Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

The relation between anti-TGBFR1 immunohistochemical reaction and low Ki67, small tumor size and high estrogen receptor expression in invasive breast cancer.

Most breast cancers are derived from the luminal epithelium, which composes the inside of the breast ductal structure. Ductal carcinoma in situ (DCIS) leads to invasive ductal carcinoma, but noncancerous intraductal proliferative lesions are also a risk factor for ductal carcinoma. The transforming growth factor beta (TGFB) signaling pathway behaves as a tumor suppressor in the early stage of cancer, and conversely as a tumor growth factor in invasive stages in several cancers. In this study, we performed immunohistochemistry with an antibody that detects the cytoplasmic region of TGFB receptor 1 (TGFBR1) and elucidated TGFBR1 protein expression in luminal epithelial cells of noncancerous breast ducts and in several cases of DCIS and invasive carcinoma. TGFBR1 expression was higher in noncancerous breast tissue than in cancerous tissue, and a difference in expression was also seen among histological subtypes. Comparing the expression level of TGFBR1 in cancer cells and clinico-pathological parameters, cases expressing low TGFBR1 tended to show low estrogen receptor expression, large tumor size (≥10 mm), and a high Ki67 labeling index. These data suggested that TGFBR1 protein expression may be related to the suppression of breast cancer cell growth.

A new fluorescent anatomic pulmonary segmentectomy using PDD endoscope system and vitamin B2: evaluation in a clinical setting using living animal.

OBJECTIVE: Identification of intersegmental planes is essential for successful anatomic pulmonary segmentectomy. We have previously reported a new fluorescence technique using a PDD endoscope system™ and vitamin B2 for identification of intersegmental planes in ex vivo experiments. In the present study, we investigated and evolved this technique to perform ideal anatomic segmentectomy in a clinical setting using living pig models. METHODS: Cranial segmentectomy in the cranial lobe of the right lung was performed in six pigs using our fluorescence technique. The fluorescent cranial segmentectomy was as follows. After identification of the cranial segmental bronchus, vitamin B2 solution as a fluorescent substance was injected via bronchoscopy. The fluorescent segment was observed using a PDD endoscope system, and the identified intersegmental plane was cut using electric cautery. The operative data collected were the rates of accurate identification of the pulmonary segment and perioperative complications. The duration and light intensity of fluorescence of the target segment were recorded to provide an objective measurement of success. RESULTS: In all procedures, it was possible to identify the target segment by its clear yellow-green fluorescence. The rate of accurate identification of the pulmonary segment was 100%. The fluorescence continued for more than 1 h with adequate light intensity. No perioperative complications were encountered. No unexpected injuries of the major segmental bronchi or vessels occurred. Hemorrhage and air leakage from the transected intersegmental plane were negligible. CONCLUSIONS: Our new fluorescence technique in a clinical setting involving a PDD endoscope system™ vitamin B2 enabled accurate and safe anatomic pulmonary segmentectomy, with enough strong and long fluorescence in living pig lungs.

Expression and regulatory effects on cancer cell behavior of NELL1 and NELL2 in human renal cell carcinoma.

Neural epidermal growth factor-like like (NELL) 1 and 2 constitute a family of multimeric and multimodular extracellular glycoproteins. Although the osteogenic effects of NELL1 and functions of NELL2 in neural development have been reported, their expression and functions in cancer are largely unknown. In this study, we examined expression of NELL1 and NELL2 in renal cell carcinoma (RCC) using clinical specimens and cell lines. We show that, whereas NELL1 and NELL2 proteins are strongly expressed in renal tubules in non-cancerous areas of RCC specimens, their expression is significantly downregulated in cancerous areas. Silencing of NELL1 and NELL2 mRNA expression was also detected in RCC cell lines. Analysis of NELL1/2 promoter methylation status indicated that the CpG islands in the NELL1 and NELL2 genes are hypermethylated in RCC cell lines. NELL1 and NELL2 bind to RCC cells, suggesting that these cells express a receptor for NELL1 and NELL2 that can transduce signals. Furthermore, we found that both NELL1 and NELL2 inhibit RCC cell migration, and NELL1 further inhibits RCC cell adhesion. These results suggest that silencing of NELL gene expression by promoter hypermethylation plays roles in RCC progression by affecting cancer cell behavior.

