The Frequency of DNA Mismatch Repair Deficiency Is Very Low in Surgically Resected Lung Carcinoma.

INTRODUCTION: DNA mismatch repair (MMR) deficiency leads to changes in the length of nucleotide repeat sequences of tumor DNA. In that situation, DNA replicational errors occur and accumulate during DNA replication. As a result, this mechanism frequently affects the coding regions of oncogenes and tumor suppressor genes and causes carcinogenesis. Recently, DNA MMR deficiency has been recognized as a predictive biomarker for immunotherapy. The aim of this study is to examine the frequency of DNA MMR deficiency and clinicopathological characteristics in surgically resected lung carcinoma (LC) and their correlation. METHODS: A total of 1153 LCs were examined. Tissue microarrays were constructed. The status of MMR deficiency was evaluated by immunohistochemical analysis of MMR protein expression (hMLH1, hMSH2, hMSH6, and hPMS2). Microsatellite instability analysis, BRAF mutation, and MLH1 methylation analysis were performed for cases that showed MMR deficiency. RESULTS: Only 2 of the 1153 cases (0.17%) showed a loss of hMLH1/hPMS2 protein expression. They also had high levels of microsatellite instability (MSI-H), had neither MLH1 promoter methylation nor BRAF mutation, and were male smokers. Histopathologically, one was a squamous cell carcinoma, and the other was combined small cell carcinoma with squamous cell carcinoma. Regarding PD-L1 protein expression, one had high expression, and the other had none. CONCLUSION: The frequency of MMR deficiency was very low in LC. However, our two cases were non-adenocarcinoma and differed from previous studies. Because of its very low frequency, MMR deficiency is not a practical biomarker to predict the effect of immune checkpoint inhibitors in LC.

The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

Programmed death ligand 1 protein expression is positively correlated with the solid predominant subtype, high MIB-1 labeling index, and p53 expression and negatively correlated with epidermal growth factor receptor mutations in lung adenocarcinoma.

Programmed death ligand 1 (PD-L1) protein expression is a proposed predictive biomarker of immunotherapy; thus, identification of the clinicopathological and molecular characteristics associated with PD-L1 expression is important and necessary. We examined PD-L1 immunohistochemical expression and its relationships with the clinicopathological and molecular characteristics of patients with surgically resected nonsmall cell lung carcinoma. PD-L1 expression differed according to the histological subtype. Among 633 patients with adenocarcinoma, 523 (82.6%) had no PD-L1 expression, 78 (12.3%) low expression, and 32 (5.1%) high expression. PD-L1 expression was more common in men (p < 0.001), in smokers (p = 0.002), and in patients with a more advanced stage (p = 0.002), the solid predominant subtype (p < 0.001), no epidermal growth factor receptor(EGFR) mutations (p < 0.001), a high MIB-1 labeling index (p < 0.001), and positive p53 immunohistochemical expression (p < 0.001). In a multivariate logistic regression analysis, the solid predominant subtype (odds ratio [OR] = 4.92, 95% confidence interval [CI]: 2.72-8.89, p < 0.001), no EGFR mutations (OR = 2.27, 95% CI: 1.35-2.7, p = 0.002), a high MIB-1 labeling index (OR = 2.78, 95% CI: 1.72-4.55, p < 0.001), and p53 positivity (OR = 2.13, 95% CI: 1.34-4.36, p = 0.042) were significantly and independently associated with PD-L1 expression. The combination of the solid predominant subtype with a high MIB-1 labeling index was strongly associated with positive expression of PD-L1. In the 193 patients with squamous cell carcinoma, 92 (47.7%) had no PD-L1 expression, 57 (29.5%) low expression, and 44 (22.8%) high expression. There were no significant correlations between PD-L1 expression and the evaluated clinicopathological or molecular characteristics of these patients. These results, indicating associations of PD-L1 with various clinicopathological or molecular characteristics in adenocarcinoma but not squamous cell carcinoma, may be useful for selecting patients with a good response to immune checkpoint inhibitors.

