ADAM9 functions as a transcriptional regulator to drive angiogenesis in esophageal squamous cell carcinoma.

Hypoxia and angiogenesis play key roles in the pathogenesis of esophageal squamous cell carcinoma (ESCC), but regulators linking these two pathways to drive tumor progression remain elusive. Here we provide evidence of ADAM9's novel function in ESCC progression. Increasing expression of ADAM9 was correlated with poor clinical outcomes in ESCC patients. Suppression of ADAM9 function diminished ESCC cell migration and in vivo metastasis in ESCC xenograft mouse models. Using cellular fractionation and imaging, we found a fraction of ADAM9 was present in the nucleus and was uniquely associated with gene loci known to be linked to the angiogenesis pathway demonstrated by genome-wide ChIP-seq. Mechanistically, nuclear ADAM9, triggered by hypoxia-induced translocation, functions as a transcriptional repressor by binding to promoters of genes involved in the negative regulation of angiogenesis, and thereby promotes tumor angiogenesis in plasminogen/plasmin pathway. Moreover, ADAM9 suppresses plasminogen activator inhibitor-1 gene transcription by interacting with its transcription factors at the promoter. Our findings uncover a novel regulatory mechanism of ADAM9 as a transcriptional regulator in angiogenesis and highlight ADAM9 as a promising therapeutic target for ESCC treatment.

Identification of theranostic factors for patients developing metastasis after surgery for early-stage lung adenocarcinoma.

Rationale: Lung adenocarcinoma (LUAD) is an aggressive disease with high propensity of metastasis. Among patients with early-stage disease, more than 30% of them may relapse or develop metastasis. There is an unmet medical need to stratify patients with early-stage LUAD according to their risk of relapse/metastasis to guide preventive or therapeutic approaches. In this study, we identified 4 genes that can serve both therapeutic and diagnostic (theranostic) purposes. Methods: Three independent datasets (GEO, TCGA, and KMPlotter) were used to evaluate gene expression profile of patients with LUAD by unbiased screening approach. Upon significant genes uncovered, functional enrichment analysis was carried out. The predictive power of their expression on patient prognosis were evaluated. Once confirmed their theranostic roles by integrated bioinformatics, we further conducted in vitro and in vivo validation. Results: We found that four genes (ADAM9, MTHFD2, RRM2, and SLC2A1) were associated with poor patient outcomes with an increased hazard ratio in LUAD. Knockdown of them, both separately and simultaneously, suppressed lung cancer cell proliferation and migration ability in vitro and prolonged survival time in metastatic tumor mouse models. Moreover, these four biomarkers were found to be overexpressed in tumor tissues from LUAD patients, and the total immunohistochemical staining scores correlated with poor prognosis. Conclusions: These results suggest that these four identified genes could be theranostic biomarkers for stratifying high-risk patients who develop relapse/metastasis in early-stage LUAD. Developing therapeutic approaches for the four biomarkers may benefit early-stage LUAD patients after surgery.

Targeting positive feedback between BASP1 and EGFR as a therapeutic strategy for lung cancer progression.

Rationale: Brain metastasis in patients with lung cancer is life-threatening. However, the molecular mechanism for this catastrophic disease remains elusive, and few druggable targets are available. Therefore, this study aimed to identify and characterize proteins that could be used as therapeutic targets. Methods: Proteomic analyses were conducted to identify differentially expressed membrane proteins between brain metastatic lung cancer cells and primary lung cancer cells. A neuronal growth-associated protein, brain acid soluble protein 1 (BASP1), was chosen for further investigation. The clinical relevance of BASP1 in lung adenocarcinoma was first assessed. Tyrosine kinase activity assays and in vitro and in vivo functional assays were conducted to explore the oncogenic mechanisms of BASP1. Results: The protein levels of BASP1 were positively associated with tumor progression and poor prognosis in patients with lung adenocarcinoma. Membrane-bound BASP1 increased EGFR signaling and stabilized EGFR proteins by facilitating their escape from the ubiquitin-proteasome pathway. Reciprocally, activation of EGFR recruited more BASP1 to the plasma membrane, generating a positive feedback loop between BASP1 and EGFR. Moreover, the synergistic therapeutic effects of EGFR tyrosine kinase inhibitor and arsenic trioxide led to a reduction in the level of BASP1 protein observed in lung cancer cells with acquired resistance to EGFR inhibitors. Conclusions: The reciprocal interaction between BASP1 and EGFR facilitates EGFR signaling in brain metastatic lung cancer. Targeting the newly identified BASP1-EGFR interaction could open new venues for lung cancer treatment.

