Genetic and phenotypic determinants of morphologies in 3D cultures and xenografts of lung tumor cell lines.

We previously proposed the classification of lung adenocarcinoma into two groups: the bronchial epithelial phenotype (BE phenotype) with high-level expressions of bronchial epithelial markers and actionable genetic abnormalities of tyrosine kinase receptors and the non-BE phenotype with low-level expressions of bronchial Bronchial epithelial (BE) epithelial markers and no actionable genetic abnormalities of tyrosine kinase receptors. Here, we performed a comprehensive analysis of tumor morphologies in 3D cultures and xenografts across a panel of lung cancer cell lines. First, we demonstrated that 40 lung cancer cell lines (23 BE and 17 non-BE) can be classified into three groups based on morphologies in 3D cultures on Matrigel: round (n = 31), stellate (n = 5), and grape-like (n = 4). The latter two morphologies were significantly frequent in the non-BE phenotype (1/23 BE, 8/17 non-BE, p = 0.0014), and the stellate morphology was only found in the non-BE phenotype. SMARCA4 mutations were significantly frequent in stellate-shaped cells (4/4 stellate, 4/34 non-stellate, p = 0.0001). Next, from the 40 cell lines, we successfully established 28 xenograft tumors (18 BE and 10 non-BE) in NOD/SCID mice and classified histological patterns of the xenograft tumors into three groups: solid (n = 20), small nests in desmoplasia (n = 4), and acinar/papillary (n = 4). The latter two patterns were characteristically found in the BE phenotype. The non-BE phenotype exhibited a solid pattern with significantly less content of alpha-SMA-positive fibroblasts (p = 0.0004) and collagen (p = 0.0006) than the BE phenotype. Thus, the morphology of the tumors in 3D cultures and xenografts, including stroma genesis, reflects the intrinsic properties of the cancer cell lines. Furthermore, this study serves as an excellent resource for lung adenocarcinoma cell lines, with clinically relevant information on molecular and morphological characteristics and drug sensitivity.

Reciprocal expression of trefoil factor-1 and thyroid transcription factor-1 in lung adenocarcinomas.

Molecular targeted therapies against EGFR and ALK have improved the quality of life of lung adenocarcinoma patients. However, targetable driver mutations are mainly found in thyroid transcription factor-1 (TTF-1)/NK2 homeobox 1 (NKX2-1)-positive terminal respiratory unit (TRU) types and rarely in non-TRU types. To elucidate the molecular characteristics of the major subtypes of non-TRU-type adenocarcinomas, we analyzed 19 lung adenocarcinoma cell lines (11 TRU types and 8 non-TRU types). A characteristic of non-TRU-type cell lines was the strong expression of TFF-1 (trefoil factor-1), a gastric mucosal protective factor. An immunohistochemical analysis of 238 primary lung adenocarcinomas resected at Jichi Medical University Hospital revealed that TFF-1 was positive in 31 cases (13%). Expression of TFF-1 was frequently detected in invasive mucinous (14/15, 93%), enteric (2/2, 100%), and colloid (1/1, 100%) adenocarcinomas, less frequent in acinar (5/24, 21%), papillary (7/120, 6%), and solid (2/43, 5%) adenocarcinomas, and negative in micropapillary (0/1, 0%), lepidic (0/23, 0%), and microinvasive adenocarcinomas or adenocarcinoma in situ (0/9, 0%). Expression of TFF-1 correlated with the expression of HNF4-α and MUC5AC (P < .0001, P < .0001, respectively) and inversely correlated with that of TTF-1/NKX2-1 (P < .0001). These results indicate that TFF-1 is characteristically expressed in non-TRU-type adenocarcinomas with gastrointestinal features. The TFF-1-positive cases harbored KRAS mutations at a high frequency, but no EGFR or ALK mutations. Expression of TFF-1 correlated with tumor spread through air spaces, and a poor prognosis in advanced stages. Moreover, the knockdown of TFF-1 inhibited cell proliferation and soft-agar colony formation and induced apoptosis in a TFF-1-high and KRAS-mutated lung adenocarcinoma cell line. These results indicate that TFF-1 is not only a biomarker, but also a potential molecular target for non-TRU-type lung adenocarcinomas.

Mucin 21 is a key molecule involved in the incohesive growth pattern in lung adenocarcinoma.

