Two cases of strangulated bowel obstruction due to exposed vessel and nerve after laparoscopic and robot-assisted lateral lymph node dissection (LLND) for rectal cancer.

BACKGROUND: The majority of small bowel obstructions (SBO) are caused by adhesion due to abdominal surgery. Internal hernias, a very rare cause of SBO, can arise from exposed blood vessels and nerves during pelvic lymphadenectomy (PL). In this report, we present two cases of SBO following laparoscopic and robot-assisted lateral lymph node dissection (LLND) for rectal cancer, one case each, of which obstructions were attributed to the exposure of blood vessels and nerves during the procedures. CASE PRESENTATION: Case 1: A 68-year-old man underwent laparoscopic perineal rectal amputation and LLND for rectal cancer. Four years and three months after surgery, he visited to the emergency room with a chief complaint of left groin pain. Computed tomography (CT) revealed a closed-loop in the left pelvic cavity. We performed an open surgery to find that the small intestine was fitted into the gap between the left obturator nerve and the left pelvic wall, which was exposed by LLND. The intestine was not resected because coloration and peristalsis of the intestine improved after the hernia was released. The obturator nerve was preserved. Case 2: A 57-year-old man underwent a robot-assisted rectal amputation with LLND for rectal cancer. Eight months after surgery, he presented to the emergency room with a complaint of abdominal pain. CT revealed a closed-loop in the right pelvic cavity, and he underwent a laparoscopic surgery with a diagnosis of strangulated SBO. The small intestine was strangulated by an internal hernia caused by the right umbilical arterial cord, which was exposed by LLND. The incarcerated small intestine was released from the gap between the umbilical arterial cord and the pelvic wall. No bowel resection was performed. The umbilical arterial cord causing the internal hernia was resected. CONCLUSION: Although strangulated SBO due to an exposed intestinal cord after PL has been a rare condition to date, it is crucial for surgeons to keep this condition in mind.

A case of infective endocarditis mimicking a large mesenteric abscess.

Infective endocarditis (IE) demonstrates a broad array of clinical presentations and complications. However, IE with prominent abdominal findings is uncommon. We encountered a case of IE caused by Staphylococcus aureus that presented a large mesenteric abscess and was initially diagnosed as an intra-abdominal infection. There are few reports of IE with mesenteric abscess formation. Even if an intra-abdominal abscess is the main symptom, the possibility that it is part of a systemic infection should be considered if the causative organism is atypical or if symptoms are present in multiple organs. Physicians should always be aware of the possibility that IE may mimic other diseases, including intra-abdominal infections.

Efficacy and Safety of Ramucirumab/nab-paclitaxel for Previously Treated Advanced Gastric Cancer in Community Hospitals.

As the nanoparticle albumin-bound paclitaxel (nab-PTX) is free of ethanol and premedication, the duration of administration is shorter and patients can drive themselves to and from the hospital. In the 2018 Japanese gastric cancer treatment guidelines, ramucirumab (RAM) plus weekly nab-PTX is conditionally recommended for previously treated patients with advanced gastric cancer. Here, we retrospectively analysed the efficacy and safety of RAM+nab-PTX for such patients in community hospitals. From January 2018 to December 2019, 43 patients with metastatic and recurrent gastric cancer received RAM+nab-PTX treatment. Six patients (13.9%) were older than 80 years and 9 patients (20.9%) showed ECOG-PS 2. Progression-free survival (PFS), overall survival (OS), overall response rate (ORR), disease control rate (DCR), and adverse events (AEs) were reviewed retrospectively. Median PFS was 114 days (95% confidence interval [CI]: 84-190) and median OS was 297 days (95% CI: 180-398). ORR and DCR were 32.4% and 72.2%, respectively. The incidence rates of ≥grade 3 neutropenia and febrile neutropenia were 53.5% and 2.3%, respectively. No treatment-related deaths occurred. RAM plus nab-PTX combination therapy demonstrated manageable toxicity even patients who were elderly or had an ECOG-PS 2. This treatment is useful in community hospital settings.