Immunological evaluation of a novel HLA-A2 restricted phosphopeptide of tumor associated Antigen, TRAP1, on cancer therapy.

The tumor necrosis factor receptor associated protein 1 (TRAP1) is a mitochondria chaperon protein that has been previously implicated as a target for cancer therapy due to its expression level is linked to tumor progression. In this study, an immunodominant phosphopeptide of TRAP1 was identified from an HLA-A2 gene transfected mouse cancer cell line using mass spectrometry, and a synthetic phosphopeptide was generated to evaluate the potency on cancer immunotherapy. In the transporter associated with antigen processing (TAP) deficient cell, the conjugated phosphate group plays a critical role to enhance the binding affinity of phosphopeptide with HLA-A2 molecule. On the basis of immunological assay, immunization of synthetic phosphopeptide could induce a high frequency of IFN-γ-secreting CD8+ T cells in HLA-A2 transgenic mice, and the stimulated cytotoxic T lymphocytes showed a high target specificity to lysis the epitope-pulsed splenocytes in vivo and the human lung cancer cell in vitro. In a tumor challenge assay, vaccination of the HLA-A2 restricted phosphopeptide appeared to suppress the tumor growth and prolong the survival period of tumor-bearing mice. These results suggest that novel phosphopeptide is naturally presented as a HLA-A2-restricted CTL epitope and capable of being a potential candidate for the development of therapeutic vaccine against high TRAP1-expressing cancers.

Comparing the effects of afatinib with gefitinib or Erlotinib in patients with advanced-stage lung adenocarcinoma harboring non-classical epidermal growth factor receptor mutations.

OBJECTIVE: Approximately 10%-15% patients with epidermal growth factor receptor (EGFR) mutations harbor non-classical mutations. However, the effects of EGFR-tyrosine kinases (TKIs), particularly second-generation EGFR-TKI (afatinib) compared to first-generation EGFR-TKIs (gefitinib/erlotinib), in patients with non-classical EGFR mutations remain unknown. METHODS: We conducted this retrospective study at the China Medical University Hospital (Taichung, Taiwan) from June 2011 to July 2016. Specimens from 1632 patients were tested for EGFR mutations. We surveyed the effectiveness of afatinib and gefitinib/erlotinib in stage IIIb-IV lung adenocarcinoma patients with non-classical EGFR mutations. RESULTS: Fifty-six patients with advanced-stage (stage IIIB-IV) lung adenocarcinoma with non-classical mutations and receiving EGFR-TKI treatment had completed follow-up and were further analyzed. Afatinib versus gefitinib/erlotinib showed that the objective response rates were 62.5% versus 50.0% (p=0.35). Median progression-free survival (PFS) of 11.0 versus 3.6 months (p=0.03), respectively, was observed for the 51 non-classical EGFR mutated (excluding 5 patients with exon 20 insertions) lung adenocarcinomas. Subset analysis showed that PFS curves of afatinib were more easily distinguished in non-classical EGFR mutations lacking a combination with a classical mutation (non-classical with classical complex mutations group: median PFS, 11.0 versus 8.2 months, p=0.19; non-classical mutation alone or in combination with other non-classical mutations group: median PFS, 18.3 versus 2.8 months, p=0.07). CONCLUSIONS: Afatinib may be a first-choice EGFR-TKI for patients with advanced-stage lung adenocarcinomas harboring non-classical mutations.

Diagnostic pitfalls of discriminating lymphoma-associated effusions.

High serum lactate dehydrogenase (LDH) level, immunologic defects, enlarged mediastinal lymph nodes, and frequent hydration and diuresis in lymphoma patients may affect the development of pleural effusion (PE). The study was to assess the clinical utility of "Light criteria" and the "recommended algorithm for investigating PEs" in patients with lymphoma.The characteristics of 126 PEs of lymphoma patients who underwent diagnostic thoracentesis between January 1, 2003, and April 30, 2012, were reviewed. Using Light criteria, 29 (23%) PEs were incorrectly classified. The sensitivity for exudates in Light criteria was 88% and the specificity was only 44%. In 32 transudates, PE LDH correlated with blood LDH concentration (P < 0.001, r = 0.66). Nine transudates were misclassified as exudates (50%; 9/18) just due to PE LDH more than two-thirds the upper limits. Among the 56 bilateral PEs, 33 (59%) were exudates. Ten (63%) polymorphonuclear (PMN)-predominant exudative PEs were malignant. Infective PEs were often mononuclear (67%) rather than PMN predominant.When a patient has lymphoma with either unilateral or bilateral PE, thoracentesis for microbiological testing and cytology is imperative. Carefully clinical correlation in addition to the result from Light criteria and differential cell count is essential for prompt management.