Decreased cell adhesion has been reported as a significant negative prognostic factor of lung cancer. However, the molecular mechanisms responsible for the cell incohesiveness in lung cancer have not yet been elucidated in detail. We herein describe a rare histological variant of lung adenocarcinoma consisting almost entirely of individual cancer cells spreading in alveolar spaces in an incohesive pattern. A whole exome analysis of this case showed no genomic abnormalities in CDH1 or other genes encoding cell adhesion molecules. However, whole mRNA sequencing revealed that this case had an extremely high expression level of mucin 21 (MUC21), a mucin molecule that was previously shown to inhibit cell-cell and cell-matrix adhesion. The strong membranous expression of MUC21 was found on cancer cells using mAbs recognizing different O-glycosylated forms of MUC21. An immunohistochemical analysis of an unselected series of lung adenocarcinoma confirmed that the strong membranous expression of MUC21 correlated with incohesiveness. Thus, MUC21 could be a promising biomarker with potential diagnostic and therapeutic applications for lung adenocarcinoma showing cell incohesiveness.

Expression of protein arginine methyltransferase-5 in oral squamous cell carcinoma and its significance in epithelial-to-mesenchymal transition.

Protein arginine methyltransferases (PRMT) 5, a member of type II arginine methyltransferases, catalyzes the symmetrical dimethylation of arginine residues on histone and non-histone substrates. Although the overexpression of PRMT5 has been reported in various cancers, its role in oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, we immunohistochemically examined the expression of PRMT5 in surgically resected oral epithelial dysplasia (OED, n = 8), oral intraepithelial neoplasia (OIN)/carcinoma in situ (CIS) (n = 11) and OSCC (n = 52) with or without contiguous OED lesions. In the normal epithelium, PRMT5 was weakly expressed in the cytoplasm of basal layer cells. In OED, OIN/CIS, and OSCC, its expression consistently and uniformly increased in the cytoplasm of dysplastic and cancer cells. Moreover, nuclear and cytoplasmic localization was detected in the invasive front of cancer cells, particularly in cases showing poor differentiation or aggressive invasion patterns. The concomitant nuclear and cytoplasmic expression of PRMT5 correlated with the loss of E-cadherin and cytokeratin 17, and the upregulation of vimentin, features that are both indicative of epithelial-to-mesenchymal transition. PRMT5 may play a role from early oncogenesis through to the progression of OSCC, particularly in the aggressive mode of stromal invasion.

Pulmonary Mycobacterium heckeshornense infection in a healthy woman.

Pulmonary infection due to Mycobacterium heckeshornense (M. heckeshornense) in healthy adults without underlying diseases is very rare and optimal treatments have not yet been established. A 39-year-old woman was admitted to our hospital for further examinations following the identification of a pulmonary cavitary nodule. Acid-fast bacilli were cultured from specimens obtained by bronchofiberscopy, and identified with M. heckeshornense using nucleotide sequencing. Antimycobacterial chemotherapy was effective temporarily, while the nodular lesion subsequently worsened. The patient underwent lobectomy and has not relapsed thus far. A lung specimen showed marked granulomatous inflammation with extensive caseous necrosis and the preservation of some parts of alveolar septa within caseous necrosis, indicating an exudative process and resistance to chemotherapy. M. heckeshornense is strongly pathogenic and switching to surgical intervention needs to be considered when chemotherapy is insufficient.

Inactivating mutations and hypermethylation of the NKX2-1/TTF-1 gene in non-terminal respiratory unit-type lung adenocarcinomas.

