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The diagnostic criteria of aspiration pneumonia have not been established, and it remains an underdiagnosed entity. Diagnosis and cause investigation is essential in improving the management of aspiration pneumonia. The Japanese Respiratory Society Guidelines for the Management of Pneumonia in Adults (JRS Guidelines) show a list of risk factors for aspiration pneumonia. We developed an algorithm to aid physicians in evaluating these possible underlying factors and guide their management with a focus on aspiration pneumonia. The algorithm was developed based on the JRS Guidelines. The algorithm suggested dysphagia screening, pneumococcal and influenza vaccination, and other preventative measures for pneumonia. The algorithm was implemented in the acute setting of a general hospital among older patients admitted with pneumonia. Their outcomes were compared with a historical control group constituting similar patients from the previous year. Forty patients with pneumonia were assessed with the algorithm group, and 44 patients were included in the control group. In the algorithm group, significantly more cases (95.0% vs. 15.9%, p 0.01) underwent early screening for a swallowing disorder. Two patients in the algorithm group were diagnosed with a new condition causing aspiration pneumonia, as opposed to none in the control group. Drugs with a potential risk for aspiration were identified and discontinued in 27.5% of the patients in the algorithm group and 4.5% in the control group. In conclusion, an aspiration pneumonia cause investigation algorithm translating the JRS guideline approach into practice enhanced the rate of swallow screening and preventative measures for aspiration.

Myxofibrosarcoma (MFS) is a rare and aggressive mesenchymal malignancy characterized by complex karyotypes with heterogeneous clinical features. The standard treatment for primary MFS is curative resection; however, the utility of systemic chemotherapy and radiotherapy has not been established. Although patient-derived cancer cell lines are a key bioresource for developing novel therapies, the number of MFS cell lines available from public cell banks is limited by the rarity of the disease, and large-scale drug screening has not yet been performed. To address this issue, we aimed to establish and characterize a novel MFS cell line. We successfully established a cell line, NCC-MFS6-C1, which harbors genetic abnormalities common in MFS and exhibits aggressive phenotypes such as continuous growth, spheroid formation, and invasion in tissue culture conditions. We performed drug screening using NCC-MFS6-C1 along with five MFS cell lines established in our laboratory and clarified the response spectrum of 214 existing anticancer agents. We found that two anticancer agents, gemcitabine and romidepsin, showed considerable antiproliferative effects, and these observations were concordant with the findings of our previous report, in which these agents attenuated the proliferation of five previously reported MFS cell lines. We conclude that NCC-MFS6-C1 is a useful resource for studying MFS.

Malignant rhabdoid tumor (MRT) is a sarcoma histologically characterized by rhabdoid cells and genetically characterized by loss of function of the chromatin remodeling complex SWI/SNF induced by SMARCB1 gene deficiency. MRT mainly occurs in children, may arise in various locations, but is predominantly in the central nervous system (CNS) and kidney. Although MRT exhibits poor prognosis, standard treatment has not yet been established due to its extreme rarity. Patient-derived cancer cell lines are critical tools for basic and pre-clinical research in the development of chemotherapy. However, none of the MRT cell lines was derived from adult patients, and only one cell line was derived from the MRT of a soft tissue, despite the clinical behavior of MRT varying according to patient age and anatomic site. Herein, we reported the first cell line of MRT isolated from the soft tissue of an adult patient and named it NCC-MRT1-C1. NCC-MRT1-C1 cells showed a biallelic loss of the SMARCB1 gene. NCC-MRT1-C1 cells demonstrated rapid proliferation, spheroid formation, invasion capability in vitro, and tumorigenesis in nude mice. Screening of antitumor agents in NCC-MRT1-C1 cells resulted in the identification of six effective drugs. In conclusion, we report the first MRT cell line from the soft tissue of an adult patient. We believe that NCC-MRT1-C1 is a useful tool for developing novel chemotherapies for MRT.