Donor-derived cell-free DNA is associated with acute rejection and decreased oxygenation in primary graft dysfunction after living donor-lobar lung transplantation.

Donor-derived cell-free DNA (dd-cf-DNA) has been shown to be an informative biomarker of rejection after lung transplantation (LT) from deceased donors. However, in living-donor lobar LT, because small grafts from blood relatives are implanted with short ischemic times, the detection of dd-cf-DNA might be challenging. Our study was aimed at examining the role of dd-cf-DNA measurement in the diagnosis of primary graft dysfunction and acute rejection early after living-donor lobar LT. Immediately after LT, marked increase of the plasma dd-cf-DNA levels was noted, with the levels subsequently reaching a plateau with the resolution of primary graft dysfunction. Increased plasma levels of dd-cf-DNA were significantly correlated with decreased oxygenation immediately (p = 0.022) and at 72 hours (p = 0.046) after LT. Significantly higher plasma dd-cf-DNA levels were observed in patients with acute rejection (median, 12.0%) than in those with infection (median, 4.2%) (p = 0.028) or in a stable condition (median, 1.1%) (p = 0.001). Thus, measurement of the plasma levels of dd-cf-DNA might be useful to monitor the severity of primary graft dysfunction, and plasma dd-cf-DNA could be a potential biomarker for the diagnosis of acute rejection after LT.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods.

Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system.

Though patients with EGFR mutations are initially responsive to EGFR-tyrosine kinase inhibitors (TKIs), most tumors ultimately acquire resistance to EGFR-TKIs. The most frequently reported mechanism is EGFR T790M mutation. In this study, using a droplet digital PCR (ddPCR) system, we assessed optimal conditions for a mutation detection assay for EGFR T790M obtained from circulating cell-free DNA (cfDNA) in plasma. The advantages of locked nucleic acids (LNA) probe, short amplicon size, and blocking oligo using peptide nucleic acids (PNA) were assessed using control DNAs from cell lines to improve the sensitivity of mutation detection. T790M alleles were then analyzed using ddPCR in 59 plasma samples from 24 NSCLC patients with EGFR mutations, and compared to the T790M status which were determined thorough re-biopsies. The assessment of the optimal assay method revealed that the assay using the short amplicon can efficiently detect more fragmented-DNA. The LNA probe and PNA clamp contributed better separation between positive and negative droplets. This PNA-LNA-ddPCR clamp method can detect mutant alleles in the sample with a mutant allele content of 0.01%. In clinical plasma samples, T790M alleles were detected via ddPCR with a sensitivity of 42.8% and specificity of 97.3%. We established a highly-sensitive detection assay for the T790M allele using the PNA-LNA-ddPCR clamp method. ddPCR is a promising method for detecting non-invasive T790M mutation.

Targeting the miR-200c/LIN28B axis in acquired EGFR-TKI resistance non-small cell lung cancer cells harboring EMT features.

MicroRNA (miR)-200 family members (miR-200s) are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT). We previously reported that miR-200s were silenced through promoter methylation in acquired EGFR-tyrosine kinase inhibitor (TKI) resistant non-small cell lung cancer (NSCLC) cells harboring EMT features. In this study, we examined the functional role of miR-200s in NSCLC cells and investigated a novel approach to overcoming acquired EGFR-TKI resistance. In the analysis of NSCLC cell lines, each of the miR-200s expression-silenced cell lines showed promoter methylation. Significant correlations between miR-200c silencing and several oncogenic pathway alterations, including EMT-changes and LIN28B overexpression, were observed in the database analysis. In addition, EGFR-wild type cell lines had lower miR-200s expression levels than EGFR-mutant cell lines. The introduction of miR-200c using pre-miR-200c caused LIN28B suppression in cells with acquired EGFR-TKI resistance that harbored EMT features. Interestingly, both the introduction of miR-200c and the knockdown of LIN28B produced an antitumor effect in acquired EGFR-TKI resistance cells, whereas these manipulations were not effective in parental cells. The miR-200c/LIN28B axis plays an important role in cells with acquired resistance to EGFR-TKI that harbor EMT features and might be a useful therapeutic target for overcoming resistance.