The major driver mutations of lung cancer, EGFR mutations and EML4-ALK fusion, are mainly detected in terminal respiratory unit (TRU)-type lung adenocarcinomas, which typically show lepidic and/or papillary patterns, but are rarely associated with a solid or invasive mucinous morphology. In order to elucidate the key genetic events in non-TRU-type lung cancer, we carried out whole-exome sequencing on 43 non-TRU-type lung adenocarcinomas based on morphology (17 acinar, nine solid, and two enteric adenocarcinomas, and 15 adenocarcinomas with a mucinous morphology). Our analysis identified mutations in TP53 (16/43, 37.2%), KRAS (13/43, 30.2%), and NKX2-1/TTF-1 (7/43; 16.3%) as the top three significantly mutated genes, while the EGFR mutation was rare (1/43, 2.3%) in this cohort. Eight NKX2-1/TTF-1 mutations (five frameshift, two nonsense, and one missense) were identified, with one case harboring two distinct NKX2-1/TTF-1 mutations (one missense and one frameshift). Functional assays with the NK2 homeobox 1 (NKX2-1)/thyroid transcription factor 1 (TTF-1) mutants revealed that none of them retain the activity as a transcriptional factor. Histologically, invasive mucinous adenocarcinomas accounted for most of the NKX2-1/TTF-1 mutations (five cases), as well as one enteric and one acinar adenocarcinoma. Immunohistochemistry showed that the cohort was largely divided into TTF-1-postive/hepatocyte nuclear factor 4-α (HNF4-α)-negative and TTF-1-negative/HNF4-α-positive groups. NKX2-1/TTF-1 mutations were exclusively found in the latter, in which the gastrointestinal markers, mucin 5AC and cytokeratin 20, were frequently expressed. Bisulfite sequencing revealed that the NKX2-1/TTF-1 gene body was highly methylated in NKX2-1/TTF-1-negative cases, including those without the NKX2-1/TTF-1 mutations. The genetic or epigenetic inactivation of NKX2-1/TTF-1 may play an essential role in the development and aberrant differentiation of non-TRU-type lung adenocarcinomas.

Frequent loss of the expression of multiple subunits of the SWI/SNF complex in large cell carcinoma and pleomorphic carcinoma of the lung.

The switch/sucrose non-fermenting (SWI/SNF) complex has recently emerged as a novel tumor suppressor in various human cancers. In the present study, we analyzed the expression of multiple SWI/SNF subunits in primary non-small cell lung cancer (NSCLC). A total of 133 NSCLC, consisting of 25 squamous cell carcinomas (SCC), 70 adenocarcinomas (AD), 16 large cell carcinomas (LC), and 22 pleomorphic carcinomas (PL), were immunohistochemically examined for the expression of BRG1, BRM, BAF47, ARID1A, and ARID1B. The frequency at which reductions in the expression of BRG1 were observed was significantly higher in the LC-PL group (13/38, 34.2%) than in the SCC-AD group (7/95, 7.4%). Similarly, the frequency at which reductions in the expression of BRM were observed was significantly higher in the LC-PL group (17/38, 44.7%) than in the SCC-AD group (14/95, 14.7%). The loss of the expression of ARID1A, ARID1B, and BAF47 was observed only in a fraction of NSCLC cases. Furthermore, the frequency at which the concurrent loss of multiple subunits of the SWI/SNF complex was observed was significantly higher in the LC-PL group (10/38, 26.3%) than in the SCC-AD group (8/95, 8.4%). Collectively, these results indicate that the loss of the SWI/SNF complex was related to dedifferentiation in NSCLC.

Immunohistochemical analysis of the expression of E-cadherin and ZEB1 in non-small cell lung cancer.

We performed an immunohistochemical analysis of the expression of zinc-finger E-box binding homeobox 1 (ZEB1), a master regulator of epithelial-mesenchymal transition (EMT), and determined its relationship with E-cadherin in 157 non-small cell lung carcinomas (93 adenocarcinomas, 36 squamous cell carcinomas, 18 large cell carcinomas, and 10 pleomorphic carcinomas). Although the expression of E-cadherin was low in the subset of adenocarcinomas (10%) and squamous cell carcinomas (11%), ZEB1 expression was only observed in one case of squamous cell carcinoma and none of the adenocarcinomas. In contrast, the low expression of E-cadherin (50% and 90%, respectively) and the positive expression of ZEB1 (11% and 50%, respectively) were more frequently observed in poorly differentiated carcinomas (large cell carcinomas and pleomorphic carcinomas). Overall, the expression of ZEB1 was inversely correlated with that of E-cadherin. Furthermore, the distribution of ZEB1-positive cancer cells was more restricted than in the area in which the expression of E-cadherin was lost, and the former was detected within the latter. We concluded that the expression of ZEB1 was not necessarily associated with the low expression of E-cadherin in lung adenocarcinomas and squamous cell carcinomas. The expression of ZEB1 correlated with an undifferentiated and/or sarcomatoid morphology that may occur in the late stage of EMT.

