HNF1A POU Domain Mutations Found in Japanese Liver Cancer Patients Cause Downregulation of HNF4A Promoter Activity with Possible Disruption in Transcription Networks.

Hepatocyte nuclear factor 1A (HNF1A) is the master regulator of liver homeostasis and organogenesis and regulates many aspects of hepatocyte functions. It acts as a tumor suppressor in the liver, evidenced by the increased proliferation in HNF1A knockout (KO) hepatocytes. Hence, we postulated that any loss-of-function variation in the gene structure or composition (mutation) could trigger dysfunction, including disrupted transcriptional networks in liver cells. From the International Cancer Genome Consortium (ICGC) database of cancer genomes, we identified several HNF1A mutations located in the functional Pit-Oct-Unc (POU) domain. In our biochemical analysis, we found that the HNF1A POU-domain mutations Y122C, R229Q and V259F suppressed HNF4A promoter activity and disrupted the binding of HNF1A to its target HNF4A promoter without any effect on the nuclear localization. Our results suggest that the decreased transcriptional activity of HNF1A mutants is due to impaired DNA binding. Through structural simulation analysis, we found that a V259F mutation was likely to affect DNA interaction by inducing large conformational changes in the N-terminal region of HNF1A. The results suggest that POU-domain mutations of HNF1A downregulate HNF4A gene expression. Therefore, to mimic the HNF1A mutation phenotype in transcription networks, we performed siRNA-mediated knockdown (KD) of HNF4A. Through RNA-Seq data analysis for the HNF4A KD, we found 748 differentially expressed genes (DEGs), of which 311 genes were downregulated (e.g., HNF1A, ApoB and SOAT2) and 437 genes were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed that the DEGs were involved in several signaling pathways (e.g., lipid and cholesterol metabolic pathways). Protein-protein network analysis suggested that the downregulated genes were related to lipid and cholesterol metabolism pathways, which are implicated in hepatocellular carcinoma (HCC) development. Our study demonstrates that mutations of HNF1A in the POU domain result in the downregulation of HNF1A target genes, including HNF4A, and this may trigger HCC development through the disruption of HNF4A-HNF1A transcriptional networks.

Hybrid QM/MM Free-Energy Evaluation of Drug-Resistant Mutational Effect on the Binding of an Inhibitor Indinavir to HIV-1 Protease.

A human immunodeficiency virus-1 (HIV-1) protease is a homodimeric aspartic protease essential for the replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analogues have been developed. However, serious drug-resistant mutants have emerged. For understanding the molecular mechanism of the drug resistance, an accurate examination of the impacts of the mutations on ligand binding and enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of indinavir, a potent transition state analogue inhibitor, to the wild-type protein and a V82T/I84V drug-resistant mutant of the HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free-energy optimization technique which combines a highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of the MM protein environment by long-time molecular dynamics simulations. Through the free-energy calculations of protonation states of catalytic groups at the binding pocket and of the ligand-binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug resistance through the direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

Interplay among transacting factors around promoter in the initial phases of transcription.

The initiation signals are raised around the promoter by one of the general transcription factors, triggering a sequence of events that lead to mRNA transcript formation from target genes. Both specific noncoding DNA regions and transacting, macromolecular assemblies are intricately involved and indispensable. The transition between the subsequent transcriptional stages is accompanied by stage-specific signals and structural changes in the macromolecular assemblies and facilitated by the recruitment/removal of other chromatin and transcription-associated elements. Here, we discuss the choreography of transacting factors around promoter in the establishment and effectuation of the initial phases of transcription such as NDR formation, +1 nucleosome positioning, promoter DNA opening, and RNAPII promoter escape from a structural viewpoint.

Fluorodeoxyglucose positron emission tomography integrated with computed tomography to determine resectability of primary lung cancer.

PURPOSE: Fluorodeoxyglucose positron emission tomography integrated with computed tomography (FDGPET/CT) was evaluated as a routine staging technique for primary lung cancer. MATERIALS AND METHODS: We prospectively compared FDG-PET/CT in determining clinical stage and surgical indication with conventional staging not including positron emission tomography (PET). A total of 50 consecutive patients diagnosed with primary lung cancer by cytological or histological examination were studied; 20 of them underwent surgery. RESULTS: Discrepancies between the two staging methods were observed in 14 patients (28%). The stage assigned by PET increased in 12 cases (24%) and decreased in 2 (4%). PET staging was accurate in eight cases with otherwise undetected distant metastases (M1) but was incorrect in six cases, including five where it overdiagnosed nodal metastases (N). Two clinical N3 patients (4%) would have missed a chance of surgery if the surgical indication had been determined by PET staging alone. According to our criteria for surgery, other patients were assigned correctly to surgery by PET staging. The maximum standard uptake value (maxSUV) of all primary lesions ranged from 0 to 23.0 (mean +/- SD, 8.0 +/- 4.4). The mean maxSUV among surgical cases (5.8 +/- 3.6) was significantly smaller than among nonsurgical cases (9.5 +/- 4.2) (P < 0.05). CONCLUSION: Staging examination including FDG-PET/CT and brain magnetic resonance imaging ordinarily can determine the clinical stage and resectability of primary lung cancer. False-positive findings in regional lymph nodes, possibly reflecting past infectious disease, are the most important remaining problem.

