Kentaro Iwanaga

A Noninvasive System for Monitoring Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors with Plasma DNA

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are widely used to treat lung adenocarcinomas with EGFR-activating mutations. However, half of the patients acquire resistance because of the gatekeeper T790M mutation. Noninvasive mutation detection system is desired considering the difficulty in obtaining tissue specimens during disease progression. Methods: Sixty-seven plasma DNA samples from 49 patients with lung adenocarcinoma and 30 healthy volunteers were evaluated. T790M in plasma DNA was determined using the mutation-biased polymerase chain reaction (PCR) quenching probe (MBP-QP) method. The method combines MBP and genotyping, the latter based on analysis of the melting curve of the probe DNA binding the target mutated site using a fluorescence QP system. Results: The detection limit was two copies of control plasmid and 0.2 ng of genomic DNA. The mutant plasmid could be detected when it accounted for as little as 0.3% of a mixture of plasmids carrying EGFR exon 20 with or without T790M. The T790M mutation was detected in plasma DNA from 10 of 19 patients (53%) who acquired resistance, but not in nonresponders, patients responding to treatment, or those not treated with EGFR tyrosine kinase inhibitor. Other mutation detection systems, such as the nucleic acid-locked nucleic acid PCR clamp, the cycleave PCR technique, and allele-specific oligonucleotide PCR, detected T790M in three, four, and six patients, respectively, among 10 in which T790M was detected by the MBP-QP method. Conclusions: The MBP-QP method is simple, sensitive, and—intriguingly—reflective of clinical course, compared with the other three mutation-detection systems. Thus, the MBP-QP method is an ideal noninvasive monitoring system for detecting T790M in plasma samples.

Application of a Highly Sensitive Detection System for Epidermal Growth Factor Receptor Mutations in Plasma DNA

Introduction: Detection of epidermal growth factor receptor (EGFR) mutations is indispensable to determine an appropriate lung cancer treatment. Although retreatment often prolongs survival, how to select the appropriate population for retreatment has not been clarified. Methods: We used novel methods to identify EGFR mutations: wild inhibiting polymerase chain reaction (PCR) and quenched probe system (WIP-QP) for exon 19 deletions and mutation-biased PCR and quenched probe system for L858R. After the detection limits were determined, we examined DNA isolated from lung cancer specimens and circulating plasma DNA samples of 39 adenocarcinoma patients whose primary tumors harbored EGFR exon 19 deletions or L858R. Results: Detection limit was 0.005 to 0.04 ng in genomic DNA and 0.1% to 0.3% in mutant plasmids. The results of cancer tissue specimens were identical to those with existing systems (nucleic acid-locked nucleic acid PCR clamp or cycleave PCR), except for two samples that showed both exon 19 deletions and L858R. One of the two samples was confirmed to harbor L858R mutation by allele-specific oligonucleotide PCR; the other one did not. Exon 19 deletions and L858R were detected in 44.7% and 8.7% of patients, using plasma DNA, among those who carried the identical abnormalities in primary tumors all of cases that evidenced pathological stage IV except for one patient, suggesting that EGFR mutations might be preferentially detected in plasma DNA obtained from patients in advanced stages. Serial monitoring of these mutations with T790M, a gate keeper mutation, demonstrated correlation with disease state. Conclusions: Our novel detection systems for EGFR mutations could be useful not only at the beginning of treatment but also for monitoring using plasma DNA for deciding appropriate treatment, including rechallenge with EGFR-tyrosine kinase inhibitors.

Hsp90 inhibitors cause G2/M arrest associated with the reduction of Cdc25C and Cdc2 in lung cancer cell lines

Purpose: Hsp90, a molecular chaperone, is involved in folding, assembly, maturation, and stabilization of the client proteins which regulate survival of cancer cells, and thus Hsp90 inhibitors may be potential molecular targeting agents for cancer treatment. We investigated whether Hsp90 inhibitors have therapeutic value in lung cancer. Methods: First, expression levels of Hsp90 in lung cancer cells were examined by western blotting and immunohistochemical analyses. Next, the effect of Hsp90 inhibitors, geldanamycin and 17-allylaminogeldanamycin (17-AAG), on lung cancer cell growth was examined. Results: Remarkable high expression of Hsp90 protein in lung cancer cell lines and a more intense signal for Hsp90 by immunohistochemistry in males, patients with smoking index over 600, and squamous cell carcinoma were observed. Both Hsp90 inhibitors dose dependently inhibited the growth of lung cancer cell lines and induced G2/M arrest concomitant with decreased protein levels of Cdc25C and Cdc2. Moreover, combination of an Hsp90 inhibitor and irradiation had an additive effect on cell growth inhibition and reduction of Cdc25C and Cdc2 protein levels. Conclusion: Hsp90 inhibitor is thus a therapeutic tool for lung cancer based on its target proteins, which are involved in tumor progression and antiproliferative activity in lung cancer cells.