The significance of Hippo pathway protein expression in oral squamous cell carcinoma.

Introduction: The Hippo pathway consists of mammalian sterile 20-like kinase 1/2 (MST1/2), large tumor suppressor 1/2 (LATS1/2), and yes-associated protein (YAP)1. Herein, we present the first report on the significance of major Hippo pathway protein expression in oral squamous cell carcinoma (OSCC). Methods: The analyses included oral epithelial dysplasia (OED, n = 7), carcinoma in situ (CIS, n = 14), and oral squamous cell carcinoma (OSCC, n = 109). Results: Cytoplasmic expression of MST1, LATS1, and LATS2 was low in OED, CIS, and OSCC. The cytoplasmic expression of MST2 was high in OED (5/7 cases), CIS (9/14 cases), and poorly differentiated OSCC (8/8 cases) but was low/lost in a proportion of differentiated OSCC (60/101 cases). The expression of YAP1 was associated with differentiation; low YAP expression was significantly more frequent in well-differentiated OSCC (35/71 cases), compared to moderately and poorly differentiated OSCC (11/38 cases). An infiltrative invasion pattern was associated with a high expression of MST2 and high expression of YAP1. The high expression of YAP1 was associated with features of epithelial-to-mesenchymal transition (EMT), such as the loss of E-cadherin and high expression of vimentin, laminin 5, and Slug. High expression of protein arginine methyltransferase (PRMT) 1 or 5, which positively regulates YAP activity, was associated with the high expression of YAP1 (p < 0.0001). Conclusion: Among the major Hippo pathway proteins, MST2 displayed a distinctive expression pattern in a significant proportion of differentiated OSCC, suggesting a possible differential role for MST2 depending on the course of OSCC progression. A high YAP1 expression may indicate aggressive OSCC with EMT via PRMTs at the invasive front.

Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

INTRODUCTION: HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. MATERIALS AND METHODS: HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. RESULT: HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. CONCLUSION: HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Genomic profiling of renal cell carcinoma in patients with end-stage renal disease.

The purpose of the present study was to determine the genomic profile of renal cell carcinoma (RCC) in end-stage renal disease (ESRD) by analyzing genomic copy number aberrations. Seventy-nine tumor samples from 63 patients with RCC-ESRD were analyzed by array comparative genomic hybridization using the Agilent Whole Human Genome 4 × 44K Oligo Micro Array (Agilent Technologies Inc., Palo Alto, CA, USA). Unsupervised hierarchical clustering analysis revealed that the 63 cases could be divided into two groups, Clusters A and B. Cluster A was comprised mainly of clear cell RCC (CCRCC), whereas Cluster B was comprised mainly of papillary RCC (PRCC), acquired cystic disease (ACD)-associated RCC, and clear cell papillary RCC. Analysis of the averaged frequencies revealed that the genomic profiles of Clusters A and B resembled those of sporadic CCRCC and sporadic PRCC, respectively. Although it has been proposed on the basis of histopathology that ACD-associated RCC, clear cell papillary RCC and PRCC-ESRD are distinct subtypes, the present data reveal that the genomic profiles of these types, categorized as Cluster B, resemble one another. Furthermore, the genomic profiles of PRCC, ACD-associated RCC and clear cell papillary RCC admixed in one tissue tended to resemble one another. On the basis of genomic profiling of RCC-ESRD, we conclude that the molecular pathogenesis of CCRCC-ESRD resembles that of sporadic CCRCC. Although various histologic subtypes of non-clear cell RCC-ESRD have been proposed, their genomic profiles resemble those of sporadic PRCC, suggesting that the molecular pathogenesis of non-CCRCC-ESRD may be related to that of sporadic PRCC.

Solitary fibrous tumor with aneurysmal bone cyst-like change in the middle cranial fossa: illustrative case.

