Keita Mori

Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System

Background Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method. Methods Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer’s protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems. Results CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0–291 cells/7.5 mL) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system. Conclusions The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.

Assessment of mutational profile of Japanese lung adenocarcinoma patients by multitarget assays: A prospective, single‐institute study

Integration of mutational profiling to identify driver genetic alterations in a clinical setting is necessary to facilitate personalized lung cancer medicine. A tumor genotyping panel was developed and the Shizuoka Lung Cancer Mutation Study was initiated as a prospective tumor genotyping study. This study reports the frequency of driver genetic alterations in Japanese lung adenocarcinoma patients, and clinicopathologic correlations with each genotype.

MicroRNA-26a inhibits TGF-β-induced extracellular matrix protein expression in podocytes by targeting CTGF and is downregulated in diabetic nephropathy

Aims/hypothesisThe accumulation of extracellular matrix (ECM) is a characteristic of diabetic nephropathy, and is partially caused by profibrotic proteins TGF-β and connective tissue growth factor (CTGF). We aimed to identify microRNAs (miRNAs) targeting CTGF on podocytes in diabetic nephropathy.MethodsWe investigated miRNAs targeting CTGF on podocytes with miRNA array analysis and identified a candidate miRNA, miR-26a. Using overexpression and silencing of miR-26a in cultured podocytes, we examined changes of ECM and its host genes. We further investigated glomerular miR-26a expression in humans and in mouse models of diabetic nephropathy.ResultsmiR-26a, which was downregulated by TGF-β1, was expressed in glomerular cells including podocytes and in tubules by in situ hybridisation. Glomerular miR-26a expression was downregulated by 70% in streptozotocin-induced diabetic mice. Transfection of miR-26a mimics in cultured human podocytes decreased the CTGF protein level by 50%, and directly inhibited CTGF expression in podocytes, as demonstrated by a reporter assay with the 3′-untranslated region of the CTGF gene. This effect was abolished by a mutant plasmid. miR-26a mimics also inhibited TGF-β1-induced collagen expression, SMAD-binding activity and expression of its host genes CTDSP2 and CTDSPL. Knockdown of CTDSP2 and CTDSPL increased collagen expression in TGF-β-stimulated podocytes, suggesting that host genes also regulate TGF-β/SMAD signalling. Finally, we observed a positive correlation between microdissected glomerular miR-26a expression levels and estimated GFR in patients with diabetic nephropathy.Conclusions/interpretationThe downregulation of miR-26a is involved in the progression of diabetic nephropathy both in humans and in mice through enhanced TGF-β/CTGF signalling.

Pleiotrophin triggers inflammation and increased peritoneal permeability leading to peritoneal fibrosis

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.

Prognostic impact of serum CYFRA 21–1 in patients with advanced lung adenocarcinoma: a retrospective study

BackgroundSerum CYFRA 21–1 is one of the most important serum markers in the diagnosis of non-small cell lung cancer (NSCLC), especially squamous-cell carcinoma. However, it remains unknown whether pretreatment serum CYFRA 21–1 values (PCV) may also have prognostic implications in patients with advanced lung adenocarcinoma.MethodsWe retrospectively reviewed the data of 284 patients (pts) who were diagnosed as having advanced lung adenocarcinoma and had received initial therapy.ResultsOf the study subjects, 121 pts (43%) had activating epidermal growth factor receptor (EGFR) mutations (Mt+), while the remaining 163 pts (57%) had wild-type EGFR (Mt-). Univariate analysis identified gender (male/ female), ECOG performance status (PS) (0-1/ ≥2), PCV (<2.2 ng/ml/ ≥2.2 ng/ml), EGFR mutation status (Mt+/ Mt-), pretreatment serum CEA values (<5.0 ng/ml/ ≥5.0 ng/ml), smoking history (yes/ no) and EGFR-TKI treatment (yes/ no) as prognostic factors (p = .008, p < .0001, p < .0001, p < .0001, p = .036, p = .0012, p < .0001 respectively). Coxs multivariate regression analysis identified PCV < 2.2ng/ml as the only factor significantly associated with prolonged survival (p < .0001, hazard ratio: 0.43, 95% CI 0.31-0.59), after adjustments for PS (p < .0001), EGFR mutation status (p = .0069), date of start of initial therapy (p = .07), gender (p = .75), serum CEA level (p = .63), smoking history (p = .39) and EGFR-TKI treatment (p = .20). Furthermore, pts with Mt+ and PCV of <2.2 ng/ml had a more favorable prognosis than those with Mt+ and PCV of ≥2.2 ng/ml (MST: 67.0 vs. 21.0 months, p < .0001), and patients with Mt- and PCV of <2.2 ng/ml had a more favorable prognosis than those with Mt- and PCV of ≥2.2 ng/ml (MST: 24.1 vs. 10.2 months, p < .0001).ConclusionPCV may be a potential independent prognostic factor in both Mt+ and Mt- patients with advanced lung adenocarcinoma.

