Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors.

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8+ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8+ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8+ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8+ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8+ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8+ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.

Combination of Skeletal Muscle Mass and Density Predicts Postoperative Complications and Survival of Patients With Non-Small Cell Lung Cancer.

BACKGROUND: Few studies have assessed the comprehensive skeletal muscle depletion associated with loss of muscle quantity (sarcopenia) and reduced muscle quality in cancer patients. This study aimed to clarify the impact of skeletal muscle depletion on outcomes after non-small cell lung cancer surgery. METHODS: Data for 341 patients with pathologic stages 1 to 3A non-small cell lung cancer who underwent lobectomy and mediastinal lymph node dissection from 2009 to 2013 were retrospectively reviewed. The integrative pectoralis muscle index (IPMI) was assessed by multiplying the normalized pectoralis muscle area (area/body mass index) and mean radiodensity on chest images. Postoperative outcomes were compared among sex-specific quartiles of IPMI. The trend of continuous and categorical variables was analyzed using the Jonckheere-Terpstra test and the Cochrane-Armitage test, respectively. RESULTS: Respiratory strength declined with decreasing quartiles of IPMI (P < 0.001). The risk of major complications escalated with the decrease of IPMI among four quartiles (7.1 %, 16.7 %, 18.4 %, and 22.4 %; P = 0.008). The hospital stay was prolonged for patients with reduced IPMI (P = 0.001). Patients in the lowest and highest quartiles had the worst and best 5-year overall survival, respectively, compared with those in the two intermediate quartiles of IPMI (67.0 %, 87.9 %, and 81.2 %, respectively; P=0.001). Multivariate analysis identified the lowest quartile of IPMI as an independent poor prognostic factor (hazard ratio, 1.88; 95 % confidence interval, 1.11-3.19; P = 0.020). CONCLUSION: Comprehensive skeletal muscle profiling, including morphometric mass and componential density on chest imaging, has the potential to refine risk stratification and prognostication in non-small cell lung cancer.

A novel NGS-based diagnostic algorithm for classifying multifocal lung adenocarcinomas in pN0M0 patients.

The classification of multifocal lung adenocarcinomas (MLAs), including multiple primary lung adenocarcinomas (MPLAs) and intrapulmonary metastases (IPMs), has great clinical significance in staging and treatment determination. However, the application of molecular approaches in pN0M0 MLA diagnosis has not been well investigated. Here, we performed next-generation sequencing (NGS) analysis in 45 pN0M0 MLA patients (101 lesion pairs) who were initially diagnosed as having MPLA by comprehensive histologic assessment (CHA). Five additional patients with intrathoracic metastases were used as positive controls, while 197 patients with unifocal lung adenocarcinomas (425 random lesion pairs) were used as negative controls. By utilizing a predefined NGS criterion, all IPMs in the positive control group could be accurately classified, whereas 13 lesion pairs (3.1%) in the negative control cohort were misdiagnosed as IPMs. Additionally, 14 IPM lesion pairs were diagnosed in the study group, with at least 7 misdiagnoses. We thus developed a refined algorithm, incorporating both NGS and histologic results, that could correctly diagnose all the known MPLAs and IPMs. In particular, all IPMs identified by the refined algorithm were diagnosed to be IPMs or suspected IPMs by CHA reassessment. The refined algorithm-diagnosed MPLAs patients also had significantly better progression-free survival than the refined algorithm-diagnosed IPMs (p < 0.0001), which is superior to conventional NGS or CHA diagnoses. Overall, we developed an NGS-based algorithm that could accurately distinguish IPMs from MPLAs in MLA patients. Our results demonstrate a promising clinical utility of NGS to complement traditional CHA-based MLA diagnosis and help determine patient staging and treatment.

Respiratory strength and pectoralis muscle mass as measures of sarcopenia: Relation to outcomes in resected non-small cell lung cancer.

