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Supplementary Figures S1-8. Figure S1: Flowchart for mutation call. Figure S2: Quantification of performance of ddPCR analysis for EGFR T790M mutation. Figure S3: Analytical sensitivity of 0.001% for the ddPCR assay. Figure S4: Determination of false-positive events from wild-type control DNA and normal human genomic DNA. Figure S5: Determination of false-positive events in genomic DNA from formalin-fixed paraffin-embedded (FFPE) A549 cells. Figure S6: Detection of EGFR T790M mutant alleles in patients with EGFR T790M-containing primary tumors. Figure S7: Comparison of T790M mutation detection in 6 samples by different operators. Figure S8: Reproducibility of ddPCR analysis from 16 T790M-positive samples.

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 (R2 = 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. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

Supplementary Materials and Methods, Reference and Supplementary Figure Legends

Supplementary Tables S1-5. Table S1: Primers and probes used for droplet digital PCR (ddPCR). Table S2: Determination of the limit of blank (LOB) from wild-type control DNA, normal human genomic DNA, and EGFR wild-type A549 genomic DNA. Table S3: Determination of the limit of blank (LOB) using genomic DNA from adjacent normal tissue samples of EGFR-activating mutation positive adenocarcinoma and EGFR wild-type squamous cell carcinoma. Table S4: Concordance between ddPCR and deep sequencing of EGFR T790M mutation. Table S5: Distribution of amplifiable DNA concentrations in the 377 analyzed samples.

The bronchopulmonary vascular bifurcation patterns in the upper lobe of the left lung are diverse. Therefore, it is important for general thoracic surgeons to understand the detailed anatomy of the pulmonary segments when performing thoracoscopic anatomical pulmonary resection. This study aimed to analyze the bronchovascular patterns of the left upper lobe and summarize the anatomical information associated with pulmonary anatomical pulmonary resection.We reviewed the anatomical patterns of pulmonary vessels and the left lung bronchus of 539 patients using computed tomography imaging data including those obtained using three-dimensional computed tomography. We herein report the anatomic structure in the left upper lobe.Regarding the superior division bronchi, a pattern of trifurcation into BThe bifurcation patterns of the bronchovascular region in the upper lobe of the left lung were clarified. These results should be carefully noted when performing anatomical pulmonary resection.

The subsuperior segmental bronchi (B*) forms the subsuperior segment (S*) between the superior (S6) and basal segment (S7, S8, S9, S10) of the lung. However, the anatomical planes of S* remains undefined. The present study clarified the anatomical features of S*. We reviewed the anatomical patterns of pulmonary vessels and the left lung bronchus in 539 patients using three-dimensional computed tomography. We report the anatomic structure in S*. A total of 537 patients were analyzed. B* was observed in 129 (24.0%) patients. The intersegmental vein between S6 and S* was complete in all cases. The absence of intersegmental veins of S* was observed in 77 (14.3%) patients, reaching 59.7% of B* cases. Twenty-two (4.1%) cases of B* diverged from the trunk of the basal bronchus, and about half of the B* branched to the dorsolateral (n = 77, 14.3%) or dorsal (n = 2, 0.37%) direction. Our study revealed the branching patterns of B* and anatomical intersegmental veins of S*. Our results provide useful information regarding anatomical segmentectomy including or adjusting to the left S*.

Purpose: Single-port video-assisted thoracoscopic (VATS) pulmonary wedge resection was reported in 2004. We started using single-port VATS (SPVATS) pulmonary wedge resection in 2017 and compared results between conventional three-port VATS (VATS group) and SPVATS (SPVATS group). Methods: We identified 145 consecutive patients with VATS group and SPVATS group. Perioperative characteristics including pain and the number of stapler cartridges used were examined as the surgical outcomes, retrospectively. Results: In all, 66 cases of SPVATS group and 79 cases of VATS group pulmonary wedge resection were compared. The rate of epidural anesthesia (p

Fogging of the thoracoscopic lens affects a surgeon’s ability to maintain a clear operating field. In uniportal video-assisted thoracoscopic surgery, the thoracoscopic lens tends to fog when the surgeon does not hold a suction instrument. Thus, a suction instrument needs to be held by the surgeon’s nondominant hand to remove surgical smoke, mist, and moisture. Here, we describe a simple, easy and cost-effective surgical smoke ventilation technique for uniportal video-assisted thoracoscopic surgery using a suction catheter to solve the problem. We present this technique and comment on its advantages, including decreased cost and improved surgical visualization.