BACKGROUND: Solitary fibrous tumor (SFT) is a rare mesenchymal tumor known for its propensity for recurrence and metastasis. Furthermore, aneurysmal bone cyst (ABC) is a benign osteolytic lesion. ABC-like areas can be seen in bone tumors that have undergone hemorrhagic cystic change. They are formed by disruptions in the osseous circulation caused by the associated lesion. The most common associated lesions are giant cell tumor, chondroblastoma, osteoblastoma, osteosarcoma, chondromyxoid fibroma, and fibrous dysplasia. There has been no reported case of SFT being the associated lesion. OBSERVATIONS: A 42-year-old woman presented with a 6-month history of headache and impaired memory. Radiological examinations revealed a 50-mm cystic lesion with multiple fluid levels arising from the left temporal bone. Total resection of the tumor was conducted, and postoperative course was uneventful. Histopathological examination was consistent with SFT with ABC-like change. LESSONS: This is the first documented case of SFT with ABC-like change in the cranial fossa. This should be considered a differential diagnosis when treating a lesion in the cranial fossa, such as in this case, to achieve complete resection of the tumor and have close follow-up postoperatively.

Downregulation of SAV1 plays a role in pathogenesis of high-grade clear cell renal cell carcinoma.

BACKGROUND: Clinical outcome of patients with high-grade ccRCC (clear cell renal cell carcinoma) remains still poor despite recent advances in treatment strategies. Molecular mechanism of pathogenesis in developing high-grade ccRCC must be clarified. In the present study, we found that SAV1 was significantly downregulated with copy number loss in high-grade ccRCCs. Therefore, we investigated the SAV1 function on cell proliferation and apoptosis in vitro. Furthermore, we attempted to clarify the downstream signaling which is regulated by SAV1. METHODS: We performed array CGH and gene expression analysis of 8 RCC cell lines (786-O, 769-P, KMRC-1, KMRC-2, KMRC-3, KMRC-20, TUHR4TKB, and Caki-2), and expression level of mRNA was confirmed by quantitative RT-PCR (qRT-PCR) analysis. We next re-expressed SAV1 in 786-O cells, and analyzed its colony-forming activity. Then, we transfected siRNAs of SAV1 into the kidney epithelial cell line HK2 and renal proximal tubule epithelial cells (RPTECs), and analyzed their proliferation and apoptosis. Furthermore, the activity of YAP1, which is a downstream molecule of SAV1, was evaluated by western blot analysis, reporter assay and immunohistochemical analysis. RESULTS: We found that SAV1, a component of the Hippo pathway, is frequently downregulated in high-grade ccRCC. SAV1 is located on chromosome 14q22.1, where copy number loss had been observed in 7 of 12 high-grade ccRCCs in our previous study, suggesting that gene copy number loss is responsible for the downregulation of SAV1. Colony-forming activity by 786-O cells, which show homozygous loss of SAV1, was significantly reduced when SAV1 was re-introduced exogenously. Knockdown of SAV1 promoted proliferation of HK2 and RPTEC. Although the phosphorylation level of YAP1 was low in 786-O cells, it was elevated in SAV1-transduced 786-O cells. Furthermore, the transcriptional activity of the YAP1 and TEAD3 complex was inhibited in SAV1-transduced 786-O cells. Immunohistochemistry frequently demonstrated nuclear localization of YAP1 in ccRCC cases with SAV1 downregulation, and it was preferentially detected in high-grade ccRCC. CONCLUSIONS: Taken together, downregulation of SAV1 and the consequent YAP1 activation are involved in the pathogenesis of high-grade ccRCC. It is an attractive hypothesis that Hippo signaling could be candidates for new therapeutic target.

Stromal p16 Expression Helps Distinguish Atypical Polypoid Adenomyoma From Myoinvasive Endometrioid Carcinoma of the Uterus.

Atypical polypoid adenomyoma (APA) is a polypoid lesion that is comprised of atypical endometrial glands and fibromuscular stroma, which pathologists often confuse with myoinvasive endometrioid carcinoma. Here, we characterized the immunohistochemical and molecular features of the stromal components of APA to find distinct markers between APA and myoinvasive endometrioid carcinoma. First, we examined the immunohistochemical expression and gene mutations that were previously investigated in uterine and breast fibroepithelial lesions using 12 cases of APA. α-smooth muscle actin was diffusely positive in the stromal component in all cases, whereas desmin and h-caldesmon were focally expressed in 8 cases. Positive expression was also observed in 9 cases for CD10, 12 cases for estrogen receptor, 3 cases for HMGA2, and 3 cases for MDM2. All cases showed normal p53 expression and negative staining of HMGA1 and nuclear ß-catenin. No mutations in MED12 exon 2 and the TERT promoter were found in any cases. p16 was positive in all cases and showed diffuse expression in 10 cases. We assessed stromal p16 expression in 84 cases of myoinvasive endometrioid carcinoma. The stromal p16 status was negative in all myoinvasive carcinomas, but there was 1 case with focal staining. There was a significant difference in stromal p16 expression between APA and myoinvasive endometrioid carcinoma (P<0.001). Stromal p16 expression was more suggestive of APA than myoinvasive endometrioid carcinoma among endometrial fibroepithelial lesions.

