Clinical features and prognostic impact of coexisting autoimmune disease other than myasthenia gravis in resected thymomas: analysis of a Japanese multi-institutional retrospective database.

OBJECTIVES: The purpose of this study was to clarify the prevalence, clinical features and survival of patients with thymoma and non-myasthenia gravis autoimmune disease (NMAD) using a nationwide cohort. METHODS: The Japanese Association for Research on the Thymus nationwide database, which includes data from 32 institutions, was examined to clarify the prevalence and characteristics of NMAD associated with thymomas and elucidate the prognostic impact of NMAD for thymoma patients. RESULTS: Among the 2423 patients with thymomas who were surgically treated between 1991 and 2010, 114 (4.7%) were identified with NMAD. The most frequently observed NMAD was pure red cell aplasia (PRCA) in 44 (1.8%), followed by hypogammaglobulinaemia (0.5%) and rheumatic arthritis (0.5%). Twenty-eight percent of patients with NMAD had concomitant myasthenia gravis. The presence of NMAD was not an independent prognostic factor for overall survival (OS) irrespective of the type of NMAD [PRCA+: hazard ratio (HR) 1.99, 95% confidence interval 0.74-4.47; PRCA- NMAD: HR 1.28, 0.30-3.56]; however, there were more cases with advanced age and disease of the thymoma amongst PRCA+ patients and these showed a worse OS than patients with PRCA- NMAD (P < 0.001), who had an OS similar to those without NMAD (P = 0.489). The 10-year OS rates in PRCA+, PRCA- NMAD and NMAD- groups were 45.5%, 97.4% and 89.5%, respectively. The main causes of death in PRCA+ patients were the progression of thymoma and other diseases including pneumonia. CONCLUSIONS: Although the presence of NMAD itself did not significantly affect survival after surgery for thymoma, the type of NMAD was associated with different clinical features and prognosis. The NMAD+ thymomas should be separately categorized according to the presence or absence of PRCA.

Can smoking duration alone replace pack-years to predict the risk of smoking-related oncogenic mutations in non-small cell lung cancer? A cross-sectional study in Japan.

OBJECTIVE: To investigate whether smoking duration alone can replace pack-years to predict the risk of oncogenic mutations in non-small cell lung cancer (NSCLC). DESIGN: A cross-sectional study using the baseline dataset from the Japan Molecular Epidemiology for Lung Cancer Study. SETTING: Forty-three medical institutions nationwide in Japan. PARTICIPANTS: From July 2012 to December 2013, 957 patients with newly diagnosed stage I-IIIB NSCLC who underwent surgery were enrolled, and molecular analyses were performed on 876 samples (from 441 ever-smokers and 435 never-smokers). MAIN OUTCOMES MEASURED: We calculated the area under the receiver operating characteristic curve (AUC) values using logistic regression to compare between the predictive values of smoking duration and pack-years for mutational frequencies in the v-Ki-ras2 Kirsten rat sarcoma (KRAS), tumour suppressor p53 (TP53), and epidermal growth factor receptor (EGFR) genes and for cytosine-to-adenine base substitution (C>A). RESULTS: For predicting KRAS mutations, the AUC values for smoking duration and pack-years were 0.746 (95% CI 0.682 to 0.800) and 0.759 (95% CI 0.700 to 0.810), respectively (p=0.058). For predicting KRAS mutations in smokers, the AUC values for smoking duration and pack-years were 0.772 (95% CI 0.697 to 0.833) and 0.787 (95% CI 0.714 to 0.845), respectively (p=0.036). There were no significant differences between the AUC values for smoking duration and pack-years in terms of predicting TP53 and EGFR mutations and C>A. Pack-years was a significantly better predictor of KRAS mutations than smoking duration. CONCLUSION: Smoking duration was not significantly different from pack-years in predicting the likelihood of smoking-related gene mutations. Given the recall bias in obtaining smoking information, smoking duration alone should be considered for further investigation as a simpler alternative to pack-years.

Impact of somatic mutations on prognosis in resected non-small-cell lung cancer: The Japan Molecular Epidemiology for lung cancer study.