Ground-glass opacities showing an adenoma-to-carcinoma sequence in the lung.

We encountered a patient with three left lower lobe pulmonary tumors evident as discrete ground-glass opacities by computed tomography. Pathological diagnoses of the resected lesions included a focus of atypical adenomatous hyperplasia (AAH) and two localized noninvasive bronchioloalveolar carcinomas (BACs) of types A and C according to Noguchi's classification. This case supports the hypothesis of an adenoma-to-carcinoma sequence in the lung, as the coexisting lesions represented sequential adenocarcinoma progression from a precancerous lesion, AAH, to very early-stage adenocarcinoma, noninvasive BAC.

Reconstruction of the anterior chest wall after subtotal sternectomy for metastatic breast cancer: report of a case.

We describe our successful operative management of a solitary metastasis in the sternal body after modified left mastectomy. Because the primary lesion was well controlled and the sternal metastasis was isolated, we performed a subtotal sternectomy, with full-thickness resection of the anterior chest wall, including the sternal body and inferior part of the manubrium (14.5 cm x 8.5 cm x 3.0 cm). A prosthesis was created to fill the defect, by sandwiching molded bone cement (methylmethacrylate) between two layers of Prolene mesh. The prosthesis was fixed to the cut ends of the costal cartilages and the residual manubrium. The patient had an uneventful course, and her respirations were normal without paradoxical movement of the thorax or hypoxemia. The skin covering the prosthesis healed well. Thus, the creation of an artificial chest wall from methylmethacrylate and Prolene mesh is a useful technique for repairing sternal defects.

A minute small-cell lung cancer showing a latent phase early in growth.

A minute small-cell lung cancer measuring 8 x 5 mm was detected and serially imaged by computed tomography for about a year preceding resection. Although this solid nodule showed a short overall doubling time (76 days), the growth curve included an early phase without apparent growth prior to the phase of rapid growth. Accordingly, lung cancer cannot be ruled out when a small nodule (<10 mm) does not enlarge in the first several months of computed tomographic follow-up.

Epidermal growth factor receptor gene mutations in early pulmonary adenocarcinomas.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations are frequently found in pulmonary adenocarcinomas. MATERIALS AND METHODS: Various lung cancers (n=30) including 8 small adenocarcinomas were examined for EGFR gene mutations in three exons. RESULTS: Mutations were detected in 32% of adenocarcinomas. Exon 19 mutations were detected in 5 tumors, often advanced stages: 1 in Noguchi's pathologic type C, 2 in type D, and 2 in type F. Exon 21 mutations were detected in 3 tumors, all small adenocarcinomas in type C, at pathologic stage IA. CONCLUSION: We suspect that exon 21 mutations are early events in small bronchioloalveolar carcinomas, while exon 19 mutations are later events occurring in adenocarcinomas of various types.

Cavernous hemangioma of the rib diagnosed preoperatively by percutaneous needle biopsy.

A 74-year-old man presented to our hospital with persistent low-grade back pain that had continued for 6 months. An expansile tumor containing delicate bony trabeculae was found in the posterior portion of the left fifth rib. Preoperative examination of a percutaneous needle biopsy specimen suggested a diagnosis of hemangioma; postoperative pathology examination of the resected tumor confirmed this diagnosis. We emphasize the value of preoperative needle biopsy in determining the most suitable treatment for these rib tumors.

Resection of a dumbbell-shaped thoracic neurinoma by hemilaminectomy: a case report.

Neurinomas originating from intercostal nerve roots can grow both inside and outside of the spinal canal, forming dumbbell-shaped tumors. Such a neurinoma was discovered at the Th3 and Th4 levels in a 73-year-old woman during evaluation for breast cancer surgery. Magnetic resonance images (MRI) showed spinal cord compression by the tumor despite lack of neurologic symptoms. The tumor was resected successfully via hemilaminectomy with costotransversectomy. Postoperative course was uneventful, and no stabilization was needed after operation. Back pain was the only postoperative complication. Analgesics were administered for 1 month, and the pain resolved over 3 months. No recurrent neurinoma was found in follow-up images at 8 months. We consider hemilaminectomy safe and effective for complete resection of a dumbbell-shaped thoracic neurinoma.