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, A prospective, multi‐center, molecular epidemiology study of 876 surgically resected non‐small cell lung cancer cases, and the impact of somatic mutations (72 cancer‐associated genes) on recurrence‐free survival and overall survival (OS). A smaller number of co‐existing mutations, earlier stage, and younger age were associated with longer recurrence free survival and OS, while epidermal growth factor receptor mutations were significantly associated with improved OS.

In conventional multiportal video-assisted thoracoscopic surgery, devices such as cotton-tipped applicators are used instead of graspers to avoid injuring the fragile lung tissue while stabilizing the lung and securing the surgical visual field. However, in uniportal video-assisted thoracoscopic surgery, which requires the simultaneous use of multiple instruments, the instruments tend to interfere with each other during the procedure because they share a single incisional port. Here, we describe a simple, easy and cost-effective lung retraction technique using cotton swabs to solve the problem. We present this technique and comment on its advantages, including decreased cost and improved surgical visualization.

BACKGROUND: Atelectasis of the middle lobe after right upper lobectomy is often seen. However, the risk factors for atelectasis are uncertain. Therefore, we assessed cases in our institution and investigated risk factors for atelectasis of the middle lobe following right upper lobectomy. METHODS: We identified 354 cases in which right upper lobectomy had been performed in our institution between January 2009 and December 2018, and 342 were included in this retrospective analysis. We divided patients into two groups according to the presence of postoperative atelectasis of the middle lobe, and then preoperative clinical variables and perioperative variables were compared between the two groups. Multivariable analyses for postoperative atelectasis of the middle lobe were performed using the logistic regression model. RESULTS: Middle lobe atelectasis was detected in 59 cases (17.3%). Multivariable analysis demonstrated that the preoperative diameter of the middle lobe bronchus [P=0.012; confidence interval (CI), 0.525–0.930] and stapling of the fissure between the upper and middle lobes (P=0.004; CI, 1.997–37.050) were independent risk factors for postoperative atelectasis of the middle lobe. CONCLUSIONS: A small preoperative diameter of the middle lobe bronchus and stapling of the fissure between the upper and middle lobes are risk factors for middle lobe atelectasis following right upper lobectomy.

When a presence of significant pleural adhesion is identified at the beginning of surgery, multiple factors determine the outcome of the surgery, particularly when it is performed thoracoscopically. These factors include identification of adhesion at the beginning of the surgery, as well as procedures involved in dissection and additional incisions. If the adhesion is partial, the lack of observation during creation of the surgical field can lead to damage being caused outside the field of view due to traction. Thus, it is important to focus observations. At our department, we started performing thoracoscopic surgery in 1992. Currently, over 80% of annual surgical cases are performed thoracoscopically. We will review our thoracoscopic surgery cases that involved pleural adhesion and discuss the tools and techniques used, as well as providing additional tips for ensuring successful thoracoscopic surgery.

Case series Patient: Male, 70 • Female, 60 Final Diagnosis: Staple stump granuloma Symptoms: Abnormal shadow on computed tomography Medication: — Clinical Procedure: — Specialty: Surgery Objective: Rare disease Background: Correctly diagnosing a staple-line mass after pulmonary resection for lung malignant tumor can be difficult. Differential diagnoses of recurrence, infectious mass, granuloma, and so on must be considered, despite their rarity. We report two cases of pulmonary staple-stump granuloma after segmentectomy for lung cancer. Case Reports: Case 1 involved a 70-year-old man with small nodule in the left upper lobe identified on computed tomography (CT). Video-assisted thoracoscopic (VATS) left upper division segmentectomy was performed. Histopathological examination revealed squamous carcinoma. Follow-up CT 1 year postoperatively showed a shadow at the staple-stump, with growth evident later. CT-guided biopsy found no malignancy. However, complete left upper lobectomy was performed because of the gradually enlarging lesion. Histopathological examination revealed epithelioid granuloma. Case 2 involved a 60-year-old with suspected lung cancer in the right upper lobe. VATS right upper division segmentectomy (S2) was performed. CT at 30 months postoperatively showed a shadow at the staple line, with subsequent growth. VATS right upper lobectomy was performed. Intraoperative rapid diagnosis revealed epithelioid granuloma. These two cases were compared with five cases of staple-stump recurrence in our institution. All cases of recurrence grew concentrically or radially from the staple line with the mass surrounding the staple line. On the other hand, cases of granuloma extended along the long axis of the staple line, and 3-dimensional CT (3DCT) may help to understand the morphology. Conclusions: Although preoperative differentiation of staple-line granuloma is difficult and pathological diagnosis is important, characteristic radiologic features and 3DCT may facilitate diagnosis.

