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Supplementary Figure from Nivolumab Retreatment in Non–Small Cell Lung Cancer Patients Who Responded to Prior Immune Checkpoint Inhibitors and Had ICI-Free Intervals (WJOG9616L)

Supplementary Table from Nivolumab Retreatment in Non–Small Cell Lung Cancer Patients Who Responded to Prior Immune Checkpoint Inhibitors and Had ICI-Free Intervals (WJOG9616L)

Purpose:To explore the efficacy of retreatment with immune checkpoint inhibitors (ICI) in patients with advanced non–small cell lung cancer (NSCLC) who responded to prior ICI and had adequate ICI-free interval.Patients and Methods:Patients with advanced NSCLC who had achieved complete response (CR), partial response (PR), or stable disease for ≥6 months with prior ICI therapy preceding progression were prospectively enrolled. All patients should have had ICI-free interval ≥60 days before registration. Patients were treated with nivolumab (240 mg) every 2 weeks until progression. The primary endpoint was overall response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival, and safety (Trial Identifier, UMIN000028561).Results:Sixty-one patients were enrolled during October 2017 to February 2020, with 59 analyzed for efficacy. Regarding prior ICI, 41 patients had CR or PR. Median treatment on ICI and median ICI-free intervals were 8.1 months and 9.2 months, respectively. Twenty patients experienced immune-related adverse events (irAE) that required discontinuation of prior ICI. Nivolumab retreatment demonstrated ORR of 8.5% [95% confidence interval (CI), 2.8–18.7%] and median PFS of 2.6 months (95% CI, 1.6–2.8 months) while 5 responders had 11.1 months of median PFS. In the multivariate analysis, ICI-free interval was the only predictive factor of PFS (HR, 2.02; P = 0.02), while prior efficacy or history of irAE was not. Common adverse events were skin disorders (23%), malaise (20%), and hypoalbuminemia (15%).Conclusions:Even in patients who initially responded to prior ICI and had ICI-free interval, once resistance occurred, retreatment with nivolumab had limited efficacy.

A multistep sorting method for enrichment of rare cells, such as circulating tumor cells, in the blood without cumbersome pretreatments required by most flow cytometry-based methods, which lead to high cost and decreased detection efficiency, was developed.After only hemolysis and cell staining, cancer cells are enriched by repetitive sorting (3×) based on nuclear-positive, cytokeratin-positive, and CD45-negative expression.Experiments using spikes of PC-9 cells showed a mean recovery of 65% and mean purity of 83%, which was retained up to 72 hours after blood draw using preservative tubes. Significant differences in expression level of programmed death-ligand 1 or vimentin were observed between high- and low-expressing cell lines, concurrently with enrichment. Next-generation sequencing analysis of recovered PC-9, A549, and MDA-MB231 cells successfully detected all known mutations.This novel isolation method applicable for preserved samples with sufficient recovery and purity may be substantially beneficial for recovering cells for subsequent molecular analysis.

Multiple primary lung cancers (MPLCs) harbour various genetic profiles among the tumours, even from individuals with same non-intrinsic risk factors. Paired mutational analyses were performed to obtain a census of mutational events in MPLC and assess their relationship with non-intrinsic risk factors. Thirty-eight surgical specimens from 17 patients diagnosed as MPLC were used. Extracted DNAs were sequenced for somatic mutations in 409 cancer-associated genes from a comprehensive cancer panel. We statistically analysed the correlation between each driver mutation frequency and non-intrinsic risk factors using Fisher's exact test, and whether genetic mutations occurred concomitantly or randomly in MPLC using an exact test. Comprehensive genetic analyses suggested different mutation profiles in tumours within the same individuals, with some exceptions. EGFR, KRAS, TP53, or PARP1 mutations were concomitantly detected in some MPLC cases. EGFR mutations were significantly more frequent in never or light smokers and females. Concomitant EGFR or KRAS mutations in MPLCs were significantly more frequent than expected by chance (P = .0023 and .0049, respectively) suggesting a more prominent role of non-intrinsic risk factors in EGFR and KRAS mutations than other mutations, which occurred more randomly. Concomitant EGFR or KRAS mutations were particularly prominent in never or light smokers and males.

Background/aim To explore the safety of osimertinib plus ramucirumab in patients with EGFR-mutated lung adenocarcinoma. Patients and methods Six advanced lung adenocarcinoma patients with EGFR mutation were treated with osimertinib 80 mg/day plus ramucirumab 10 mg/kg, every two weeks. Defined dose-limiting toxicity (DLT) was assessed within the first two treatment cycles. Results Of those enrolled, five patients had both EGFR exon 20 T790M mutation and sensitizing mutation. DLT was observed in one patient (grade 3 appetite loss). During the entire period, no other severe adverse event was observed. Five patients showed partial response and one disease progression. Median progression-free survival for patients with EGFR T790M was 9.2 months. In an exploratory analysis, changes of cell-free DNA at 2 weeks predicted radiological tumor responses. Conclusion The safety results of osimertinib plus ramucirumab in Japanese lung adenocarcinoma patients with EGFR mutation will lead to further efficacy investigation.

Background/aim Molecular targeted agents have been successfully developed against solid tumors and their use is also being investigated for the treatment of malignant pleural mesothelioma (MPM). We have previously reported von Hippel Lindau (VHL) mutations detected by massive parallel sequencing technology in samples of patients with MPM. Here, we conducted an in vitro study to investigate the therapeutic approaches in VHL-mutant MPM. Materials and methods Three MPM cell lines with or without a VHL mutation were used and the effects of molecular-targeted agents on growth inhibition were evaluated. Based on the characteristics of the molecular targeted agents that exhibited growth inhibitory effect, the effects of knockdown by siRNA were also evaluated. Results NCI-H28 MPM cells harboring the VHL L89H mutation were sensitive to YC-1, known as an inhibitor of hypoxia inducible factor (HIF)-1α, and YC-1treatment induced massive apoptosis in a dose-and-time-dependent manner. Knockdown of HIF-1α by siRNA partially inhibited the growth of NCI-H28 cells, suggesting that an additional blockade may be required to completely inhibit growth signaling. Conclusion The VHL mutation may predict tumor responses to YC-1, a HIF-1α inhibitor.