Detection and discrimination of preneoplastic and early stages of lung adenocarcinoma using hnRNP B1 combined with the cell cycle- related markers p16, cyclin D1, and Ki-67

Heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, is a useful marker for early detection of lung squamous cell carcinoma because it is overexpressed in the early stages of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. In the case of adenocarcinoma, we investigated the utility of hnRNP B1 for both detection of early adenocarcinoma and discrimination of non-invasive lesion, atypical adenomatous hyperplasia (AAH) from adenocarcinoma. hnRNP B1, cyclin D1, p16, and Ki-67 were analyzed in lung adenocarcinoma tissues and divided into early and overt adenocarcinoma and AAH, using immunohistochemistry. The intensity of these molecular markers was compared among three groups and also analyzed for 4 patients who showed both adenocarcinoma and AAH. Thirty-six of 54 (67%) adenocarcinoma patients showed positive staining of hnRNP B1: 14/20 (70%) early adenocarcinoma and 22/34 (65%) overt adenocarcinoma. In contrast, overexpression of hnRNP B1 in non-invasive lesion, AAH was observed in only 9% (1/11). Overexpression of cyclin D1 and decrease of p16 were frequently observed in both adenocarcinoma and AAH. These results suggest that hnRNP B1 would be a candidate of molecular marker for detection of early lung adenocarcinoma. In addition, combined analysis of hnRNP B1 and cell cycle-related genes, such as cyclin D1 and p16, might aid in discrimination of AAH from early adenocarcinoma.

Autoantibodies against heterogeneous nuclear ribonucleoprotein B1 in CSF of MS patients.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play an important role as the autoantigens in certain autoimmune disorders including neurological diseases such as HTLV‐1–associated myelopathy/tropical spastic paraparesis and paraneoplastic neurological syndromes. To clarify their implication in multiple sclerosis (MS), we assayed antibodies (Abs) against hnRNP A and B proteins in sera and cerebrospinal fluid (CSF) of MS patients and compared the results with 25 patients with other neurological diseases (ONDs). Using recombinant hnRNP A1, A2, and B1 proteins and Western blotting for the assay, we found Abs against hnRNP B1 in CSF from 32 of 35 MS patients (91.4%) but not in any sera or CSF of the 25 OND patients. Most notably, no Abs against hnRNP B1 were found in sera of all 22 MS patients examined. Although Abs against hnRNP A1 and A2 were concomitantly found in CSF reacting with B1, their incidence and immunoreactivity were lower or weaker than those of anti–hnRNP B1 Abs. There was no correlation between the reactivity of CSF with hnRNP B1 and CSF parameters—such as the number of the cells and the IgG level—or clinical parameters—such as duration of illness and disease activity. The selective generation of Abs against hnRNP B1 in CSF was shown to be highly specific for MS, which makes them a disease marker. Ann Neurol 2004

Monitoring EGFR T790M with plasma DNA from lung cancer patients in a prospective observational study.

Use of plasma DNA to detect mutations has spread widely as a form of liquid biopsy. EGFR T790M has been observed in half of lung cancer patients who have acquired resistance to EGFR tyrosine kinase inhibitors (EGFR‐TKI). Effectiveness of monitoring T790M via plasma DNA during treatment with EGFR‐TKI has not been established as an alternative to re‐biopsy. This was a prospective multicenter observational study involving non‐small cell lung cancer patients carrying EGFR L858R or exon 19 deletions, treated with EGFR‐TKI. The primary objective was to determine whether T790M could be detected using plasma DNA in patients with progressive disease (PD). T790M was examined using the mutation‐biased PCR and quenching probe (MBP‐QP) method, a sensitive, fully‐automated system developed in our laboratory. Eighty‐nine non‐small cell lung cancer patients were enrolled from seven hospitals in Japan. Sequential examinations revealed T790M in plasma DNA among 40% of patients who developed PD. Activating mutations, such as L858R and exon 19 deletions, were detected in 40% of patients using plasma DNA, and either T790M or activating mutations were observed in 62%. Dividing into four periods (before PD, at PD, at discontinuation of EGFR‐TKI and subsequently), T790M was detected in 10, 19, 24 and 27% of patients, respectively. Smokers, males, patients having exon 19 deletions and patients who developed new lesions evidenced significantly frequent presence of T790M in plasma DNA. Monitoring T790M with plasma DNA using MBP‐QP reflects the clinical course of lung cancer patients treated with EGFR‐TKI. Detection of T790M with plasma DNA was correlated with EGFR mutation type, exon 19 deletions and tumor progression. Re‐biopsy could be performed only in 14% of PD cases, suggesting difficulty in obtaining re‐biopsy specimens in practice. Monitoring T790M with plasma DNA reflects the clinical course, and is potentially useful in designing strategies for subsequent treatment.