Distant Bystander Effect of REIC/DKK3 Gene Therapy Through Immune System Stimulation in Thoracic Malignancies.

BACKGROUND: Reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) is a tumor-suppressor gene, and its overexpression by adenovirus vector (Ad-REIC) exhibits a remarkable therapeutic effect on various human cancer types through a mechanism triggered by endoplasmic reticulum stress. MATERIALS AND METHODS: We examined the direct anti-tumor effect of Ad-REIC gene therapy on lung cancer and malignant mesothelioma cell lines in vitro, and the distant bystander effect using immunocompetent mouse allograft models with bilateral flank tumors. RESULTS: Ad-REIC treatment showed antitumor effect in many lung cancer and malignant mesothelioma cell lines in vitro. In an in vivo model, Ad-REIC treatment inhibited the growth not only of directly treated tumors but also of distant untreated tumors. By immunohistochemical analysis, infiltration of T-cells and natural killer (NK) cells and expression of the major histocompatibility complex (MHC) class I molecules were observed in bilateral tumors. CONCLUSION: Ad-REIC treatment not only had a direct antitumor effect but also an indirect bystander effect through stimulation of the immune system.

Interaction of cytokeratin 19 head domain and HER2 in the cytoplasm leads to activation of HER2-Erk pathway.

HER2 is a receptor tyrosine kinase and its upregulation via activating mutations or amplification has been identified in some malignant tumors, including lung cancers. Because HER2 can be a therapeutic target in HER2-driven malignancies, it is important to understand the molecular mechanisms of HER2 activation. In the current study, we identified that cytokeratin 19 (KRT19) binds to HER2 at the inside face of plasma membrane. HER2 and KRT19, which were concurrently introduced to a human embryonic kidney 293 T cells, revealed an association with each other and resulted in phosphorylation of HER2 with the subsequent activation of a downstream Erk-associated pathway. A binding assay revealed that both the NH2-terminal head domain of KRT19 and the COOH-terminal domain of HER2 were essential for their binding. To investigate the impact of the interaction between HER2 and KRT19 in lung cancer, we examined their expressions and localizations in lung cancers. We found that KRT19 was highly expressed in HER2-positive lung cancer cells, and KRT19 and HER2 were co-localized at the cell membrane. In conclusion, we found that KRT19 intracellularly binds to HER2, playing a critical role in HER2 activation.

Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive disease that is resistant to conventional therapies. Cell lines are useful models for studying the biological characteristics of tumors; therefore, the establishment of MPM cell lines is valuable for exploring novel therapeutic strategies for MPM. In the present study, 4 MPM cell lines (YUMC8, YUMC44, YUMC63, and YUMC64) were established, which consisted of 2 epithelioid and 2 sarcomatoid mesothelioma histological subtypes, from Japanese patients with MPM. The DNA methylation status, mutations, copy number gains, protein expression of representative genes, and the sensitivity to several drugs were examined in these 4 cell lines. Methylation of P16 was demonstrated in 3/4 cell lines, in which the protein expression of p16 was lost. Methylation of RASSF1A was observed in 3/4 cell lines. Copy number gains of EGFR, HER2 or MET were not detected in the 4 cell lines. Mutations in various genes, including EGFR, KRAS, HER2, BRAF, and PIK3CA, which are frequently detected in non-small cell lung cancer, were not detected in the 4 cell lines. microRNA-34b/c is a direct transcriptional target of p53 and is often silenced in MPM by promoter methylation. In the present study, miR-34b/c was heavily methylated in 2/4 established MPM cell lines. For cell adhesion molecules, E-cadherin expression was detected in the 2 epithelioid MPM cell lines, whereas N-cadherin expression was detected in all 4 established cell lines by western blotting. Vimentin was strongly expressed in the 2 sarcomatoid MPM cell lines. None of the established MPM cell lines demonstrated significant responses to the drugs tested, including NVP-AUY922, 17-DMAG, Trichostatin A, and Vorinostat. Although novel molecular findings were not observed in the current characterization of these MPM cell lines, these lines will be useful for future extensive analyses of the biological behavior of MPM and the development of novel therapeutic strategies.

