Kazutoshi Komiya

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.

Mina53, a novel c-Myc target gene, is frequently expressed in lung cancers and exerts oncogenic property in NIH/3T3 cells.

PurposeMina53, whose expression is directly induced by c-Myc, is overexpressed in various cancers and plays an important role in cell growth. To clarify the involvement of Mina53 in lung cancers, we investigated its expression in human lung cancer tissues as well as in various lung cancer cell lines.MethodsMina53 expression was determined by real-time RT-PCR, western blotting, and immunohistochemistry using lung cancer cell lines, normal human bronchial epithelial cells, and lung cancer tissues. Biological effects of Mina53 were evaluated by soft agar colony formation assay and tumorigenicity in nude mice using Mina53-transfected NIH/3T3 cells. cDNA microarray analysis was performed to determine the gene alteration by Mina53 and confirmation was made using real-time RT-PCR with mina53 expression plasmid or mina53 shRNA-transfected NIH/3T3 cells.ResultsWe observed that 62% of patients evidenced overexpression of Mina53 from the early clinical stages of lung cancer. Differences according to gender, smoking status, or histologic type were not statistically significant. Forced expression of Mina53 in NIH/3T3 cells induced cell transformation, and mina53-transfected NIH/3T3 clones produced tumors in nude mice, demonstrating that Mina53 has oncogenic potential. cDNA microarray revealed that 254 genes had altered expression in a mina53-transfected NIH/3T3 clone. Mina53 regulates several genes related to cell adhesion and metabolism, which have also been reported to be regulated by c-Myc. Genes regulated by Mina53, but not by c-Myc included cytokine/growth factor related genes such as EGFR, IL-6, and HGF.ConclusionOur results suggest that Mina53 plays an important role in carcinogenesis and may be a target for cancer prevention.

Expression of Mina53, a novel c-Myc target gene, is a favorable prognostic marker in early stage lung cancer.

Mina53, a novel target gene product of c-Myc, is overexpressed in various malignancies. We previously demonstrated that Mina53 is overexpressed in lung cancer patients from the early clinical stages. In this paper, the association between disease prognosis and Mina53 expression in lung cancer patients is analyzed; we found that overexpression of Mina53 in lung cancer patients is associated with favorable prognosis. Statistical analysis using the Kaplan-Meier method showed that patients with negative staining for Mina53 had significantly shorter survival than patients with positive staining for Mina53, especially in stage I or with squamous cell carcinoma. Because the major cause of death in lung cancer patients after surgery is distant metastasis, the effect on cancer cell invasiveness was analyzed for the mechanisms involved in the association with favorable outcome. Overexpression of Mina53 in H226B, a lung squamous cell carcinoma cell line, inhibited cancer cell invasion. Transfection with mina53 shRNA increased the number of invading cells. These results suggest that Mina53 immunostaining is a useful prognostic marker--especially in the early stage of lung cancer--and that Mina53 negative patients should be managed particularly carefully after surgery.

Markers that can Reflect Asthmatic Activity before and after Reduction of Inhaled Corticosteroids: A Pilot Study

Evaluation of airway inflammation is important in achieving adequate dosing of inhaled corticosteroids (ICS) for treating bronchial asthma. However, there is no evaluation tool that can be used in clinical settings. We examined biomarkers that can precisely reflect airway inflammation when ICS are decreased in stable asthmatic patients. This was a 12-week, single-arm, open-label clinical study performed at a single university hospital. Twenty-five patients (6 male and 19 female) with stable asthma were included in this study. We investigated whether the levels of nitrite and nitrate in exhaled breath condensate (EBC) increase after ICS reduction. We also investigated whether blood eosinophils, serum immunoglobulin E (IgE), high-sensitivity C reactive protein (hs-CRP), interleukin (IL)-13, IL-17, and periostin are different before and after ICS reduction. Peak expiratory flow (PEF), pulmonary function tests, asthma control test (ACT), and asthma quality of life questionnaire (AQLQ) were also examined. We considered an unscheduled hospital visit due to asthmatic symptoms and decline in average PEF over one week by more than 10% to indicate disease instability, and compared patients with stable and unstable disease for analysis. Unstable status was detected in 5 patients. Age, sex, asthma duration, ACT and AQLQ scores, and the level of serum IgE did not differ between stable and unstable groups. In the unstable group, the total concentration of nitrite and nitrate at the last visit was 9.84 (6.65–11.24) μM. Surprisingly, this was similar to the concentration at the first visit (5.58 (2.94–17.29) μM). Serum periostin before ICS reduction (141.9 [107.7–147.7] pg/mL) was higher in the unstable group than in the stable group (91.5 [78.75–103.5] pg/mL). The unstable group had a higher peripheral blood eosinophil count and wider diurnal variation of PEF at the first visit compared to the stable group. Higher eosinophils in peripheral blood and wider diurnal variation of PEF were predictive markers for unstable disease after ICS reduction. Serum periostin is another candidate for the predictive marker.

Exon 19 of EGFR mutation in relation to the CA-repeat polymorphism in intron 1.