Highlights • Tumor microenvironment (TME) was compared among multiple primary lung cancers (MPLCs). • Sex and smoking status concomitantly impacted PD-L1 expression in paired tumors. • EGFR mutations were independently associated with PD-L1 expression. • KRAS mutations altered the TMEs according to the types of co-mutations. • The number of FOXP3-positive t cells reflected histological subtypes., Introduction Multiple primary lung cancers (MPLCs) occur in common carcinogenetic risks such as lifestyle, biological aging, immune responses, hormones, and metabolism. Although MPLCs harbor various genetic profiles within the same individuals, differences in the tumor microenvironment (TME) are unclear. We investigated the impact of genetic aberrations, non-intrinsic factors, and pathological subtypes on tumor immunity. Materials and Methods In total, 73 surgically resected specimens from 32 patients with MPLC were analyzed. PD-L1 expression in tumor cells (TCs) and immune cells (ICs), CD3-positive tumor-infiltrating lymphocytes (TILs), CD8/CD3 ratios, and FOXP3-positive TILs that compose TMEs were evaluated by immunohistochemistry and classified on a score of 0–2. 38 tumors were sequenced for somatic mutations in 409 cancer-associated genes. Results Females and never or light smokers had a higher incidence of PD-L1-negative tumors and a higher concordance rate. PD-L1 positivity in TCs and ICs was significantly less frequent in EGFR-mutated than in wild-type tumors. Differences in the score of TMEs were observed between the KRAS-mutated-only tumor and the KRAS and TP53-co-mutated tumors, and between the KRAS-mutated-only tumor and the KRAS and STK11-co-mutated tumors. Significantly more FOXP3-high TILs were observed in invasive pathological subtypes than in non-invasive ones. Conclusion Comparing TMEs among MPLCs revealed that non-smokers or light smokers and females were unlikely to express PD-L1 regardless of tumor site and confirmed that the EGFR mutations and co-occurring KRAS and STK11 or TP53 mutations were associated with TME. Pathological subtypes may impact the efficacy of immune therapy due to their potential correlations with regulatory T cells.

Background Although predictive value of immune‐related adverse events (irAEs) induced by immune checkpoint inhibitors (ICIs) have been suggested by several studies, their assessments were insufficient because patients were categorized only by the occurrence of irAEs. It has not been elucidated whether irAEs also play a significant role even in responders. Materials and Methods Between December 2015 and September 2018, 106 patients with advanced non‐small cell lung cancer treated with ICIs were enrolled in our prospective biomarker study. Twenty‐three of these were responders, defined as those with complete or partial response. We investigated the proportion of irAEs among overall and responders. For responders, progression‐free survival (PFS) and overall survival of ICIs were compared between those with and without irAEs. As an exploratory analysis, we measured 41 proteins from peripheral blood before and after ICI treatment. Results The proportion of irAEs was significantly higher in responders than nonresponders (65.2% vs. 19.3%, p < .01). Among responders, clinical characteristics did not differ regardless of the occurrence of irAEs. However, there was a significant difference in PFS among responders (irAE group 19.1 months vs. non‐irAE group 5.6 months; hazard ratio: 0.30 [95% confidence interval: 0.10–0.85]; p = .02). Of 41 protein analyses, fibroblast growth factor‐2 at baseline and monocyte chemoattractant protein fold change showed significant differences between them (p < .04). Conclusion Although this is a small sample–sized study, irAE might be a predictive factor of durable efficacy, even in patients who responded to ICIs. Investigation into the significance of irAEs in responders will contribute to the establishment of optimal administration of ICI. Implications for Practice Although the predictive value of immune‐related adverse events (irAEs) induced by immune checkpoint inhibitors (ICIs) has been suggested by several studies, it has not been elucidated whether irAEs also play a significant role even in responders. This study showed that more than 60% of responders had irAEs. It demonstrated the strong correlation between irAEs and efficacy even in responders. Investigation into the significance of irAEs in responders will contribute to the establishment of optimal administration of ICI., Immune‐related adverse events might be a predictive factor of efficacy, even I patients who respond to immune checkpoint inhibitor therapy. This study investigated the significance of immune‐related adverse events in patients with non‐small cell lung cancer who had complete or partial response after treatment with immune checkpoint inhibitors.

We retrospectively investigated the impact of the tumor microenvironment (TME) on the efficacy of epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs) as first‐line treatment in 70 patients with advanced EGFR‐mutant non‐small cell lung cancer and who were seen at Osaka City University Hospital (Osaka, Japan) between August 2013 and December 2017. Using immunohistochemical staining with 28‐8 and D7U8C Abs, the tumor proportion score was assessed for programmed cell death‐1 ligand‐1 (PD‐L1), as high (50% or more) or low (less than 50%), and ligand‐2 (PD‐L2) expression, respectively. The extent of CD8+ tumor‐infiltrating lymphocytes was evaluated on a scale of 0‐3, with 0‐1 as low and 2‐3 as high. The TME of the 52 evaluable pretreatment specimens was categorized into 4 subtypes, according to the respective PD‐L1 tumor proportion and CD8+ scores, as follows: (a) high/high (13.5%, n = 7); (b) low/low (42.3%, n = 22); (c) high/low (17.3%, n = 9); and (d) low/high (26.9%, n = 14). Expression of PD‐L2 was significantly the highest in type 1 (57.1% vs 4.5% vs 11.1% vs 7.1%, respectively; P = .0090). Response rate was significantly the lowest in type 1 (14.3% vs 81.8% vs 66.7% vs 78.6%, respectively; P = .0085). Progression‐free survival was the shortest in type 1 and the longest in type 4 (median, 2.4 vs 11.3 vs 8.4 vs 17.5 months, respectively; P = .00000077). The efficacy of EGFR‐TKIs differed according to the TME, and the phenotype with high PD‐L1 and CD8+ expression might be the subset that would poorly benefit from such treatment.

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.