ADAM9 promotes lung cancer metastases to brain by a plasminogen activator-based pathway.

The transmembrane cell adhesion protein ADAM9 has been implicated in cancer cell migration and lung cancer metastasis to the brain, but the underpinning mechanisms are unclear and clinical support has been lacking. Here, we demonstrate that ADAM9 enhances the ability of tissue plasminogen activator (tPA) to cleave and stimulate the function of the promigratory protein CDCP1 to promote lung metastasis. Blocking this mechanism of cancer cell migration prolonged survival in tumor-bearing mice and cooperated with dexamethasone and dasatinib (a dual Src/Abl kinase inhibitor) treatment to enhance cytotoxic treatment. In clinical specimens, high levels of ADAM9 and CDCP1 correlated with poor prognosis and high risk of mortality in patients with lung cancer. Moreover, ADAM9 levels in brain metastases derived from lung tumors were relatively higher than the levels observed in primary lung tumors. Our results show how ADAM9 regulates lung cancer metastasis to the brain by facilitating the tPA-mediated cleavage of CDCP1, with potential implications to target this network as a strategy to prevent or treat brain metastatic disease.

Density features of screened lung tumors in low-dose computed tomography.

RATIONALE AND OBJECTIVES: Using low-dose computed tomography (LDCT), small and heterogeneous lung tumors are detected in screening. The criteria for assessing detected tumors are crucial for determining follow-up or resection strategies. The purpose of this study was to investigate the capacity of density features in differentiating lung tumors. MATERIALS AND METHODS: From July 2008 to December 2011, 48 surgically confirmed tumors (29 malignancies, comprising 17 cases of adenocarcinoma and 12 cases of adenocarcinoma in situ [AdIs], and 19 benignancies, comprising 11 cases of atypical adenomatous hyperplasia [AAH] and eight cases of benign non-AAH) in 38 patients were retrospectively evaluated, indicating that the positive predictive value (PPV) of physicians is 60.4% (29/48). Three types of density features, tumor disappearance rate (TDR), mean, and entropy, were obtained from the CT values of detected tumors. RESULTS: Entropy is capable of differentiating malignancy from benignancy but is limited in differentiating AdIs from benign non-AAH. The combination of entropy and TDR is effective for predicting malignancy with an accuracy of 87.5% (42/48) and a PPV of 89.7% (26/29), improving the PPV of physicians by 29.3%. The combination of entropy and mean adequately clarifies the four pathology groups with an accuracy of 72.9% (35/48). For tumors with a mean below -400 Hounsfield units, the criterion of an entropy larger than 5.4 might be appropriate for diagnosing malignancy. For others, the pathology is either benign non-AAH or adenocarcinoma; adenocarcinoma has a higher entropy than benign non-AAH, with the exception of tuberculoma. CONCLUSIONS: Combining density features enables differentiating heterogeneous lung tumors in LDCT.

Primary pulmonary epithelioid angiosarcoma presenting as a solitary pulmonary nodule on image.

Primary angiosarcoma of lung is a rare condition. Only about 20 cases have appeared in English published reports so far. Its rarity and consequent low index of suspicion makes clinical diagnosis difficult. Pathological diagnosis of the epithelioid variant of pulmonary angiosarcoma is particularly challenging. We report a case of primary pulmonary epithelioid angiosarcoma as a solitary pulmonary nodule in image study in a 41-year-old man with a brief review, to contribute it to the sparse literature on this disease.

A novel HLA-A2-restricted CTL epitope of tumor-associated antigen L6 can inhibit tumor growth in vivo.