Diverse involvement of isoforms and gene aberrations of Akt in human lung carcinomas.

Emerging evidence confirms a central role of Akt in cancer. To evaluate the relative contribution of deregulated Akt and their clinicopathological significance in lung carcinomas, overexpression, activation of Akt and AKT gene increases were investigated. Immunohistochemical staining for 108 cases revealed overexpression of total Akt, Akt1, Akt2 and Akt3 in 61.1, 47.2, 40.7 and 23.1%, respectively, and phosphorylated Akt in 42.6% of cases. Expression of total Akt, Akt2 and Akt3 were frequently observed in small cell carcinoma, but phosphorylated Akt and Akt1 were more frequently observed in squamous cell carcinoma. FISH analysis to evaluate gene increases of AKT1-3 revealed amplification of AKT1 in 4.2% and AKT1 increase by polysomy of chromosome 14 in 27.3% of cases. For AKT2, amplification was observed in 3.2% and polysomy of chromosome 19 in 26.3% of cases. AKT3 increase was observed in 40.0% of cases only by polysomy of chromosome 1. Although "FISH-positive" AKT1 and AKT2 gene increases (amplification/high-level polysomy) were found exclusively in the cases overexpressing total Akt, Akt1 or Akt2, respectively, AKT3 increase was irrelevant of Akt3 expression. Statistically, expressions of Akt2, p-Akt and cytoplasmic-p-Akt were correlated with lymph node metastasis (P = 0.0479, P = 0.0371 and P = 0.0310, respectively). Although AKT1 and AKT2 gene increase showed positive correlation with, or trend towards a positive correlation with tumor size (P = 0.0430, P = 0.0590, respectively), AKT3 did not. In conclusion, Akt isoforms are differentially involved in the pathological phenotype of lung carcinoma in a diverse manner. Because abnormality of Akt1/AKT1 and Akt2/AKT2 correlated with clinicopathological profiles, Akt1/2-specific targeting may open a novel therapeutic window for the group showing Akt deregulation.

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with 'gain' or 'amplified' status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.

Overexpression and gene amplification of both ERBB2 and EGFR in an esophageal squamous cell carcinoma revealed by fluorescence in situ hybridization, multiplex ligation-dependent probe amplification and immunohistochemistry.

EGFR and ERBB2 belong to the EGFR gene family. In esophageal squamous cell carcinomas (SCCs), amplification of EGFR or ERBB2 is usually mutually exclusive. EGFR amplification occurs in approximately 15% of SCCs, ERBB2 occurs in less than 5%. Here, we report the co-amplification of EGFR and ERBB2 in an ulcerative and infiltrating-type SCC that measured approximately 4.2 × 2.7 × 1.2 cm with a superficial lesion occurring in the thoracic esophagus of a 72-year-old man. Multiplex ligation-dependent probe amplification using representative tumor sections showed gain of CCND1 and coincident amplification of ERBB2 or EGFR or neither. Immunohistochemistry and fluorescence in situ hybridization revealed that the tumor comprised three cancer-cell populations: well-differentiated SCC with high-level ERBB2 amplification and ERBB2 overexpression, more infiltrative poorly-differentiated SCC with high-level EGFR amplification and EGFR overexpression, and poorly-differentiated SCC lacking any ERBB2 or EGFR abnormality. These three populations each had low-level CCND1 amplification and nuclear cyclin D1 overexpression. This histological topology and gene amplification combinations suggested that genetic instability first produced CCND1 amplification, and then ERBB2 or EGFR gene amplification occurred. It is further speculated that during cancer progression and clonal selection indecisive predominance of either clone caused the rare co-amplification of ERBB2 and EGFR in a single chimeric tumor.

Thyroidectomy using pure natural orifice transluminal endoscopic surgery in a porcine model.

Surgical procedures for thyroid disease that provide cosmetically acceptable results are in demand. Natural orifice transluminal endoscopic surgery (NOTES) is performed through natural orifices and thus avoids incision of the body wall. This study aimed to develop an incision-free surgical procedure for thyroid lobectomy using pure NOTES with an oral approach. In six pig carcasses, an incision was made between the mandible and subcutaneous tissue under direct vision. After subcutaneous dissection and identification of the hyoid bone, the operative field was developed under endoscopic view. After the thyrohyoid membrane was identified, dissection was continued along the thyroid cartilage until the cricoid cartilage was identified and the thyroid isthmus was reached. An original retractor was inserted between dissected tissues to lift and fix the carcass. The thyroid gland was successfully removed through the incision. Similar macroscopic and histological findings were observed on the normal and treated sides, with no damage to the recurrent laryngeal nerves. The times required for securing the operative field and thyroidectomy improved with each operation. This study suggests the feasibility and safety of using pure NOTES for thyroidectomy through a subcutaneous route with an original retractor.