Genetic alterations in lung adenocarcinoma with a micropapillary component.

Pulmonary adenocarcinoma (PA) with a micropapillary component (PA-MPC) is known as an aggressive subtype of PA. The molecular profiles of PA-MPC have not been well characterized. the pathological reports of patients who underwent surgical resection for lung cancer between April, 2004 and May, 2012 were reviewed. Of the 674 patients diagnosed with PA, 28 were found to have MPC. A total of 138 resected PAs without MPC were selected in the same period to serve as age-, gender- and smoking status-matched controls to the PA-MPC group. Mutational status was determined by the following two methods: SNaPshot assay based on multiplex polymerase chain reaction (PCR), primer extension and capillary electrophoresis that was designed to assess 38 somatic mutations in 8 genes [AKT1, BRAF, endothelial growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), mitogen-activated protein kinase kinase 1, neuroblastoma RAS viral oncogene homolog, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) and phosphatase and tensin homolog]; and a PCR-based sizing assay that assesses EGFR exon 19 (deletions), EGFR exon 20 (insertions) and human epidermal growth factor receptor 2 exon 20 (insertions). echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene (EML4-ALK) was screened by ALK immunohistochemistry and confirmed using the reverse transcription PCR assay and the break-apart fluorescence in situ hybridization assay. Regarding genetic alterations, 13 (46.4%) of the 28 PA-MPCs harbored mutually exclusive mutations: 9 (32.1%) EGFR mutations, 1 (3.6%) KRAS mutation and 3 (10.7%) EML4-ALK fusion genes. PAs without MPC harbored 42 (30.4%) EGFR mutations, 17 (12.3%) KRAS mutations, 3 (2.2%) EML4-ALK fusion genes and 1 (0.7%) PIK3CA mutation. EML4-ALK fusion genes appeared to occur significantly more frequently in PA-MPCs compared with PAs without MPC (P=0.027). Although the sample size was small, our study suggests that the molecular pathogenesis of PA-MPC may be different from that of other adenocarcinomas.

Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

Primary pulmonary melanoma: a report of two cases.

Malignant melanoma is a refractory malignancy with a dismal prognosis. It generally arises from the skin in most cases, and cases of primary pulmonary malignant melanoma are rare and often behave aggressively. We have treated two cases of localized primary pulmonary malignant melanoma using surgical resection. Pulmonary malignant melanomas often metastasize to the brain and liver; one of our cases exhibited metastasis to the cecum at about 8 months after surgery. Because cutaneous melanomas often carry activating mutations in the BRAF gene (V600E), we performed a BRAF mutational analysis using direct sequencing for both of these tumors arising from the lung. However, no BRAF mutations were detected. We detected a p53 mutation, which was thought to be a potential somatic mutation, in one of the two cases using a sequencing panel targeting 20 lung cancer-related genes. Although we also checked the expression of programmed death ligand 1 (PD-L1) on the surface of the tumor cells by immunohistochemical testing, neither of our two cases expressed PD-L1. Further molecular analyses may uncover the characteristics of primary pulmonary malignant melanomas.

Downregulation of TBXAS1 in an iron-induced malignant mesothelioma model.

