Cluster analysis of long COVID in Japan and association of its trajectory of symptoms and quality of life.

BACKGROUND: Multiple prolonged symptoms observed in patients who recovered from COVID-19 are defined as long COVID. Although diverse phenotypic combinations are possible, they remain unclear. This study aimed to perform a cluster analysis of long COVID in Japan and clarify the association between its characteristics and background factors and quality of life (QOL). METHODS: This multicentre prospective cohort study collected various symptoms and QOL after COVID-19 from January 2020 to February 2021. This study included 935 patients aged ≥18 years with COVID-19 at 26 participating medical facilities. Hierarchical cluster analysis was performed using 24 long COVID symptom at 3 months after diagnosis. RESULTS: Participants were divided into the following five clusters: numerous symptoms across multiple organs (cluster 1, n=54); no or minor symptoms (cluster 2, n=546); taste and olfactory disorders (cluster 3, n=76); fatigue, psychoneurotic symptoms and dyspnoea (low prevalence of cough and sputum) (cluster 4, n=207) and fatigue and dyspnoea (high prevalence of cough and sputum) (cluster 5, n=52). Cluster 1 included elderly patients with severe symptoms, while cluster 3 included young female with mild symptoms. No significant differences were observed in the comorbidities. Cluster 1 showed the most impaired QOL, followed by clusters 4 and 5; these changes as well as the composition of symptoms were observed over 1 year. CONCLUSIONS: We identified patients with long COVID with diverse characteristics into five clusters. Future analysis of these different pathologies could result in individualised treatment of long COVID. TRIAL REGISTRATION NUMBER: The study protocol is registered at UMIN clinical trials registry (UMIN000042299).

Levels of environmental contamination with SARS-CoV-2 in hospital rooms and salivary viral loads of patients with coronavirus disease 2019.

BACKGROUND: Clarifying the presence of viable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rather than SARS-CoV-2 viral RNA in inpatient rooms is important for infection control of coronavirus disease 2019 (COVID-19). In this study, we investigated levels of viral RNA and viable virus on environmental surfaces and in patient saliva. METHODS: Environmental samples from 23 sites in hospital rooms were collected every other day until patient discharge. Saliva specimens and samples from the inner surface of patient masks were also collected. Additionally, environmental samples were collected from 46 sites in hospital rooms on discharge day. The samples were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and plaque assays. RESULTS: The 10 enrolled cases were classified as mild COVID-19, and patients were discharged after 6-9 days. The viral RNA was detected in 12.4% (105/849) of serially collected environmental samples during hospitalization, whereas viable virus was detected only in 0.47% (4/849), which were from sinks and tap levers. Although all patients recovered, three cases retained viable virus in the last saliva specimen collected. In the 15 discharged rooms, viral RNA was detected in 6.6% (45/682) of the samples, and viable virus was detected in only one sample from the sink. CONCLUSIONS: Although environmental surfaces surrounding patients with COVID-19 were frequently contaminated with viral RNA, the presence of viable virus was rare and limited only to areas around sinks. These results suggest that contact infection risk via fomites in hospital rooms is extremely rare.

COVID-19 vaccine effectiveness against severe COVID-19 requiring oxygen therapy, invasive mechanical ventilation, and death in Japan: A multicenter case-control study (MOTIVATE study).