Clonal profiling of mixed lobular and ductal carcinoma revealed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew.

Diagnosis and treatment of low-grade osteosarcoma: experience with nine cases.

BACKGROUND: Low-grade osteosarcoma, including low-grade central osteosarcoma and parosteal osteosarcoma, is an extremely rare variant, and the diagnosis is occasionally difficult. In this article we present cases of low-grade osteosarcomas that should be reviewed by a clinical oncologist. PATIENTS AND METHODS: Nine cases of histologically diagnosed Broder grade 1 osteosarcoma were retrospectively reviewed. The pathological diagnoses included parosteal osteosarcoma, low-grade central osteosarcoma, and low-grade chondroblastic osteosarcoma in four, four, and one cases, respectively. RESULTS: Duration from initial surgical intervention including biopsy to final diagnosis as low-grade osteosarcoma was a mean of 9.4 months. The initial benign diagnoses on biopsy specimens included fibrous dysplasia in three cases, chondroblastoma in one case, and a giant cell tumor in one case. The average number of histological examinations was 1.8. Low-grade osteosarcomas are well suited for biological reconstruction: seven cases were reconstructed by frozen autografts, distraction osteogenesis, or vascularized bone grafts. CONCLUSION: Low-grade osteosarcomas can be misdiagnosed as benign lesions, especially fibrous dysplasia. If the diagnosis of a low-grade osteosarcoma is not established on the basis of radiologic findings, care should be exercised, even when a biopsy suggests a benign lesion. Low-grade osteosarcomas should be treated with wide excision, even after an intralesional excision. Biological reconstruction might be a better option for low-grade osteosarcomas.

Gene amplification of ESR1 in breast cancers--fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study.

Oestrogen receptor-alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ERα-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the 'HSR-like' signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5'- and 3'-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3' to 5' sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a 'gain' of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma.

Genomic copy-number alterations of MYC and FHIT genes are associated with survival in esophageal squamous-cell carcinoma.

Esophageal squamous-cell carcinoma (ESCC) is one of the most common cancers and is associated with a poor prognosis. Studies are warranted on the clinical relevance of its genomic copy-number alterations (CNA) as prognosticators for ESCC. In the present study, we first screened recurrent CNA by array-based comparative genomic hybridization using an in-house focused bacterial artificial chromosome-based array for 108 loci in 45 ESCC specimens. We detected 14 regions showing recurrent (>20%) CNA (4 losses and 10 gains) by array-based comparative genomic hybridization in the first cohort. Among them, loss of 3p14.2 and gain of 8q24.21 for the FHIT and MYC genes, respectively, and the accumulation of those two CNA (higher FM-CNA scores) were significantly associated with a worse overall survival (OS) in the first cohort (P = 0.0273, P = 0.0356 and P = 0.0089, respectively). In the independent validation cohort of 92 resected ESCC cases, loss of FHIT, gain of MYC and higher FM-CNA scores determined by a quantitative genomic PCR-based copy-number analysis were associated with a worse OS (P = 0.0011, P = 0.0104 and P = 0.0008, respectively) and disease-free survival (P = 0.0038, P = 0.0132 and P = 0.0021, respectively). In addition, the Cox model showed the presence of either CNA to be an independent prognosticator for OS and disease-free survival in the validation cohort (P = 0.0120 and P = 0.0255, respectively). These results suggest that CNA of MYC and FHIT are poor prognostic markers, and risk stratification based on the copy-number status of those genes is useful to select the optimal treatment strategy in resected ESCC patients.

LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer.

BACKGROUND: Long interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed. METHODS: Formalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples. RESULTS: LINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC. CONCLUSIONS: LINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

A novel combined radiological method for evaluation of the response to chemotherapy for primary bone sarcoma.