Pulmonary inflammatory myofibroblastic tumor resected by video-assisted thoracoscopic surgery: Report of a case.

Pulmonary inflammatory myofibroblastic tumor (IMT) is rare. A 38-year-old woman visited our hospital 2 days after experiencing transient anterior chest pain. Chest radiography showed a nodule suggestive of adenocarcinoma in the middle lobe; however, transbronchial lung biopsy and computed tomography (CT)-guided percutaneous needle cytology showed signs of inflammation. To obtain a definitive diagnosis we performed a wedge resection using video-assisted thoracoscopic surgery and removed the nodule completely. The pathologic diagnosis made during surgery was IMT. The longest dimension of the nodule was 28 mm. Immunohistochemical examination showed proliferating spindle cells, which were positive for vimentin and smooth muscle actin, but negative for desmin, CD34, cytokeratin, epithelial membrane antigen, S100 protein, and neuron specific enolase. These findings were consistent with the staining pattern of IMT previously reported. Careful follow-up is necessary to detect any sign of local recurrence and distant metastases.

Role of preoperative chemotherapy for non-small-cell lung cancer: a meta-analysis.

The efficacy of preoperative chemotherapy for improving postoperative survival in patients with non-small-cell lung cancer (NSCLC) is controversial. We therefore conducted a meta-analysis of the published phase III randomized clinical trials (RCTs) to quantitatively evaluate the survival benefit of preoperative chemotherapy. After searching the MEDLINE database from 1980 to 2005, five studies were selected for the present meta-analysis. DerSimonian-Laird random effects analysis was used to estimate the hazard ratio (HR) of patients who underwent preoperative chemotherapy at the time points of 1, 3, and 5 years after surgery. A total of 564 patients in stages IB-IIIA served as the data sources. Preoperative chemotherapy was assigned to a total of 281 patients, while surgery alone was assigned to 283 patients. The combined HRs at 1, 3, and 5 years after resection were 0.65 (95% CI, 0.45-0.94), 0.83 (95% CI, 0.74-0.93), and 0.85 (95% CI, 0.70-1.04), respectively, for preoperative chemotherapy compared to surgery alone. The combined survival differences at 1 and 3 years time point were significant, while the difference at 5 years after resection was not significant. When only the 122 stage IIIA patients were analyzed, none of the HR at any time point was significant. In conclusion, the present meta-analysis suggests that the benefit of preoperative chemotherapy for patients with NSCLC is unclear, especially for stage IIIA patients.

Quantitative detection of lung cancer cells by fluorescence in situ hybridization: comparison with conventional cytology.

STUDY OBJECTIVE: The aim of this study was to clarify whether fluorescence in situ hybridization (FISH) can diagnose lung cancer in various clinical specimens in comparison with conventional cytology. DESIGN: Prospective study. SETTING: University hospital in a metropolitan area. PATIENTS: Fifty consecutive patients with abnormal chest radiography or CT scan findings were enrolled. The patients included 32 men and 18 women, with an average age of 64 years. The final definitive diagnosis was made by histologic examination, as follows: 38 primary lung cancers (24 adenocarcinomas, 8 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas); 1 metastatic renal cell carcinoma; and 11 benign lesions. METHODS: Four types of clinical specimens were analyzed. Cells obtained by transbronchial brushing and transbronchial fine-needle aspiration using a fiberoptic bronchoscope under fluoroscopy, CT scan-guided percutaneous needle biopsy, and bronchial washings. On every examination, duplicate slides were made for analyses of conventional cytology and FISH. RESULTS: Classifications according to conventional cytology were as follows: class I, 4 patients; class II, 15 patients; class IIIa, 3 patients; class IIIb, 5 patients; and class V, 23 patients. A classification higher than class IIIb was considered to be positive for cancer. For cytology, we found no false-positive cases and 11 false-negative cases. The specificity was 100%, and the sensitivity was 71.8%. By FISH, 34 cases showed aberrant copy numbers in either chromosome 3 or 17. We found no false-positive cases and five false-negative cases. The specificity was 100%, and the sensitivity was 87.1%. CONCLUSION: The ability of FISH to detect aneusomic lung cancer cells is superior to conventional cytology for the diagnosis of lung cancer.