OBJECTIVES: Physical biomarkers to stratify patients with lung cancer into subtypes predictive of outcome beyond tumor-related characteristics are underexplored. This study was designed to investigate the clinical utility of preoperative sarcopenia based on respiratory strength and pectoralis muscle mass to predict the risk of death. METHODS: This retrospective study included 346 consecutive patients undergoing curative-intent resection of non-small cell lung cancer from 2009 to 2013. Respiratory strength and muscle mass were assessed by peak expiratory flow rate and pectoralis muscle index (pectoralis muscle area/body mass index) using preoperative spirometry and chest axial images, respectively. Sarcopenia cutoff points were defined by gender-specific medians of peak expiratory flow rates and pectoralis muscle indices. Survival was compared between patients with sarcopenia and patients without. RESULTS: Sarcopenia was present in 98 patients (28.3%) and was significantly associated with advancing age (P < .001). Patients with sarcopenia exhibited worse 5-year overall survival compared with patients without sarcopenia (69.9% vs 87.2%, P < .001). Multivariate analysis revealed that sarcopenia was an independent adverse prognostic factor (hazard ratio, 1.88; 95% confidence interval, 1.09-3.24; P = .023) after adjustment for gender, age, smoking status, coronary heart disease, diffusing capacity for carbon monoxide, neutrophil-to-lymphocyte ratio, albumin, histologic type, and pathologic stage. CONCLUSIONS: Preoperative sarcopenia as identified by the criteria of low respiratory strength and reduced pectoralis muscle mass is significantly associated with poor overall survival. This may help to develop more individualized management strategies and optimize longitudinal care for patients.

Immuno-genomic profiling of biopsy specimens predicts neoadjuvant chemotherapy response in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is primarily treated with platinum-based neoadjuvant chemotherapy (NAC). Some ESCCs respond well to NAC. However, biomarkers to predict NAC sensitivity and their response mechanism in ESCC remain unclear. We perform whole-genome sequencing and RNA sequencing analysis of 141 ESCC biopsy specimens before NAC treatment to generate a machine-learning-based diagnostic model to predict NAC reactivity in ESCC and analyzed the association between immunogenomic features and NAC response. Neutrophil infiltration may play an important role in ESCC response to NAC. We also demonstrate that specific copy-number alterations and copy-number signatures in the ESCC genome are significantly associated with NAC response. The interactions between the tumor genome and immune features of ESCC are likely to be a good indicator of therapeutic capability and a therapeutic target for ESCC, and machine learning prediction for NAC response is useful.

Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1.

An important factor associated with primary resistance to immune-checkpoint therapies (ICT) is a "cold" tumor microenvironment (TME), characterized by the absence of T cell infiltration and a non-inflammatory milieu. Whole-exome and RNA sequencing to predict neoantigen expression was performed on the LLC1 cell line which forms "cold" tumors in mice. Dendritic cell (DC)-based vaccination strategies were developed using candidate neoantigen long peptides (LPs). A total of 2536 missense mutations were identified in LLC1 and of 132 candidate neoantigen short peptides, 25 were found to induce CD8+ T cell responses. However, they failed to inhibit LLC1 growth when incorporated into a cancer vaccine. In contrast, DCs pulsed with LPs induced CD4+ and CD8+ T cell responses and one of them, designated L82, delayed LLC1 growth in vivo. By RNA-Seq, CD38 was highly expressed by LLC1 tumor cells and, therefore, anti-CD38 antibody treatment was combined with L82-pulsed DC vaccination. This combination effectively suppressed tumor growth via a mechanism relying on decreased regulatory T cells in the tumor. This study demonstrated that an appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant "cold" tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells.

Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy.

To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

Advances in personalized neoantigen vaccines for cancer immunotherapy.

Immunotherapy, which targets T cell inhibitory receptors (immune checkpoints), is now being widely used to treat a variety of types of cancer combined with surgery, chemotherapy, or radiotherapy. However, immune checkpoint inhibitors are highly dependent on the ability to present diverse tumor antigens to T cells. Neoantigens, arising from somatic mutations and specifically targeting tumor cells, have the potential to stimulate a highly specific immune anti-tumor response. Technological advances such as genomic sequencing and bioinformatics algorithms for epitope prediction have directly facilitated the development of neoantigen vaccines for individual cancers. Currently, several preclinical studies and early clinical trials using neoantigen in combination with checkpoint inhibitors have resulted in robust T cell responses and antitumor action. In the future, efforts will be made to optimize effective personalized neoantigen vaccines targeting individual tumors and to elucidate the immune mechanisms underlying tumor evolution.