BACKGROUND: In the treatment of bone sarcoma, evaluation of chemotherapeutic effects is extremely important. In this study, we compared radiological evaluations and histological response, and developed a combined radiological scoring system for assessing the response to chemotherapy. METHODS: A total of 79 patients with primary bone sarcomas were examined by X-ray photography (Xp), angiography, MRI, 201Tl scintigraphy (Tl) and 99mTc-MIBI scintigraphy (MIBI) to evaluate their response to preoperative chemotherapy. Patients were classified as responders and non-responders according to radiological images. All resected tumors were evaluated histologically and classified as a good response (≥90% necrosis) or a poor response (≤90% necrosis). The sensitivity, specificity, accuracy, and kappa values in radiological evaluation were calculated. Furthermore, we developed a combined radiological scoring system that correlated the results of radiological images with histological response. RESULTS: Sensitivity, specificity, and accuracy were 90.9%, 38.2%, and 67.5% (K = 0.31), respectively, for Xp; 91.7%, 33.3%, and 66.7% (K = 0.27) for angiography; 81.0%, 67.6%, and 75.0% (K = 0.49) for MRI; 78.9%, 72.4%, and 76.1% (K = 0.51) for Tl; 85.3%, 69.2%, and 78.3% (K = 0.55) for MIBI; and 93.3%, 76.5%, and 86.1% (K = 0.71) for combined radiological scoring. CONCLUSIONS: Combined radiological evaluations showed high correlation with histological response for assessing the effects of chemotherapy.

Use of 99mTc-MIBI scintigraphy in the evaluation of the response to chemotherapy for osteosarcoma: comparison with 201Tl scintigraphy and angiography.

BACKGROUND: In the treatment of osteosarcoma, the chemotherapeutic effect influences decisions regarding the surgical margin, continuation of chemotherapy, and choice of anticancer drugs for further chemotherapy. Therefore, it is necessary to evaluate the response to chemotherapy in the middle of the chemotherapy course. In this study, we investigated the use of (99m)Tc-hexakis-2-methoxyisobutyl-isonitrile ((99m)Tc-MIBI) scintigraphy in evaluating the response to chemotherapy of patients with osteosarcoma in comparison with (201)Tl scintigraphy and angiography. METHODS: A total 45 patients with osteosarcoma were examined using (99m)Tc-MIBI scintigraphy, (201)Tl scintigraphy, and angiography to evaluate their response to chemotherapy. The percentage reduction in the uptake ratios (ΔUR) calculated as 100 × [(prechemotherapy value - postchemotherapy value)/prechemotherapy value] were compared with histological assessments. Similarly, changes in tumor vascularity assessed by angiograms were compared with histological assessments. On the angiographic findings, the results were classified as complete response (CR), partial response (PR), no change (NC), or progressive disease (PD). On the images, ΔUR in (99m)Tc-MIBI ≥ 30% and ΔUR in (201)Tl ≥ 30% were classified as responder, as were CR and PR in angiograms. The therapeutic effect was assessed by histopathological examination of the resected specimens. The tumors of poor responders showed less than 90% necrosis, whereas the tumors of good responders showed 90% or more necrosis. RESULTS: Sensitivity, specificity, and accuracy were 73.9, 84.2, and 78.6%, respectively, for (99m)Tc-MIBI (κ = 0.57); 80.0, 61.1, and 72.1%, respectively, for (201)Tl (κ = 0.42); and 91.7, 35.0, and 65.9%, respectively for angiography (κ = 0.28). CONCLUSION: (99m)Tc-MIBI could be effectively used to predict the final response to chemotherapy of osteosarcoma.

Suitability of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration versus Paired Transbronchial Biopsy Specimens for Evaluating Programmed Death Ligand-1 Expression in Stage III and IV Lung Cancer: A Comparative Retrospective Study.