Colorectal cancer (CRC) is caused by genetic alterations, and comprehensive sequence analyses have revealed the mutation landscapes. In addition to somatic changes, genetic variations are considered important factors contributing to tumor development; however, our knowledge on this subject is limited. Familial adenomatous polyposis coli (FAP) is an autosomal‐dominant inherited disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. FAP patients are classified into two major groups based on clinical manifestations: classical FAP (CFAP) and attenuated FAP (AFAP). In this study, we established 42 organoids from three CFAP patients and two AFAP patients. Comprehensive gene expression analysis demonstrated a close association between IFN/STAT signaling and the phenotypic features of FAP patients. Genetic disruption of Stat1 in the mouse model of FAP reduced tumor formation, demonstrating that the IFN/STAT pathway is causally associated with the tumor‐forming potential of APC‐deficient tumors. Mechanistically, STAT1 is downstream target of KRAS and is phosphorylated by its activating mutations. We found that enhanced IFN/STAT signaling in CFAP conferred resistance to MEK inhibitors. These findings reveal the crosstalk between RAS signaling and IFN/STAT signaling, which contributes to the tumor‐forming potential and drug response. These results offer a rationale for targeting of IFN/STAT signaling and for the stratification of CRC patients.

Background: Although it has been known that N-glycan moiety in immunoglobulin G (IgG) fragment crystallizable (Fc) region, which affects antigen dependent cellular cytotoxicity (ADCC) activity is different between healthy individuals and cancer patients, the impact of N-glycan structure on cancer treatment is unknown. Here, we evaluated the putative ADCC activity by the N-glycan moiety of IgG-Fc using serum samples and analyzed its association with clinical benefits from immune checkpoint inhibitors (ICIs) in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: Serum samples from healthy volunteers (HVs) were obtained from Tohoku Medical Megabank and chronic obstructive pulmonary disease (COPD) patients were enrolled as a smoking-related non-cancer disease cohort. Serum samples from advanced NSCLC patients receiving ICIs until disease progression or unacceptable toxicity were collected prior treatment. Three IgG fractions (area 1, 2 and 3) corresponding to three peaks identified by affinity chromatography using TSKgel FcR-IIIA-NPR column (Tosoh, Japan) were quantified and the putative ADCC activity was estimated based on the ratio of area 3, which associates with highest ADCC activity, to the whole area (area 3 (%)). Mann-Whitney U-test, Kaplan-Meier methods and the log-rank test were conducted for statistical analyses using JMP Pro software (ver. 14.0). Results: Forty-two COPD and ninty-six NSCLC patients were registered in the study between Jan 2016 and May 2021 at Wakayama Medical University. Characteristics of the patients were as follows: median age, 70 (range, 49-91); male. 78%; smoker, 79%; previous treatment ≥1, 77%; performance status 0-1, 82%; stage IV, 69%; squamous/non-squamous/unknown, 29/69/2%; nivolumab/pembrolizumab/atezolizumab, 46/42/12%. The objective response rate was 25.0% (24/96). The median progression free survival (PFS) and median overall survival (OS) was 72 days (95%CI, 49 to 127) and 310 days (95% CI, 216 to 453), respectively. The area 3 (%) was significantly lower in NSCLC patients than both HVs and COPD patients (P=0.0002 and p Conclusion: Our results suggest that evaluation of the N-glycan moiety of IgG-Fc in peripheral blood has a potential as a prognostic biomarker in advanced NSCLC patients treated with ICIs. Citation Format: Jun Oyanagi, Yasuhiro Koh, Yasuyuki Akiyama, Atsushi Morimoto, Koichi Sato, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Atsushi Hayata, Yuichi Ozawa, Hiroaki Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. N-glycan moiety of IgG-Fc region is a prognostic biomarker for advancednon-small cell lung cancertreated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 516.

Background: To overcome resistance to EGFR TKI osimertinib in EGFR-mutated lung cancer is critically needed. We performed a biomarker study utilizing serially collected plasma samples in a phase I study to investigate the safety and efficacy of the combination of osimertinib and afatinib in patients who failed osimertinib (jRCTs051180008), in which a total of 13 patients were enrolled and the overall response rate and the median progression-free survival were 7.7% and 2.4 months, respectively, to access the trajectory of the mutational landscape and its association with the efficacy. Methods: Peripheral blood samples were collected from the patients in K2 EDTA vacutainers (Becton Dickinson) before and four weeks after administration of the combination therapy and upon disease progression. Plasma was isolated immediately after the blood draw and stored at -80℃ until use. Cell-free DNA (cfDNA) was extracted from the plasma and used for next-generation sequencing library construction using AVENIO ctDNA Surveillance Kit (Roche Diagnostics) to assess genetic alterations in 197 cancer-related genes. Sequencing was performed on Nextseq500 system (Illumina) followed by mutation analyses using an Avenio Oncology Analysis Server (Roche Diagnostics). Results: Mutations in EGFR gene were detected in 10 out of 13 cfDNA samples (77%) including T790M/cis-C797S mutations before treatment and among those harboring T790M/cis-C797S mutations, 2 patients had progressive disease (PD) and 1 had stable disease (SD), indicating little benefit from the combination therapy in this genotype. Although other mutations in genes such as TP53 and/or KRAS were detected before treatment, no relevant associations were observed between tumor response or progression-free survival. One patient who had SD harbored CNTN5 mutation at PD, suggesting the potential involvement with the acquired resistance. Early emergence of EGFR G796S mutation observed in one patient after the initiation of the treatment along with basal L858R/T790M mutations may confer early resistance to the treatment. We also observed the emergence of MET amplification in 2 patients and ERBB2 amplification in 1 patient at PD, strongly suggesting the potential mechanisms responsible for resistance. Conclusions: This signal seeking biomarker study using cfDNA to better understand the association of mutational status and the treatment efficacy of the combined treatment was feasible and informative despite the small-scale study, supporting the advantage of further implementation. Citation Format: Yasuhiro Koh, Satoru Miura, Jun Oyanagi, Hiroshige Yoshioka, Koichi Azuma, Hidenobu Ishii, Kayoko Kibata, Kenichi Koyama, Shunsuke Teraoka, Yuichi Ozawa, Takaaki Tokito, Toshio Shimokawa, Takayasu Kurata, Nobuyuki Yamamoto, Hiroshi Tanaka. Longitudinal cell-free DNA analysis in phase I study evaluating afatinib in combination with osimertinib in patients who failed prior osimertinib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5153.