Prognostic significance of low pectoralis muscle mass on preoperative chest computed tomography in localized non-small cell lung cancer after curative-intent surgery.

OBJECTIVES: The impact of sarcopenia on the outcome in patients following resection of non-small cell lung cancer is yet to be fully determined. This study aimed to evaluate the clinical utility of a computed tomography-based pectoralis muscle assessment, which reflects sarcopenia, to predict the risk of postoperative outcomes. MATERIALS AND METHODS: This retrospective study included 347 consecutive patients undergoing curative-intent resection of non-small cell lung cancer from 2009 to 2013. The pectoralis muscle index (pectoralis muscle area/body mass index) was assessed at the level of the fourth thoracic vertebra on chest axial images. The primary outcomes were compared between the lowest gender-specific quintile (sarcopenia) and the other quintiles according to the index. The prognostic significance of low pectoralis muscle index was calculated by the Cox proportional hazards regression model. A propensity score matching analysis was performed to adjust the differences in clinical characteristics. RESULTS: Sixty-nine patients were identified with sarcopenia according to the lowest gender-specific quintile of pectoralis muscle index. Patients with sarcopenia exhibited worse 5-year overall survival rate compared with patients without sarcopenia (64.2 % vs. 86.7 %, P < 0.001). Even in stage I non-small cell lung cancer, the rate of 5-year overall survival in the sarcopenia group was lower than that in the non-sarcopenia group (74.2 % vs. 92.4 %, P = 0.001). Multivariate analysis revealed that low pectoralis muscle index was independently associated with adverse overall survival (hazard ratio: 2.09, 95 % confidence interval: 1.20-3.62, P = 0.009). After propensity score matching, the prognostic impact of sarcopenia based on low pectoralis muscle index was also robust for overall survival (hazard ratio: 3.23, 95 % confidence interval: 1.38-7.60, P = 0.007). CONCLUSIONS: Low pectoralis muscle index was significantly associated with poor long-term outcomes in patients with localized non-small cell lung cancer after curative surgery. This may help assist preoperative risk stratification and longitudinal management after surgery.

Outcomes of marginal donors for lung transplantation after ex vivo lung perfusion: A systematic review and meta-analysis.

OBJECTIVE: Ex vivo lung perfusion (EVLP) is reportedly a useful strategy that permits marginal donor lungs to be evaluated and reconditioned for successful lung transplantation (LTx). This systematic review and meta-analysis was performed to evaluate the outcomes of EVLP conducted for marginal donor lungs. METHODS: We searched PubMed, the Cochrane Library, and Embase to select studies describing the results of LTx following EVLP for marginal donor lungs compared with standard LTx without EVLP. We performed a meta-analysis to examine donor baseline characteristics, recipient baseline characteristics, and postoperative outcomes. RESULTS: Of 1380 studies, 8 studies involving 1191 patients met the inclusion criteria. Compared with the non-EVLP group (ie, standard LTx without EVLP), the EVLP group (ie, EVLP of marginal donors following LTx) had similar donor age and sex and recipient baseline age, sex, body mass index, bridge by ventilator/extracorporeal life support/extracorporeal membrane oxygenation, and rate of double LTx but more abnormal donor lung radiographs (P = .0002), a higher smoking history rate (P = .03), and worse donor arterial oxygen tension/inspired oxygen fraction (P < .00001). However, there were no significant differences in outcomes between the EVLP and non-EVLP groups with respect to the length of postoperative intubation, postoperative extracorporeal life support/extracorporeal membrane oxygenation use, length of intensive care unit stay, length of hospital stay, 72-hour primary graft dysfunction of grade 3, 30-day survival, or 1-year survival (all P values > .05). CONCLUSIONS: Posttransplant outcomes were similar between EVLP-treated LTx and standard LTx without EVLP, although the quality of donor lungs was worse with EVLP-treated LTx.