Objectives: Cancer cells usually escape tumor-reactive T-cell responses using immune checkpoint proteins, such as programmed death protein-1 (PD-1) and its ligand, programmed death ligand-1 (PD-L1). These proteins can be blocked by immune checkpoint inhibitors (ICIs); the decision on ICI-based first-line treatment for advanced lung cancers depends on the PD-L1 levels in tumor specimens. Determining the PD-L1 expression conventionally requires histological specimens from resected tumors and core biopsy specimens. Non-small cell lung cancer (NSCLC) is usually diagnosed at stage III or IV; therefore, only small biopsy specimens, such as those obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are available. However, the suitability of EBUS-TBNA specimens determining the PD-L1 expression levels in advanced lung cancers remains unclear. Materials and Methods: Here, we investigated the concordance rate of PD-L1 expression between EBUS-TBNA and matched transbronchial biopsy (TBB) specimens. Using the 22C3 anti-PD-L1 antibody (immunohistochemistry), we determined the PD-L1 expression levels in paired specimens obtained from 69 patients (50 with advanced NSCLC and 19 with small cell lung cancer [SCLC]), as well as the efficacy of ICIs in these patients. Results: The concordance rate of PD-L1 expression between the EBUS-TBNA and TBB specimens was 78.3%. The κ values referent to the PD-L1-positive expression rate between EBUS-TBNA and TBB specimens were 0.707 and 0.676 at cutoff limits of ≥1% and ≥50%, respectively. Among the 19 SCLC patients, 16 (84.2%) exhibited no PD-L1 expression in both EBUS-TBNA and TBB specimens. Notably, the progression-free survival of patients with ≥50% PD-L1 expression in the paired specimens who received ICI treatment was 8.3 months. Conclusion: Collectively, our results validate the use of EBUS-TBNA specimens for the determination of the PD-L1 expression levels in the context of NSCLC and SCLC.

Clinicopathological significance of platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor-A expression, PDGF receptor-ß phosphorylation, and microvessel density in gastric cancer.

BACKGROUND: Angiogenesis is important in the growth and metastasis of various kinds of solid tumors, including gastric cancers. The angiogenic process is triggered by several key growth factors, including vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B, that are secreted by tumors. Our aim was to define: i) the expression pattern of VEGF-A and PDGF-B in tumor cells and the activation of PDGF receptor (PDGFR)-ß tyrosine kinase in stromal cells of human gastric adenocarcinomas; and ii) the relationship between VEGF-A and PDGF-B expression and microvessel density (MVD), to determine if there is a rationale for a new therapeutic strategy. METHODS: A series of 109 gastric adenocarcinoma cases that had undergone surgical resection was examined immunohistochemically using antibodies against VEGF-A, PDGF-B, and CD34, followed by further examination of PDGFR-ß phosphorylation by immunoblotting analysis. RESULTS: MVD was higher in diffuse-type than intestinal-type cancers (p < 0.001). VEGF-A overexpression correlated to PDGF-B overexpression in both the intestinal-type (p < 0.005) and diffuse-type (p < 0.0001) groups, indicating that VEGF-A and PDGF-B are secreted simultaneously in the same tumor, and may thus play important roles together in angiogenesis. However, several differences between intestinal-type and diffuse-type cancers were observed. In the diffuse-type cancer group, higher MVD was related to the PDGF-B proportion (p < 0.05) and VEGF-A overexpression (p < 0.05), but not to PDGF-B overexpression or the VEGF-A proportion. On the other hand, in the intestinal-type cancer group, higher MVD was correlated to overexpression (p < 0.005), intensity (p < 0.05), and proportion (p < 0.05) of PDGF-B, but not of VEGF-A. In addition, phosphorylation of PDGFR-ß was correlated with depth of cancer invasion at statistically significant level. CONCLUSIONS: Our results indicate that PDGF-B, which is involved in the maintenance of microvessels, plays a more important role in angiogenesis in intestinal-type gastric carcinomas than VEGF-A, which plays a key role mainly in the initiation of new blood vessel formation. In contrast, VEGF-A has a critical role for angiogenesis more in diffuse-type cancers, but less in those of intestinal type. Thus, a therapy targeting the PDGF-B signaling pathway could be effective for intestinal-type gastric carcinoma, whereas targeting VEGF-A or both VEGF-A and PDGF-B signaling pathways could be effective for diffuse-type gastric carcinomas.

Gene amplification of ERBB2 and EGFR in adenocarcinoma in situ and intramucosal adenocarcinoma of Barrett's esophagus.