BACKGROUND: To report the follow up data and clinical outcomes of the JME study (UMIN 000008177), a prospective, multicenter, molecular epidemiology examination of 876 surgically resected non-small-cell lung cancer (NSCLC) cases, and the impact of somatic mutations (72 cancer-associated genes) on recurrence-free survival (RFS) and overall survival (OS). METHODS: Patients were enrolled between July 2012 and December 2013, with follow up to 30th November 2017. A Cox proportional hazards model was used to assess the impact of gene mutations on RFS and OS, considering sex, smoking history, age, stage, histology, EGFR, KRAS, TP53, and number of coexisting mutations. RESULTS: Of 876 patients, 172 had ≥2 somatic mutations. Median follow-up was 48.4 months. On multivariate analysis, number of coexisting mutations (≥2 vs 0 or 1, HR = 2.012, 95% CI: 1.488-2.695), age (≥70 vs <70 years, HR = 1.583, 95% CI: 1.229-2.049), gender (male vs female, HR = 1.503, 95% CI: 1.045-2.170) and pathological stage (II vs I, HR = 3.386, 95% CI: 2.447-4.646; ≥III vs I, HR = 6.307, 95% CI: 4.680-8.476) were significantly associated with RFS, while EGFR mutation (yes vs no, HR = 0.482, 95% CI: 0.309-0.736), number of coexisting mutations (≥2 vs 0 or 1, HR = 1.695, 95% CI: 1.143-2.467), age (≥70 vs <70 years, HR = 1.932, 95% CI: 1.385-2.726), and pathological stage (II vs I, HR = 2.209, 95% CI: 1.431-3.347; ≥III vs I, HR = 5.286, 95% CI: 3.682-7.566) were also significant for OS. CONCLUSION: A smaller number of coexisting mutations, earlier stage, and younger age were associated with longer RFS and OS, while EGFR mutations were significantly associated with improved OS.

Adenocarcinoma with fetal features invading the right superior sulcus treated with neoadjuvant chemoradiotherapy followed by complete video-assisted thoracoscopic right upper lobectomy: a case report.

BACKGROUND: Fetal adenocarcinoma of the lung is a rare lung neoplasm that accounts for only 0.5% of all primary lung cancers. Because of its rarity, effective treatments for the management of the tumor are poorly understood. We herein report a case of adenocarcinoma with fetal features of the lung with invasion of the right superior sulcus that was treated with neoadjuvant chemoradiotherapy followed by surgical resection. CASE PRESENTATION: A 54-year-old man was referred to a medical institution due to right inner forearm pain. On computed tomography of the chest, a 56-mm mass with invasion of right superior sulcus was discovered. Bronchoscopic biopsy revealed non-small cell lung carcinoma. We performed concurrent chemotherapy (2 cycles of cisplatin and vinorelbine) and thoracic radiation therapy (40 Gy in 20 fractions). As the result of extreme tumor reduction after neoadjuvant chemoradiotherapy, we could perform right upper lobectomy by complete video-assisted thoracoscopic surgery. Since no viable cancer cells were detected from the pathological examination of the resected tissue, the specimen obtained by bronchoscopic biopsy was reexamined by immunohistochemistry. The analysis supported a pathologic diagnosis of adenocarcinoma with fetal features. CONCLUSIONS: We experienced a case of adenocarcinoma with fetal features of the lung in which the patient showed a complete response to neoadjuvant chemoradiotherapy. In addition, the tumor invading the right superior sulcus was completely resected by video-assisted thoracoscopic lobectomy. Neoadjuvant chemoradiotherapy followed by surgery may be also an effective treatment for advanced-stage high-grade fetal adenocarcinoma of the lung, similarly to other subtypes of advanced-stage primary lung cancer.

Programmed Death-ligand 1 Expression With Clone 22C3 in Non-small Cell Lung Cancer: A Single Institution Experience.