Mapping mammalian replication domains using the ion torrent semiconductor sequencing platform.

Here, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method.

Tumor spread through air spaces is a useful predictor of recurrence and prognosis in stage I lung squamous cell carcinoma, but not in stage II and III.

OBJECTIVES: Tumor spread through air spaces (STAS) is a newly identified invasion pattern in lung adenocarcinoma. This study aimed to analyze and validate the clinical impact of tumor STAS in surgically resected lung squamous cell carcinoma (SQCC). MATERIALS AND METHODS: We retrospectively reviewed 220 patients with lung SQCC. Tumor STAS was defined as detached tumor cells within the air spaces in the lung parenchyma beyond the edge of the main tumor. Statistical analyses were conducted to investigate the proportion of STAS and the relationship between the presence of STAS and clinicopathological factors, including clinical outcome. RESULTS: STAS was identified in 42 of 220 patients (19.1%). The patients with STAS had a significantly worse 5-year recurrence-free survival (RFS) and 5-year overall survival (OS) than those without STAS (5-year RFS: 37.4% vs. 68.4%; p = 0.0006; 5-year OS: 50.2% vs. 71.4%, p = 0.0078) in stage I, but not in stage II and III. A multivariate analysis showed that the presence of STAS was an independent predictive factor of recurrence (hazard ratio = 3.27; 95% confidence interval, 1.7-6.29; p = 0.0004) and an independent prognostic factor (hazard ratio = 3.01; 95% confidence interval, 1.54-5.89; p = 0.0013) in stage I, but not in stage II and III. CONCLUSION: We found that STAS was detected in 19.1% of surgical resected SQCC, and it was associated with recurrence and worse survival in stage I SQCC, but not in stage II and III SQCC. Therefore, we suggest that STAS is a useful predictor of recurrence and prognosis in stage I lung SQCC.

ß-NMR measurements of molecular-scale lithium-ion dynamics in poly(ethylene oxide)-lithium-salt thin films.

ß-detected NMR (ß-NMR) has been used to study the molecular-scale dynamics of lithium ions in thin films of poly(ethylene oxide) (PEO) containing either lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) or lithium trifluoroacetate (LiTFA) salts at monomer-to-salt ratios (EO/Li) of 8.3. The results are compared with previous ß-NMR measurements on pure PEO and PEO with lithium triflate (LiOTf) at the same loading [McKenzie et al., J. Am. Chem. Soc. 136, 7833 (2014)]. Activated hopping of 8Li+ was observed in all of the films above ∼250 K, with the hopping parameters strongly correlated with the ionicity of the lithium salt rather than the polymer glass transition temperature. The pre-exponential factor increases exponentially with ionicity, while the activation energy for hopping increases approximately linearly, going from 6.3±0.2 kJ mol-1 in PEO:LiTFA to 17.8±0.2 kJ mol-1 in PEO:LiTFSI. The more rapid increase in the pre-exponential factor outweighs the effect of the larger activation energy and results in 8Li+ hopping being fastest in PEO followed by PEO:LiTFSI, PEO:LiOTf, and PEO:LiTFA.

The Clinical Impact of Solid and Micropapillary Patterns in Resected Lung Adenocarcinoma.

