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Noninvasive diagnostics using circulating tumor cells (CTCs) are expected to be useful for decision making in precision cancer therapy. AXL, a receptor tyrosine kinase is associated with tumor progression, epithelial‐to‐mesenchymal transition (EMT), and drug resistance, and is a potential therapeutic target. However, the epithelial markers generally used for CTC detection may be not enough to detect AXL‐expressing CTCs due to EMT. Here, we evaluated the detection of AXL‐expressing CTCs using the mesenchymal marker vimentin with a microcavity array system. To evaluate the recovery of cancer cells, spike‐in experiments were performed using cell lines with varying cytokeratin (CK) or vimentin (VM) expression levels. With high CK and low VM‐expressing cell lines, PC‐9 and HCC827, the recovery rate of AXL‐expressing cancer cells was 1%‐17% using either CK or VM as markers. Whereas, with low CK and high VM‐expressing cell lines, MDA‐MB231 and H1299, it was 52%‐75% using CK and 72%‐88% using VM as a marker. For clinical evaluation, peripheral blood was collected from 20 non–small cell lung cancer patients and CTCs were detected using CK or VM as markers in parallel. Significantly more AXL‐expressing single CTCs were detected in VM‐positive than CK‐positive CTCs (P, We established the detection of AXL‐expressing in circulating tumor cells of lung cancer patients incorporating vimentin as a CTC marker in addition to cytokeratin. Significantly more AXL‐expressing CTCs were detected in patients who had prior treatment history than those did not, suggesting the involvement of AXL in drug resistance.

Purpose: Malignant pleural mesothelioma (MPM) is a rare and aggressive malignancy with poor prognosis. Patients with MPM who do not respond to standard first-line chemotherapy have limited treatment options. We evaluated the efficacy and safety of nivolumab, an immune checkpoint inhibitor, for the treatment of advanced or metastatic MPM. Patients and Methods: Japanese patients with unresectable, advanced, or metastatic MPM resistant or intolerant to ≤2 regimens of chemotherapy and ≥1 measurable lesion(s) were enrolled. Patients received nivolumab 240 mg intravenously every 2 weeks until progressive disease or unacceptable toxicity. The primary endpoint was objective response rate by central assessment according to the Modified Response Evaluation Criteria in Solid Tumors. Adverse events (AEs) and treatment-related AEs (TRAEs) were evaluated. Results: Thirty-four patients were enrolled between July 2016 and October 2016. Median follow-up was 16.8 (range: 1.8–20.2) months. Ten (29%, 95% confidence interval, 16.8–46.2) patients showed a centrally assessed objective response. The objective response rates were 26% (7/27), 67% (2/3), and 25% (1/4) patients for epithelioid, sarcomatoid, and biphasic histologic subtypes, respectively. Median duration of response was 11.1 months with a 68% disease control rate. Median overall survival and progression-free survival were 17.3 and 6.1 months, respectively. The objective response rate was 40% with programmed death-ligand 1 expression ≥1% and 8% with Conclusions: Nivolumab met the primary endpoint as second- or third-line treatment for patients with MPM and showed promising efficacy with manageable toxicity. See related commentary by Mansfield and Zauderer, p. 5438

Although programmed cell death-ligand-1 (PD-L1) expression in tumor tissue has been established as predictive biomarker for the anti-programmed cell death-1 (PD-1) antibody treatment of non-small-cell lung cancer (NSCLC), additional biomarkers are critically needed. We evaluated serum proteins relevant to immune checkpoint blockade in patients with NSCLC treated with nivolumab to identify novel non-invasive predictive biomarkers.Patients with advanced NSCLC, who had failed at least one prior chemotherapy regimen, received nivolumab monotherapy (3 mg/kg, Q2W) until progressive disease (PD) or unacceptable toxicity was observed. Blood samples were collected at baseline and week 4. Fifty-seven serum protein levels were quantified with a Milliplex MAP assay. The associations of both clinical benefit (CB) and the onset of immune related adverse events (irAEs) with serum proteins levels were evaluated.Thirty-eight patients with advanced NSCLC were enrolled in the study, with 38 and 32 paired serum samples at baseline and week 4 being available for efficacy analysis and irAE analysis, respectively. In durable CB (DCB) patients compared with non-DCB patients, the baseline serum levels of BMP-9 were significantly higher, whereas the follistatin, IL-8, IP-10, and TNF-α levels were significantly lower. In irAE patients compared with non-irAE patients the serum levels of G-CSF and RANTES at week 4 were significantly higher, whereas the levels of leptin were significantly lower. A multivariate analysis revealed that follistatin and IP-10 were statistically associated with DCB (p 0.05) and RANTES was associated with irAE onset (p 0.05). In a subset of irAE-developed patients, RANTES levels decreased after steroid administration, supporting its involvement in irAE.Serum proteins have the potential to be predictive markers for DCB and irAEs onset in patients with NSCLC treated with nivolumab. In addition, antitumor activity and irAEs may not be regulated by the same mechanisms.