Epidermal growth factor receptor (EGFR) mutations in lung cancer enhance tyrosine kinase activity and increase sensitivity to the EGFR tyrosine kinase inhibitor, gefitinib. Mutation analysis of the EGFR gene is therefore indispensable for predicting gefitinib response. We investigated a CA‐repeat polymorphism in the EGFR gene related to EGFR mutations. Because an increasing number of CA‐repeats at intron 1 of the EGFR gene has been reported to reduce transcription activity, we examined the relationship between EGFR mutations and this CA‐repeat polymorphism. EGFR mutations at exon 19 were closely associated with shorter CA‐repeat length in the shorter allele, but this was not the case for EGFR mutations at exons 18 or 21. Increased intrinsic EGFR mRNA expression in non‐cancerous lung tissues from lung adenocarcinoma patients was also significantly associated with shorter CA‐repeat length. A higher frequency of EGFR mutations at exon 19 was associated with shorter CA‐repeat length only in patients with high levels of EGFR mRNA expression. To determine the phenotypes of cells possessing shorter CA‐repeats, an in vitro study using human bronchial epithelial cells with different CA‐repeat lengths was performed; more rapid cell growth and activated EGF/EGFR signaling were found more often in the cells having both shorter CA‐repeats and increased EGFR mRNA expression. These results suggest that CA‐repeat length in the EGFR gene may be a genetic factor related to cancer in the case of EGFR mutations at exon 19. The mechanism likely involves enhanced intrinsic expression of EGFR mRNA and activated EGF/EGFR signaling that accompany shorter CA‐repeats. (Cancer Sci 2008; 99: 1180–1187)

SPARC is a possible predictive marker for albumin-bound paclitaxel in non-small-cell lung cancer

Objectives Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) produced good tumor response in cases with lung squamous cell carcinoma, one of the most difficult cancers to treat. Secreted protein acidic and rich in cysteine (SPARC) binds to albumin, suggesting that SPARC plays an important role in tumor uptake of nab-paclitaxel. There is as yet no predictive marker for cytotoxic agents against non-small-cell lung cancer (NSCLC), and hence we believed that SPARC expression might be associated with tumor response to nab-paclitaxel. Patients and methods We studied stromal SPARC reactivity and its association with clinicopathological characteristics in 200 cases of NSCLC using a custom tissue microarray fabricated in our laboratory by immunohistochemical staining. We also investigated the relationship between stromal SPARC reactivity and tumor response to nab-paclitaxel using biopsy or surgical specimens obtained from advanced or recurrent lung cancer patients. Results High SPARC stromal reactivity (>50% of optical fields examined) was detected in 16.5% of cases and intermediate SPARC reactivity (10%–50%) in 56% of cases. High expression in cancer cells was rare (five cases). Stromal SPARC level was correlated with smoking index, squamous cell carcinoma, and vessel invasion. Furthermore, patients with high stromal SPARC reactivity in biopsy specimens such as transbronchial lung biopsy or surgical specimens tended to respond better to nab-paclitaxel. Conclusion Stromal SPARC was detected by immunohistochemical staining in ∼70% of NSCLC cases, and good tumor response to nab-paclitaxel was correlated with high stromal SPARC reactivity. SPARC may be a useful predictive marker for selecting patients likely to respond favorably to nab-paclitaxel treatment.

Phase II trial of weekly nab-paclitaxel for previously treated advanced non-small cell lung cancer: Kumamoto thoracic oncology study group (KTOSG) trial 1301.

OBJECTIVES We performed an open-label, multicenter, single-arm phase II study (UMIN ID 000010532) to prospectively evaluate the efficacy and safety of nab-paclitaxel for previously treated patients with advanced non-small cell lung cancer (NSCLC). METHODS Patients with advanced NSCLC who experienced failure of prior platinum-doublet chemotherapy received weekly nab-paclitaxel (100mg/m(2)) on days 1, 8, and 15 of a 21-day cycle until disease progression or the development of unacceptable toxicity. The primary end point of the study was objective response rate (ORR). RESULTS Forty-one patients were enrolled between September 2013 and April 2015. The ORR was 31.7% (90% confidence interval, 19.3%-44.1%), which met the primary objective of the study. Median progression-free survival was 4.9 months (95% confidence interval, 2.4-7.4 months) and median overall survival was 13.0 (95% confidence interval, 8.0-18.0 months) months. The median number of treatment cycles was four (range, 1-17) over the entire study period, and the median dose intensity was 89.1mg/m(2) per week. Hematologic toxicities of grade 3 or 4 included neutropenia (19.5%) and leukopenia (17.1%), with no cases of febrile neutropenia being observed. Individual nonhematologic toxicities of grade 3 or higher occurred with a frequency of <5%. CONCLUSION Weekly nab-paclitaxel was associated with acceptable toxicity and a favorable ORR in previously treated patients with advanced NSCLC. Our results justify the undertaking of a phase III trial comparing nab-paclitaxel with docetaxel in this patient population.

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.

Severe eosinophilic pneumonia presenting during gemcitabine adjuvant chemotherapy

Gemcitabine is widely accepted as the standard treatment for pancreatic cancer, but it can cause unpredictable side effects. Acute respiratory distress syndrome is a rare complication with gemcitabine, but is sometimes fatal. We describe a cured case of acute, severe gemcitabine-induced pulmonary toxicity. The patient was a 76-year-old man with pancreatic cancer who was receiving adjuvant gemcitabine chemotherapy after surgery. The patient received gemcitabine 1,000 mg/m2 on days 1, 8, and 15 for three 4-week cycles, with intervals of 1 week. He developed severe general fatigue on day 1 of the third cycle. Computed tomography showed diffuse ground-glass opacity with pleural effusion. There was no increase in β-D-glucan, and cytomegalovirus antigenemia assays were negative. No bacteria or acid-fast bacilli were found. The number of eosinophils in bronchoalveolar lavage fluid was increased. Considering these data, we diagnosed eosinophilic pneumonia induced by gemcitabine. The patient was immediately treated with a steroid and neutrophil elastase inhibitor under respiratory supportive therapy. After 4 weeks, his pulmonary symptoms were markedly improved. Physicians should be cognizant of the possible association of serious pulmonary toxicity with gemcitabine treatment. A delay in diagnosis and treatment could lead to a fatal outcome.