Synovial sarcoma (SS) is a rare and aggressive mesenchymal malignancy driven by a unique chromosomal translocation that generates the expression of the SS18:SSX fusion protein. It occurs at almost any anatomical site and most commonly in young adults. The standard curative treatment for primary SS is a wide surgical resection combined with radiotherapy and/or neoadjuvant chemotherapy. The prognosis of SS varies among patients, with the 5 years survival rate ranging from 50 to 60% in adults and 90% in children. Although patient-derived cell lines are a useful resource for the development of new therapies, only a few are available from public cell banks. Therefore, this study aimed to establish and characterize a novel SS cell line. We successfully established a novel cell line, NCC-SS5-C1, harboring an SS18-SSX1 fusion gene. NCC-SS5-C1 cells demonstrated constant growth and invasion ability. We performed integrative drug screening using eight SS cell lines, including NCC-SS5-C1 cells, and examined the response spectrum of existing anticancer agents. We conclude that NCC-SS5-C1 is a useful resource for studying SS.

Dedifferentiated liposarcoma (DDLPS) is morphologically characterized by well-differentiated liposarcomas associated with high-grade non-lipogenic sarcoma and molecularly characterized by the coamplification of MDM2 and CDK4(12q14-15). DDLPS is highly aggressive, and effective systemic chemotherapy has not been developed yet. In this study, we established a novel DDLPS cell line, NCC-DDLPS6-C1, as a potential tool for the development of novel therapies. NCC-DDLPS6-C1 cells were established from surgically resected tumor tissues of a patient with DDLPS. Amplification and overexpression of MDM2 and CDK4 were observed in NCC-DDLPS6-C1 cells. NCC-DDLPS6-C1 cells proliferated rapidly, invaded aggressively, and formed spheroids. Moreover, NCC-DDLPS6-C1 cells formed tumors in mice. These observations suggested that the malignant potentials that may reflect the original features of DDLPS were retained in the NCC-DDLPS6-C1. Anticancer drugs that significantly reduced the proliferation of NCC-DDLPS6-C1 cells were identified by drug library screening. Thus, NCC-DDLPS6-C1 may recapitulate the original genotypes and phenotypes, and we conclude that the NCC-DDLPS6-C1 cell line is a useful resource for the study of DDLPS.

Ewing sarcoma (ES) is a small round cell sarcoma that is characterized by the unique gene translocation EWSR1-FLI1. It is the second most common primary bone and soft tissue malignancy in children and adolescents. It constitutes 10-15% of all bone sarcomas and is highly aggressive and rapidly recurring. Although intensive treatments have improved the clinical outcome of ES patients, 20-25% of them exhibit metastases during diagnosis. Thus, the prognoses of these patients remain poor. Cell lines are pivotal resources to investigate the molecular background of disease progression and to develop novel therapeutic modalities. In this study, we established and characterized a novel ES cell line, NCC-ES2-C1. The presence of the EWSR1-FLI1 fusion gene in these cells was confirmed in the NCC-ES2-C1 cells. Furthermore, these cells exhibited constant proliferation, and invasion, but did not form tumors in mice. We screened the anti-tumor effects of 214 anti-cancer drugs in NCC-ES2-C1 cells and found that the drugs which effectively reduced the proliferation of NCC-ES2-C1 cells. We concluded that NCC-ES2-C1 cells are a useful resource to study functions of the EWSR1-FLI1 fusion gene, investigate phenotypic changes caused by genes and proteins, and evaluate the anti-tumor effects of novel drugs.

Supplementary Figure 3 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 7 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 1 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Materials and Figure Legends 1-5 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure Legends 1-8 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Methods from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 5 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 8 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 6 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 2 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 3 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure 4 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure 5 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic HrasG12V (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer. [Cancer Res 2008;68(14):5699–705]

The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, ΔNp63α, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that ΔNp63α functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing ΔNp63α. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of ΔNp63α decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that ΔNp63α maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of ΔNp63α together with p53 inactivation in SCCs. Cancer Res; 70(10); 4034–44. ©2010 AACR.