Patients treated with nivolumab often experience its unique adverse events, called immune-related adverse events (irAEs). Regarding the mechanisms of immune-checkpoint inhibitors (ICIs), the occurrence of irAEs may also reflect antitumor responses. Here, we report the clinical correlation between irAEs and efficacy in NSCLC patients treated with nivolumab.Between December 2015 and February 2017, 38 advanced NSCLC patients were treated in our institution. All the patients were enrolled in our single-institutional, prospective, observational cohort study (UMIN000024414). IrAEs were defined as having a potential immunological basis that required more frequent monitoring and potential intervention. We divided the patients into two groups (irAEs group or no-irAEs group) and evaluated the objective response rate (ORR) and progression-free survival (PFS).The median age of the patients was 68.5 years (range 49-86 years); male/female ratio was 28/10; squamous/non-squamous cell carcinoma cases were 10/28; performance status was 0-1/2/3, 7/26/5. Among the overall population, ORR was 23.7% and median PFS was 91days. At the data cutoff, 14 irAEs were observed. The most common irAE was interstitial pneumonia (n=5). Other irAEs were hypothyroidism (n=4), hyperthyroidism, hypopituitarism, liver dysfunction, rash, and elevated thyroid stimulating hormone levels (n=1, each). Patients with irAEs had significantly higher ORRs compared with no-irAE patients (63.6% versus 7.4%, p0.01). Similarly, the PFS among irAE patients was longer (median: not reached [95% confidence interval {CI}: 91days to not applicable]) than no-irAE patients (median 49days [95% CI: 36-127days], hazard ratio [HR] 0.10 [95% CI: 0.02-0.37, p0.001]). Landmark analysis of patients who achieved PFS ≥60days demonstrated similar tendencies, but this was not significant (HR 0.28 [95% CI: 0.04-1.46], p=0.13).There was a correlation between irAE and efficacy in NSCLC patients treated with nivolumab.

Background Blockade of the programmed death receptor-1 (PD-1) pathway is effective against solid tumors including lung cancer. PD-ligand 1 (PD-L1) expression on tumor tissue serves as a predictive biomarker for the efficacy of PD-1 pathway blockade. Here, we evaluated the expression of PD-L1 on circulating tumor cells (CTCs) in patients with lung cancer. Materials and Methods Peripheral whole blood (3 mL) was collected from patients, and CTCs and PD-L1 expression were detected using a microcavity array (MCA) system. Immunohistochemistry for PD-L1 detection was also performed using matched tumor tissues. Results Sixty-seven patients with lung cancer were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University Hospital. The characteristics of the patients were as follows: median age, 71 years (range, 39-86 years); male, 72%; stage II to III/IV, 14%/85%; non–small-cell lung cancer/small-cell lung cancer/other, 73%/21%/6%. CTCs were detected in 66 of 67 patients (median, 19; range, 0-115), and more than 5 CTCs were detected in 78% of patients. PD-L1-expressing CTCs were detected in 73% of patients, and the proportion score of PD-L1-expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Tumor tissues were available from 27 patients and were immunostained for PD-L1, and no correlation was observed between tumor tissues and CTCs based on the proportion score (R2 = 0.0103). Conclusion PD-L1 expression was detectable on CTCs in patients with lung cancer, and intra-patient heterogeneity was observed. No correlation was observed between PD-L1 expression in tumor tissues and CTCs.