Primary carcinosarcoma of the ureteropelvic junction associated with ureteral duplication: A case report.

RATIONALE: Primary carcinosarcoma of the upper urinary tract is rare. Ureteral duplication is one of the most common urinary tract malformations. Additionally, the association between ureteral duplication and malignancy is unknown. To the best of our knowledge, no cases of malignant tumors diagnosed as carcinosarcoma with ureteral duplication have been reported. We herein report the case of a patient with carcinosarcoma of the ureteropelvic junction associated with incomplete ureteral duplication. PATIENT CONCERNS: A 60-year-old Japanese woman presented with painless gross hematuria. She had a history of total hysterectomy and chemotherapy for endometrioid carcinoma 5 years before. She had no history of occupational chemical exposure. DIAGNOSES: Radiographic imaging revealed right incomplete ureteral duplication, hydronephrosis, and a polypoid tumor in the ureteropelvic junction of the lower moiety of the right kidney. Urine cytology showed a small amount of degenerated atypical epithelial and nonepithelial cells. The transureteral biopsy specimen showed dysplastic urothelial cells and atypical myoid spindle cells. These findings were indefinite for malignancy. INTERVENTIONS: The patient underwent right nephroureterectomy. Pathological examination of the resected tumor showed a biphasic neoplasm composed of carcinomatous and sarcomatous components. The sarcomatous component was immunohistochemically positive for vimentin, desmin, h-caldesmon, and α-SMA and negative for pancytokeratin (AE1/AE3), low molecular weight cytokeratin (CAM 5.2), EMA, E-cadherin, GATA3, uroplakin 2, and p63. Based on these findings, we diagnosed the tumor as carcinosarcoma. OUTCOMES: The postoperative course was uneventful. No additional therapy was administered. The patient has remained alive without recurrence for 21 months since surgery. LESSONS: Carcinosarcoma can arise from ureteral duplication. Although the majority of carcinosarcomas of the upper urinary tract are diagnosed at an advanced stage and have a poor prognosis, some can have a less aggressive course. Further studies are needed to determine the association between ureteral duplication and malignancy.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Clinical features of pulmonary aspergillosis associated with interstitial pneumonia.

OBJECTIVE: We retrospectively investigated the clinical features of pulmonary aspergillosis associated with interstitial pneumonia. METHODS: We reviewed the medical records of all patients treated for interstitial pneumonia with or without pulmonary aspergillosis at our institution between April 2006 and August 2012 and evaluated the clinical features as well as risk and prognostic factors for pulmonary aspergillosis associated with interstitial pneumonia. RESULTS: Among 539 patients with interstitial pneumonia, 15 who suffered from pulmonary aspergillosis were identified. The median age was 69.2±7.0 years, and fourteen patients were men. The subtypes of pulmonary aspergillosis were chronic pulmonary aspergillosis (n=14) and invasive pulmonary aspergillosis (n=1). The forms of interstitial pneumonia included idiopathic pulmonary fibrosis (n=9), rheumatoid arthritis-related interstitial pneumonia (n=4) and pleuroparenchymal fibroelastosis (n=2). The underlying conditions were emphysema (n=9) and a history of oral corticosteroid and/or immunosuppressive use (n=4). Home oxygen therapy (HOT) was administered in 11 patients. Following the diagnosis of pulmonary aspergillosis, all patients were treated with antifungal drugs. Ten patients (66.6%) died. A comparison of the interstitial pneumonia patients with and without pulmonary aspergillosis showed that the presence of emphysema, use of HOT and death were significantly associated with pulmonary aspergillosis. CONCLUSION: Pulmonary aspergillosis is one of the major complications of interstitial pneumonia and its prognosis is poor. Therefore, providing careful monitoring and proper treatment is extremely important.