Supplementary Figure 1 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Malignant peripheral nerve sheath tumors (MPNSTs) are a rare subtype of mesenchymal tumors that arise from the sheath of peripheral nerves. MPNSTs exhibit strong metastatic potential, leading to poor clinical outcomes. The clinical utility of radiation therapy and chemotherapy is marginal with respect to overall survival and effective systematic therapies for MPNSTs are still needed to improve patient outcome. Although patient-derived cell lines are an essential tool for the development of novel therapies, only a limited number of cell lines have been reported and are available from cell banks. Thus, we established the novel MPNST cell line, NCC-MPNST6-C1, using surgically resected MPNST tissue. The NCC-MPNST6-C1 cells retained copy-number alterations similar to those of the original tumors and demonstrated constant proliferation, spheroid formation, and invasion capability in vitro, which reflected the malignant features of the original tumor tissue. While the NCC-MPNST6-C1 cells did not exhibit tumorigenesis in nude mice, their use in drug screening resulted in anti-cancer agents with low IC50 values. Hence, we conclude that the NCC-MPNST6-C1 cell line is a useful resource for the study of MPNSTs.

Giant cell tumor of bone (GCTB) is a rare osteolytic intermediate bone tumor that harbors a pathogenic H3F3A gene mutation and exhibits characteristic histology. The standard curative treatment for GCTB is complete surgical resection, but it frequently results in local recurrence and, more rarely, metastasis. Therefore, effective multidisciplinary treatment is needed. Although patient-derived tumor cell lines are promising tools for preclinical and basic research, there are only four available cell lines for GCTB in public cell banks. Thus, the aim of this study was to establish a novel GCTB cell line. Using surgically resected tumor tissues from a patient with GCTB, we established a cell line named NCC-GCTB4-C1. The cells harbored the typical H3F3A gene mutation and exhibited constant proliferation and invasive capabilities. After characterizing NCC-GCTB4-C1 cell behaviors, we conducted high-throughput screening of 214 anti-tumor drugs and identified seven effective drugs. Comparing the results of high-throughput screening using NCC-GCTB4-C1 cell line with the results using NCC-GCTB1-C1, NCC-GCTB2-C1, and NCC-GCTB3-C1 cell lines that we previously established, four drugs were in common effective. This study showed potential drugs for the treatment of GCTB. These data indicate that NCC-GCTB4-C1 has the potential to be a powerful tool in preclinical and basic research on GCTB.

Pleomorphic liposarcoma (PLPS) is a highly malignant subtype of liposarcoma. It is histologically characterized by the presence of pleomorphic lipoblasts and can be accompanied by morphological foci that demonstrate differentiation to other histological lineages. PLPS is rare and accounts for only 5% of all liposarcomas. PLPS exhibits poor prognosis; distant metastases develop in 30-50% of patients after curative surgical resection, tumor-associated mortality occurs in up to 50% of patients, and effective chemotherapies for PLPS have not been established. The histological accompaniment of other morphological foci is an important prognostic factor for PLPS, and the development of chemotherapies for PLPS considering the histological morphology is necessary. Patient-derived cancer cell lines are critical tools for basic and pre-clinical research to understand diseases and develop chemotherapies. However, only two PLPS-derived cell lines have been reported, and their donor tumor specimens did not histologically accompany morphological foci other than lipoblasts. Thus, there is a need to establish patient-derived PLPS cell lines from various histological morphologies. Here, we report a novel PLPS cell line from a tumor specimen that histologically accompanied pleomorphic and bone-forming foci, and named it NCC-PLPS2-C1. NCC-PLPS2-C1 cells demonstrated constant proliferation, spheroid formation, and invasion capability in vitro. Screening of antitumor agents in NCC-PLPS2-C1 cells showed that bortezomib, romidepsin, and trabectedin were effective against NCC-PLPS2-C1. In conclusion, we report the first PLPS cell line from a tumor specimen that was morphologically accompanied by pleomorphic and born-forming foci. We believe that NCC-PLPS2-C1 will be useful for the development of novel chemotherapies for PLPS.

Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft-tissue sarcoma genetically characterized by the presence of the FUS-CREB3L2 gene fusion. While LGFMS exhibits indolent features during its early stages, the rates of recurrence, metastasis, and death from the disease are high. Presently, the role of FUS-CREB3L2 gene fusions in the unique features of LGFMS is not clear, and there is no modality to improve the clinical outcomes of patients with LGFMS; thus, extensive studies on LGFMS are required. Patient-derived cancer cell lines are critical tools for cancer research. However, no cell line has been established for LGFMS. Here, we aimed to develop a novel cell line for LGFMS and successfully established it using surgically resected tumor tissues. The cells, named NCC-LGFMS1-C1, possessed the same fusion genes as their original tumor and visible copy number variations. The cells had a fibroblastic appearance, formed spheroids when they were seeded in a low-attachment dish, and exhibited constant growth and invasion. Additionally, we demonstrated the feasibility of high-throughput drug screening using these cells. In conclusion, the NCC-LGFMS1-C1 cell line is a useful tool for studying LGFMS.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastasizing tumor. GCTB is characterized by the presence of unique giant cells and a recurrent mutation in the histone tail of the histone variant H3.3, which is encoded by H3F3A on chromosome 1. GCTB accounts for ~ 5% of primary bone tumors. Although GCTB exhibits an indolent course, it has the potential to develop aggressive behaviors associated with local recurrence and distant metastasis. Currently, complete surgical resection is the only curative treatment, and novel therapeutic strategies are required. Patient-derived cancer cell lines are critical tools for basic and pre-clinical research. However, only a few GCTB cell lines have been reported, and none of them are available from public cell banks. Therefore, we aimed to establish novel GCTB cell lines in the present study. Using curetted tumor tissues of GCTB, we established two cell lines and named them NCC-GCTB2-C1 and NCC-GCTB3-C1. These cells harbored a typical mutation in histones and exhibited slow but constant growth, formed spheroids, and had invasive capabilities. We demonstrated the utility of these cell lines for high-throughput drug screening using 214 anticancer agents. We concluded that NCC-GCTB2-C1 and NCC-GCTB3-C1 cell lines were useful for the in vitro study of GCTB.

Background Spontaneous pneumomediastinum is characterized by the presence of interstitial air in the mediastinum without any underlying disease. Some cases of spontaneous pneumomediastinum have been reported in the past, although only few reports are available, and its management remains uncertain. This study reviewed our experience in the diagnosis and treatment of spontaneous pneumomediastinum. Methods A retrospective study of 71 cases treated for spontaneous pneumomediastinum at the Yodogawa Christian Hospital between April 2005 and March 2020 was conducted. Results The patients’ mean age was 19.3 years (range, 7–48 years). A triggering event was noted in 69% of the cases. Seventy-six percent of the patients were admitted to the hospital, and 24% were outpatients. Treatment included analgesia, rest, antibiotics, and/or oxygen therapy. Thirty-six patients (51%) were treated with antibiotics. None of the cases presented any complications, including mediastinitis or worsening respiratory condition. Two patients (3%) had a recurrence of spontaneous pneumomediastinum. Conclusions All the patients, with or without antimicrobial treatment and hospitalization, had favorable outcomes. We should therefore reconsider the need for hospitalization and antimicrobial therapy for patients with mediastinitis prophylaxis.

In older adults, community-acquired pneumonia (CAP) is often aspiration-related. However, as aspiration pneumonia (AP) lacks clear diagnostic criteria, the reported prevalence and clinical management vary greatly. We investigated what clinical factors appeared to influence the diagnosis of AP and non-AP in a clinical setting and reconsidered a more clinically relevant approach. Medical records of patients aged ≥75 years admitted with CAP were reviewed retrospectively. A total of 803 patients (134 APs and 669 non-APs) were included. The AP group had significantly higher rates of frailty, had higher SARC-F scores, resided in institutions, had neurologic conditions, previous pneumonia diagnoses, known dysphagia, and were more likely to present with vomiting or coughing on food. Nil by mouth orders, speech therapist referrals, and broad-spectrum antibiotics were significantly more common, while computed tomography scans and blood cultures were rarely performed; alternative diagnoses, such as cancer and pulmonary embolism, were detected significantly less. AP is diagnosed more commonly in frail patients, while aspiration is the underlying aetiology in most types of pneumonia. A presumptive diagnosis of AP may deny patients necessary investigation and management. We suggest a paradigm shift in the way we approach older patients with CAP; rather than trying to differentiate AP and non-AP, it would be more clinically relevant to recognise all pneumonia as just pneumonia, and assess their swallowing functions, causative organisms, and investigate alternative diagnoses or underlying causes of dysphagia. This will enable appropriate clinical management.