Background: Though PD-ligand 1 (PD-L1) expression on tumor tissue has been established as companion diagnostics in non-small-cell lung cancer (NSCLC) for anti-PD-1 treatment, additional biomarkers are critically needed. While immune related adverse events (irAEs) have been reported to be associated with efficacy, there are no established biomarkers to predict irAEs onset. Here, we measured multiple serum proteins in NSCLC patients treated with immune checkpoint inhibitors (ICIs) and explored the potential of predicting clinical response and irAE onset. Patients and Methods: Advanced NSCLC patients received nivolumab, pembrolizumab or atezolizumab until progressive disease (PD) or unacceptable toxicity. Serum samples were collected at baseline and after the treatment (at week 4 or week 6). Durable clinical benefit (DCB) was defined as patients whose response was lasting over 6 months. Using Luminex technology, serum levels of 41 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were measured. All statistical analyses were carried out using JMP Pro software (ver. 14.0). Cut-off value of serum protein levels were estimated with receiver operating characteristic curve. Results: Ninety-eight patients were registered in the study between January 2016 and May 2019 at Wakayama Medical University Hospital and 97 were included in the final analysis. Demographics of the patients were as follows: median age 70 (range, 49 to 91); male 78%; stage III/IV 31/69%; squamous/non-squamous/unknown 29/69/2%; previous treatment: 0/≥1 22/78%; ICI: nivolumab/pembrolizumab/atezolizumab 46/41/13%. Objective response rate was 24.7% (24/97), and disease control rate was 46.4% (45/97). Among 41 serum proteins measured serially, IL-8 levels at baseline were significantly lower in DCB patients than non-DCB patients in entire cohort (p < 0.05). Among those, Kaplan-Meier analysis revealed lower levels of IL-8 were significantly associated with longer PFS (cut-off, 11.9217 pg/ml; median PFS, 293 vs 59 days; p < 0.01). In second or later line subset, GRO-α, IL-8, IP-10, PLGF and VEGF-D were significantly lower in DCB patients than non-DCB-patients at baseline (p < 0.05). Lower levels of those proteins except for VEGF-D were significantly associated with longer PFS ((cut-off, 1.073 ng/ml, 11.9217 pg/ml, 480.8 pg/ml, and 14.83 pg/ml; median PFS, 183 vs 49, 293 vs 49, 133 vs 42 , and 153 vs 49 days; 95% CI, 56-322 days, 69 days-not reached, 58-211 days and 52-293 days; p < 0.05, 0.01, 0.05, 0.01). Follistatin which we previously reported to be associated with DCB upon nivolumab treatment, also showed significant association with longer PFS both in entire cohort (cut-off, 572.5; median PFS, 160 vs 59 days; 95% CI, 68-322 days, p < 0.05) and 2nd or later line subset (cut-off, 572.5; median PFS, 100 vs 49 days; 95% CI, 39-322 days, p < 0.05), though it was not significantly different between DCB and non-DCB. Regarding irAEs, GRO-α levels were significantly increased between baseline and the second sampling point in patients who acquired irAEs both in entire cohort and 2nd or later line subset (p < 0.01). Conclusions: We identified potential serum protein markers associated with clinical benefit and irAEs in advanced NSCLC treated with ICIs by multiplex protein-based assay. Citation Format: Jun Oyanagi, Yasuhiro Koh, Hiroaki Akamatsu, Koichi Sato, Shunsuke Teraoka, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Yuichi Ozawa, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Detection of serum protein levels for predicting clinical benefit in advanced non-small-cell lung cancer patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A052. doi:10.1158/1535-7163.TARG-19-A052

Purpose: Circulating tumor cells (CTCs) have a potential for the noninvasive decision-making. AXL, a receptor tyrosine kinase is associated with promoting cell proliferation, migration, epithelial-to-mesenchymal transition and drug resistance. We previously reported that the establishment of the detection method of AXL-expressing CTCs by an automated microcavity array (MCA) system incorporating mesenchymal marker vimentin (VM). Here we analyzed the clinicopathological correlation with VM-positive AXL-expressing CTCs in advanced non-small-cell lung cancer (NSCLC) patients. Methods: For detecting CTCs, 6 mL of peripheral blood collected from patients was divided into two portions and used for CTC identification in parallel incorporating markers cytokeratin (CK) and VM, respectively. An automated MCA system was used for enrichment and staining for CD45, DAPI, CK or VM with the additional staining for AXL. DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. All statistical analyses were carried out using GraphPad Prism 6 and Mann-Whitney U test, Fisher’s exact test, and rog-rank test were performed accordingly. A p value Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Koichi Sato, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Clinical significance of vimentin-positive AXL-expressing circulating tumor cells in advanced non-small-cell lung cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A032. doi:10.1158/1535-7163.TARG-19-A032