INTRODUCTION: Since the new adenocarcinoma (ADC) classification was presented in 2011, several authors have reported that patients with solid (S) and/or micropapillary (MP) predominant patterns showed a worse prognosis. On the other hand, there are several patients who have S and/or MP patterns even if their patterns are not predominant. However, the evaluation of these patients is uncertain. METHODS: A total of 531 ADCs were examined. We classified the patients into five subgroups according to the proportion of S and/or MP patterns: (1) both patterns absent (S-/MP-), (2) S predominant (S pre), (3) MP predominant (MP pre), (4) S pattern present although not predominant and MP pattern absent (S+ not pre/MP-), and (5) MP pattern present although not predominant (MP+ not pre). RESULTS: Of the 531 ADCs, 384 (72.3%) were classified as S-/MP-, 55 (10.4%) as S pre, 11 (2.1%) as MP pre, 42 (7.9%) as S+ not pre/MP-, and 39 (7.3%) as MP+ not pre. In a univariate analysis, the recurrence-free survival (RFS) and overall survival differed significantly among the five subgroups (p < 0.01 and p < 0.01, respectively). In a multivariate analysis, patients with S-/MP- had significantly higher RFS rates than did those with other subgroups. On the other hand, patients with MP pre had lower RFS rates than did those with other subgroups. CONCLUSION: Patients with S and/or MP patterns have a poorer prognosis even if their patterns are not predominant. The S and/or MP patterns must be treated at the time of diagnosis.

Direct visualization of replication dynamics in early zebrafish embryos.

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

[Intravascular papillary endothelial hyperplasia of the lung].

We report a rare case of pulmonary intravascular papillary endothelial hyperplasia. The patient was a 63-year-old male. Multiple lung nodules were noted on chest computed tomography( CT) at preoperative check for gastric cancer. Metastatic lung tumor was suspected, and partial resection of the right lung was performed. Histopathologic examination revealed papillary proliferation lined by endothelial cells and a hematoma. Immunohistochemically, the endothelial cells were positive for CD31/CD34 and factor VIII related antigen.

ß-NMR measurements of lithium ion transport in thin films of pure and lithium-salt-doped poly(ethylene oxide).

ß-Detected nuclear spin relaxation of (8)Li(+) has been used to study the microscopic diffusion of lithium ions in thin films of poly(ethylene oxide) (PEO), where the implanted lithium ions are present in extremely low concentration, and PEO with 30 wt % LiCF3SO3 over a wide range of temperatures both above and below the glass transition temperature. Recent measurements by Do et al. [Phys. Rev. Lett. 2013, 111, 018301] found that the temperature dependence of the Li(+) conductivity was identical to that of the dielectric α relaxation and was well described by the Vogel-Fulcher-Tammann relation, implying the α relaxation dominates the Li(+) transport process. In contrast, we find the hopping of Li(+) in both samples in the high temperature viscoelastic phase follows an Arrhenius law and depends significantly on the salt content. We propose that the hopping of Li(+) between cages involves motion of the polymer but that it is only for long-range diffusion where the α relaxation plays an important role.

The correlation of the International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) classification with prognosis and EGFR mutation in lung adenocarcinoma.

BACKGROUND: The purpose of this study was to validate the prognostic effect and the frequency of mutations in the gene expressing epidermal growth factor receptor (EGFR) in lung adenocarcinoma of Japanese patients, on the basis of the new adenocarcinoma classification proposed by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society. METHODS: The new classification was used to reclassify 486 adenocarcinomas. The percentage of each histopathologic subtype and the predominant pattern were determined. EGFR mutation was also investigated. The relationship between these results and clinicopathologic backgrounds was investigated statistically. RESULTS: No patients with adenocarcinoma in situ or minimally invasive adenocarcinoma died within the follow-up periods, followed by patients with lepidic predominant. Patients with papillary or acinar predominant, or invasive mucinous adenocarcinoma, showed almost similar overall survival (OS). The patients with solid predominant and micropapillary predominant showed the worst OS. Multivariate analysis showed that the new classification was an independent predictor of OS. The frequency of EGFR mutation was adenocarcinoma in situ (62%), minimally invasive adenocarcinoma (60%), lepidic (77%), acinar (49%), papillary (50%), solid (28%), micropapillary (43%), and invasive mucinous adenocarcinoma (0%). CONCLUSIONS: This new adenocarcinoma classification is a very useful predictive marker to plan and determine a therapeutic strategy for lung adenocarcinoma.

Positive intraoperative pleural lavage cytology is a predictive marker of disease recurrence in stage I lung adenocarcinoma.