Association of HER-2 overexpression with prognosis in nonsmall cell lung carcinoma: a metaanalysis.

BACKGROUND: Prognostic implications of overexpression of the HER-2 gene in nonsmall cell lung carcinoma (NSCLC) are a matter of controversy. Many conflicting results have been reported from different laboratories. METHODS: A metaanalysis of published studies was performed for this quantitative review of the effects of HER-2 overexpression on survival among patients with NSCLC. Of 44 articles initially selected, 20 articles fulfilled eligibility criteria. DerSimonian-Laird random effects analysis was used to estimate the effects of HER-2 overexpression on survival differences (the survival rate among patients without HER-2 overexpression minus the survival rate among patients with HER-2 overexpression) at endpoints of 1 years, 3 years, and 5 years after resection of NSCLC. RESULTS: In total, 2579 patients were included in the final analysis. Overall, HER-2 positivity differed according to histologic type and included 38% of patients with adenocarcinoma, 16% of patients with squamous cell carcinoma, and 18% of patients with large cell carcinoma (P < 0.0001). The combined survival differences in patients with NSCLC at 1 year, 3 years, and 5 years, respectively, were 2.7% (95% confidence interval [95% CI], 1.3-6.7%; P = 0.1787), 15.2% (95% CI, 5.8-24.5%; P = 0.0015), and 16.4% (95% CI, 7.9-14.8%; P = 0.0001), suggesting significant poorer survival at 3 years and 5 years among patients with HER-2 overexpression. In patients with adenocarcinoma, the combined survival difference at 5 years was 26.0% (95% CI, 16.0-36.1%; P < 0.0001), suggesting a particularly strong survival impact for HER-2 overexpression. CONCLUSIONS: A significant, unfavorable prognostic effect of HER-2 overexpression in NSCLC was evident from the metaanalysis. However, because several studies that found no significant difference were excluded by the current eligibility criteria, caution is needed in interpreting the results.

History of limited resection for non-small cell lung cancer.

A retrospective study of limited resection for lung cancer in a large number of patients was first reported in the 1970s. The reported outcome of segmentectomy was comparable to that of standard lobectomy. The North American Lung Cancer Study Group (LCSG) performed a randomized controlled clinical trial to compare limited resection (segment or wedge) with lobectomy for T1N0 (stage IA) non-small cell lung cancer (NSCLC) in the 1980s. The study revealed a significant excess of intrathoracic recurrence rate and a tendency to poorer survival in the limited resection group. Since then, limited resection has not been considered the standard operation for lung cancer. However, this situation is gradually changing, because the recent introduction of chest computed tomography (CT) to mass surveys has made peripherally located lung cancer detectable at the earliest stage. Several recent non-randomized studies of intentional limited resection from Japan demonstrated good outcomes comparable to those of lobectomy. Organ-sparing wedge resection without systematic dissection of lymph nodes may be suitable for some types of small lung cancers detected only by CT. Our meta-analysis of published data comparing survival rates after limited resection and lobectomy for stage I lung cancer revealed that limited resection was comparable to lobectomy. Further studies are necessary to define precise targets of intentional limited resection for lung cancer.

Cytokine responsiveness in cultured human small airway epithelial cells in relation to lung transplantation.

BACKGROUND: We report a new method for collecting and establishing small airway epithelial cells (SAEC). This method enables the evaluation of the cytokine responsiveness of SAEC, which is still unknown. In this study we evaluated intercellular adhesion molecule-1 (ICAM-1) expression on SAEC stimulated with several inflammatory cytokines and compared it with that on large airway epithelial cells (LAEC). MATERIALS AND METHODS: LAEC and SAEC were treated with IFN-gamma, TNF-alpha, IL-1beta, or their combination. ICAM-1 expression under various conditions was quantified by flow cytometry. Furthermore, immunocytochemical staining was performed to determine intranuclear displacement of signal transducer and activator transcription 1 (Stat1) during ICAM-1 expression by various cytokine stimulations. RESULTS: 1) ICAM-1 expression on both LAEC and SAEC was significantly increased by IFN-gamma stimulation alone and synergistically enhanced by IFN-gamma plus TNF-alpha or IL-1beta stimulation, 2) intranuclear displacement of Stat1 in SAEC by the stimulation with IFN-gamma plus TNF-alpha or IL-1beta was recognized earlier in comparison with that by IFN-gamma stimulation alone. CONCLUSION: The previously unknown peripheral cytokine responsiveness and its mechanisms of SAEC were revealed by this study, which contributes to the understanding of chronic lung allograft rejection recognized around small airways.