Background: We have previously reported that serum IL-8 and G-CSF were correlated with clinical benefit and immune-related adverse events (irAE), respectively in non-small-cell lung cancer (NSCLC) patients treated with nivolumab. Here, we measured multiple serum proteins serially in NSCLC patients treated with pembrolizumab and explored the potential of predicting clinical response or irAE onset. This study was registered at UMIN (ID: 000024414). Patients and Methods: Advanced NSCLC patients received pembrolizumab monotherapy (200 mg/body, q3W) until progressive disease (PD) or unacceptable toxicity. Serum samples were collected at baseline and at week 6. Best response was classified into partial response (PR), stable disease (SD), or progressive disease (PD) according to RECIST v1.1. Using LuminexTM xMapTM technology, serum levels of 41 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were measured. All statistical analyses were carried out using JMP Pro software (ver. 13.0) and Mann-Whitney U test were performed accordingly. A p value Results: Thirty-seven patients were registered in the study between March 2017 and October 2018 at Wakayama Medical University Hospital and 32 were included in the final analysis. Demographics of the patients were as follows: median age 70 (range, 50 to 91); male 75%; smoker 88%; stage III/IV, 22/78%; squamous/non-squamous, 31/69%, previous treatment; 0/1≤, 47/53%. Objective response rate was 28% and disease control rate was 56% in the entire cohort and 40% and 67%, respectively in the first-line subset. Among 41 serum proteins measured, no serum protein at baseline was associated with efficacy or irAE onset in the entire cohort. Levels of serum VEGF-C and sCD40L at baseline, however, in the first-line subset were found significantly lower in the patient who had PR and SD than those who did not. RANTES was also found significantly lower in patients who had PR in the 2nd or later line subset. With regard to irAE prediction, changes of HB-EGF levels between baseline and week 6 were significantly smaller in irAE patients in the entire cohort and changes of MCP-1 levels were significantly smaller in irAE patients in second or later line subset. The serum proteins identified in the current study were not overlapped with those identified in advanced NSCLC patients treated with nivolumab in the previous study. Conclusions: We identified potential serum protein markers associated with clinical benefit and irAE from pembrolizumab treatment in advanced NSCLC by multi-analyte protein-based assay. Our results suggest that serum proteins associated with efficacy and irAE may vary among different anti-PD-1 antibodies and also between in the first-line setting and later one. Citation Format: Jun Oyanagi, Yasuhiro Koh, Shunsuke Teraoka, Kuninobu Kanai, Atsuhsi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Predictive significance of serum protein levels in advanced non-small-cell lung cancer patients treated with pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 416.

Purpose: Noninvasive diagnostics has been developed over the last decade and we have previously reported that CTCs can be utilized for evaluating molecular features of NSCLC. AXL, a receptor tyrosine kinase is linked to epithelial-to-mesenchymal transition (EMT) leading to cancer progression and regarded as a potential therapeutic target. However, many of current technologies rely on epithelial markers to detect CTCs and that makes it difficult to detect AXL-expressing CTCs. Here, we established the detection of AXL expression on CTCs using MCA system. Methods: Preclinical experiments were performed using NSCLC cell lines H1299, PC9, and HCC827 and a breast cancer cell line MDA-MB231 with varying cytokeratin (CK) and AXL expression levels. The cells were spiked into 3 ml of peripheral blood from healthy donors, then enriched using MCA system, and detected by staining for CD45, DAPI, CK or vimentin (VM) with the addition staining for AXL. For clinical evaluation, 3ml of peripheral blood was collected from advanced NSCLC patients. Results: DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. In spike-in experiments, when CK was used as a marker, AXL expression was detected in 5 and 17% of high CK-expressing HCC827 and PC9 cells, respectively and detected in 52 and 75% of low CK-expressing H1299 and MDA-MB231 cells, respectively. On the other hand, when VM was used as a marker, AXL expression was detected in 72 and 88% of high VM-expressing MDA-MB231 and H1299 cells, respectively whereas detected in 1 and 7% of PC9 and HCC827 cells with low VM expression, respectively. Twenty-four patients were enrolled in the clinical study. The patient characteristics were as follows: median age 69.5 years (range, 49-84); male 88%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 63/33/4%. Both CK and VM staining in 17 patients, only CK in 6 patients, and only VM in 1 patient were performed. CK-positive single CTCs were detected in all patients (median, 4; range, 1-50) and AXL-expressing CK-positive single CTCs were detected in 26% of patients (median, 0; range, 0-1). On the other hand, VM-positive single CTCs were detected in 94% of patients (median, 6.5; range, 0-128) and AXL-expressing VM-positive single CTCs were detected in 89% of patients (median, 1; range, 0-106). Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive (p < 0.001). Notably, all of identified CTC clusters were positive for only VM, not CK and AXL-expressing CTC clusters were detected in 33% of patients (median, 0; range, 0-22). Conclusion: Our data indicate that incorporating VM staining is necessary to detect AXL-positive CTCs due to EMT. Further clinical evaluation is warranted for validating the method and the clinical significance of AXL-positive CTCs. Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Jun Oyanagi, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Detection of AXL-expressing circulating tumor cells (CTCs) in non-small-cell lung cancer (NSCLC) patients using an automated microcavity array (MCA) system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 414.