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma (RMS) that has fascicular spindle cell and/or sclerosing morphology. SsRMS has a diverse molecular background and is categorized into three groups: congenital/infantile ssRMS with a gene fusion involving the NCOA2 and VGLL2, ssRMS with the MYOD1 mutation, and ssRMS with no recurrent identifiable genetic alterations. Because ssRMS is a newly defined disease concept of RMS, the optimal treatment methods have not been determined. This results in unfavorable prognosis and consequently signals the urgent need for continuous research. Patient-derived cell lines are essential tools in basic and translational research. However, only two ssRMS cell lines with the MYOD1 mutation have been reported to date. Thus, we established a novel ssRMS cell line named NCC-ssRMS2-C1 using a surgically resected tumor tissue from an adult ssRMS patient. NCC-ssRMS2-C1 cells retained the copy number alterations corresponding to the original tumor and are categorized into the group with no recurrent identifiable genetic alterations. NCC-ssRMS2-C1 cells demonstrated constant proliferation, spheroid formation, and capability for invasion in vitro, reflecting the malignant features of the original tumor tissue. In a drug screening test, ssRMS demonstrated remarkable sensitivity to romidepsin, trabectedin, actinomycin D, and bortezomib. Hence, we conclude that the NCC-ssRMS2-C1 cell line is the first ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations, and it will be a useful resource in both basic and translational studies for ssRMS.

Background Histone deacetylase inhibitors (HDACi) are used to treat patients with cutaneous T-cell lymphoma (CTCL), but they show limited efficacy. Hence, combination therapies should be explored to enhance the effectiveness of HDACis. Objective This study was conducted to identify novel therapeutic targets that can be combined with HDACis for treating CTCL. Methods We performed a global kinome profiling assay of three CTCL cell lines (HH, MJ, and Hut78) with three HDACis (romidepsin, vorinostat, and belinostat) using the PamChip® microarray. The three cell lines were co-treated with romidepsin and an inhibitor against the tyrosine kinase pathway. Results Principal component analysis revealed that kinome expression patterns were mainly related to the cell origin and were not affected by the drugs. Few kinases were commonly activated by the HDACis. Most identified kinases were Src-associated molecules, such as annexin A2, embryonal Fyn-associated substrate, and progesterone receptor. Phosphorylated Src was not observed in any untreated cell lines, whereas Src phosphorylation was detected in two of the three cell lines after HDACi treatment. Ponatinib, a Src inhibitor, significantly enhanced romidepsin-induced apoptosis not only in HH, MJ, and Hut78 cells, but also in Myla and SeAx CTCL cell lines. Conclusion The Src pathway is a possible target for combination therapy involving HDACis for CTCL.

Synovial sarcoma (SS) is defined as a monomorphic blue spindle cell sarcoma showing variable epithelial differentiation, and is characterized by a specific fusion gene, SS18-SSX. Although SS is rare, it accounts for approximately 8% of all soft tissue sarcomas, which occupies a significant proportion of soft tissue tumors. The prognosis of SS is unfavorable, with 5-year survival rate of 50-60%, and only a few anti-cancer agents are recommended for its treatment. Thus, we need to urgently establish novel treatment methods. Patient-derived cell lines are essential tools in basic research and pre-clinical studies. However, there are only 4 publicly available SS cell lines. Therefore, we established a novel SS cell line, NCC-SS4-C1, using surgically resected tumor tissues of a patient with SS. The cell line maintained the characteristic fusion gene, SS18-SSX1, and copy number alteration, in concordance with the original tumor. The cells also exhibited moderate cell proliferation, invasion ability, and spheroid formation ability. Moreover, a drug-screening test using 4 SS cell lines, including NCC-SS4-C1, demonstrated the significant anti-proliferative effects of ALK and HDAC inhibitors. Thus, we concluded that the NCC-SS4-C1 cell line is a useful tool for basic and pre-clinical studies of SS.