BACKGROUND: In recent years, the anti-programmed cell death 1 (PD-1) drug pembrolizumab (Keytruda) was approved for treatment of unresectable advanced non-small cell lung cancer (NSCLC) as first- or second-line therapy depending on the clone 22C3-programmed death-ligand 1 (PD-L1) immunohistochemical expression score by the companion diagnostic assay. We herein evaluated 22C3-PD-L1 expression of NSCLC in a single institution experience and compared it with clinicopathologic features. MATERIALS AND METHODS: We assessed 22C3-PD-L1 expressions of 411 patients with NSCLC from our institution, including in past specimens. Programmed death-ligand 1 immunohistochemistry (IHC) testing was performed using the PD-L1 clone 22C3 pharmDx kit (Agilent Technologies/Dako, Carpinteria, CA, USA). Patients were separated into 3 groups with <1% (no expression), 1% to 49% (low expression), or ⩾50% (high expression) positive tumor cells. RESULTS: In all, 137 patients (33%) did not express PD-L1, 155 (38%) showed low expression, and 119 (29%) demonstrated high expression. Archival samples showed lower PD-L1 expression than that of recent samples, and the ratios of no expression case significantly increased by using paraffin blocks embedded particularly in more than 4 years ago. Programmed death-ligand 1 positivity was significantly associated with male sex, smoking, higher tumor grade, squamous cell carcinoma in histologic type, wild-type EGFR, and ALK rearrangement positive. CONCLUSIONS: The rate of 22C3-PD-L1 expression of NSCLC detected in this study was similar to the frequencies of the previous reports, although the ratio of expression case decreased when using old paraffin blocks.

A High PD-L1 Expression in Pulmonary Pleomorphic Carcinoma Correlates with Parietal-pleural Invasion and Might Predict a Poor Prognosis.

Objective Pleomorphic carcinoma (PC) is a rare pulmonary epithelial malignant tumor with a poor prognosis. The objective of the present study was to investigate the programmed death-ligand 1 (PD-L1) expression in PC and its correlation between the clinicopathological factors and prognosis. Methods Clinical and pathological data of 35 patients with surgically resected PC encountered from 2002 to 2016 at our institution were collected. The PD-L1 expression on tumor cells was evaluated via immunohistochemistry (clone 22C3). We examined the correlation between the PD-L1 expression and patients' clinicopathological factors and their prognosis. Results A high PD-L1 expression (≥50%) was seen in 21 (60%) patients, and parietal-pleural invasion was significantly correlated with a high PD-L1 expression (p=0.012). The 5-year overall survival and relapse-free survival were 68.2% and 43.2%, respectively. Tumor size ≥50 mm (p=0.021), lymph node metastasis (p=0.023), and a high PD-L1 expression (p=0.047) were correlated with a short relapse-free survival. Since lymph node metastasis was an independent risk factor of a poor overall survival (p=0.012), patients with a high PD-L1 expression also tended to have a worse overall survival than those with low levels (p=0.081). Conclusion A high PD-L1 expression is frequently seen in PC. The PD-L1 expression is associated with parietal-pleural invasion and might indicate a poor prognosis.

PIK3CA mutation as a distinctive genetic feature of non-small cell lung cancer with chronic obstructive pulmonary disease: A comprehensive mutational analysis from a multi-institutional cohort.

OBJECTIVES: Non-small cell lung cancer (NSCLC) and chronic obstructive pulmonary disease (COPD) have been proposed to have a mutual developmental mechanism, but their association has not been fully understood. We aimed to examine the association of the mutational landscape of NSCLC with co-morbid COPD. MATERIALS AND METHODS: A total of 197 surgical specimens of early stage NSCLC were retrospectively collected from two independent sources, namely, the Japan Molecular Epidemiology for Lung Cancer Study and the Osaka City University Hospital cohort from 2010 to 2013. COPD and its severity were defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines and grading system. For molecular profiling of NSCLC patients with COPD, the extracted DNAs were deep-sequenced using next generation sequence technologies for somatic mutations in a maximum 72 cancer-associated genes. Logistic regression analysis was performed to evaluate the impact of COPD on the somatic mutations. RESULTS: The COPD group (n=77), including 56 GOLD 1 and 21 GOLD 2 or 3 patients, had 58 squamous cell lung carcinoma (SCC) cases and 19 adenocarcinoma cases. The non-COPD group (n=120) had 53 SCC cases, 64 adenocarcinoma cases, and three cases with other histology. The frequency of PIK3CA mutation was significantly higher in the COPD group than in the non-COPD group (10.4% vs. 1.7%, p=0.015). Meanwhile, NFE2L2 mutation was observed only in SCC cases, with no difference in the frequency between the two groups (17.2% vs. 17.0%). In the multivariate logistic regression model with consideration for COPD status, age, smoking dose, pathological stage, and histology, significantly more PIK3CA mutation was observed in the presence of COPD (odds ratio=5.31, 95% CI: 1.03-27.29, p=0.046). CONCLUSIONS: PIK3CA mutation is a distinctive genetic feature of NSCLC with COPD, regardless of age, smoking dose, pathological stage, and histology.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR.