Background: Anti-PD-1 antibody nivolumab has become a new standard treatment for pretreated, advanced non-small cell lung cancer (NSCLC). Although PD-L1 expression on tumor tissue has a predictive value, significance of its expression on circulating tumor cells (CTCs) is unknown. Here, we conducted a sequential evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients who received nivolumab at Wakayama Medical University Hospital were enrolled in the study (UMIN000024414). Nivolumab was administered 3 mg/kg bi-weekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in an EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4 and week 8. CTCs were detected using automated microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam). Results: Thirty-eight patients were registered in the study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73 %; stage IV 100 %; squamous/non-squamous, 30/65 %. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval [CI]: 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman's correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥50 % have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p Conclusions: Sequential monitoring of PD-L1 expression on CTCs during nivolumab treatment was successfully conducted. PD-L1 expression on CTCs at baseline was a strong predictor in efficacy. Predictive significance of PD-L1-positive CTCs should be evaluated in a larger validation cohort. Citation Format: Yasuhiro Koh, Hiroaki Akamatsu, Keita Mori, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Masayuki Higuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Predictive impact of sequential evaluation of PD-L1-expressing circulating tumor cells in NSCLC patients treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5595.

Background: Blockade of programmed death receptor-1 (PD-1) pathway is effective against various malignancies. Although PD-ligand 1 (PD-L1) expression on tumor tissue has been established as companion diagnostics in non-small cell lung cancer (NSCLC), additional biomarkers to enrich the patients likely to benefit from the therapy are critically needed. Here, we conducted a serial evaluation of multiple serum cytokines, growth factors, and angiogenesis factors relevant to immune checkpoint blockade in NSCLC patients treated with nivolumab. Patients and Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Serum samples were collected in a serum separation tube (Venoject II autosep, TERUMO) at baseline and at week 4. Best response was classified into partial response (PR), stable disease (SD), or progressive disease (PD) according to RECIST v1.1. Using LuminexTM xMapTM technology, serum levels of 54 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were analyzed. All statistical analyses were carried out using JMP Pro software (ver. 13.0) and Mann-Whitney U test and Spearman's test were performed accordingly. A p value Citation Format: Jun Oyanagi, Yasuhiro Koh, Hiroaki Akamatsu, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Serial evaluation of multiple serum protein levels in non-small lung cancer patients treated with nivolumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A057.

Background: Nivolumab (anti-PD-1 antibody) has become a new standard treatment in pretreated, advanced non-small cell lung cancer (NSCLC). Although strong expression of PD-L1 on tumor tissue has predictive value, significance of its expression on circulating tumor cell (CTC) is unknown and its status can be changed during the treatment. Here, we conducted a serial evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients who receive nivolumab at Wakayama Medical University Hospital were enrolled in this prospective observational study (registered at UMIN (000024414)). Nivolumab was administered 3 mg/kg biweekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in a EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4, and week 8. CTCs were detected using microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam). Results: Thirty-eight patients were registered in this study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73%; stage IV 100%; squamous/non-squamous, 30/65%. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval (CI): 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman’s correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥ 50 have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Keita Mori, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Masayuki Higuchi, HIsashige Kanbara, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Sequential tracking of PD-L1 expression on circulating tumor cells in NSCLC patients treated with nivolumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A056.