Rebiopsy for patients with non‐small‐cell lung cancer after epidermal growth factor receptor‐tyrosine kinase inhibitor failure

Although third‐generation epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI) can overcome T790M‐mediated resistance in non‐small‐cell lung cancer (NSCLC), rebiopsy to confirm T790M status is occasionally difficult. We aimed to investigate the current tendency and the limitations of rebiopsy in clinical practice. This study included 139 consecutive NSCLC patients with EGFR mutations, who had experienced progressive disease (PD) after EGFR‐TKI treatment. We retrospectively reviewed patient characteristics, tumor progression sites and rebiopsy procedures. Of 120 patients (out of the original 139) who were eligible for clinical trials, 75 (63%) underwent rebiopsy for 30 pleural effusions, 32 thoracic lesions, four bone, two liver, and seven at other sites. Rebiopsy procedures included 30 thoracocentesis, 24 transbronchial biopsies, 13 computed tomography (CT)‐guided needle biopsies and 8 other procedures. Of the 75 rebiopsied patients, 71 (95%) were pathologically diagnosed with malignancy; and 34 (45%) had available tissue samples for EGFR analyses. Of the 75 biopsied patients, 61 (81%) were analyzed for EGFR mutation, using tissue or cytology samples; T790M mutations were identified in 20 (33%) of the 61 patients. Of the 120 patients, 45 (38%) did not undergo rebiopsy, because of inaccessible tumor sites (n = 19), patient refusal (n = 6) or decision of physician (n = 10). In conclusion, among patients with EGFR mutations who had PD after EGFR‐TKI treatment, 63% underwent rebiopsy. Most rebiopsy samples were diagnosed with malignancy. However, tissue samples were less available and T790M mutations were identified less frequently than in previous studies. Skill and experience with rebiopsy and noninvasive alternative methods will be increasingly important.

Regorafenib Versus Trifluridine/Tipiracil for Refractory Metastatic Colorectal Cancer: A Retrospective Comparison

Micro‐Abstract It is unclear which drug should be administered first for refractory metastatic colorectal cancer, regorafenib or trifluridine/tipiracil (TAS‐102). We retrospectively evaluated 200 patients who had received regorafenib or TAS‐102 at 2 institutions to compare these 2 drugs in terms of efficacy and safety. Our results suggest that regorafenib and TAS‐102 have similar efficacy but different toxicities, which could guide the agent choice. Background: Regorafenib and trifluridine/tipiracil (TAS‐102) both prolong survival for patients with refractory metastatic colorectal cancer. However, it is unclear which drug should be administered first. Materials and Methods: We retrospectively evaluated the data from patients who had received regorafenib or TAS‐102 at 2 institutions from May 2013 to March 2015. The inclusion criteria were disease refractory or intolerant to fluoropyrimidines, oxaliplatin, irinotecan, anti‐vascular endothelial growth factor antibodies, and anti‐epidermal growth factor receptor (EGFR) antibodies (if KRAS exon 2 wild‐type), and no previous treatment with regorafenib or TAS‐102. Results: A total of 146 and 54 patients received regorafenib and TAS‐102, respectively. The baseline characteristics were similar between the 2 groups, except for a history of irinotecan and anti‐EGFR therapy and high alkaline phosphatase levels. The median progression‐free survival and overall survival were 2.1 months and 6.7 months, respectively, with regorafenib and 2.1 months and 6.5 months, respectively, with TAS‐102 (progression‐free survival hazard ratio 1.20, P = .27; overall survival hazard ratio, 1.01, P = .97). The analysis of overall survival for patients after the approval of TAS‐102 in Japan was similar to the overall survival for the entire population. The frequency of hand–foot syndrome and increased aspartate aminotransferase, alanine aminotransferase, and bilirubin levels was higher and the frequency of neutropenia, leukopenia, anemia, nausea, and febrile neutropenia was lower with regorafenib than with TAS‐102. No remarkable differences were found in the efficacy and safety of TAS‐102 between patients with and without previous regorafenib and vice versa. Conclusion: Regorafenib and TAS‐102 had similar efficacy but resulted in different toxicities, which could guide the agent choice.