Malignant mesothelioma is an aggressive and therapy-resistant neoplasm arising from mesothelial cells. Evidence suggests that the major pathology associated with asbestos-induced mesothelioma is local iron overload. In the present study, we induced iron-induced mesothelioma in rats based on previous reports. Ten Wistar rats were given ferric saccharate and nitrilotriacetate i.p. for 5 days a week. Five of the ten rats exhibited widespread mesotheliomas in the peritoneum and tunica vaginalis. The tumor cells showed positive immunostaining for calretinin, wilms tumor-1, podoplanin and the oxidative DNA marker 8-hydroxy-2'-deoxyguanosine. In three of the five rats with mesothelioma, array-based comparative genomic hybridization analysis identified a common chromosomal deletion mapped to the chromosomal 4q31 locus, which encompasses the TBXAS1 gene. Downregulation of the TBXAS1 gene was confirmed using quantitative PCR. TBXAS1 gene expression was also reduced in three of four human malignant pleural mesothelioma cell lines compared with normal bronchial epithelial cells. Immunohistochemistry revealed that TBXAS1 expression was weakly positive and positive in five and three out of eight human malignant mesothelioma samples, respectively. In conclusion, TBXAS1 gene expression was downregulated in rats with iron-induced mesothelioma. The relationship between iron overload and TBXAS1 downregulation should be pursued further.

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib.

Afatinib is an irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR-TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non-small cell lung cancer. In this study, we established various afatinib-resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib-resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827-ACR, was established from one of the MET amplified-cell lines. Several afatinib-resistant cell lines including HCC827-ACR displayed epithelial-to-mesenchymal transition (EMT) features and epigenetic silencing of miR-200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib-resistant cells and found that MET amplification, EMT, and stem cell-like features are observed in cells with acquired resistance to EGFR-TKIs. This finding may provide clues to overcoming resistance to EGFR-TKIs.

Hsp90 inhibitor NVP-AUY922 enhances the radiation sensitivity of lung cancer cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors.

Acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is a critical issue that needs to be overcome in the treatment of patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. EGFR and AKT are client proteins of the 90-kDa heat shock protein (Hsp90). Therefore, it was hypothesized that the use of Hsp90 inhibitors might allow the resistance to EGFR-TKIs to be overcome. Furthermore, Hsp90 inhibitors are known to function as radiosensitizers in various types of cancer. In the present study, we evaluated the radiosensitizing effect of the novel Hsp90 inhibitor, NVP-AUY922 (AUY), on NSCLC cell lines harboring EGFR activating mutations and showing acquired resistance to EGFR-TKIs via any of several mechanisms. We used HCC827 and PC-9, which are NSCLC cell lines harboring EGFR exon 19 deletions, and gefitinib-resistant sublines derived from the same cell lines with T790M mutation, MET amplification or stem-cell like properties. AUY was more effective against the gefitinib-resistant sublines with T790M mutation and MET amplification than against the parental cell lines, although the subline with stem cell-like properties showed more than a 10-fold higher resistance to AUY than the parental cell line. AUY exerted a significant radiosensitizing effect on the parental cell line and the MET-amplified subline through inducing G2/M arrest and inhibition of non-homologous end joining (NHEJ). In contrast, the radiosensitizing effect of AUY was limited on the subline with stem cell-like properties, in which it did not induce G2/M arrest or inhibition of NHEJ. In conclusion, combined inhibition of Hsp90 plus radiation was effective, and therefore a promising treatment alternative for overcoming major EGFR-TKI resistance, such as that induced by T790M mutation or MET amplification. However, other approaches are required to overcome minor resistance to EGFR-TKIs, such as that observed in cells with stem cell-like properties.