Background: Blockade of programmed death receptor-1 (PD-1) pathway is effective against solid tumors including lung cancer. Although PD-ligand 1 (PD-L1) expression on tumor tissue is expected as a potent predictive biomarker, its detection remains challenging due to its dynamic and unstable status. Circulating tumor cells (CTCs) have potential as an alternative material for non-invasive and real-time diagnosis. Here, we evaluated the PD-L1 expression on CTCs in patients with lung cancer and investigated the agreement between tumor tissues and CTCs. Material and methods: CTCs were captured and immune-stained using microcavity array system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression on CTCs was evaluated by addition of the process of PD-L1 immunocytochemistry. For CTCs detection, 3 ml of peripheral whole blood was collected from the patients who consented in written form and PD-L1 immunohistochemistry was performed using corresponding tumor tissues. Results: Sixty-seven lung cancer patients were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University. Patient characteristics were as follows: median age 71 (range, 39 to 86); male 72%; stage II-III/IV, 15/85%; non-small cell lung cancer (NSCLC)/small cell lung cancer (SCLC)/Other, 73/21/6%. CTCs were detected in 66 out of 67 patients (median 19; range, 0 to 115) and more than 5 CTCs were detected in 78% of patients. PD-L1-expressing CTCs were detected in 73% of patients and the proportion score (PS) of PD-L1-expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Significantly more PD-L1-expressing CTCs were detected in patients without EGFR mutations than those with EGFR mutations (P = 0.0433). Tumor tissues were available from 28 patients and were immune-stained for PD-L1. Seven showed the PS of PD-L1-expressing tumor cells < 1%, 11 showed 1-49%, and 10 showed ≥ 50%. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on PS (R2 = 0.0034). Three adenocarcinoma cases with PD-L1-positive tumor tissue did not harbor any PD-L1-expressing CTCs and conversely, three adenocarcinoma cases with PD-L1-negative tumor tissue harbored PD-L1-expressing CTCs, showing the discrepancy between tumor tissues and CTCs. It is also noteworthy that SCLC patients had perfect agreement on PD-L1 expression between tumor tissues and CTCs. Conclusions: PD-L1 expression was detectable on CTCs in lung cancer patients and intra-patient heterogeneity of its expression was observed. There was no agreement between tumor tissues and CTCs on PD-1 expression. Further investigation is warranted to better understand the clinical significance of PD-L1-expressing CTCs. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Satomi Yagi, Satoshi Kambayashi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Keiichiro Akamatsu, Masayuki Higuchi, Hisashige Kanbara, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. PD-L1 expression on circulating tumor cells and its comparison with tumor tissues in Japanese lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2017-3778

11541 Background: PD-L1 expression on tumor tissue is associated with response to PD-1 blockade in NSCLC. Here, we conducted a serial evaluation of PD-1-expressing circulating tumor cells (CTCs) as a potential real-time diagnostic modality in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected for CTC evaluation at baseline and at week 4. CTCs were detected using microcavity array system (Hitachi Chemical Co., Ltd, Chikusei, Japan). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs. This study was registered at UMIN (ID: 000024414). Results: Thirty patients were registered in the study between January 2016 and September 2016 at Wakayama Medical University Hospital and 29 were included in the analysis. Demographics of the patients were as follows: median age 70 (range, 49 to 86); male 73 %; stage IV, 100 %; squamous/non-squamous, 27/73 %. At baseline, CTCs were detected in all patients (median, 15; range, 1 to 90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs ranged from 6% to 100%, indicating intrapatient heterogeneity. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on TPS (R2 = 0.0035). Overall response rate was 25% (7/29), and disease control rate was 54% (15/29). Total CTC count was significantly decreased after nivolumab treatment at week 4 (p < 0.05), but no significant change was observed in PD-L1 TPS on CTC. Patients harboring CTCs with PD-L1 TPS 50% or more at baseline were significantly more likely to achieve non-PD than those harboring CTCs with TPS less than 50% (p < 0.05). Conclusions: This is the first report on a serial monitoring of PD-L1 expression on CTCs in patients treated with nivolumab. PD-L1-expressing CTCs are suggested to hold potential for predicting clinical benefit. Clinical trial information: 000024414.