Deep immunophenotyping at the single-cell level identifies a combination of anti-IL-17 and checkpoint blockade as an effective treatment in a preclinical model of data-guided personalized immunotherapy.

BACKGROUND: Although immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each patient's cancer-immune interactions in the tumor microenvironment (TME) to prevent suppression of antitumor immune responses. To demonstrate the feasibility of this kind of approach, we developed combination therapy for a preclinical model guided by deep immunophenotyping of the TME. METHODS: Gastric cancer cell lines YTN2 and YTN16 were subcutaneously inoculated into C57BL/6 mice. YTN2 spontaneously regresses, while YTN16 grows progressively. Bulk RNA-Seq, single-cell RNA-Seq (scRNA-Seq) and flow cytometry were performed to investigate the immunological differences in the TME of these tumors. RESULTS: Bulk RNA-Seq demonstrated that YTN16 tumor cells produced CCL20 and that CD8+ T cell responses were impaired in these tumors relative to YTN2. We have developed a vertical flow array chip (VFAC) for targeted scRNA-Seq to identify unique subtypes of T cells by employing a panel of genes reflecting T cell phenotypes and functions. CD8+ T cell dysfunction (cytotoxicity, proliferation and the recruitment of interleukin-17 (IL-17)-producing cells into YTN16 tumors) was identified by targeted scRNA-Seq. The presence of IL-17-producing T cells in YTN16 tumors was confirmed by flow cytometry, which also revealed neutrophil infiltration. IL-17 blockade suppressed YTN16 tumor growth, while tumors were rejected by the combination of anti-IL-17 and anti-PD-1 (Programmed cell death protein 1) mAb treatment. Reduced neutrophil activation and enhanced expansion of neoantigen-specific CD8+ T cells were observed in tumors of the mice receiving the combination therapy. CONCLUSIONS: Deep phenotyping of YTN16 tumors identified a sequence of events on the axis CCL20->IL-17-producing cells->IL-17-neutrophil-angiogenesis->suppression of neoantigen-specific CD8+ T cells which was responsible for the lack of tumor rejection. IL-17 blockade together with anti-PD-1 mAb therapy eradicated these YTN16 tumors. Thus, the deep immunological phenotyping can guide immunotherapy for the tailored treatment of each individual patient's tumor.

Tumor location may affect the clinicopathological features and prognosis of thymomas.

BACKGROUND: The current staging systems do not consider the tumor location of thymomas, and its clinical relevance is poorly understood. This study aimed to evaluate the impact of tumor location on the clinicopathological features and prognosis of thymomas. METHODS: We performed a retrospective review of patients at our institution who underwent an extended thymectomy for a thymoma from 1976 to 2015. The tumor location was classified as either the superior or inferior mediastinum based on the maximum tumor diameter. The clinicopathological characteristics of the thymoma were also evaluated. Kaplan-Meier estimates and Cox proportional hazards models were used to analyze the survival outcomes and risk factors for recurrence. RESULTS: A total of 194 patients with thymoma were eligible for this study. Compared with the inferior mediastinum group (n = 167), the superior mediastinum group (n = 27) had a higher frequency of myasthenia gravis (MG), advanced Masaoka-Koga staging, disease progression and recurrence (P < 0.05). The Kaplan-Meier analysis demonstrated thymomas in the superior mediastinum had worse survival outcomes that included overall survival, progression-free survival and disease-free survival (P < 0.05). The multivariate analysis showed tumor location was an independent prognostic factor for all the survival outcomes (P < 0.05). Furthermore, the tumor location (P = 0.004) and Masaoka-Koga stage (P < 0.001) were the only two independent risk factors for recurrence in the multivariate analysis. CONCLUSIONS: The clinicopathological features of thymomas on MG, Masaoka-Koga staging, disease progression, and recurrence were different between locations of superior and inferior mediastinum locations. Thymomas in the superior mediastinum tended to be associated with worse survival and increased recurrence.