A 60-year-old man had a malignant left lower lung tumour with no metastases and underwent video-assisted thoracoscopic left lower lobectomy and lymphadenectomy. Pathological examination led to a diagnosis of capicua transcriptional repressor (CIC)-rearranged sarcoma. He has had 3.5 years of recurrence-free survival. CIC-rearranged sarcoma is a Ewing-like sarcoma that shows pathological findings similar to Ewing sarcoma. Most of CIC-rearranged sarcoma is CIC-double homeobox 4 protein (DUX4) fusion. Pulmonary CIC-rearranged sarcoma is extremely rare and has an unfavourable prognosis. However, complete resection can produce prognosis of long-term survival, and thus, surgery is an important option.

Background Thrombus of the pulmonary vein (PV) stump is reportedly more frequent after left upper lobectomy than after other lobectomies, risking postoperative cerebral infarction (PCI). We have performed central vascular ligation before vascular dissection to improve the safety of surgical stapling. The effects of central vascular ligation in terms of PV stump thrombus and PCI are uncertain. Methods This study retrospectively reviewed the records of patients who underwent left upper lobectomy at a single center (Hokkaido Cancer Center, Sapporo, Japan) between November 1, 2008 and July 31, 2018. Relationships between PCI, PV stump thrombus and background characteristics were investigated and analyzed. Results All 208 cases of left upper lobectomy underwent central vascular ligation of the PV during this study. PCI occurred in 8 of the 208 patients. PV stump thrombus was detected in 14 of the 132 patients. In comparison with no-PCI cases, PCI cases showed significantly higher rates of comorbidity with cardiovascular disease (P=0.02), double cancer within 5 years (P=0.04), intraoperative hyperthermic chemotherapy (P=0.02), postoperative intrapleural inflammation (P=0.02) and postoperative PV stump thrombus (P=0.04). Presence of both comorbid cardiovascular disease (odds ratio, 18.4) and intraoperative hyperthermic chemotherapy (odds ratio, 30.4) was associated with higher risk of PV stump thrombus than presence of none of these factors. Cerebral infarction within 30 days postoperatively was seen in only 2 of the 208 cases (1.0%). Conclusions Central ligation of the PV may prevent postoperative early cerebral infarction. Further study of the effectiveness of this method for preventing PV stump thrombus is needed.

To identify and clarify the comprehensive anatomic patterns in the left lower lobe (LLL). Using computed tomography (CT) imaging data, including that obtained using three-dimensional CT, we reviewed the anatomic patterns of the pulmonary vessels and bronchi in the left lungs of 539 patients, focusing on the LLL. The two-stem type in A6 was observed in 131 (24.7%) patients and the three-stem type in A6 was observed in 11 (2.1%) patients. The independent two-stem type in B6 was observed in four (0.75%) patients. The B7 with independent branching from the basal bronchi was observed in 42 (7.9%) patients. B* was observed in 129 (24.0%) patients and B* was accompanied by A* in all patients. An extrapericardial common trunk of the left pulmonary veins was identified in five patients (0.93%). We identified various bronchovascular patterns in the LLL of a large number of patients. Our results provide useful information for anatomic pulmonary resection, especially segmentectomy.