Background Viral infections are a major cause of chronic obstructive pulmonary disease (COPD) exacerbations. Toll-like receptor 3 (TLR3) reacts with double-stranded RNA (dsRNA) and participates in the immune response after viral infection. In the present study, we examined whether cigarette smoke, which is involved in the pathogenesis of COPD, enhances mucin production via the TLR3-epidermal growth factor receptor (EGFR) pathway in airway epithelial cells. Methods We studied the effects of cigarette smoke extract (CSE) on signal transduction and the production of mucin 5AC (MUC5AC) in NCI-H292 cells and differentiated primary human bronchial epithelial cells stimulated with a synthetic dsRNA analogue, polyinosinic-polycytidylic acid [poly(I:C)], used as a TLR3 ligand. Results CSE significantly potentiated the production of MUC5AC in epithelial cells stimulated with poly(I:C). Antibodies to EGFR or EGFR ligands inhibited CSE-augmented MUC5AC release in poly(I:C)-treated cells. Treatment with poly(I:C) or CSE alone increased the phosphorylation of EGFR and extracellular signal-regulated kinase (ERK). However, after poly(I:C) stimulation, CSE did not enhance EGFR phosphorylation, but did augment ERK phosphorylation. EGFR inhibitors and an ERK inhibitor inhibited the augmented release of MUC5AC. In addition, treatment with N -acetylcysteine, an antioxidant, inhibited the CSE-augmented phosphorylation of ERK and MUC5AC. Conclusions These data show that cigarette smoke increases TLR3-stimulated MUC5AC production in airway epithelial cells, mainly via ERK signaling. The effect might be mediated in part by oxidative stress. Modulation of this pathway might be a therapeutic target for viral-induced mucin overproduction in COPD exacerbation.

Viral infection often triggers asthma exacerbation and contributes to airway remodeling. Cell signaling in viral infection is mainly mediated through TLR3. Many mediators are involved in airway remodeling, but matrix metalloproteinases (MMPs) are key players in this process in asthma. However, the role of TLR3 activation in production of MMPs is unknown. In this study, we examined the effects of polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3, on production of MMPs in human lung fibroblasts, with a focus on nitrosative stress in TLR3 modulation of MMP production. After lung fibroblasts were treated with poly(I:C), production of MMP-1, -2, and -9 and inducible NO synthase (iNOS) was assessed. The roles of NF-κB and IFN regulatory factor-3 (IRF-3) in the poly(I:C)-mediated production of MMPs and the responsiveness to poly(I:C) of normal lung fibroblasts and asthmatic lung fibroblasts were also investigated. Poly(I:C) augmented production of MMPs and iNOS in fibroblasts, and an iNOS inhibitor diminished this production of MMPs. Poly(I:C) stimulated translocation of NF-κB and IRF-3 into the nucleus in fibroblasts and inhibition of NF-κB or IRF-3 abrogated the poly(I:C)-induced increase in both iNOS expression and release of MMPs. Poly(I:C)-induced production of iNOS and MMPs was greater in asthmatic fibroblasts than in normal fibroblasts. We conclude that viral infection may induce nitrosative stress and subsequent MMP production via NF-κB– and IRF-3–dependent pathways, thus potentiating viral-induced airway remodeling in asthmatic airways.

Background and purpose: Immune-checkpoint blockade with anti-programmed death-1 (PD-1) antibodies is rapidly emerging for the treatment of human malignancies including lung cancer. Although programmed death-ligand 1 (PD-L1) has been studied as a predictive biomarker, detection and evaluation of PD-L1 expression level on tissue samples remain challenging due to its dynamic and unstable expression. Thus the diagnostic tool for real-time monitoring of PD-L1 expression is critically needed. Here, we assessed the expression pattern of PD-L1 on circulating tumor cells (CTCs) by using microcavity array (MCA) system in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Experimental procedure: PD-L1 staining on CTCs was established using NSCLC cell lines H820, H441, A549 and H23 expressing varying levels of PD-L1 spiked in the peripheral blood obtained from healthy donors. For clinical evaluation, 3 ml of peripheral whole blood was collected from 20 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Cells were captured and immuno-stained by using the automated MCA system (Hitachi Chemical Co., Ltd). CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression level on CTCs was visualized by addition of PD-L1 immunocytochemistry procedure. High-resolution fluorescent images were obtained using fluorescence microscope (Carl Zeiss Microscopy Co., Ltd). Results: Characteristics of 20 lung cancer patients enrolled in clinical study were as follows: median age 74 (range, 48 to 84); male 60%; stage III/IV, 10/90%; NSCLC/SCLC, 70/30%. More than 2 CTCs were identified in 14 patients (median 22.5; range, 4 to 71), and PD-L1 positive CTCs were detected in 12 patients (median 5; range, 2 to 15). No correlation was detected between the number of total CTCs and that of PD-L1 positive CTCs in each patient (R2 = 0.05). We found a total of 25 CTC clusters from 20 patients, of which PD-L1 expression was both homogenous and heterogeneous. It is noteworthy that clustered CTCs have larger proportion of PD-L1 positive CTCs per whole clustered CTCs than that of non-clustered CTCs (24/54, 44% versus 51/347, 15%, respectively). We further focused on CTC-interacting white blood cells, which intensively bound with aggregated CTCs rather than single CTC (12/54, 22% versus 43/337, 13%, respectively). Our data implicate that PD-L1 expression on CTC correlates with aggregation of CTCs (p < 0.05). Conclusions: Our results showed that PD-L1 expression on CTCs was detectable and there is intrapatient heterogeneity of its expression in patients with advanced lung cancer. Further investigation is warranted to better understand the biological importance of the correlation between PD-1 expression and CTC aggregation and CTC bound to white blood cells. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Differential expression of PD-L1 on circulating tumor cells among patients with advanced lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2257.