INTRODUCTION: Since the SARS-CoV-2 Omicron variant became dominant, assessing COVID-19 vaccine effectiveness (VE) against severe disease using hospitalization as an outcome became more challenging due to incidental infections via admission screening and variable admission criteria, resulting in a wide range of estimates. To address this, the World Health Organization (WHO) guidance recommends the use of outcomes that are more specific to severe pneumonia such as oxygen use and mechanical ventilation. METHODS: A case-control study was conducted in 24 hospitals in Japan for the Delta-dominant period (August-November 2021; "Delta") and early Omicron (BA.1/BA.2)-dominant period (January-June 2022; "Omicron"). Detailed chart review/interviews were conducted in January-May 2023. VE was measured using various outcomes including disease requiring oxygen therapy, disease requiring invasive mechanical ventilation (IMV), death, outcome restricting to "true" severe COVID-19 (where oxygen requirement is due to COVID-19 rather than another condition(s)), and progression from oxygen use to IMV or death among COVID-19 patients. RESULTS: The analysis included 2125 individuals with respiratory failure (1608 cases [75.7%]; 99.2% of vaccinees received mRNA vaccines). During Delta, 2 doses provided high protection for up to 6 months (oxygen requirement: 95.2% [95% CI:88.7-98.0%] [restricted to "true" severe COVID-19: 95.5% {89.3-98.1%}]; IMV: 99.6% [97.3-99.9%]; fatal: 98.6% [92.3-99.7%]). During Omicron, 3 doses provided high protection for up to 6 months (oxygen requirement: 85.5% [68.8-93.3%] ["true" severe COVID-19: 88.1% {73.6-94.7%}]; IMV: 97.9% [85.9-99.7%]; fatal: 99.6% [95.2-99.97]). There was a trend towards higher VE for more severe and specific outcomes. CONCLUSION: Multiple outcomes pointed towards high protection of 2 doses during Delta and 3 doses during Omicron. These results demonstrate the importance of using severe and specific outcomes to accurately measure VE against severe COVID-19, as recommended in WHO guidance in settings of intense transmission as seen during Omicron.

ALK-positive lung adenocarcinoma in a patient with rheumatoid arthritis with long-term treatment for organizing pneumonia: A case report.

RATIONALE: Rheumatoid arthritis (RA) causes inflammation in various organs including the lungs. Pulmonary manifestations include inflammation of the pleura, vasculature, airway, and parenchyma, including interstitial lung disease (ILD). RA-organizing pneumonia (OP) is the third most common cause of RA-ILD. Cases of OP coexisting/complicated with lung cancer have been reported. Therefore, lung cancer can represent a diagnostic challenge, especially in patients with underlying pulmonary diseases including OP. PATIENT CONCERNS: An 81-year-old woman with a 12-year history of RA-OP underwent multiple transbronchial lung biopsies (TBLBs), all of which resulted in no malignant findings. She was treated with prednisolone (PSL) depending on the deteriorated infiltrations. At admission, chest computed tomography (CT) images showed exacerbation of left S8 consolidation on chest CT. Additionally, her RA activity was exacerbated, and PSL dose was increased to 30 mg/day, which resulted in improved dyspnea and consolidation. Accordingly, PSL dose was gradually decreased. However, 6 months later, when PSL dose was 11 mg/d, due to a worsening of consolidation and the joint symptoms of RA, PSL dose was increased to 20 mg/d and tacrolimus 2 mg/d was administered. 3 months after the increase in PSL dose, dyspnea improved and PSL dose was reduced to 15 mg/d; however, she was admitted to our hospital because of low back pain. DIAGNOSIS: Spinal magnetic resonance imaging showed bone metastases in the third and fifth lumbar vertebrae, and lung cancer was suspected as the primary tumor on CT. INTERVENTIONS: TBLB was performed on the left B8 infiltrate, which showed no evidence of malignancy in the previous TBLB. OUTCOMES: Pathological examination of TBLB on the left B8 revealed an adenocarcinoma that was positive for anaplastic lymphoma kinase. LESSONS: Physicians should be aware of the development of lung cancer in regions with OP, even after a partial response to corticosteroid therapy.

Comprehensive and long-term surveys of COVID-19 sequelae in Japan, an ambidirectional multicentre cohort study: study protocol.