While PD-1/L1 inhibitors are characterized by durable tumor control, they also prolong survival without prolongation of progression-free survival (PFS) in part of patients. However, little is known about the factors and mechanisms involved in this. Between December 2015 and September 2018, 106 patients with advanced non-small cell lung cancer treated with ICI monotherapy were enrolled in a prospective-observational study. Sixty-nine of whom progressed or died within 6 months after ICI initiation were defined as patients without durable clinical benefit (NDBs). Clinical factors and 39 serum proteins before ICI initiation and at the time of progressive disease (PD) were explored for an association with overall survival (OS) and OS after PD (OS-PD). As a result, median PFS, OS, and OS-PD were 44 days [95% confidence interval (CI): 39-56), 211 days (95% CI: 158-425), and 193 days (95% CI: 118-349), respectively. By multivariate analysis for OS, CRP ( 1.44 mg/dl) [HR 2.59 (95% CI:1.33-5.04), P = 0.005] and follistatin ( 685 pg/ml) [HR 2.29 (95% CI:1.12-4.69), P = 0.023] before ICI initiation were significantly predictive. Notably, no serum protein at the time of PD was predictive for OS-PD. There were also no serum predictive factors of OS in the 33 patients with durable clinical benefit. In conclusion, serum levels of CRP and follistatin before ICI initiation, not at the time of PD, are predictive for OS in NDBs, suggesting long-term survivor in NDBs are predetermined by the immune status before ICI initiation.

BACKGROUND Programmed cell death ligand 1 is a biomarker of immune checkpoint inhibitors (ICIs) for treating advanced non-small-cell lung cancer (NSCLC). Here, we evaluated serum proteins from patients with advanced NSCLC treated with ICIs to determine their potential as noninvasive predictive biomarkers for efficacy and immune-related adverse events (irAEs). PATIENTS AND METHODS Patients with advanced NSCLC who received nivolumab or pembrolizumab monotherapy until disease progression or unacceptable toxicity were integrated with previously reported nivolumab-treated patients. Blood samples were collected serially from baseline until the disease progressed. Serum protein levels were quantified using the Luminex assay. Associations of clinical benefit (CB) and onset of irAEs with serum protein levels were evaluated. RESULTS Sixty-three patients with advanced NSCLC were studied, and we used 63 and 47 paired serum samples at baseline and the second sampling point, respectively, for efficacy analysis. Baseline growth-regulated oncogene 1 (GRO-1) levels were significantly lower in durable CB (DCB) patients than in non-DCB patients (P < .05). Changes in monocyte chemoattractant protein 1 (MCP-1) levels significantly decreased between baseline and the second sampling point (P < .05). Patients with the low GRO-1/decreased MCP-1 subtype showed significantly longer progression-free survival (PFS) and overall survival (OS) than the high GRO-1/increased MCP-1 subgroup did (median PFS, not reached vs. 47 days, P < .0001; median OS, 985 days vs. 148 days, P = .0002, respectively). Elevated GRO-1 levels were associated with immune-related adverse event onset. CONCLUSIONS Serum GRO-1 and MCP-1 levels can identify patients with advanced NSCLC who are likely to benefit from ICI treatment. Time-course tracing of these protein levels might be valuable in ICI treatment.

CD24, a heavily glycosylated glycosylphosphatidylinositol–anchored surface protein, inhibits phagocytosis as potently as CD47. The relationship between such anti‐phagocytic factors and the immune response with immune–checkpoint inhibitors (ICI) remains unexplored. We evaluated CD24 and CD47 tumor proportion scores (TPS) in 68 of the 106 patients with advanced non–small‐cell lung cancer who participated in a prospective observational study of ICI treatment. We also explored the impact of CD24 TPS and CD47 TPS on ICI efficacy and serum cytokine changes. CD24 positivity (TPS ≥ 1) was negatively associated with progression–free survival (PFS) of ICI when PD‐L1 TPS was, We evaluated tumor proportion score (TPS) of CD24 and CD47 in 68 patients with advanced, non–small‐cell lung cancer who participated in a prospective observational study for treatment with ICI, and we also explored the impact of CD24 TPS and CD47 TPS on the efficacy of ICI and serum cytokine alterations. CD24 positivity (TPS ≥ 1) was negatively associated with progression–free survival of ICI when PD‐L1 TPS

Background: Structure of N-linked glycan in immunoglobulin G (IgG) fragment crystallizable (Fc) region is associated with antigen-dependent cellular cytotoxicity (ADCC) activity. Although it has been known that N-glycan moiety is different between healthy individuals and cancer patients, the relationship between the N-glycan structure and treatment response in cancer patients is unknown. Here, we evaluated the putative ADCC activity by the N-glycan moiety of IgG-Fc using peripheral blood and analyzed its association with clinical benefits from immune checkpoint inhibitors (ICIs) in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: Advanced NSCLC patients received nivolumab, pembrolizumab or atezolizumab treatment until disease progression or unacceptable toxicity. Serum samples were collected at baseline. Tumor responses were classified into complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) based on RECIST v1.1. Durable clinical benefit (DCB) was defined as patients whose response was lasting over 6 months. Three IgG fractions (area 1, 2 and 3) corresponding to three peaks identified by affinity chromatography using TSKgel FcR-IIIA-NPR column (Tosoh, Japan) were quantified and the putative ADCC activity was estimated based on the ratio of area 3, which associates with highest ADCC activity, to the whole area. All statistical analyses were carried out using JMP Pro software (ver. 14.0). Mann-Whitney U-test was conducted. Time to event analysis was conducted using Kaplan-Meier methods and the log-rank test. Results: Ninety-seven patients were registered in the study between Jan 2016 and Nov 2018 at Wakayama Medical University. Characteristics of the patients were as follows: median age, 70 (range, 49-91); male. 78%; smoker, 79%; previous treatment ≥1, 77%; performance status 0-1, 82%; stage III/IV, 31/69%; squamous/non-squamous/unknown, 29/69/2%; nivolumab/pembrolizumab/atezolizumab, 46/42/12%. The objective response rate was 25.0% (24/96) and the disease control rate was 55.2% (53/96). The DCB rate was 45.4% (44/96). The median progression free survival (PFS) was 72 days (95%CI, 49 to 127) and median overall survival (OS) was 310 days (95% CI, 229 to 475). When time-to-event analysis was conducted by dividing into two groups at the median of the ratio of area 3 to the whole area, OS was significantly longer in patient with higher ratio than those with lower one in both entire cohort (median OS, 952 vs 482; 95% CI, 482 days-not reached and 233-744 days; log rank p=0.0292) and 2nd line cohort (median OS, 952 vs 292; 95% CI, 453 days-not reached and 120-692 days; log rank p=0.0010). Evaluation of IgG fraction was not associated with tumor response or PFS in this study cohort. Conclusion: Our results suggest that evaluation of the N-glycan moiety of IgG-Fc in peripheral blood has a potential as a prognostic biomarker in advanced NSCLC patients treated with ICIs. Citation Format: Jun Oyanagi, Yasuhiro Koh, Yasuyuki Akiyama, Atsushi Morimoto, Koichi Sato, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Atsushi Hayata, Yuichi Ozawa, Hiroaki Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Clinical impact of evaluating serum IgG fractions in advanced non-small cell lung cancer treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P014.