Background and Purpose: Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. Material and method: The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell. Results: Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p = 0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p = 0.0001, n = 17). Conclusions Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort. Citation Format: Satomi Yagi, Yasuhiro Koh, Hiroaki Akamatsu, Woong Kim, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2244.

e23036Background: Immune-checkpoint blockage with anti-PD-1 antibodies is effective against human malignancies including lung cancer. Although the expression of PD-L1 on tumor cells is expected as ...

Circulating tumor cells (CTCs) are associated with prognosis of patients with advanced solid tumors including lung cancer and reflect characteristics of the respective primary tumor and its metastatic deposits. Assessment of CTCs is expected to improve effectiveness of anticancer therapy and to sophisticate our knowledge to cancer metastasis. We previously reported that detection of CTCs using microcavity array (MCA) system yielded the superior sensitivity than FDA-approved CellSearch system in patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Here we developed the automated CTCs detection device and evaluated its performance using preclinical spike-in model and blood samples from NSCLC and SCLC patients. The feasibility study on the assessement of PD-L1 expression level was also performed. To evaluate the recovery of CTCs preclinically, NSCLC cells, H1975, A549, H441 and PC-14 were spiked into 6 mL of peripheral whole blood obtained from healthy volunteers. Then, cells were captured and immuno-stained by using automated MCA system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. Additionally, normal blood cells and cancer cells were distinguished according to their size. For clinical evaluation, 6 mL of peripheral whole blood was collected from 10 healthy donors and 30 advanced lung cancer patients prior to the initiation of chemotherapy. Head-to-head comparison with CellSearch system was also conducted. PD-L1 immunostaining was established in a preclinical spike-in study using NSCLC cell lines, H820, A549, H441, and H23 with varying PD-L1 expression levels and tested as an exploratory objective in a subset of patients enrolled in a clinical study. We confirmed that up to 90% of spiked-in tumor cells were recovered by the automated MCA system, suggesting that the detection sensitivity by this automated device is on par with that by previous detection procedure. Characteristics of 30 lung cancer patients in clinical study were as follows: median age 71 (range, 48 to 85); male 70%; stage III/IV 17/83%; NSCLC/SCLC 83/17%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 in 6 mL of peripheral blood, respectively. More than 2 CTCs were detected in 77% of patients with advanced lung cancer (n = 23/30) and more than 5 CTCs were detected in 50% of patients (n = 15/30) (median CTC count 5.5). Significantly more CTCs were detected by the automated MCA system than by CellSearch system. Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those without one (median CTC count, 8 versus 4, p = 0.058). No difference in CTC counts between NSCLC and SCLC was observed in this study cohort. PD-L1 expression was assessed in a subset of patients and intra-patient heterogeneity of PD-L1 staining among CTCs was observed in patients who harbor PD-L1-positive CTCs. Our results suggest that the automated MCA device for size-based enrichment and isolation of CTCs has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This also enables us to deal with more samples owing to its automated procedure and higher throughput. Further clinical evaluation including PD-L1 expression will be performed in an expansion cohort and the biology of CTCs will be investigated utilizing this device. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Nobuyuki Yamamoto. Evaluation of a novel automated device for size-based enrichment and isolation of CTCs in patients with advanced lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B138.