INTRODUCTION: The rapid spread of COVID-19 posed a global burden. Substantial number of people died of the disease in the acute phase of infection. In addition, a significant proportion of patients have been reported to suffer from post-acute phase symptoms, sequelae of COVID-19, which may negatively influence the quality of daily living and/or socioeconomic circumstances of the patients. However, no previous study has comprehensively and objectively assessed the quality of life of patients by using existing international scales. Further, evidence of socioeconomic consequences among patients with COVID-19 is scarce. To address the multidimensional issues from sequelae of COVID-19, evidence from comprehensive surveys beyond clinical perspectives is critical that investigates health, and social determinants of disease progression as well as socioeconomic consequences at a large scale. METHODS AND ANALYSIS: In this study, we plan to conduct a nationwide and comprehensive survey for the sequelae of COVID-19 in a total of 1000 patients diagnosed at 27 hospitals throughout Japan. This study will evaluate not only the health-related status of patients from clinical perspectives but also the Health-related Quality of Life (HRQoL) scores, socioeconomic status and consequences to discuss the sequelae of the disease and the related risk factors. The primary endpoint is the frequency of long-term complications of COVID-19 infection. The secondary endpoints are risk factors for progression to sequelae of COVID-19 infection. The study will provide robust and important evidence as a resource to tackle the issues from the sequelae of COVID-19 from the multi-dimensional perspectives. ETHICS AND DISSEMINATION: This trial was approved by the Keio University School of Medicine Ethics Committee (20200243, UMIN000042299). The results of this study will be reported at a society meeting or published in a peer-reviewed journal.

Synchronous Primary Lung Adenocarcinoma and Hepatocellular Carcinoma Successfully Treated with a Combination of Atezolizumab, Bevacizumab, Carboplatin, and Paclitaxel.

Chemotherapy for multiple primary malignancies is challenging. We herein report a case of synchronous primary lung adenocarcinoma and hepatocellular carcinoma (HCC). A 72-year-old man was admitted for the evaluation of an abnormal shadow on his lung. Computed tomography revealed a lung nodule in the right upper lobe and multiple liver masses. He was diagnosed with synchronous primary lung adenocarcinoma and HCC. Atezolizumab, bevacizumab, carboplatin, and paclitaxel (ABCP) chemotherapy was efficacious for both tumors. ABCP chemotherapy may be a potential treatment option for synchronous primary lung adenocarcinoma and HCC.

SHOC2 Is a Critical Modulator of Sensitivity to EGFR-TKIs in Non-Small Cell Lung Cancer Cells.

EGFR mutation-positive patients with non-small cell lung cancer (NSCLC) respond well to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI); however, treatment with EGFR-TKIs is not curative, owing to the presence of residual cancer cells with intrinsic or acquired resistance to this class of drugs. Additional treatment targets that may enhance the efficacy of EGFR-TKIs remain elusive. Using a CRISPR/Cas9-based screen, we identified the leucine-rich repeat scaffold protein SHOC2 as a key modulator of sensitivity to EGFR-TKI treatment. On the basis of in vitro assays, we demonstrated that SHOC2 expression levels strongly correlate with the sensitivity to EGFR-TKIs and that SHOC2 affects the sensitivity to EGFR-TKIs in NSCLC cells via SHOC2/MRAS/PP1c and SHOC2/SCRIB signaling. The potential SHOC2 inhibitor celastrol phenocopied SHOC2 depletion. In addition, we confirmed that SHOC2 expression levels were important for the sensitivity to EGFR-TKIs in vivo. Furthermore, IHC showed the accumulation of cancer cells that express high levels of SHOC2 in lung cancer tissues obtained from patients with NSCLC who experienced acquired resistance to EGFR-TKIs. These data indicate that SHOC2 may be a therapeutic target for patients with NSCLC or a biomarker to predict sensitivity to EGFR-TKI therapy in EGFR mutation-positive patients with NSCLC. Our findings may help improve treatment strategies for patients with NSCLC harboring EGFR mutations. IMPLICATIONS: This study showed that SHOC2 works as a modulator of sensitivity to EGFR-TKIs and the expression levels of SHOC2 can be used as a biomarker for sensitivity to EGFR-TKIs.

Analysis of clinical outcomes in elderly patients with impaired swallowing function.