Background Pulmonary visceral subpleural hematoma is rare. We report visceral subpleural hematoma of the left diaphragmatic surface following left upper division segmentectomy. This very rare case was difficult to distinguish from thoracic abscess. Case presentation A 68-year-old man with hypertension had undergone video-assisted thoracoscopic left upper division segmentectomy for suspected lung carcinoma. Deep vein thrombosis of the lower leg was identified and edoxaban, a so-called novel oral anticoagulant, was started on postoperative day 7. The chest drainage tube was removed on postoperative day 12 because of persistent air leakage, but fever appeared the same day. Computed tomography revealed a cavity with mixed air and fluid, so antibiotics were started on suspicion of abscess. Computed tomography-guided drainage was attempted, but proved unsuccessful. Fever continued and surgical investigation was therefore performed. Visceral subpleural hematoma was identified under the diaphragmatic surface of the left basal lung. We excised the pleura, then performed drainage and applied running sutures. The parenchyma and visceral pleura were covered with polyglycolic acid sheet and fibrin glue. Edoxaban was restarted on postoperative day 12 of video-assisted thoracoscopic surgery and no recurrence of hematoma has been revealed. Conclusions Visceral subpleural hematoma after thoracic surgery is extremely rare. Furthermore, correct diagnosis was difficult and surgery offered a good diagnostic and therapeutic procedure.

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, Highlights • An ultrasensitive multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction was developed. • This multiplex mutation detection of common mutations of EGFR is a feasible alternative. • A rare pretreatment EGFR mutation (T790M) with a mutant allele frequency below 0.1% was detected via this multiplex assay. Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKI therapy. In this article, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction with 0.01% sensitivity. All 33 EGFR-activating mutations detected in FFPE samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation was detected in the same reaction. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations.

Purpose Chronic or recurrent pericardial effusion is often associated with malignant disease. However, there have been few reports of the long-term outcomes after video-assisted thoracoscopic (VATS) pericardiectomy. We have performed it since 1992, and report our procedure and outcomes. Methods Patients who underwent VATS pericardiectomy were investigated. Results In all, 29 patients (12 men; median age: 61 (23-88) years) were evaluated; 8 had no malignancies and 21 did. Preoperative performance status (PS) scores were as follows: 1, 11 patients; 2, 10 patients; 3, 5 patients; and 4, 2 patients. One patient with malignancy died intraoperatively. PS improved significantly after the procedure (p = 0.0163). Median survival times were 5360 days in the nonmalignant group, 160 days in the malignant group, 209 days in breast cancer patients, and 62 days in other malignancy patients. The nonmalignant group had significantly longer survival than the malignant group (p = 0.0015). Most cases had uneventful postoperative courses. No recurrent pericardial effusions have been observed. Conclusion In cases of nonmalignant pericardial effusion, long-term survival is expected following VATS pericardiectomy. Malignant pericardial effusion has a poor prognosis, but most cases maintain good PS. However, early postoperative death may occur, and it is important to select patients carefully.

Objectives In 2015, we reported the outcomes of patients undergoing intentional limited resection (ILR) for non-small-cell lung cancer (NSCLC) from a retrospective, multi-institutional large database in Japan. Here, we analyse the clinicopathological characteristics of the patients extracted from this database with late recurrence and compare them with those with early recurrence. Methods Of 1538 patients in the database with cT1aN0M0 NSCLC, 92 (6%) had recurrence. In this study, early recurrence was defined as recurrence within 5 years and late recurrence as recurrence beyond 5 years after surgery. We compared the clinicopathological characteristics and post-recurrence survival (PRS) between patients with early and late recurrence. Results Of the 92 patients with recurrence, 21 (23%) had late recurrence. Compared with the early recurrence group, there were significantly more adenocarcinomas and local recurrences in the late recurrence group (P = 0.04 for both). The 3- and 5-year PRS rates were 53 and 24%, respectively, and the median PRS period was 38 months. There were no significant differences in the PRS curves between patients with early and late recurrence (P = 0.12). Only 3 patients (0.2%) had recurrence more than 10 years after ILR. Of the 21 late-recurrence patients, 17 (81%) had tumours with a consolidation/tumour ratio (CTR) >0.25. Conclusions Late recurrence occurred in 21 (23%) of 92 patients with recurrence after ILR for cT1aN0M0 NSCLC. Late recurrence was more likely to involve adenocarcinoma and local recurrence. It is thus considered reasonable to follow patients with a CTR >0.25 for 10 years after ILR.