A fully integrated, automated and rapid detection system for KRAS mutations.

KRAS mutations are detected in tumors of various organs, and they are also markers of resistance for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and monoclonal antibodies against the EGFR. Thus, the accurate and rapid detection of KRAS mutations is crucial, not only for screening, but also for the prediction of the efficacy of molecular-targeted therapy. The aim of the present study was to establish a novel automated detection system for KRAS mutations. One hundred and thirty-six lung adenocarcinoma patients were genotyped for KRAS mutations with both the conventional direct sequence (DS) method and with the newly developed quenching probe (QP) method that obtains data automatically within 60 min. The detection limit of the QP method using a control plasmid containing the KRAS mutation was 50 copies, and 10% mutant plasmid was detected in the mixture of wild-type and mutants. The results obtained by the QP and DS methods were identical in all but two of the 136 cases. The two differentially identified samples, which consisted of substantially fewer lung cancer cells, were positive according to the QP method but negative as determined by DS for KRAS mutations. These findings characterize the QP method as an accurate and rapid detection system for KRAS mutations.

Phase II trial of gefitinib plus pemetrexed after relapse using first-line gefitinib in patients with non-small cell lung cancer harboring EGFR gene mutations

OBJECTIVES Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (i.e., EGFR-TKIs) improve the survival of lung cancer patients harboring EGFR mutations. Despite the initial efficacy of EGFR-TKIs, the disease progression caused by acquired resistance to these inhibitors is inevitable. T790M mutations represent a major resistance mechanism to EGFR-TKIs but can be overcome using osimertinib. The IMPRESS trial revealed that the continuation of EGFR-TKI beyond progressive disease (PD) concurrent with platinum-doublet chemotherapy was not beneficial. However, various clinical trials have suggested that EGFR-TKI beyond PD plus single-agent chemotherapy may be a possible treatment strategy. MATERIALS AND METHODS This study was a single-arm phase II trial. Patients with EGFR-activating mutations (del19 and L858R) that progressed using first-line gefitinib treatment were enrolled and treated with gefitinib beyond PD plus pemetrexed 500 mg/m2 q3w. The primary endpoint was progression-free survival (PFS). Mutation-biased polymerase chain reaction quenching probe, which is the original method for detecting T790M mutations in cell-free plasma DNA, was used prior to treatment. RESULTS Thirty-six patients were enrolled between May 1, 2013, and March 31, 2016. One patient was excluded before starting the treatment. Among the 35 patients, 15 patients had del19 mutations, and 20 patients had L858R mutations; 33 patients were evaluable for response by using radiographic findings. The median PFS was 6.7 months (95% confidence interval: 4.4-7.7 months). Nineteen patients were T790M positive. No significant difference in PFS was found in a subgroup analysis of EGFR mutation status and T790M positivity. All toxicities were tolerable. CONCLUSION Gefitinib plus pemetrexed treatment following relapse using gefitinib in patients with Non-small cell lung cancer harboring EGFR mutations demonstrated preferable PFS with mild toxicity. This combination therapy may be considered for platinum-unfit patients without T790M with disease progression using first-line gefitinib. (This clinical trial was registered in UMIN-CTGR as UMIN000010709).