Background: In the past decade, three-dimensional organoid culture derived from various tumors has been applied to cancer research. Compared to the conventional two-dimensional cell culture, organoid culture recapitulates in vivo system and furthermore, its high establishment rate may hold the potential to help decision-making for precision medicine. However, it remains challenging to establish organoids from advanced thoracic malignancies due to a limited amount of biopsy specimens. Here, we report the attempt to establish organoids from endoscopic biopsy specimens and pleural effusions in patients with advanced thoracic malignancies Methods: Organoid culture was attempted using biopsy specimens or pleural effusions from patients who underwent transbronchial biopsy, thoracoscopic biopsy or thoracentesis. After confirming the presence of malignant cells by rapid on-site evaluation, biopsy specimens were digested with collagenase and dispase at 37°C followed by being harvested and cultured in Matrigel. As for pleural effusions, mononuclear cells were isolated by ficoll density gradient centrifugation and CD45+ cells were depleted using anti-CD45 magnetic beads. Then, the resultant cells were cultured in Matrigel. Next-generation sequencing was performed to confirm cancer-related somatic mutations using Ion PGM system. Cell viability assay was performed by resazurin assay. Results:142 patients were registered in the study between May 2019 and October 2020 at Wakayama Medical University. Fifty-one cases were excluded because of negative diagnosis for malignancy, retraction of the consent or risk of bleeding associated with biopsy. We attempted to culture organoids using 98 samples from 91 patients. The types of malignancies were as follows: non-small cell lung cancers (NSCLC)/SCLC/others, 80/8/3. A total of 33 organoids were successfully cultured with sustainable proliferation and 16 were successfully cryopreserved. Established organoids were confirmed to harbor the same mutations as detected by clinical testing with tissue samples and to form tumors in a xenotransplantation model. HLCO-75, established from pleural effusion at disease progression after osimertinib treatment was genotyped and found to gain ERBB2 V777L mutation in exon 20 in addition to original EGFR L858R mutation. Marked growth-inhibitory effect by poziotinib, a HER2 tyrosine kinase inhibitor with IC50 value 6 nM was observed whereas not by TAS6417, another EGFR/HER2 exon 20 inhibitor, suggesting the potential of organoid culture for decision-making in cancer precision medicine. Conclusion: Our results suggest that organoid culture is feasible from small biopsy specimens and these organoids could be applied for personalized medicine. Citation Format: Jun Oyanagi, Yasuhiro Koh, Koichi Sato, Masanori Tanaka, Katsuyuki Furuta, Takeya Sugimoto, Ryota Shibaki, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Hiroaki Akamatsu, Atsushi Hayata, Atsushi Hayata, Yuichi Ozawa, Hiroki Ueda, Nobuyuki Yamamoto. Establishment of organoids derived from patients with advanced thoracic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2976.

The laminin γ2 chain, a subunit of laminin-332 (α3β3γ2), is a molecular marker for invasive cancer cells, but its pathological roles in tumor progression remain to be clarified. It was recently found that the most N-terminal, domain V (dV) of γ2 chain has activities to bind CD44 and stimulate tumor cell migration and vascular permeability. In the present study, we prepared a mAb recognizing γ2 dV. Immunoblotting with this antibody, for the first time, showed that proteolytic fragments containing dV in a range of 15-80 kDa were highly produced in various human cancer cell lines and lung cancer tissues. In immunohistochemistry of adenocarcinomas and squamous cell carcinomas of the lung, this antibody immunostained the cytoplasm of invasive tumor cells and adjacent stroma much more strongly than a widely used antibody recognizing the C-terminal core part of the processed γ2 chain. This suggests that the dV fragments are highly accumulated in tumor cells and stroma compared to the processed γ2 protein. The strong tumor cell staining with the dV antibody correlated with the tumor malignancy grade. We also found that the laminin β3 and α3 chains were frequently overexpressed in tumor cells and tumor stroma, respectively. The cytoplasmic dV detection was especially prominent in tumor cells infiltrating stroma, but low in the cells surrounded by basement membranes, suggesting that the active tumor-stroma interaction is critical for the aberrant γ2 expression. The present study suggests important roles of laminin γ2 N-terminal fragments in tumor progression.