Background Safety is of vital importance for lung resection. The dissection of pulmonary vessels is associated with vascular injury and bleeding, and identification of the vessels is necessary. The most common abnormal branching pattern of the left pulmonary artery is the mediastinal lingular artery. However, a mediastinal basal pulmonary artery is very rare. A case of abnormal branching from the left pulmonary artery to S8 which was diagnosed intraoperatively, and, thus, its dissection was avoided, is reported. Case presentation A 76-year-old woman with rheumatoid arthritis was diagnosed with left upper lung adenocarcinoma and visited our hospital. Contrast CT was not performed due to renal dysfunction, and abnormal branching of the left pulmonary artery was not identified. Video-assisted thoracoscopic left upper lobectomy and lymphadenectomy were performed. After the upper pulmonary vein was dissected and tissue around it was detached carefully, a pulmonary mediastinal branch from the left main pulmonary artery was identified descending between the upper pulmonary vein and upper bronchus. It was possible to separate the interlobar fissure safely and preserve A8. On retrospective examination, non-contrast CT showed A8. Conclusions Although preoperative identification of left pulmonary mediastinal branches was difficult by non-contrast CT, a careful surgical procedure preserved the left pulmonary mediastinal A8.

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 (R2 = 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. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

We conducted a retrospective investigation in patients who underwent surgery for T3 lung cancer with invasion of chest wall and other adjacent organs. Fifty patients who underwent surgery for T3 lung cancer with invasion of adjacent organs were investigated. The chest wall was the most frequently invaded organ, followed by the pericardium and mediastinal pleura. In cases of chest wall invasion, patients underwent en bloc resection and, when a complete resection was judged feasible, extrapleural resection. Six patients with superior sulcus tumor underwent surgery after preoperative treatment. The 5-year-survival was 55.7%. A significant difference in 5-year survival was observed between complete resection and incomplete resection in patients with lung cancer with chest wall invasion( p0.047).

The operative morbidity rate in elderly patients with lung cancer is high in comparison to nonelderly patients, probably because of the increase in comorbidities that occurs with aging. However, previous reports were retrospective and were performed at single institutions; thus, the preoperative comorbidities and operative morbidity could not be fully assessed. We conducted a multi-institutional prospective observational study of elderly patients (>75 years of age) with a completely resected non–small cell lung cancer. From March 2014 to April 2015, 264 patients from 22 hospitals affiliated with the National Hospital Organization in Japan were prospectively registered in the present study. The primary end point was operative morbidity (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0). The secondary end points were operative mortality and the risk factors for operative morbidity. Preoperative comorbidities were assessed according to the Adult Comorbidity Evaluation-27 index. The mean age at the time of surgery was 79.3 years (range 75-90 years). Forty-one percent of the patients were >80 years of age. Twenty-six percent underwent sublobar resection. The incidence of morbidities of any grade was 43.2% (90% confidence interval: 38.2%-48.2%). Respiratory system-related morbidity (19.3%), followed by cardiovascular system-related morbidity (10.2%), was the most common morbidity. The in-hospital mortality rate was 1.1% (3 of 264 patients). A multivariate analysis of the risk factors for operative morbidity showed that both Adult Comorbidity Evaluation-27 grade and the blood loss volume were significant factors. The results of the present prospective multi-institutional study should be used as a reference in the surgical treatment of elderly patients with lung cancer.

Hybrid supramolecular capsules self-assemble by simultaneously forming hydrogen and metal-ligand coordination bonds on mixing a C2-symmetrical cavitand (calix[4]resorcinarene-based cavitands with ureide and terminal 4-pyridyl units) with platinum or palladium complexes ([Pt(OTf)2] or [Pd(OTf)2] with chelating bisphosphines) in 1:1 ratio. Hemicapsular assemblies formed in the presence of excess amounts of cavitand relative to the platinum or palladium complexes are identified as intermediates in the above self-assembly process by 2D-NOESY spectroscopy. External-anion-assisted encapsulation of a neutral guest, 4,4'-diiodobiphenyl, inside the hybrid supramolecular capsules accompanied conformational changes in the hydrogen-bonding moieties. The in/out exchange ratio of the encapsulated guest depends on the bite angle of the bisphosphine ligand. Addition of DMSO accelerates guest exchange by weakening the hydrogen bonds in the encapsulation complex. Therefore, variations in the structure of the metal complex and amount of polar solvent exert dual control on the dynamics of the guest exchange.

