.

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (P<0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with β1 integrins and distributed at the tumor cell periphery and cell–cell contacts, suggesting that CD9 modifies β1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

Diagnostic Accuracy of Noninvasive Genotyping of EGFR in Lung Cancer Patients by Deep Sequencing of Plasma Cell-Free DNA

BACKGROUND Genotyping of EGFR (epidermal growth factor receptor) mutations is indispensable for making therapeutic decisions regarding whether to use EGFR tyrosine kinase inhibitors (TKIs) for lung cancer. Because some cases might pose challenges for biopsy, noninvasive genotyping of EGFR in circulating tumor DNA (ctDNA) would be beneficial for lung cancer treatment. METHODS We developed a detection system for EGFR mutations in ctDNA by use of deep sequencing of plasma DNA. Mutations were searched in >100 000 reads obtained from each exon region. Parameters corresponding to the limit of detection and limit of quantification were used as the thresholds for mutation detection. We conducted a multi-institute prospective study to evaluate the detection system, enrolling 288 non-small cell lung cancer (NSCLC) patients. RESULTS In evaluating the performance of the detection system, we used the genotyping results from biopsy samples as a comparator: diagnostic sensitivity for exon 19 deletions, 50.9% (95% CI 37.9%-63.9%); diagnostic specificity for exon 19 deletions, 98.0% (88.5%-100%); sensitivity for the L858R mutation, 51.9% (38.7%-64.9%); and specificity for L858R, 94.1% (83.5%-98.6%). The overall sensitivities were as follows: all cases, 54.4% (44.8%-63.7%); stages IA-IIIA, 22.2% (11.5%-38.3%); and stages IIIB-IV, 72.7% (60.9%-82.1%). CONCLUSIONS Deep sequencing of plasma DNA can be used for genotyping of EGFR in lung cancer patients. In particular, the high specificity of the system may enable a direct recommendation for EGFR-TKI on the basis of positive results with plasma DNA. Because sensitivity was low in early-stage NSCLC, the detection system is preferred for stage IIIB-IV NSCLC.

Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).

Monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor DNA of heterogeneous mutant EGFR genes in lung cancer

OBJECTIVES Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatic effects on EGFR-mutant non-small-cell lung cancer (NSCLC). However, most patients experience disease recurrences, approximately half of which are T790M-mediated. Monitoring EGFR status with re-biopsy has spatiotemporal limitations. PATIENTS AND METHODS EGFR circulating tumor DNA (ctDNA) in serial plasma samples was amplified and 10(5) of them were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 10(5)EGFR cell free DNA (cfDNA). RESULTS PM scores of various EGFR mutations showed dynamic, case-specific changes during EGFR-TKI treatments in 52 patients. The effects of the treatment on EGFR ctDNA were evaluated in 38 patients with elevated pre-treatment PM scores. The ctDNA responses correlated well with radiologic responses in radiologic good responders, whereas correlation was poor in non-responders. In addition to the peaks for the most prevalent ctDNA, small peaks of ctDNA with different types of activating EGFR mutations or the T790M mutation (early T790M ctDNA) appeared transiently in 10.5% and 26.3%, respectively. Early T790M ctDNA disappeared in all patients, including 7 who eventually developed acquired resistance accompanied by elevated levels of T790M ctDNA. CONCLUSIONS Monitoring ctDNA is useful in evaluating treatment responses and monitoring driver oncogene status in NSCLC. ctDNA revealed clonal heterogeneity and genetic processes of cancer evolution in individual patients. The simple presence of the T790M mutation may be insufficient to confer EGFR-TKI resistance to tumor cells.

A retrospective analysis of 335 Japanese lung cancer patients who responded to initial gefitinib treatment.