Objectives Low-frequency epidermal growth factor receptor (EGFR) T790M mutation could be detected by ultrasensitive methods in EGFR tyrosine kinase inhibitor (TKI)-naive non-small cell lung cancer (NSCLC). However, the impact of pretreatment T790M (preT790M) on the efficacy of EGFR-TKIs and on resistance remains unclear. Materials and methods Two independent cohorts consisting of advanced EGFR-mutated NSCLC patients treated with first-line EGFR-TKIs, a derivation cohort that started treatment between August 2013 and July 2016 (cohort A, n = 44) and a validation cohort between August 2016 and December 2017 (cohort B, n = 22), were examined in this study. Among these, 28 patients underwent re-biopsy at disease progression. DNAs from pretreatment tumor biopsy samples and re-biopsy samples were assessed to detect T790M by the Cobas EGFR Mutation Test v2 (Cobas) and for quantitating T790M by droplet digital polymerase chain reaction (ddPCR). Results Detection rates of preT790M were 40.9% (18/44) in cohort A and 45.5% (10/22) in cohort B by ddPCR, and none by Cobas. A cutoff value of 0.3% for dividing into high- vs. low-preT790M allele frequency was determined by receiver operating characteristic curve analysis in cohort A. Progression-free survival (PFS) was significantly shorter in the high- preT790M group (n = 12) than in the low-preT790M (n = 6) and negative (n = 26) groups (combined low-preT790M) (median: 6.9 vs. 13.8 months, P = 0.00073). These observations were validated in cohort B [median: 6.2 (n = 5) vs. 15.3 months (n = 17), P = 0.0029]. In 28 paired biopsies, Cobas detected post-progression T790M in 60% (3/5) of the high-preT790M, in 57% (4/7) of the low-preT790M, and in 56% (9/16) of the negative-preT790M groups. Conclusion EGFR-mutated NSCLC with high preT790M had significantly shorter PFS on EGFR-TKIs. However, preT790M abundance may not necessarily confer post-TKI T790M resistance.

e21048 Background: The cGAS/STING pathway is an innate immune pathway that promotes cytokine production in response to cytoplasmic DNA, and its activation is important for the induction of anti-tumor immunity. However, predictivity of cGAS/STING tumor expression on the efficacy of PD-1/L1 inhibitors and subsequent immune responses (e.g., changes in serum cytokines) remain to be elucidated. Non-small cell lung cancer with PD-L1 tumor proportion score (TPS) of 50% or higher respond well to immune checkpoint inhibitor (ICI) monotherapy, but there is still a poor response group among them, and the extraction of such patients is an urgent issue. Methods: This is a post hoc analysis of prospective biomarker study, which enrolled 106 patients with advanced non-small cell lung cancer (NSCLC) who were treated with ICI monotherapy between December 2015 and September 2018. We investigated in 68 patients with preserved evaluable tissue samples taken before start of ICI treatment. cGAS, STING, and PD-L1 expression in tumors were stained by immunohistochemistry. cGAS and STING were evaluated by H-score. Using peripheral blood which was collected by the observational study, 41 serum proteins at the time of PD-1/L1 inhibitors initiation and in 4 – 6 weeks later were quantified. Results: The median cGAS and STING H-SCORE were 220 (5 – 300) and 190 (0 – 300), respectively. There were no differences in cGAS or STING H-SCORE between PD-L1 high (TPS ≥50) and low (TPS < 50) groups ( p= 0.990 and 0.283). Cases were divided into two groups according to median of the H-SCORE, respectively, and compared. Unexpectedly, cGAS high (H-SCORE ≥220) patients showed significantly shorter progression free survival of ICI when PD-L1 TPS ≥50 (median progression free survival (PFS); 143 days vs. not reached, p = 0.028) and progression free rate at 18 months was 7 and 53%, while no association was observed when PD-L1 TPS < 50 (median PFS; 47 vs. 61 days, p= 0.798). STING tumor expression was not associated with PFS regardless of PD-L1 TPS. In cytokine analysis, cGAS high was associated with significantly higher serum concentrations of TGF-β1 and β2 before ICI initiation (47.5 vs. 22.3hg/l, p= 0.023; 2118 vs. 882rg/ml, p= 0.037), and H-SCORE of cGAS, not STING, were significantly correlated with TGF-β1 and β2 basal levels (r = 0.447, p= 0.009; r = 0.373, p= 0.033). Analysis about fold change from baseline to 4 – 6 weeks later of 41 cytokines revealed that leptin significantly increased in cGAS high tumor while no difference was seen in all of cytokines between STING high and low tumor. Conclusions: Tumor expression (H-Score) of cGAS, not STING, is a candidate for predictor of poor response to ICI monotherapy in NSCLC with PD-L1 high (TPS ≥50). cGAS tumor expression may be associated with TGF-β producing, immune-suppressive tumor microenvironment in NSCLC. Clinical trial information: UMIN000024414.

Background: Multiple primary lung cancers (MPLC) are reported to occur in 0.2-20 % of all primary lung cancer cases. MPLC are considered to harbor various genetic profiling patterns among the tumors even within the same individuals, though they developed from the same occupational and environmental exposure. In this study, we performed paired mutational analyses to clarify genetic mutations in MPLC and its relationship with environmental factors. Materials and methods: We obtained 38 surgical specimens from 17 patients who were pathologically diagnosed as MPLC and underwent surgery at Osaka City University Hospital between October 2007 and March 2019. In addition to the clinicopathological features, the patients' age, sex, smoking history, body mass index, radiologic features and surgical procedures were reviewed. Four of the 17 MPLC patients had metachronous lesions, whereas 13 patients only had synchronous lesions. For genetic profiling of MPLC patients, the extracted DNAs were deep-sequenced on Ion S5 sequencer (Thermo Fisher) for somatic mutations in 409 cancer-associated genes (Ion AmpliSeq Comprehensive Cancer Panel). The study was approved by institutional review board of Osaka City University Hospital and written informed consent was obtained from all of the patients. For statistically analyses, we assumed genetic mutations occurred by chance and calculated the frequency of concomitant mutations in multiple tumors within the same individuals based on a previous study (JME study; Kawaguchi T et al, J Clin Oncol 2016). Comparing the assumed frequency of mutations with the actual data, statistically analyses whether mutations would occur concomitantly or randomly were performed using an exact test. Results: Deep sequencing was successfully performed in all of the specimens with the mean coverage of 823. Results from comprehensive genetic analyses suggested that the mutation profiles differed among the tumors within the same individuals, whereas EGFR, KRAS, TP53, and PARP1 mutations were concomitantly detected in multiple tumors within the same individuals. In this study, the frequency of EGFR mutations was significantly higher in never or light -smokers and females. The occurrence of concomitant EGFR or KRAS mutations in multiple tumors within the same individuals was significantly more frequent than expected by chance (EGFR, P = .0023; KRAS, P = .0049), suggesting environmental factors play more role in EGFR and KRAS mutations than the other mutations including TP53 which occurred more randomly in individual tumors. Concomitant EGFR or KRAS mutations were particularly prominent in never or light-smokers and males. Conclusions: Results from our MPLC study confirmed that the development of EGFR or KRAS-mutated tumors were strongly related to environmental factors such as sex and smoking history, and suggested that the other mutations may occur randomly. Citation Format: Motohiro Izumi, Jun Oyanagi, Kenji Sawa, Mitsuru Fukui, Koichi Ogawa, Yoshiya Matsumoto, Yoko Tani, Tomohiro Suzumura, Tetsuya Watanabe, Hiroyasu Kaneda, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Yasuhiro Koh. Mutational landscape of multiple primary lung cancers and its correlation with environmental factors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2333.