A precise preoperative diagnosis of in situ or minimally invasive carcinoma may identify patients who can be treated by limited resection. Although some clinical trials of limited resection for lung cancer have started, it will take a long time before the results will be published. We have already reported a large-scale study of limited resection. We herein report the data for a subclass analysis according to differences in pathology.Data from multiple institutions were collected on 1710 patients who had undergone limited resection (segmentectomy or wedge resection) for cT1N0M0 non-small cell carcinoma. The disease-free survival (DFS) and recurrence-free proportion (RFP) were analyzed. Small cell carcinomas and carcinoid tumors were excluded from this analysis. Adenocarcinomas were sub-classified into four groups using two factors, the ratio of consolidation to the tumor diameter (C/T) and the tumor diameter alone.The median patient age was 64 (20-75) years old. The mean maximal diameter of the tumors was 1.5 ± 0.5 cm. The DFS and RFP at 5 years based on the pathology were 92.2 and 94.7 % in adenocarcinoma (n = 1575), 76.3 and 82.4 % in squamous cell carcinoma (SqCC) (n = 100), and 73.6 and 75.9 % in patients with other tumors (n = 35). The prognosis of adenocarcinoma in both groups A (C/T ≤0.25 and tumor diameter ≤2.0 cm) and B (C/T ≤0.25 and tumor diameter2.0 cm) was good. In SqCC, only segmentectomy was a favorable prognostic factor. In the groups with other pathologies, large cell carcinomas were worse in prognosis (the both DFS and RFP: 46.3 %).Knowing the pathological diagnosis is important to determine the indications for limited resection. Measurement of the tumor diameter and C/T was useful to determine the indications for limited resection for adenocarcinoma. Limited resection for adenocarcinomas is similar with a larger resection, while the technique should be performed with caution in squamous cell carcinoma and other pathologies.

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 β (ERβ) 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 ERβ 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 ERβ expression. Little association was observed between HPV and NSCLC.

肺類上皮性血管内皮腫は本邦での症例報告数が50例程度の稀な腫瘍である．われわれは肝転移を認めた肺類上皮性血管内皮腫の1切除例を経験したので報告する．症例は67歳，女性で他疾患経過観察中に胸部異常影を指摘され当院を受診した．胸部X線および胸部CTで右肺野に比較的境界明瞭な結節影を認めた．確定診断に至らず，胸腔鏡下肺部分切除術を施行．病理組織検査では，腫瘍細胞は肺胞内に充満するように存在し，空胞を有する異型細胞の増殖を認めた．免疫組織染色では血管内皮細胞マーカーであるCD31，CD34で腫瘍細胞が陽性となり肺類上皮性血管内皮腫の診断となった．その後早期に肝転移を生じ，肝部分切除術を施行した．外科的切除以外に有効な治療法が確立されておらず，胸腹部CT，脳MRIなどの画像検査を含め注意深い経過観察が必要である．

8549Background: We previously reported molecular profiling as a primary endpoint in a prospective multicenter molecular epidemiology study, collecting 876 surgically resected NSCLC and examining 72...

大腸癌の根治度A, B手術後に腹膜再発をきたした18例のうち7例 (手術群) に可及的転移巣摘出とMMC腹腔内散布を行った. このうち4例はCEA値上昇のみで試験開腹, 3例は腫瘤触知ないし画像診断で腫瘍摘除を行った. 残る11例 (非手術群) は姑息手術, 化学療法などで治療された. 初回手術時のCEA値上昇は手術群86%, 非手術群54.5%で, 再発診断は非手術群ではCEA値上昇を契機とする例はなかった. 初回手術後無再発期間は手術群33か月で非手術群16.2か月より長かった. 手術群の再発後50%生存期間は2年8か月と非手術群の5 か月より長く, 11年以上無病生存例もあった. 以上より大腸癌手術後, 腹膜再発が疑われた場合, 画像診断で局在が特定できなくても診断的開腹のうえ積極的に腫瘍摘除を図る意義はあることを述べた.