Japan is the world's leading aging society, and increasing medical expenses for elderly people is an urgent issue. Since aspiration pneumonia in elderly people with impaired swallowing function is a huge problem in Japan, their expected long-term clinical course should be clarified. Accordingly, we collected data from 991 elderly (≥75 years old) patients whose swallowing function was evaluated by Kitasato Institute Hospital's speech therapists (January 1, 2010 to December 31, 2017). We analyzed the relationship between swallowing function and the subjects' long-term prognosis. To clarify the prognostic factors of patients with dysphagia, we obtained their clinical information (age, gender, activities of daily living, nutritional status, availability of alternative feeding pathways such as percutaneous endoscopic gastrostomy, and cognitive function). We confirmed 372 death cases and stratified the cases into three groups using Fujishima's swallowing ability grade, which is used to predict elderly people's real-world life expectancy. Results showed the median survival days were 331 and 952 days in Groups I (Grades 1-3, n = 308) and II (Grades 4-6, n = 153), respectively, whereas the median survival days for Group III (Grades 7-10, n = 530) could not be calculated. We conducted a multivariate analysis using the Cox proportional hazards model with Group I, which revealed that initial grade and percutaneous endoscopic gastrostomy were significant prognostic factors for the subjects' long-term survival. Nevertheless, further discussion is necessary, particularly to determine advanced care planning regarding indications for alternative feeding pathways in elderly patients with severe dysphagia, since percutaneous endoscopic gastrostomy could significantly prolong their survival.

Alternative splicing of APOBEC3D generates functional diversity and its role as a DNA mutator.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family members have cytidine deaminase activity and can induce cytosine to uracil transition in nucleic acid. The main function of APOBEC3 (A3) proteins is to trigger an innate immune response to viral infections. Recent reports have shown that several APOBEC family proteins such as A3B can induce somatic mutations into genomic DNA and thus promote cancer development. However, the role of A3D on somatic mutations is unclear. Here, we identified the alternative splicing of A3D, and investigated each splice variant's subcellular localization and role in DNA mutagenesis. We identified four A3D variants, which all have one or two cytidine deaminase domains. The full-length form of A3D (variant 1) and truncated forms of A3D (variant 2, 6, 7) showed the ability to induce C/G to T/A transitions in foreign DNA and genomic DNA and retained antiretroviral activity. Furthermore, we demonstrated that A3D and A3B could induce deletions that are possibly repaired by microhomology-mediated end joining (MMEJ). Taken together, our experiments illustrated that alternative splicing generates functional diversity of A3D, and some variants can act as DNA mutators in genomic DNA.

A phase II trial of induction of erlotinib followed by cytotoxic chemotherapy for EGFR mutation-positive non-squamous non-small cell lung cancer patients.

BACKGROUND: No consensus has been reached regarding the treatment order and timing of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and cytotoxic chemotherapy administration for EGFR mutation-positive non-small cell lung cancer (NSCLC) patients. METHODS: In this phase II trial, chemotherapy-naïve patients harboring activating EGFR mutations with stage IIIB/IV or post-surgical recurrent non-squamous NSCLC were enrolled. Patients were treated with erlotinib induction at 150 mg/day for 3 months. This was followed by cytotoxic chemotherapy with platinum plus pemetrexed, with or without bevacizumab, when the induction erlotinib achieved a CR or PR. The primary end point was the 1-year progression-free survival (PFS) rate, while the secondary end points were the response rate (RR), PFS, safety, and overall survival (OS). RESULTS: Twenty patients were enrolled in this study. The median age was 63 years. Eighteen patients had stage IV disease, and 2 patients had recurrent disease. Eleven patients achieved a PR after induction of erlotinib and 9 out of 11 patients were switched to chemotherapy. The 1-year PFS rate was 45.0% (90% CI 26.8-63.2), the overall RR was 55.0%, and the median PFS was 10.7 months in the intention-to-treat (ITT) population. Grade 3-4 adverse events were reported for 40% of the patients, including patients with leukopenia (10%), neutropenia (20%), and interstitial pneumonitis, bacterial pneumonia, rash, and nausea (all 5%). CONCLUSIONS: The primary end point of this study was not achieved. However, the therapy was well tolerated and may be a treatment option for a future study with patients responsive to short-term erlotinib treatment. CLINICAL TRIALS REGISTRATION NUMBER: UMIN ID: 000013125.

[Pulmonary tuberculosis following anti-tumor necrosis factor-α therapy in Crohn's disease:a case report].