Abstract 4680: DNA dependent protein kinase as a new molecular target for treatment of non-small cell lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prognosis with non-small cell lung cancer (NSCLC) has improved with the recent introduction of molecular targeted therapy. However, it is limited to patients with epidermal growth factor receptor (EGFR) mutations, who benefit from EGFR tyrosine kinase inhibitors (EGFR-TKI). A new molecular target is needed with other NSCLC patients. In general, chemotherapeutic agents cause DNA double-strand breaks (DNA DSB), leading to apoptosis of cancer cells; DNA repair reverses their effect, resulting in resistance to chemotherapy. DNA DSB repair can be mediated via two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). More than 90% of DSBs in mammalian cells are repaired by NHEJ, which is accomplished by core protein components including Ku70/Ku86, DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, Artemis, and Cernunnos-XLF. We recently reported that NK314, a dual inhibitor of topoisomerase IIα (Top2β) and DNA-PK, evidences a potent anti-tumor effect in adult T-cell leukemia-lymphoma (ATL). NK314 induced DNA DSBs and inhibited DNA repair mediated through degradation of the DNA-PK catalytic subunit (DNA-PKcs). In the present study, we examined the effect of NK314 in NSCLC cell lines and assessed DNA-PK as a possible molecular target for NSCLC. Twenty separate NSCLC cell lines were used, with IC50 varying from 46-193 nM. Levels of DNA-PKcs protein varied among the cell lines, whereas those of Ku70 and Ku86 did not. The inhibitory effect of cell growth was closely related to level of DNA-PKcs protein, but not to levels of other DNA-damage-activated protein kinases such as ATM or ATR. Establishment of predictive markers concomitantly with the development of a novel molecular target is indispensable for clinical application. Because detection of DNA-PKcs in peripheral blood is difficult, we investigated an alternative marker for predicting DNA-PKcs inhibitor. We previously found that heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, interacts with DNA-PK, and we established a system for detecting hnRNP B1 mRNA in peripheral blood. We therefore examined the suitability of hnRNP B1 as an alternative biomarker for DNA-PK inhibitors. Close correlation between levels of DNA-PKcs and hnRNP B1 proteins was established using Western blot analysis with NSCLC cell lines (p=0.995). These results suggest that DNA-PK is a novel molecular target for NSCLC, and detection of hnRNP B1 might serve as an alternative molecular marker for the amount of DNA-PK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4680. doi:1538-7445.AM2012-4680

Abstract 1906: Mechanisms of resistance to EGFR tyrosine kinase inhibitors involving HER-family ligands and receptors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Known mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor (EGFR-TKI), including the second mutation of EGFR, T790M, MET amplification, and HGF overexpression, explain approximately 60% of cases, but the remaining mechanisms have not been clarified. To investigate novel mechanisms of acquired resistance to EGFR-TKI, we examined HER-family ligands and HER-family receptor status in non-small cell lung carcinoma (NSCLC) cell lines (n=13). Cell lines were divided into two groups with respect to EGFR-TKI sensitivity: highly sensitive (HCC827, PC-9, HCC2935, and HCC4006) and resistant (H1395, H1666, H596, H1437, A549, H226B, H1299, H460, H1792). We examined expression levels of eleven HER-family ligands\_EGF, TGFβ, amphiregulin (AREG), betacellulin (BTC), HB-EGF, epigen (EPGN), epiregulin (EREG), and neuregulin (NRG) 1, NRG2, NRG3, and NRG4\_in these cell lines. NRG2, NRG4, and EPGN were expressed at 39.5-, 31.4-, and 6.5-fold higher levels, respectively, in EGFR-TKI primary resistant NSCLC cell lines than in highly sensitive cell lines, whereas TGFA, EGF, and AREG were expressed at 0.22-, 0.3-, and 0.34-fold lower levels. Phosphorylation of EGFR was elevated in sensitive cell lines compared to resistant cell lines, but HER2-4 receptors did not evidence a difference in phsophorylation. These results suggest that the EGFR pathway is activated in sensitive cell lines, and HER2-4, which binds to EPGN, EREG, NRG2, and NRG4, might be activated in resistant cell lines. We prepared cell lines HCC827GR and HCC827ER, derived from HCC827, which acquired resistance to gefitinib or erlotinib, respectively. We analyzed HER-family ligands and receptors using these cell lines. Compared to parent cells, EPGN, EREG, HB-EGF, TGFA, NRG1, and AREG were up-regulated in HCC827GR. Phosphorylation status of EGFR, HER2, and HER3 was not altered, but that of HER4 was increased in HCC827GR. In HCC827ER, EGFR binding ligands AREG, BTC, and EGF were down-regulated, while phosphorylation of EGFR, HER2, and HRE3 was decreased. However, NRG2 was remarkably elevated and HER4 and its downstream STAT5 were phosphorylated. These results indicate that HER4 and its binding ligands may contribute to acquired resistance to EGFR-TKI. Considering these results, it is possible that the HER4-STAT5 pathway might be common to primary and acquired resistance to EGFR-TKI. We continue to investigate its significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1906. doi:1538-7445.AM2012-1906