症例は63歳の女性で, 盲腸原発の粘液癌, n2 (+), p2 (S状結腸近傍) に対し回盲部切除+D3郭清+腹膜転移摘除およびマイトマイシン10mg散布施行した. 1年3か月後よりCEA値上昇がみられ, CT, MRI, 内視鏡検査にて異常所見はなかったが, 十分な説明と同意のもとに2年3か月後試験的開腹術を施行した. 手術創下, 腹壁, 吻合部, ダグラス窩に計5個の腫瘤をみとめ, これらを摘出した. 初回手術より5年3か月後, CT上, 脾臓腫瘤をみとめ脾臓摘出術を施行した. この際の洗浄細胞診はclass Vであった. マイトマイシン10mg腹腔内散布した. これら腫瘍はすべて粘液癌であった. 術後5'DFUR 800mg/日を6年間投与し, 初回手術から13年6か月の現在無治療であるが無病健存である.

Four distinct cDNAs (NtPT1, NtPT2, NtPT3, and NtPT4) encoding phosphate transporters were isolated from tobacco ( Nicotiana tabacum L.). They show high degrees of similarity to known high-affinity phosphate transporters in higher plants. Northern blot analysis using probes specific to NtPT1 and NtPT2 and to NtPT3 and NtPT4 indicated that large quantities of these four transcripts accumulated under phosphate-deficient conditions. The transcripts of NtPT1 / 2 were detected in immature leaves, mature leaves, old leaves, stems, and roots, while those of NtPT3 / 4 were detected only in old leaves and roots. When tobacco was grown under phosphate-deficient conditions, the transcripts of NtPT1 / 2 and NtPT3 / 4 were systemically enhanced after reduction of the total phosphate concentration in each organ. On the other hand, when phosphate was supplied to phosphate-deficient plants, the transcripts of NtPT1 / 2 and NtPT3 / 4 decreased systemically before an increase in total phosphate concentration. These results suggest that high-affinity phosphate transporter genes are controlled at the level of transcription by two regulatory systems: one responds to the internal phosphate status and the other to the external phosphate status.

進行再発乳癌20例に対しpaclitaxel 50～80mg/m2,週1回1時間投与を行った. (50mg/m2が6例, 80mg/m2が14例)6投2休を1サイクルとして3サイクルを上限として投与した.投与回数の中央値は10回,奏効率50%, time to progressionの中央値は14.5週で,薬物有害反応としては重篤な急性過敏症は認めず,白血球減少,知覚障害は軽度で,軽度の倦怠感が認められたが外来での投与が十分可能であった. Anthracycline,またはdocetaxel投与歴のある進行再発乳癌に対しても有効率は変わらず,本来QOLを重視する必要がある進行再発乳癌に対して有用であると考えられた.

A precise preoperative diagnosis of 'very early' lung carcinoma may identify patients who can undergo curative surgery with limited resections.Data from a multi-institutional project were collected on 1737 patients who had undergone limited resections (segmentectomy or wedge resection) for T1N0M0 non-small-cell carcinomas. As it was expected, this study was predominantly including ground glass nodules. Computed tomography was used to obtain the ratio of consolidation to the maximal tumour diameter to determine invasive potential of the tumours. Overall and disease-free survivals and recurrence-free proportions were analysed.Median age was 64 years. Mean maximal diameter of the tumours was 1.4±0.5 cm. Overall and recurrence-free survivals after limited lung resection were 94.0 and 91.1% at 5 years, respectively. Recurrence-free proportions were 93.7% at 5 years. Unfavourable prognostic factors in overall survival were lymph node metastasis, interstitial pneumonia, male gender, older age, comorbidities (cardiac disease, diabetes etc.) and consolidation/tumour ratio (C/T)≤0.25. C/T≤0.25 predicted good outcomes especially in cT1aN0M0 disease. In a subclass analysis of cT1N0M0 squamous cell carcinomas, wedge resection was the only unfavourable prognostic factor in both overall and disease-free survivals.If the patient was 75 years old or younger and was judged fit for lobectomy, limited resection for cStage I non-small-cell lung cancer (NSCLC) showed excellent outcomes and was not inferior to the reported results of lobectomy for small-sized NSCLC. The carcinomas with C/T≤0.25 rarely recur and are especially good candidates for limited resection.