Low truncal muscle area on chest computed tomography: a poor prognostic factor for the cure of early-stage non-small-cell lung cancer†.

OBJECTIVES: Depletion in skeletal muscle is closely associated with limited physical ability and high mortality. In this study, we evaluated the prognostic significance of skeletal muscle depletion in patients with early-stage non-small-cell lung cancer. METHODS: A retrospective analysis of patients with pathological stages I-II lung cancer, who underwent curative resection between 2009 and 2013, was conducted. The truncal muscle index (TMI) (area/height2) at the first lumbar vertebral level was measured by preoperative axial computed tomography. Overall survival and recurrence-free survival were compared between the lowest gender-specific quartile of the TMI and the other quartiles. RESULTS: A total of 314 subjects were included in the study. The cumulative 5-year recurrence-free and overall survival rates were significantly shorter in patients with lower TMIs (69% vs 83.5%, P = 0.028; 64.8% vs 80.1%, P = 0.003, respectively). In multivariable models, the TMI was identified as an independent prognostic factor for overall survival (P = 0.017, hazard ratio 1.84, 95% confidence interval 1.12-3.05), after adjusting for age, gender, preoperative serum albumin, carcinoembryonic antigen, neutrophil to lymphocyte ratio and pathological stage. CONCLUSIONS: A low preoperative TMI was associated with a poor postoperative outcome in patients with early-stage non-small-cell lung cancer. This factor may be included in the preoperative assessment of patients, for whom surgical intervention is considered.

Idiopathic pulmonary fibrosis in East Asian.

Idiopathic pulmonary fibrosis (IPF) is a rare lung disease with a prognosis that can be worse than that of many cancers. Recent studies have improved our understanding of IPF and new treatment options have become available. However, most studies are conducted predominantly in Western countries while few are conducted in East Asian countries. The distribution, effectiveness of treatment, and prognosis for IPF differ among Westerners and East Asians, but whether the heterogeneity of IPF in East Asians is the result of ethnic differences and geographic variability is unclear. This study highlights the current prevalence of IPF and its characteristics in the East Asian population and it provides valuable information to understand the current clinical status of patients with IPF in light of recent advances in its diagnosis and treatment.

Role of fibulin-3 in lung cancer: in vivo and in vitro analyses.

Lung cancer was the most commonly diagnosed cancer in 2008 worldwide. The level of fibulin-3 expression was found to be decreased in many cancer types due to aberrant promoter methylation and is correlated with poor survival of patients. However, the role of fibulin-3 and which form of fibulin-3 is expressed in lung cancer cells remain unclear. Therefore, pathologic and functional studies were carried out to determine the role of fibulin-3 in suppressing lung cancer both in vivo and in vitro. In the present study, we found that the levels of fibulin-3 mRNA and protein were lower in cancer tissues than in normal tissues. Downregulation of fibulin-3 mRNA in tumor tissues was associated with an increase in fibulin-3 promoter methylation. Circulating fibulin-3 was significantly associated with tumor progression, survival rate of lung cancer patients, and the number of circulating tumor cells (CTCs). To examine the effects of exogenous expression of fibulin-3 in vitro, lung cancer A549 cells were transfected with the pEGFP-C1-fibulin-3 expression vector. Relative to the untreated cells, fibulin-3-expressing cells exhibited lower proliferation and mobility as determined by MTT and Transwell assays, respectively. To conclude, our results suggest that fibulin-3 negatively modulates the invasiveness of lung cancer cells via regulation of p38-MAPK and MMP-2/9.

Brachial plexus palsy, a rare delayed complication of the Nuss procedure for pectus excavatum: a case report.

We report a rare complication after the Nuss procedure for the correction of pectus excavatum in a 15-year-old adolescent boy. He began to have delayed right brachial plexus injury on the 15th postoperative day. Careful physical check-up revealed a painful and enlarged subaxillary lymph node. He was successfully treated using anti-inflammatory medications and physical therapy.