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 novolumab 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, Koichi Sato, Shunsuke Teraoka, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Predictive significance of serum proteins on clinical outcome in advanced non-small-cell lung cancer patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4305.

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

Cancer-associated fibroblasts (CAFs) play critical roles in the tumor progression. However, it remains unclear how cancer cells migrate in the three-dimensional (3D) matrix of cancer tissues and how CAFs support the cancer invasion. Here we propose a novel mechanism of fibroblast-dependent cancer cell invasion in the 3D collagen matrix. Human cancer cell lines from the pancreas (Panc-1), lung (A549) and some other organs actively adhered to normal fibroblasts and primary lung CAFs in cultures. To show its significance in tumor invasion, we designed a new invasion assay in which homogeneous microspheroids consisting of cancer cells and fibroblasts were embedded into collagen gel. Time-lapse experiments showed that cancer cells adhered to and quickly migrated on the long protrusions of fibroblasts in the 3D collagen matrix. Fibroblast-free cancer cells poorly invaded the matrix. Experiments with function-blocking antibodies, siRNAs, and immunocytochemistry demonstrated that cancer cells adhered to fibroblasts through integrin α5β1-mediated binding to fibronectin on the surface of fibroblasts. Immunochemical analyses of the co-cultures and lung cancers suggested that cancer cells could acquire the migratory force by the fibronectin/integrin signaling. Our results also revealed that the fibroblast-bound fibronectin was a preferential substrate for cancer cells to migrate in the collagen matrix.

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.

Background: Involvement of tumor immune microenvironment including programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) in biological properties of EGFR-mutant non-small cell lung cancer (NSCLC) remains elusive. Correlation of EGFR mutational status and PD-L1 expression and its impact on anti-PD-1/PD-L1 therapies have been intensively investigated but the relationship between pretreatment EGFR T790M (preT790M) and PD-L1 status has not yet been tackled. We have previously reported that EGFR-TKI-naïve patients with high preT790M had significantly shorter progression-free survival (PFS) upon EGFR-TKI treatment. In this study we investigated the association between preT790M status of tumor and immune microenvironment and its impact on efficacy of EGFR-TKI. Materials and methods: We had previously assessed preT790M by droplet digital PCR in 44 advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016. For the current study, tumor proportion score (TPS) for PD-L1 and PD-L2 expression was assessed immunohistochemically using 28-8 antibody and D7U8C antibody, respectively. CD8-positive tumor-infiltrating lymphocytes (TILs) in tumor specimens were also evaluated. Results: PD-L1/PD-L2 TPS and CD8-positive TILs were evaluable in 39 of 44 pretreatment tumor specimens. PD-L1 TPS of ≥50%, 1-49% and Conclusion: Results of our study suggest that there may exist the subtypes in EGFR-mutated NSCLC according to PD-L1 status and tumor immune microenvironment. Differential efficacy of EGFR-TKIs based on these subtypes further implies the potential impact of PD-L1 status and tumor immune microenvironment in EGFRblockade along with preT790M. Citation Format: Yoshiya Matsumoto, Kenji Sawa, Mitsuru Fukui, Jun Oyanagi, Motohiro Izumi, Koichi Ogawa, Tomohiro Suzumura, Tetsuya Watanabe, Hiroyasu Kaneda, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Yasuhiro Koh. Association between low-frequency pretreatment EGFR T790M mutation and tumor immune microenvironment in patients with EGFR-mutated non-small cell lung cancer [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 4906.

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: T790M mutation in EGFR accounts for nearly 50% of the acquired mechanism of resistance to EGFR-tyrosine kinase inhibitors (TKIs). Previous studies suggested that EGFR T790M mutation was also detected in a considerable number of EGFR-TKI-naïve non-small-cell lung cancer (NSCLC) patients utilizing ultrasensitive detection methods such as droplet digital PCR (ddPCR). Here we investigated the significance of low-frequency pretreatment EGFR T790M mutation (preT790M) and its association with cancer molecular heterogeneity in the efficacy of EGFR-TKIs. Materials and methods: Fifty-two advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016 were enrolled in the study. DNAs from tumor biopsies at diagnosis were available from 44 patients for detecting preT790M by the cobas® EGFR Mutation Test v2 (cobas) and ddPCR (RainDance Technologies) and those from 33 were available for assessing the actionable mutations in 50 genes by next-generation sequencing (NGS). NGS was performed on the Ion PGM using Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Paired biopsies before EGFR-TKIs treatment and at disease progression (PD) were obtained from 15 patients to assess the T790M mutation by cobas. Results: The overall detection rate of preT790M by ddPCR was 40.9% (18/44), while not detected in any case by cobas. The median progression free survival (mPFS) was 10.0 months for the patients with preT790M and 13.5 months for those without preT790M (p=0.288), respectively. When divided into 3 categories based on the frequency of preT790M such as high with T790M allele frequency (AF) >0.3%, low with AF ≤0.3% and undetected, 12 patients with high AF had a relatively shorter mPFS than 6 with low AF (p=0.090) and 26 without detectable preT790M (p=0.046) (7.7 vs 17.1 vs 13.5 months, respectively). NGS revealed 18 additional coexisting actionable mutations in 15 out of 33 patients: TP53 (n=7), PIK3CA (n=5), CTNNB1 (n=3) and uncommon EGFR (n=3). There was a trend for patients with higher preT790M AF to harbor less additional coexisting mutations (27% with high AF, 60% with low AF and 53% without preT790M, p=0.320). In 15 paired biopsies, T790M mutation was detected in 60% (9/15) at PD by cobas in the clinical setting; 67% (2/3) with high AF, 50% (1/2) with low AF and 60% (6/10) without detectable preT790M, suggesting T790M mutation after first-line EGFR-TKIs developed from both clonal selection and secondary acquisition models. Conclusion: Results of our study indicated that EGFR-mutated NSCLCs with higher AF preT790M >0.3% had significantly shorter duration of response to EGFR-TKIs. In addition, the AF of preT790M in EGFR-mutated NSCLC may be associated with coexisting actionable mutation load potentially affecting the efficacy of EGFR-TKIs. Citation Format: Yoshiya Matsumoto, Kenji Sawa, Jun Oyanagi, Mitsuru Fukui, Naoki Yoshimoto, Tomohiro Suzumura, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Kazuto Hirata, Yasuhiro Koh. Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors [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 2613.