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M).

Assessment of ALK gene rearrangement in lung cancer using a new rapid automated SureFISH (Dako Omnis) assay.

The ALK fluorescence in situ hybridisation (FISH) method is the examination essential for pathological diagnosis and choice of molecular-targeted therapy in ALK-rearranged lung cancer. Here, for detection of ALK gene rearrangement in patients with lung cancer, we evaluated the rapid FISH technology (ALK SureFISH), a newly developed assay for the automated staining platform Dako Omnis, using 21 formalin-fixed paraffin-embedded (FFPE) samples. All cases could be evaluated with the SureFISH method. SureFISH provided excellent quality signals without any background staining. The SureFISH assay was able to offer a rapid turnaround time (approximately 3.5 hours) and was 100% concordant with prior Vysis FISH results in our laboratory.

Analysis of the impact of lung cancer treatment on nontuberculous mycobacterial lung diseases.

BACKGROUND: Although nontuberculous mycobacteria (NTM) lung diseases can occur in association with lung cancer, no study has evaluated the effect of lung cancer treatment on NTM lung diseases. Therefore, the present study aimed to retrospectively examine the effect of lung cancer treatment on NTM lung diseases. METHODS: Patients diagnosed with NTM lung diseases in combination with cytologically or histologically proven lung cancer between January 1, 2010 and October 31, 2014 were enroled. The clinical history of eligible patients was retrospectively reviewed. RESULTS: Seven hundred twenty-eight patients were diagnosed with NTM lung diseases. Among these patients, 29 (3.9%) also had lung cancer. Of the 29 patients with NTM and lung cancer, 62% had Mycobacterium avium complex as the pathogenic organism. The most common lung cancer histology was adenocarcinoma (62.1%). Anti-cancer cytotoxic chemotherapy was administered to seven patients, and the two patients who did not receive NTM treatment showed worsening of their NTM lung disease. CONCLUSION: Whether NTM lung disease should be treated during anti-cancer chemotherapy has not been not clarified by this study. Induction of anti-NTM therapy should be made after careful consideration, because the duration of anti-NTM treatment is long and anti-mycobacterial drugs have extensive effects on anti-cancer drugs. However, we think that anti-NTM therapy should be introduced after consideration of the worsening of symptoms and radiological findings associated with NTM lung disease.

A Case of Solitary Well-Differentiated Papillary Mesothelioma with Invasive Foci in the Pleura.

Well-differentiated papillary mesothelioma (WDPM) is a rare, distinct tumor consisting of mesothelial cells with a papillary architecture, bland cytological features, and a tendency toward superficial spread without invasion. Rare cases with superficial invasion are termed WDPM with invasive foci. We report a case of solitary WDPM with invasive foci in the pleura. A 61-year-old woman presented with a lung adenocarcinoma. A small papillary lesion measuring 29 × 10 × 8 mm was incidentally found in the parietal pleura during a lobectomy for the lung adenocarcinoma. The fibrovascular core of the small papillary lesion was surrounded by a single layer of cuboidal cells with mild to moderate atypia and large nucleoli. Atypical mesothelial cells focally invaded the submesothelial layer. The cells of the papillary lesion were positive for cytokeratins and mesothelial markers. The Ki67 index was <1 %. The lesion did not show p16 loss on fluorescence in situ hybridization. We could not detect atypical mesothelial cells in the specimen from an extrapleural pneumonectomy. WDPM with invasive foci is prone to multifocality; however, our case represents a solitary case in the pleura.