OBJECTIVES: This study aimed at analysing the relationship between the pleural lavage cytology (PLC) status and clinicopathological characteristics, including the outcome of examined patients and tumour recurrence sites in surgically resected stage I non-small-cell lung carcinoma. METHODS: From April 2002 to August 2012, PLC was performed immediately after thoracotomy in 428 consecutive patients undergoing pulmonary resection for lung cancer. The relationship between clinicopathological characteristics and the PLC status was retrospectively analysed. RESULTS: The frequency of PLC-positive results was 4.4%, and larger tumour size, stage IB and pleural invasion were found more frequently in PLC-positive patients. Patients with a PLC-positive status had significantly worse disease-free survival (DFS) than those with a PLC-negative status (PLC positive versus PLC negative: hazard ratio [HR] = 2.79, 95% confidence interval [CI]: 1.4-5.57, P < 0.004; 5-year DFS: 46.6 vs 76.5%). With regard to the PLC status and histological type, adenocarcinoma was associated with a worse DFS in PLC-positive patients when compared with PLC-negative patients (5-year DFS: 38.1 vs 81.1%, P < 0.001). In multivariate analysis, PLC status remained significantly associated with DFS in patients with a PLC-positive status having an increased risk of recurrence, compared with PLC-negative patients (HR = 2.494, 95% CI: 1.241-5.011, P = 0.01) only in the case of adenocarcinoma. CONCLUSIONS: Our current study showed the clinicopathological characteristics associated with PLC status and demonstrated that PLC status was an independent predictor of increased recurrence in stage I lung adenocarcinoma.

Changes in NAD content induced by nicotinic acid-related compounds in K562 cells during differentiation.

Two nicotinic acid-related compounds were found to induce erythroid differentiation in K562 cells. We investigated the changes in nicotinamide adenine dinucleotide (NAD) content induced by nicotinic acid-related compounds during differentiation. The NAD content was reduced by a treatment with nicotinic acid and isonicotinic acid. These results provide important clues toward elucidating the erythroid differentiation mechanism.

[Comparison of HER2 immunohistochemical results for advanced gastric cancer obtained on using 3 different antibodies].

We compared the results of immunohistochemical assessment of HER2 expression in 107 samples of advanced gastric cancer on using 3 currently used antibodies. Expression was scored as 0 to 3+, and equivocal or discordant cases were subjected to fluorescence in situ hybridization(FISH)analysis. HER2 scores of 2+or 3+were noted in 16.8% of cases(18/ 107)using SV2-61g, in 29.9% of cases(32/107)using Dako HercepTest, and in 34.6% of cases(37/107)using 4B5. The results of the HER2 test differed according to the antibodies used for immunohistochemistry preceding FISH analysis, and the HER2 positive rates after the FISH analysis were 14.0%(15/107)using SV2-61g, 19.6% (21/107)using Dako HercepTest, and 22.4% (24/107)using 4B5. Thus, therapeutic decisions might be considerably influenced by the antibody used for the HER2 test.

Prognostic impact and initial recurrence site of lymphovascular and visceral pleural invasion in surgically resected stage I non-small-cell lung carcinoma.

OBJECTIVES: This study aimed to analyse and validate the prognostic impact and effect of the initial recurrence site of lymphovascular and visceral pleural invasion (VPI) on survival outcomes for Stage I non-small-cell lung carcinoma (NSCLC). METHODS: We retrospectively reviewed 433 patients undergoing resection of Stage I NSCLC. The relationship between the clinicopathological background and the pathological variables, lymphovascular invasion (LVI) and VPI, was evaluated by univariate and multivariate analyses. RESULTS: Lymphovascular and VPI was observed in 41 and 45 patients, respectively. On univariate analysis, the presence of LVI was associated with a significant decrease in relapse-free survival (RFS) (P < 0.001) and overall survival (OS) (P < 0.001). The RFS of the patients of Stage IB with LVI was worse than the RFS of those of Stage IIA (T2aN1 and T2bN0)/IIB (T3N0), and similar to the RFS of those of Stage IIB (T2bN1). The presence of VPI was also associated with a significant decrease in RFS (P < 0.001) and OS (P = 0.01). On multivariate analysis, LVI was found to be an independent predictor of both decreased RFS and decreased OS. However, VPI was not an independent predictor of both. Recurrence was seen in 68 patients. As an initial recurrence site, distant recurrence was seen in 32 patients and local recurrence, in 36. The proportion of local recurrence was significantly higher in the patients with VPI than in those without VPI compared with between the patients with LVI and those without LVI. CONCLUSIONS: We propose that LVI and/or VPI may be a candidate marker to determine adjuvant therapy or a more careful follow-up for these patients.