Progression-free survival, post-progression survival, and tumor response as surrogate markers for overall survival in patients with extensive small cell lung cancer

Objectives: The effects of first-line chemotherapy on overall survival (OS) might be confounded by subsequent therapies in patients with small cell lung cancer (SCLC). We examined whether progression-free survival (PFS), post-progression survival (PPS), and tumor response could be valid surrogate endpoints for OS after first-line chemotherapies for patients with extensive SCLC using individual-level data. Methods: Between September 2002 and November 2012, we analyzed 49 cases of patients with extensive SCLC who were treated with cisplatin and irinotecan as first-line chemotherapy. The relationships of PFS, PPS, and tumor response with OS were analyzed at the individual level. Results: Spearman rank correlation analysis and linear regression analysis showed that PPS was strongly correlated with OS (r = 0.97, p < 0.05, R 2 = 0.94), PFS was moderately correlated with OS (r = 0.58, p < 0.05, R 2 = 0.24), and tumor shrinkage was weakly correlated with OS (r = 0.37, p < 0.05, R 2 = 0.13). The best response to second-line treatment, and the number of regimens employed after progression beyond first-line chemotherapy were both significantly associated with PPS ( p ≤ 0.05). Conclusion: PPS is a potential surrogate for OS in patients with extensive SCLC. Our findings also suggest that subsequent treatment after disease progression following first-line chemotherapy may greatly influence OS.

Mutant allele frequency predicts the efficacy of EGFR-TKIs in lung adenocarcinoma harboring the L858R mutation

BACKGROUND Whether the mutant allele frequency (MAF) may also have predictive implications for tyrosine kinase inhibitor (TKI) therapy in patients with advanced epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (AELAd) remains unknown. PATIENTS AND METHODS Based on a biobanking system in conjunction with our institution, we assessed EGFR mutation status using pyrosequencing (Py) and by outsourcing laboratory tests, such as the Cycleave (Cy) and the Scorpion ARMS (A). RESULTS Out of 705 patients enrolled in the Shizuoka Lung Cancer Mutation Study between July 2011 and March 2013, 102 AELAd patients were identified as carrying the L858R mutation (L858Rm) using Py to analyze histological specimens. Of these 102 patients, the EGFR mutation status was assessed using both Py and Cy in 48 patients: the median MAF of L858R (MAFLR) was 18.5% (range: 8%-82%), and 45 patients (94%) were identified as having an L858Rm using both Py and Cy. Three patients (6%) with discrepant L858Rm findings were only identified using Py. The plotting of a receiver operating characteristic curve to identify the discordance in L858Rm findings showed that the area under the curve for MAFLR was 0.967 (95% confidence interval: 0.91-1) and that an MAFLR of 9% resulted in high sensitivity (100%) and specificity (99%). Also, 29 patients with AELAd, excluding those with postoperative recurrences, had their L858R status assessed using Cy or A. The median age, 69 years (range: 47-84 years); male/female, 14 (48%)/15 (52%); smokers/never-smokers 13 (45%)/16 (55%); ECOG PS 0-1/2-3, 26 (90%)/3 (10%); stage IIIB/IV, 4 (14%)/25 (86%); median MAFLR, 18% (range: 8%-63%). Patients with an MAFLR of ≤9% had a significantly shorter progression-free survival (PFS) period after TKI therapy than those with an MAFLR of >9% (mPFS: 92 versus 284 days, P = 0.0027). CONCLUSION The MAF may be a potential predictive factor of TKI treatment efficacy in patients with AELAd carrying the L858Rm.

Interstitial Lung Disease Associated with Gefitinib in Japanese Patients with EGFR-mutated Non-small-cell Lung Cancer: Combined Analysis of Two Phase III Trials (NEJ 002 and WJTOG 3405)

OBJECTIVE Interstitial lung disease associated with gefitinib is a critical adverse reaction. When geftinib was administered to EGFR-unknown patients, the interstitial lung disease incidence rate was approximately 3-4% in Japan, and usually occurs during the first 4 weeks of treatment. However, it has not been fully investigated in EGFR-mutated patients. METHODS We collected clinical records of participants of two Phase III trials (WJTOG 3405 and NEJ 002), which compared gefitinib with platinum doublet chemotherapy. All patients were EGFR mutated, chemo-naïve and had good performance status. RESULTS A total of 402 patients were enrolled in this study. In the gefitinib arm, 10 (5.0%) of 201 patients developed interstitial lung disease, of whom five (2.5%) were Grade 3 or greater, with two deaths (1.0%). In contrast, only one patient developed interstitial lung disease (Grade 1) in the chemotherapy arm. With regard to gefitinib, smoking history was significantly associated with developing interstitial lung disease (odds ratio 0.18; 95% confidence interval: 0.05-0.74; P = 0.01). The cumulative incidence rate of interstitial lung disease was similar in the 0-4, 5-8 and 9-12 week time periods. However, between smokers and never-smokers, cumulative incidence rates in the first 4 weeks were significantly different (4.7% versus 0%, P = 0.03). Three of 10 patients developed interstitial lung disease after 8 weeks of gefitinib administration (days 135, 171 and 190, respectively). CONCLUSIONS Among EGFR-mutated patients, the incidence of interstitial lung disease associated with gefitinib was not different from that in previous reports. Smoking history was associated with developing interstitial lung disease, and smokers had a higher incidence rate of interstitial lung disease in the first 4 weeks.