BACKGROUND Gefitinib treatment results in considerably better progression-free survival compared with that of platinum doublets in the first line treatment of nonsmall-cell lung cancer (NSCLC) carrying an activating epidermal growth factor receptor (EGFR) mutation. Some patients who respond to gefitinib have an overall survival (OS) of more than 5 years, whereas other initial responders do less well. Although there has been considerable effort made to elucidate the mechanisms of acquired resistance, there have only been a few studies that addressed the effect of clinical backgrounds and treatment histories on the survival of the patients who had responded to an EGFR-tyrosine kinase inhibitor (TKI). In this study, we especially focused on the clinical benefit of EGFR-TKI administration after progression. PATIENTS AND METHODS We retrospectively analyzed consecutive patients with advanced NSCLC who were diagnosed before October 2010, treated with gefitinib after July 2002, and responded to it. The primary objective of this study was to evaluate how clinical backgrounds and treatment histories influence survival of the patients who respond to gefitinib. The secondary objectives were to evaluate the safety of long-term gefitinib use and to establish the optimal treatment sequence using a dynamic treatment regimen analysis (DTRA). RESULTS A total of 335 patients were recruited. Twenty-eight (8.4%) patients survived more than 5 years. Sixty-five and 93 patients received gefitinib as rechallenge and beyond progressive disease (BPD), respectively. A statistically significant difference in OS was observed between the patients who underwent gefitinib rechallenge and those who did not rechallenge (median: 1272 days vs. 774 days; p < 0.001), a result supported by a DTRA. Patients treated with gefitinib BPD also showed a tendency of longer survival. CONCLUSIONS Gefitinib rechallenge and BPD played a central role in long term survival of the patients who initially responded to gefitinib.

Gene therapy utilizing the Cre/loxP system selectively suppresses tumor growth of disseminated carcinoembryonic antigen-producing cancer cells.

Recent clinical trials of cancer gene therapy have shown encouraging results for controlling localized tumors. However, to control metastatic or disseminated tumor cells, further modification of vectors is required to enhance specificity and infectivity against targets. We investigated whether utilization of the Cre recombinase(Cre)/loxP system contributes to enhanced antitumor effects together with minimal adverse reactions in specific gene therapy against disseminated carcinoembryonic antigen (CEA)‐producing cancer cells in the peritoneal cavity of mice. CEA‐producing cancer would be a good therapeutic target because it is found in lung, stomach and colon sites, which account for most cancers. We constructed a pair of recombinant adenoviral vectors (Ads), one of which expresses the Cre gene under the control of the CEA promoter (Ad.CEA‐Cre); the other expresses the herpes simplex virus thymidine kinase (HSV‐TK) gene (Ad.lox‐TK), or the β‐galactosidase gene (β‐gal) by Cre (Ad.lox‐β‐gal). Intraperitoneal coinjection of Ad.CEA‐Cre and Ad.lox‐β‐gal into mice with peritonitis carcinomatosa by CEA‐producing tumor cells showed selective expression of the β‐gal gene in tumor foci. Coinfection of Ad.CEA‐Cre and Ad.lox‐TK followed by ganciclovir (GCV) administration significantly suppressed the total tumor weight in the peritoneal cavity of the mice to 13% of that of the untreated mice and 22% of that of the mice treated with Ad.CEA‐TK/GCV, an Ad that expressed the HSV‐TK gene driven by the CEA promoter alone. Moreover, treatment with Ad.CEA‐Cre and Ad.lox‐TK/GCV completely suppressed tumors in 4 of 10 (40%) mice without significant weight loss, although 2 of 10 mice treated with Ad.CAG‐TK/GCV, an adenovirus vector that strongly but nonspecifically expressed the TK gene, died due to severe side effects including diarrhea, weight loss and liver dysfunction. These findings suggest that cell type‐specific gene therapy using the Cre/loxP system is effective against disseminated cancer cells without significant side effects.

Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a myc-max binding motif

Recent clinical trials of gene therapy for patients with thoracic cancers have shown that these treatments were well tolerated with minimal side effects and that we need to further enhance specificity as well as efficiency of gene transfer to target cancer cells. We previously reported that myc‐overexpressing SCLC cell lines became selectively sensitive to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV‐TK) gene under the control of the Myc‐Max response elements (a core nucleotide sequence, CACGTG) and that this construct (MycTK) could be utilized to develop a novel treatment against chemo‐radio‐resistant SCLC. We report here in vivo antitumor effects and safety of a replication‐deficient adenoviral vector containing the Myc‐Max binding motif (AdMycTK) on SCLC cells. In vitro infection with AdMycTK selectively rendered myc‐overexpressing SCLC cell lines 63‐ to 307‐fold more sensitive to GCV. In vivo injections with AdMycTK followed by GCV administration markedly suppressed the growth of myc‐overexpressing tumors established in the subcutis or in the peritoneal cavity of athymic mice. On the other hand, infection with AdMycTK did not significantly affect either in vitro GCV sensitivity of the cells expressing very low levels of the myc genes or the growth of their subcutaneous tumors. Moreover, we observed no apparent side effects of this treatment including body weight loss or biochemical abnormalities in contrast to the treatment with AdCATK that conferred strong but nonspecific expression of the HSV‐TK gene. These results suggested that AdMycTK/GCV therapy is effective on SCLC patients whose tumors overexpress myc family oncogenes.

Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4-ALK fusion gene.

Introduction: Using cell line-based assays, the secretion of erythroblastic leukemia viral oncogene homologue (ERBB) ligands has been reported to contribute to resistance against crizotinib in lung cancer with the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase fusion gene. However, it is difficult to predict the role of the ligands in each patient. Here, we report an analysis of the mechanism of resistance behind crizotinib resistance using a primary culture of cancer cells from pleural effusion of an anaplastic lymphoma kinase-positive lung cancer patient who was clinically resistant to crizotinib. Methods: Primary cancer cells were prepared as cancer tissue-originated spheroids (CTOSs) according to previously described methods. CTOSs were maintained in StemPro medium, and a sensitivity assay was performed under growth factor-free conditions, or under stimulation with epidermal growth factor (EGF) or neuregulin-1/heregulin. The effect of treatment with crizotinib alone or a combination of crizotinib and erlotinib was examined. Results: Cancer cells (LB53) were established to be CTOSs from a patient who was clinically resistant to crizotinib. The CTOSs were sensitive to crizotinib under growth factor-free conditions in vitro, whereas resistant under stimulation with EGF or neuregulin-1. These ligands rescued the inhibition of intracellular signaling by crizotinib. Pleural effusion from the patient also activated EGF receptor signaling to the similar extent of EGF stimulation. The resistance to crizotinib by EGF was reversed by blocking EGF receptor signaling by erlotinib in vitro. Conclusion: Stimulation by ERBB ligands is suggested to be responsible for resistance to crizotinib in this patient. The CTOS method may enable analysis of resistance mechanism for targeted therapy in individual patients.

Dynamics of circulating tumor DNA represented by the activating and resistant mutations in epidermal growth factor receptor tyrosine kinase inhibitor treatment.

Circulating tumor DNA (ctDNA) is an emerging field of cancer research. For lung cancer, non‐invasive genotyping of EGFR is the foremost application. The activating mutations represent the ctDNA from all cancer cells, and the T790M‐resistant mutation represents that from resistant cells. We examined the ctDNA dynamics of EGFR mutations by using deep sequencing with a massively parallel DNA sequencer. We obtained 190 plasma samples from 57 patients at various times during the treatment course and classified them according to treatment status. The mutation detection rate of exon 19 deletion/L858R in plasma was high at the initiation of treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI; P = 0.001), suppressed during EGFR‐TKI treatment before disease progression, and elevated after the onset of disease progression (P = 0.023). The mutation detection rate of T790M was low until the onset of disease progression and elevated thereafter (P = 0.01). Samples across the development of disease progression were obtained from 10 patients and showed a correlation between increased ctDNA level and disease progression. Decreased ctDNA level in response to the initiation of EGFR‐TKI was observed in 4 of 6 eligible patients. In two patients, the ctDNA dynamics suggested the presence of cancer cell populations only with the T790M mutation. In another patient, the T790M ctDNA represented cell subpopulations that respond to cytotoxic agents differently from the major population. Considering the high incidence, ctDNA could be a clinical parameter to complement information from image analyses.