Anti-tumor necrosis factor-α (TNF-α) is a principal treatment for Crohn's disease (CD). However, it increases the susceptibility to tuberculosis (TB) infection, and therefore, screening examination prior to treatment initiation is crucial. Here, we report the case of a patient with CD who developed pulmonary TB following anti-TNF-α therapy, despite negative screening. A 19-year-old female who had no history of TB or had traveled to TB-endemic regions was diagnosed with CD. After negative TB screening with chest X-ray and interferon-gamma release assay, the patient was initiated on oral prednisolone and pH-dependent mesalazine. The treatment was changed to infliximab (IFX) because of the inadequate response observed to prednisolone;however, she developed pulmonary TB only 10 weeks after the initiation of IFX. The standard short-course anti-TB regimen was initiated to treat pulmonary TB, whereas IFX was discontinued and replaced with budesonide. Our case suggests that the risk of developing TB should not be excluded, despite the initial negative TB screening, particularly when a patient develops respiratory symptoms during anti-TNF-α therapy.

Erlotinib as second- or third-line treatment in elderly patients with advanced non-small cell lung cancer: Keio Lung Oncology Group Study 001 (KLOG001).

The aim of this study was to assess the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), as second- or third-line treatment for elderly Japanese patients with non-small-cell lung cancer (NSCLC). The patients eligible for this phase II trial were aged ≥70 years, had stage III/IV or recurrent NSCLC, and had previously received 1 or 2 chemotherapy regimens that did not include EGFR-TKIs. The patients received erlotinib at a dose of 150 mg/day. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. A total of 38 patients with a median age of 76 years were enrolled. The majority of the patients were men (66%), had an Eastern Cooperative Oncology Group performance status of 1 (58%), stage IV disease (66%) and adenocarcinoma (74%). Of the 35 patients, 13 (34%) had tumors with EGFR mutations. The ORR was 26.3% (95% confidence interval: 12.1-40.5%) and the disease control rate was 47.4%. The median PFS was 3.7 months and the median OS was 17.3 months. The grade 3 adverse events observed included rash (13%), diarrhea (5%), interstitial pneumonitis (5%), anorexia (3%) and gastrointestinal bleeding (3%). Grade 4 or 5 adverse events were not observed. The median OS did not differ significantly between patients aged <75 years (14.9 months) and those aged ≥75 years (19.0 months; P=0.226). Therefore, erlotinib was found to be effective and well-tolerated in elderly patients with previously treated NSCLC.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases.

Dynamic Transport and Cementation of Skeletal Elements Build Up the Pole-and-Beam Structured Skeleton of Sponges.

Animal bodies are shaped by skeletons, which are built inside the body by biomineralization of condensed mesenchymal cells in vertebrates [1, 2] and echinoderms [3, 4], or outside the body by apical secretion of extracellular matrices by epidermal cell layers in arthropods [5]. In each case, the skeletons' shapes are a direct reflection of the pattern of skeleton-producing cells [6]. Here we report a newly discovered mode of skeleton formation: assembly of sponges' mineralized skeletal elements (spicules) in locations distant from where they were produced. Although it was known that internal skeletons of sponges consist of spicules assembled into large pole-and-beam structures with a variety of morphologies [7-10], the spicule assembly process (i.e., how spicules become held up and connected basically in staggered tandem) and what types of cells act in this process remained unexplored. Here we found that mature spicules are dynamically transported from where they were produced and then pierce through outer epithelia, and their basal ends become fixed to substrate or connected with such fixed spicules. Newly discovered "transport cells" mediate spicule movement and the "pierce" step, and collagen-secreting basal-epithelial cells fix spicules to the substratum, suggesting that the processes of spiculous skeleton construction are mediated separately by specialized cells. Division of labor by manufacturer, transporter, and cementer cells, and iteration of the sequential mechanical reactions of "transport," "pierce," "raise up," and "cementation," allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges.

A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation.

Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.

CCAAT/enhancer binding protein ß is dispensable for development of lung adenocarcinoma.