DNA copy number gains in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with an extremely poor prognosis. The incidence of MPM is increasing as a result of widespread exposure to asbestos. The molecular pathogenesis of MPM remains unclear. The present study analyzed the frequency of various genomic copy number gains (CNGs) in MPM using reverse transcription-quantitative polymerase chain reaction. A total of 83 primary MPMs and 53 primary lung adenocarcinomas were analyzed to compare the CNGs of EGFR, KRAS, MET, FGFR1 and SOX2. In MPM, the CNGs of EGFR, KRAS, MET, FGFR1 and SOX2 were detected in 12 (14.5%), 8 (9.6%), 5 (6.0%), 4 (4.8%) and 1 (1.2%) of the samples, respectively. In lung adenocarcinomas, the CNGs of EGFR, KRAS, MET, FGFR1 and SOX2 were detected in 21 (39.6%), 12 (22.6%), 5 (9.4%), 10 (18.9%) and 0 (0.0%) of the samples, respectively. The CNGs of EGFR, KRAS and FGFR1 were significantly less frequent in the MPMs compared with the lung adenocarcinomas (P=0.0018, 0.048 and 0.018, respectively). Overall, the MPMs exhibited these CNGs less frequently compared with the lung adenocarcinomas (P=0.0002). The differences in CNGs between the two tumor types suggested that they are genetically different.

A new human lung adenocarcinoma cell line harboring the EML4-ALK fusion gene.

OBJECTIVE: The echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients with non-small cell lung cancer. To the best of our knowledge, there are only three cell lines harboring the EML4-ALK fusion gene, which have contributed to the development of therapeutic strategies. Therefore, we tried to establish a new lung cancer cell line harboring EML4-ALK. METHODS: A 61-year-old Japanese female presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was treated with chemotherapy, her disease progressed with massive pleural effusion. Because the EML4-ALK rearrangement was found in a biopsied specimen using fluorescence in situ hybridization, she was treated with crizotinib. She did well for 3 months. RESULTS: Tumor cells were obtained from the malignant pleural effusion before treatment with crizotinib. Cells continued to proliferate substantially for several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein and genes were identified in ABC-11 cells using fluorescence in situ hybridization and immunohistochemistry, respectively. ABC-11 cells were sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and AP26113), as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Phosphorylated ALK protein and its downstream signaling were suppressed by treatment with crizotinib in western blotting. Furthermore, we could transplant ABC-11 cells subcutaneously into BALB/c nu/nu mice. CONCLUSIONS: We successfully established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene. This cell line could contribute to future research of EML4-ALK-positive lung cancer both in vivo and in vitro.

Presence of the minor EGFR T790M mutation is associated with drug-sensitive EGFR mutations in lung adenocarcinoma patients.

The T790M mutation in the epidermal growth factor receptor (EGFR) gene is known to be associated with the acquired resistance of lung adenocarcinoma patients to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). The minor T790M mutant allele is occasionally detected in EGFR-TKI-naive tumor samples, yet findings concerning the clinical impact of the minor T790M mutation vary among previous studies. In the present study, we assessed the clinical impact of the minor T790M mutation using a novel, highly sensitive assay combining high-resolution melting (HRM), mutant-enriched PCR and co-amplification at a lower denaturation temperature (COLD)-PCR. We determined the T790M mutational status in 146 surgically resected lung adenocarcinomas without a history of EGFR-TKI treatment using mutant-enriched COLD-HRM (MEC-HRM) and standard HRM assays. The sensitivities of the MEC-HRM and standard HRM assays for the detection of T790M-mutant alleles among wild-type alleles were 0.01 and 10%, respectively. Although the T790M mutation was not detected using a standard HRM assay, we identified 19 (13%) T790M mutations using the MEC-HRM assay and defined these 19 mutations as minor T790M mutations. The proportion of T790M alleles was <0.1% in 17 (84%) of the 19 samples. Multivariate analyses revealed that a minor T790M mutation was significantly associated with the presence of EGFR exon 19 deletions or the L858R mutation (both of which are drug-sensitive EGFR mutations) (P=0.04). In conclusion, the minor EGFR T790M mutations were present in 13% of EGFR-TKI-naive surgically resected lung adenocarcinomas and were associated with drug-sensitive EGFR mutations.