A phosphatase has been purified 1767-fold, to a final specific activity of 53.0 (μmol pyruvate produced per min per mg protein), from the bulb of Allium cepa L. The enzyme has high specificity for a phosphoenolpyruvate (PEP) substrate. The molecular mass of the enzyme is approximately 240 kDa, as determined by gel column chromatography. Although 52 and 42 kDa polypeptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), their N-terminal amino acid sequences were the same. We deduce, therefore, that the enzyme consists of four polypeptide subunits of 52 kDa. The purified enzyme hydrolyzes a wide variety of phosphate esters, and the lowest K m was obtained with PEP (0.44 mM). The enzyme consumed 1 mol of PEP, with the release of 1 mol of phosphate and 1 mol of pyruvate, with no ATP formation during the enzymatic reaction in the presence of ADP and Mg 2+ . Therefore, the enzyme is thought to be a PEP phosphatase. A cDNA that encodes PEP phosphatase, the deduced amino acid sequence of which resembles that of the plant acid phosphatases, was also isolated from onion bulb RNA.

In the food industry, syrups containing a high concentration of sugar used for fruit preservation is abundantly discharged as a food processing waste and disposed by incineration, resulting in the rise of the manufacturing cost and environmental pollution. This study demonstrates how waste syrup can be utilized as carbon source for production of docosahexaenoic acid (DHA) and astaxanthin by the thraustochytrid strain, Aurantiochytrium sp. KH105. The strain could grow in culture medium containing 3-50% waste syrup, and the maximum yields of DHA and astaxanthin were 207.6 mg/L (at 50%) and 1.1 mg/L (at 25%), respectively. After the optimization of culture medium composition by response surface method, DHA and astaxanthin yields increased by 2.1 and 1.5 fold, respectively. When the waste syrup was treated with activated charcoal, citrate concentration in the syrup was reduced and the astaxanthin yield increased by 2.3 fold. This study shows that the waste syrup can be effectively used for the functional lipid production by the thraustochytrid.

BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency ( CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Purpose: The resistance of the epidermal growth factor receptor (EGFR) to tyrosine kinase inhibitors (TKIs) 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 EGFR T790M mutation was also detected in TKI-naive NSCLCs in a small cohort and the prevalence of a de novo EGFR T790M mutation in patients with EGFR-mutant NSCLC remains unclear. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment EGFR T790M mutation in a larger cohort. Experimental design: ddPCR was established as follows: EGFR 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 EGFR. Surgically resected tumor tissues from 373 early-stage NSCLC patients with EGFR-activating mutations detected by Cycleave PCR method were then examined for the presence of pretreatment EGFR T790M mutation using ddPCR. Results: Preclinical model data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of ∼0.001%. The overall incidence of the pretreatment EGFR T790M mutation was 79.9% (298/373) and the frequency ranged from 0.009% to 26.9%, while only five patients were diagnosed positive for EGFR T790M mutation by Cycleave PCR method. The EGFR 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 to the others. Among the 69 patients who had co-incident mutations in EGFR and p53, 61 patients (88.4%) had the EGFR T790M mutation (p = 0.096). Moreover, all 11 patients with mutations in PIK3CA and EGFR had the EGFR T790M mutation (p = 0.097). These results, while not statistically significant, suggest that tumors with the pretreatment EGFR T790M mutation may manifest greater genomic complexity. Conclusions: To our knowledge, this is the largest study thus far using an ultra-sensitive method for detecting the pretreatment EGFR T790M mutation that has demonstrated associations between the incidence of the pretreatment T790M mutation and clinicopathological as well as genetic features in early-stage NSCLC. The ultra-sensitive ddPCR assay revealed that pretreatment EGFR T790M mutation 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 EGFR T790M mutation on treatment with EGFR-TKIs, allowing the possibility of developing strategies for personalized cancer therapies in NSCLC patients. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultrasensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using picodroplet digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 617. doi:10.1158/1538-7445.AM2015-617

3528 Background: There have been few reports of surgical outcome for pulmonary metastasis (PM) of colorectal cancer (CRC) (CRC-PM) in the era of modern effective chemotherapy including oxaliplatin....

7516 Background: Driver mutations are critical for lung cancer development as well as therapeutic targets. Its associations with environmental factors, sex hormones and virus remain elusive. Method...