Lung cancer is the leading cause of cancer death worldwide. Although disruption of normal proliferation and differentiation is a vital component of tumorigenesis, the mechanisms of this process in lung cancer are still unclear. A transcription factor, C/EBPß is a critical regulator of proliferation and/or differentiation in multiple tissues. In lung, C/EBPß is expressed in alveolar pneumocytes and bronchial epithelial cells; however, its roles on normal lung homeostasis and lung cancer development have not been well described. Here we investigated whether C/EBPß is required for normal lung development and whether its aberrant expression and/or activity contribute to lung tumorigenesis. We showed that C/EBPß was expressed in both human normal pneumocytes and lung adenocarcinoma cell lines. We found that overall lung architecture was maintained in Cebpb knockout mice. Neither overexpression of nuclear C/EBPß nor suppression of CEBPB expression had significant effects on cell proliferation. C/EBPß expression and activity remained unchanged upon EGF stimulation. Furthermore, deletion of Cebpb had no impact on lung tumor burden in a lung specific, conditional mutant EGFR lung cancer mouse model. Analyses of data from The Cancer Genome Atlas (TCGA) revealed that expression, promoter methylation, or copy number of CEBPB was not significantly altered in human lung adenocarcinoma. Taken together, our data suggest that C/EBPß is dispensable for development of lung adenocarcinoma.

A phase II study of biweekly paclitaxel and carboplatin in elderly patients with advanced non-small cell lung cancer.

PURPOSE: The number of elderly patients with advanced non-small cell lung cancer (NSCLC) is increasing. Although several studies have suggested the benefit of chemotherapy with a platinum doublet for elderly patients with advanced NSCLC, this treatment is still controversial in this age group. To evaluate the efficacy and tolerability of combination chemotherapy with biweekly paclitaxel and carboplatin for elderly patients with advanced NSCLC, we conducted a multicenter, non-randomized, open label, phase II trial. METHODS: We recruited patients aged ≥70 years with clinical stage IIIB and IV NSCLC and ECOG performance status (PS) of 0-2. Patients received paclitaxel (90 mg/m(2)) and carboplatin (AUC = 2.5) on day 1 and 15, every 4 weeks. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Sixty-five patients (median age 79 years; range 70-87 years) were enrolled. Forty-nine patients were men, and 48 were stage IV. The PS was 0, 1, and 2 in 28, 33, and 4 patients, respectively. The histological type of NSCLC was non-squamous in 69.3 % and squamous cell carcinoma in 30.7 % of patients. The median number of treatment cycles was 3 (range 1-6). The response rate was 29.4 % (95 % CI 18.7-43.0), and the disease control rate was 78.0 % (95 % CI 64.8-87.2). Median PFS and OS were 3.8 months (95 % CI 1.9-5.3) and 17.3 months (95 % CI 10.4-25.1), respectively. The most common grade 3 or 4 toxicities were neutropenia (27 %), leukopenia (15 %), infection (10 %), and anemia (8 %). CONCLUSION: The combination of biweekly paclitaxel and carboplatin was effective and well tolerated in elderly patients with advanced NSCLC.

FOXD1 expression is associated with poor prognosis in non-small cell lung cancer.

AIM: Clinical microarray datasets were analyzed to search for new therapeutic targets and prognostic markers of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Microarray datasets from 90 lung cancer specimens, were analyzed with focus on the FOXD1 gene. Levels of FOXD1 mRNA were assessed in lung cancer cell lines and these levels were correlated with survival. RESULTS: FOXD1-knockdown led to suppression of cell proliferation. Moreover, patients with high FOXD1 expression survived for a significantly shorter time than those with low FOXD1 expression. CONCLUSION: The expression status of FOXD1 is a novel prognostic factor and may lead to new treatment strategies for NSCLC.

Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma.

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non-small-cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI-2) expression in these clinical samples. In contrast to TMPRSS4, TFPI-2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI-2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI-2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5-aza-2'-deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI-2 by decreasing its methylation in vitro. The TFPI-2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P = 0.02). We found a novel molecular mechanism that TFPI-2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI-2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC.

ß-catenin contributes to lung tumor development induced by EGFR mutations.

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that ß-catenin is essential for development of EGFR-mutated lung cancers. ß-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated ß-catenin, leading to an increase in ß-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of ß-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the ß-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that ß-catenin plays an essential role in lung tumorigenesis and that targeting the ß-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.