Prospective Analysis of Oncogenic Driver Mutations and Environmental Factors: Japan Molecular Epidemiology for Lung Cancer Study.

PURPOSE: Oncogenic driver mutations are critical for lung cancer development and serve as therapeutic targets. However, their associations with environmental factors are not fully understood. We aimed to elucidate the relationship between tumor developmental biology and exposure to environmental factors. PATIENTS AND METHODS: This was a prospective, multicenter, molecular epidemiology study. Eligible patients were those with newly diagnosed stages I to IIIB non-small-cell lung cancer (NSCLC) who underwent surgery. The tumors were examined for somatic mutations in 72 cancer-associated genes by targeted deep sequencing, estrogen receptor ß (ERß) expression using immunohistochemical staining, and infection with any of 37 types of human papillomavirus (HPV) using a polymerase chain reaction-based microarray system. Detailed information on patient demographics and environmental factors was obtained from a comprehensive questionnaire. RESULTS: From July 2012 to December 2013, 957 patients were enrolled, and molecular analyses were performed on 876 samples (from 441 ever- and 435 never-smokers). Oncogenic driver mutations in P53 and KRAS increased proportionally with smoking status, whereas mutations in EGFR and SMAD4 decreased. KRAS mutations in smokers and SMAD4 mutations were observed more frequently in proportion to body mass index. TP53 and NFE2L2 mutations were observed more frequently in advanced NSCLC stages. As for never-smokers, no environmental factors were significantly associated with mutational changes. EGFR mutations and TP53 mutations were observed more frequently in women and in men, respectively. Mutations in these two genes were also potentially associated with ERß expression. Only three patients (0.3%) were HPV positive. CONCLUSION: The mutational spectrum is associated with smoking, body mass index, and other environmental factors, as well as with ERß expression. Little association was observed between HPV and NSCLC.

A Zbtb7a proto-oncogene as a novel target for miR-125a.

In our previous study, we showed that miR-125a directly targeted a WT1 oncogene, which was overexpressed in leukemia and various kinds of solid tumors including lung, breast, gastric, and colon cancers, and brain tumors and was deeply involved in leukemogenesis and tumorigenesis and that miR-125a knockout mice overexpressed WT1 and developed myeloproliferative disease. It had been also reported that miR-125a is downregulated in leukemia and various types of solid tumors such as lung cancers, suggesting its tumor suppressor function. Therefore, it is important to elucidate what is target(s) of miR-125a for understandings of such functions although few target genes for it are known. In the present study, Zbtb7a oncogene was identified as a potential target for miR-125a by gene expression profiling in miR-125a knockout mice combined with bioinformatics target prediction. EGFP-3'UTR reporter assay showed that miR-125a suppressed Zbtb7a expression through its direct binding to the Zbtb7a-3'UTR. Zbtb7a knockdown by siRNA suppressed cell proliferation and induced G1 cell cycle arrest and apoptosis in lung cancer cells. Furthermore, miR-125a expression showed a negative correlation with Zbtb7a expression in non-small cell lung cancer tissues. The present study showed for the first time that Zbtb7a was a direct target for miR-125a and was involved in cell cycle progression and apoptosis of lung cancer cells. These results also demonstrated that deregulation of miR-125a-Zbtb7a signaling was associated with the development and progression of lung cancer. © 2015 Wiley Periodicals, Inc.

[CLINICAL STUDY OF 29 CASES OF ENDOBRONCHIAL TUBERCULOSIS].

[Background] Endobronchial tuberculosis (EBTB) is defined as a tuberculosis infection of the tracheobronchial tree and is often misdiagnosed as bronchial asthma or bron- chitis owing to a lack of typical imaging findings. [Aim] The aim of this study was to elucidate the clinical characteristics of EBTB. [Method] We retrospectively studied EBTB patients hos- pitalized at the National Hospital Organization Kinki-chuo Chest Medical Center (Sakai City, Japan) between January 2005 and April 2014. [Result] A total of 29 patients (8 men and 21 women) were enrolled in this study. The patients' ages ranged from 17 to 86 years. Cough was the most frequently reported symptom. The interval between the appearance of symptoms and an EBTB diagnosis was significantly longer than usual when there was an initial misdiagnosis of bronchial asthma. The most frequent finding of fiber-optic bronchoscopy performed after more than 1 month of treatment was a V-type scar based on Arai's classification system. [Conclusion] A misdiagnosis of EBTB as bronchial asthma leads to a significant delay in correct diagnosis and treat- ment. EBTB must be included in the differential diagnoses of chronic cough and airway constriction sound.