Vaccines utilizing cytotoxic T lymphocyte (CTL) epitopes are promising for the treatment of cancer and chronic infectious diseases. Tumor-associated antigen L6 (TAL6) is overexpressed in some epithelial cancer cells. In this report, we detected TAL6 expression in breast cancer tissue using quantitative reverse-transcriptase-polymerase chain reaction. We found that >80% of breast tumor tissue highly expressed TAL6 compared with adjacent normal breast tissue. To identify CTL epitopes from TAL6, we synthesized 18 peptides for HLA-A2-binding assay based on the MHC-binding motif using 4 computer prediction programs. Positive binders identified by ELISA were immunized in HLA-A2 transgenic (A2 Tg) mice. Two peptides, peptide 2 and peptide 5, induced T-cell responses in A2 Tg mice. To confirm whether these peptides could be processed and presented to induce T-cell responses in vivo, A2 Tg mice were immunized with plasmid DNA encoding TAL6. We found that both peptides 2 and 5 stimulated splenocytes from TAL6-immunized mice to secrete interferon-γ. However, only peptide 5 could induce expression of the cytolytic molecule CD107a on CD8+ T cells after immunization. Furthermore, peptide 5-immunized A2 Tg mice could inhibit the growth of TAL6-positive tumors (EL4/TAL6/HLA-A2) in A2 Tg mice but not in wild-type mice. These results demonstrate that the TAL6-derived CTL epitope could induce HLA-A2-restricted immunity against TAL6-expressing tumor cells.

Vascular endothelial growth factor C as a predictor of early recurrence and poor prognosis of resected stage I non-small cell lung cancer.

INTRODUCTION: Stage I non-small cell lung cancer (NSCLC) is potentially curable after completely resection, but early recurrence may infl uence prognosis. This study hypothesises that vascular endothelial growth factor C (VEGF-C) plays a key role in predicting early recurrence and poor survival of patients with stage I NSCLC. MATERIALS AND METHODS: The expression of VEGF-C was immuno-histochemically (IHC) analysed in tumour samples of primary stage I NSCLC and correlated to early recurrence (< 36 months), disease-free survival, and overall survival in all 49 patients. RESULTS: Early recurrence was identifi ed in 16 patients (33%), and the early recurrence rate in strong and weak VEGF-C activity was significantly different (P = 0.016). VEGF-C was also an independent risk factor in predicting early recurrence (HR = 3.98, P = 0.02). Patients with strong VEGF-C staining also had poor 3-year disease-free survival (P = 0.008) and overall survival (P = 0.007). CONCLUSION: Strong VEGF-C IHC staining could be a biomarker for predicting early recurrence and poor prognosis of resected stage I NSCLC, if the results of the present study are confirmed in a larger study. A more aggressive adjuvant therapy should be used in this group of patients.

Impact of the new lung cancer staging system for a predominantly advanced-disease patient population.

INTRODUCTION: To investigate the feasibility and clinical impact of the 7th edition of the "Tumor, Node, Metastasis" (TNM) classification scheme in lung cancer as proposed by the International Association for the Study of Lung Cancer (IASLC) for non-small cell lung cancer. METHODS: We evaluated the feasibility of the new staging system in our routine biweekly multidisciplinary lung cancer staging conference compared with the 6th TNM staging in a prospective manner from April 2008 to June 2009. The impact of IASLC staging versus the 6th TNM staging was observed at three levels: change in substaging, staging, and clinical management (based on the discussion within the staging conference). RESULTS: From 348 patients discussed during these conferences, 226 eligible non-small cell lung cancer patients newly diagnosed within the study period were reviewed and clinically staged. The majority were elderly (median age, 67 years) and men (58%). Of these, 23 patients had different staging, and four patients had different substaging in the IASLC staging compared with the 6th TNM staging. An impact on clinical management was seen in 2.7% (6 of 226) of these patients because of coding ipsilateral different-lobe metastasis as T4 instead of M1. CONCLUSIONS: The new staging system was clinically feasible and resulted in some (27 of 226, 12%) differences in staging. An impact on clinical decision making was occasionally seen within our institutional practice. Further studies are needed to investigate the comprehensive and long-term impact of the new staging system.

Angiosarcoma with pulmonary metastasis presenting with spontaneous bilateral pneumothorax in an elderly man.

Cutaneous angiosarcoma is a rare and invasive endothelial-derived sarcoma that occurs most frequently in the scalp and facial skin of elderly men. It is frequently accompanied by thin-walled cavitary pulmonary metastasis, and is often obscure on chest radiograph. We report a case of angiosarcoma of the scalp with cystic metastasis to the lung in a 63-year-old man, presenting as recurrent bilateral spontaneous pneumothorax. Lung metastasis was missed at the first episode of pneumothorax because the lung-expanded chest radiograph showed no significant abnormality. Two months later, bilateral pneumothorax recurred, and high-resolution computed tomography revealed multiple cystic, cavitary and nodular lesions. Pulmonary metastasis was confirmed by video-assisted thoracoscopic surgery, and pleurodesis was performed. After the operation, the patient received chemotherapy; no recurrence of pneumothorax was found during 6 months of follow-up. Pneumothorax in the elderly should be differentiated from malignant metastatic lung tumors.