We examined 11 cases of carcinoma arising from Barrett's esophagus consisting of two adenocarcinomas in situ (ACIS), two intramucosal adenocarcinomas, and seven overt invasive adenocarcinomas. Overexpression of p53 (implying a mutation of the p53 gene), ERBB2, and EGFR was measured by immunohistochemistry, and gene amplification of ERBB2 and EGFR was measured by fluorescence in situ hybridization (FISH). In all cases of ACIS and the intramucosal adenocarcinomas, almost all cancer cells overexpressed p53, however the populations overexpressing ERBB2 and EGFR varied in different cases: in one ACIS, ERBB2 was coexpressed in all the cancer cells, in the other ACIS and one intramucosal adenocarcinoma, ERBB2 was overexpressed in about 50% and only 10% of the p53-positive cells respectively. EGFR was co-expressed in 20% in the other intramucosal adenocarcinoma. Protein overexpression of ERBB2 or EGFR corresponded to the amplification of their respective genes on a cell by cell basis. These gene amplifications, however, were not found in the seven invasive adenocarcinomas. Thus we speculate that the gene amplification occurred late in the dysplasia-carcinoma sequence probably after the mutation of p53. Furthermore, new clonal expansion accompanied by tumor invasion might have extinguished the originally amplified genes in these tumors.

Semi-comprehensive analysis of gene amplification in thymic malignant tumors using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

Research on the amplification of oncogenes in thymic malignant tumor is limited. In this study, we aimed to determine the gene amplification status of receptor tyrosine kinases and other cell regulator genes in thymic malignant tumors, with a view toward the future introduction of molecular targeted therapy. In addition, we examined the usefulness of multiplex, ligation-dependent probe amplification (MLPA) in the semi-comprehensive detection of these gene amplifications. The participants of this study were nine patients with thymic carcinoma and one patient with atypical carcinoid who underwent resection at our department from 1999 to 2016. Twenty-four oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, BRAF, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR) were analyzed for amplification by MLPA. In cases where amplification by MLPA was suspected, confirmation was performed by fluorescence in situ hybridization (FISH). Immunostaining for detected oncoproteins and p53 were performed in cases with confirmed oncogene amplification. MYC (2/10, 20%) and MDM2 (1/10, 10%) amplifications were detected using MLPA and FISH. Immunostaining in both cases was positive. The MDM2-amplified tumor relapsed and spread rapidly after operation despite the use of post-operative chemo-radiotherapy. MYC amplification may be involved in the carcinogenesis of thymic malignant tumors. In addition, MDM2 amplification may be a concern in the increased malignancy.

EGFR-dependent and independent activation of Akt/mTOR cascade in bone and soft tissue tumors.

To gain the insight into the involvement of signaling mediated by the mammalian target of rapamycin (mTOR) in the phenotype and biological profiles of tumors and tumor-like lesions of the bone and soft tissue, we analyzed the expression and phosphorylation (activation) of mTOR and its correlation with the status of upstream and downstream modulator proteins Akt, p70S6-kinase (S6K), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which we refer to collectively as mTOR cassette proteins. Immunohistochemical analysis of 140 cases showed activation of Akt in 55% (61% in malignant and 27% in benign), and mTOR expression in 61% (66% in malignant and 39% in benign). The preponderance of mTOR activation was found in tumors of peripheral nerve sheath (malignant peripheral nerve sheath tumor and schwannoma), skeletal muscle origin (rhabdomyosarcoma), and in those exhibiting epithelial nature (chordoma and synovial sarcoma). Together with the result of immunoblotting analysis, it was shown that many of those particular tumors with mTOR activation exhibited activation of Akt, S6K, and 4E-BP1, suggesting the constitutive activation of the Akt/mTOR pathway. In addition, although activation of the Akt/mTOR pathway was largely independent of activation of epidermal growth factor receptor (EGFR), mutation of EGFR was frequently accompanied by constitutive activation of Akt-mTOR-S6K/4E-BP1. By clinicopathological analysis, activation of Akt correlates with statistically higher probability of metastasis. We conclude that mTOR-mediated signaling proteins function not only in the proliferation of the tumor cells, but also in the differentiation and/or maintenance of morphological phenotypes in tumors of rhabdomyoblastic and nerve sheath cell origin. Furthermore, mTOR signaling may also modulate morphogenesis of tumors exhibiting epithelial nature. Additionally, activated Akt may have a function in metastasis. Overall, these results suggest that inhibitors of mTOR cassette may be useful as novel components of combined chemotherapy for a defined subset of bone and soft tissue sarcomas.