New IASLC/ATS/ERS classification and invasive tumor size are predictive of disease recurrence in stage I lung adenocarcinoma.

INTRODUCTION: The purpose of this study is to analyze and validate the prognostic impact of the new lung adenocarcinoma (ADC) classification proposed by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society and invasive tumor size in stage I lung ADC of Japanese patients. METHODS: We reclassified 191 stage I ADCs according to the new classification. The percentage of each histological subtype and the predominant type were determined. In addition, both total tumor size and invasive tumor size were examined. The relationship between these results and clinicopathological backgrounds was investigated statistically. RESULTS: The 5-year disease-free survival (DFS) of adenocarcinoma in situ and minimally invasive adenocarcinoma was 100%; lipidic-predominant ADCs, 94.9%; papillary-predominant ADCs, 85.4%; acinar-predominant ADCs, 89.7%; and solid-predominant ADCs, 54%. The predominant growth pattern was significantly correlated with DFS (p < 0.001, overall). With regard to tumor size, total tumor size was not correlated with DFS (p = 0.475, overall), however, invasive tumor size was significantly correlated with DFS (≤ 0.5 cm/ > 0.5 cm, ≤ 1 cm/ >1 cm, ≤ 2 cm/>2 cm, ≤ 3 cm/ >3 cm, 100%/91.5%/85.9%/80.8%/66.7%% in 5-year DFS) (p = 0.006, overall). A multivariate analysis showed solid-predominant and invasive tumor size were independent predictors of increased risk of recurrence (solid versus nonsolid: hazard ratio = 4.08, 95% confidence interval:1.59-10.5, p = 0.003; invasive tumor size: hazard ratio = 2.04, 95% confidence interval:1.14-3.63, p = 0.016). CONCLUSION: : The new International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society ADC classification and invasive tumor size are very useful predictors of recurrence of stage I ADCs in Japanese patients.

Comparison of HER2 immunohistochemical results using a monoclonal antibody (SV2-61γ) and a polyclonal antibody (for Dako HercepTest) in advanced gastric cancer.

We compared a monoclonal antibody (SV2-61γ) and a polyclonal antibody (Dako HercepTest) in immunohistochemical assessments of human epidermal growth factor receptor 2 (HER2) expression in 73 samples of advanced gastric cancer. Results were scored as 0 to 3+, and equivocal or discordant (SV2-61γ/Dako HercepTest = 0/2+, 0/3+, 1+/3+ or 2+/3+) cases were subjected to fluorescence in situ hybridization (FISH) analysis. The frequencies of HER2 scores of 2+ or 3+ were 15.1% (11/73) using SV2-61γ and 38.4% (28/73) using Dako HercepTest. All of the equivocal or discordant cases with a HER2 score of 3+ using Dako HercepTest exhibited amplification of the HER2 gene regardless of the HER2 score determined with SV2-61γ. The results of the HER2 tests differed according to the antibodies used for immunohistochemistry that preceded FISH analysis, being 15.1% (11/73) using SV2-61γ and 23.3% (17/73) using Dako HercepTest. Thus, therapeutic decisions might be markedly influenced by the selection of antibody used in the HER2 test.