Life-threatening and Rapidly Growing Teratoma in the Anterior Mediastinum.

This report describes the case of a 23-year-old man with a mediastinal teratoma. Five months before admission, a chest radiograph during a routine health checkup was normal. Four months before admission, the patient developed sudden onset of mild right-sided chest pain. He gradually developed dyspnea and was admitted to our hospital. Computed tomography revealed a giant tumor that was markedly compressing the right atrium. Urgent surgery was performed, and a ruptured, benign mature teratoma was diagnosed. Mature mediastinal teratomas are benign tumors, but they can rupture and have the potential to grow rapidly, potentially leading to life-threatening complications.

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms' tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3' end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms.

Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR.

PURPOSE: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non-small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort. EXPERIMENTAL DESIGN: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR. RESULTS: Our data revealed a linear performance for this ddPCR method (R(2) = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others. CONCLUSIONS: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs.

Molecular analysis of human papillomavirus in never-smokers with non-small cell lung cancer.

The causes of lung cancer in never-smokers remain unclear. The potential contribution of human papillomavirus (HPV) to the carcinogenesis of non-small cell lung cancer (NSCLC) has been reported. In 2008, a prospective registry of never-smokers with NSCLC was established at the Kinki-Chuo Chest Medical Center, Sakai, Osaka, Japan. Never-smokers with NSCLC were consecutively enrolled onto the registry. Of these patients, 114 with large tumor specimens, the majority of which were surgical tissues, were selected. In total, 23 of the most clinically relevant HPV types were assayed using polymerase chain reaction amplification of the viral genome. Following exclusion of samples with suboptimal quality, DNA was extracted from 96 formalin-fixed paraffin-embedded samples. These 96 cases consisted of 82 females (85.4%) and 14 males (14.6%), with a median age of 67 years (range, 29-83). Almost all cases (93.8%) were of the adenocarcinoma histological subtype. Despite confirmation of the quality and amount of DNA, HPV type 6 was detected in only one case (1.1%). Furthermore, no other samples examined were positive for any other HPV types. The results therefore suggest that HPV does not play a major role as the driving oncogenic event in never-smokers with NSCLC.

Small calcifying fibrous pseudotumor of the heart confined to the epicardium.

A calcifying fibrous pseudotumor (CFPT) is a rare benign lesion that often presents in the upper and lower extremities of children and young adults. In the present report, we describe a case of a small CFPT arising from the epicardium (visceral pericardium) in a 32-year-old woman. The tumor presented as a 25-mm polypoid mass protruding into the pericardial cavity, without extending into the myocardium. A complete resection was performed, and the patient has not experienced any relapse for more than 2 years. On histological examination, the lesion contained densely hyalinized collagen with psammomatous and dystrophic calcifications, as well as patchy chronic inflammatory infiltrate. The localization in the epicardium with no involvement of the myocardium was confirmed by the elastic stain. Amyloid was negative by the Congo red stain. On immunohistochemical analysis, the lesional cells indicated diffuse positive staining for vimentin and factor XIIIa and focal positive staining for CD34, but did not indicate positive staining for other pertinent antigens such as cytokeratins, calretinin, desmin, α-smooth muscle actin, ALK, and estrogen and progesterone receptors as well as IgG4 in plasma cells. To our knowledge, only three cases of CFPT in the heart have been reported in the literature, all of which developed in young females as a large mass involving the epicardium; the lesion also extended to the parietal pericardium in two cases. Moreover, all cases presented with few symptoms, despite the large lesion. In the present case, the CFPT developed also in a young woman, but the lesion was much smaller than those previously published and was localized in the visceral serous membrane of the heart. The findings of this case suggest a potential preferable site of origin of CFPTs of the heart.