Laminin gamma2 chain is a subunit of the heterotrimeric basement membrane protein laminin-332 (alpha3beta3gamma2). The gamma2 chain is highly expressed by human cancers at the invasion fronts and this expression correlates with poor prognosis of the cancers. Our previous study showed that the gamma2 chain is expressed as a monomer form in invading carcinoma cells. However, the role of the gamma2 protein in tumor invasion remains unknown. Here, we demonstrate that the monomeric gamma2 chain promotes invasive growth of human cancer cells in vivo. First, we analyzed regulatory factors for the gamma2 chain expression using 2 gastric carcinoma cell lines. It was found that tumor necrosis factor-alpha, by itself or in a combination with transforming growth factor-beta1, strongly induced the secretion of the monomeric gamma2 chain. In addition, epidermal growth factor families appeared to function as the gamma2 chain inducers in human cancers. Next, we established T-24 bladder carcinoma cell lines expressing the full-length or the short arm of the laminin gamma2 chain. When these cell lines were i.p. injected into nude mice, they produced larger tumors in the abdominal cavity and showed much stronger invasive growth onto the diaphragms than the control cell line. The gamma2-expressing T-24 cells often produced ascites fluid, but scarcely the control cells. In culture, the gamma2-expressing cells migrated through Matrigel more efficiently than the control cells. These findings imply that the gamma2 monomer is induced in human cancers by inflammatory and stromal cytokines and promotes their invasive growth in vivo.

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.

Laminin γ2 (Lmγ2) chain, a subunit of laminin-332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lmγ2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor-promoting activity of Lmγ2 remains unknown. Here we investigated the interaction between Lmγ2 and vascular endothelial cells. When treated with an N-terminal proteolytic fragment of γ2 (γ2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lmγ2 or treatment with γ2pf stimulated T-24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, γ2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, γ2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, γ2pf induced delocalization of VE-cadherin and β-catenin from the intercellular junction. As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat. These data suggest that the interaction between γ2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lmγ2 seem to support the aberrant growth of cancer cells.

Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the γ2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the γ2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the γ2 chain.

Epithelial-mesenchymal transition (EMT) is a crucial event in tumor invasion and metastasis. However, most of past EMT studies have been conducted in the conventional two-dimensional (2D) monolayer culture. Therefore, it remains unclear what invasive phenotypes are acquired by EMT-induced cancer cells. To address this point, we attempted to characterize EMT cells in more physiological, three-dimensional (3D) collagen gel culture. EMT was induced by treating three human carcinoma cell lines (A549, Panc-1 and MKN-1) with TGF-ß. The TGF-ß treatment stimulated these cells to overexpress the invasion markers laminin γ2 and MT1-MMP in 2D culture, in addition to the induction of well-known morphological change and EMT marker expression. EMT induction enhanced cell motility and adhesiveness to fibronectin and collagen in 2D culture. Although EMT cells showed comparable cell growth to control cells in 2D culture, their growth rates were extremely suppressed in soft agar and collagen gel cultures. Most characteristically, EMT-induced cancer cells commonly and markedly extended invasive protrusions in collagen gel. These protrusions were mainly supported by microtubules rather than actin cytoskeleton. Snail-introduced, stable EMT cells showed similar protrusions in 3D conditions without TGF-ß. Moreover, these protrusions were suppressed by colchicine or inhibitors of heat shock protein 90 (HSP-90) and protein phosphatase 2A. However, MMP inhibitors did not suppress the protrusion formation. These data suggest that EMT enhances tumor cell infiltration into interstitial stroma by extending microtubule-based protrusions and suppressing cell growth. The elevated cell adhesion to fibronectin and collagen and high cell motility also seem important for the tumor invasion.

Epithelial tumor cells often change their epithelial phenotype to mesenchymal one when they invade into surrounding stromal tissue. This change, called epithelial-mesenchymal transition (EMT), is thought to be a crucial event in tumor progression. EMT is known to be regulated by environmental factors. However, it is unclear how EMT contributes to invasive growth of tumor cells. To address this question, we characterized EMT-induced tumor cells under various conditions in vitro. EMT was induced in three human carcinoma cell lines of the lung, pancreas and stomach by treating with TGF-beta and/or TNF-alpha. The EMT treatment of these cell lines induced expression of the invasion marker laminin gamma2 chain and MT1-MMP, in addition to other well-known phenotypes such as the morphological change and vimentin expression. EMT induction enhanced cell motility in monolayer culture. Although EMT cells showed comparable cell growth with the control cells in monolayer culture, their growth rates were extremely suppressed in collagen gel and soft agar culture conditions. Most characteristically, EMT-induced tumor cells commonly and markedly extended invadopodia-like protrusions in collagen gel. Such protrusion formation in collagen gel was effectively blocked by the TGF-beta/Smad pathway inhibitor SB431542 but scarcely suppressed by the synthetic and natural MMP inhibitors, TAPI-1 and TIMP-2, respectively. These data suggest that EMT may enhance interstitial tumor infiltration without cell growth by promoting prominent invadopodia formation and cell motility. The TGF-beta-induced cytoskeletal change seems to play a more critical role than MMP expression in the tumor invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2011-3360