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To date, few clinical observations have been reported for changes of alternative angiogenic factors (other than vascular endothelial growth factor [VEGF]) in human gliomas under bevacizumab (Bev). The elucidation of the mechanism of action and resistance is imperative to overcome resistance to Bev and develop a more effective therapy. This study aims to investigate the status and change of alternative angiogenic factors regarding Bev usage.The present study used 56 glioblastoma tissues obtained at 3 different settings: tumors of initial resection (naive Bev group), tumors resected following Bev therapy (effective Bev group), and recurrent tumors after Bev therapy (refractory/autopsied Bev group). The expression of alternative angiogenic factors including basic fibroblast growth factor (bFGF), placental growth factor (PlGF), Angiopoietin1 (Ang-1), Angiopoietin2(Ang-2) and EphrinA2 were investigated via immunohistochemistry. In large tumor vasculature (diameter >15μm), the expression of PlGF was significantly higher in the effective Bev group than naive Bev group. The expression of Ang-2 was persistently suppressed in the refractory/autopsied Bev group. In microtumor vasculature (diameter

Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of β1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo. These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics. Significance: This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing β1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance.

Background Preoperative neoadjuvant bevacizumab (neoBev) reduces enhancement and perifocal edema by inhibition of angiogenesis and vascular permeability for glioblastoma (GBM). The aim of this study was to investigate safety and efficacy of neoBev for newly diagnosed GBM through an exploratory prospective multi-center study. METHODS & PATIENTS 15 patients with newly diagnosed GBM were enrolled in the present study. Eligibility was a patient with a brain tumor representing with ring-shaped enhancement and perifocal edema on magnetic resonance imaging (MRI). Based on neuroimage of typical GBM, neoBev and temozolomide (TMZ) were administered prior to craniotomy. Two weeks after neoBev, the tumor volume on T1-weighted gadolinium enhancement (T1Gd) and fluid attenuated inversion recovery (FLAIR) were assessed. Three to four weeks after neoBev and TMZ administration, patients underwent surgical resection. The primary endpoint was feasibility and safety, and the secondary endpoint was efficacy. Adverse events including systemic toxicity and wound healing delay during radiation (RT), TMZ, and Bev combined therapy were carefully monitored throughout clinical course including extent of resection. RESULTS Average of tumor regression rate two weeks after neoBev on T1Gd and FLAIR were 37.0% and 54.0%, respectively. Cerebral blood flow and cerebral blood volume were reduced after neoBev. Patients underwent surgical resection safely without excess blood loss due to less vascular and less degree of brain swelling. Two patients underwent awake surgery without any complications. Postoperative MRI showed that the all tumors were totally removed except one with multiple invasive tumors. Histological diagnosis of all patients was GBM, IDH-1 wild-type. Postoperative course was uneventful without neurological deficit and adverse effects except one postoperative hematoma in the resection cavity and one wound infection due to wound dehiscence. CONCLUSION Preoperative neoBev for newly diagnosed GBM might contribute to safe surgery. Clinical outcome of this therapeutics is now currently under investigation.

Objective PET scans are useful for the diagnosis and treatment planning of glioma. Introduced in 2013, bevacizumab (Bev) treatment efficacy can be difficult to determine using MRI imaging. We instead performed PET scans to determine the efficacy of Bev-based glioblastoma treatment. Methods Eighty glioblastoma patients treated with Bev from July 2013 to December 2021 were included. The patients were divided into the following three groups: the first group received Bev added to the Stupp regimen after biopsy or subtotal resection (first-dose group), the second group received Bev added to the Stupp regimen after biopsy followed by tumor resection (neoadjuvant group), and the third group received the Stupp regimen after tumor resection followed by TMZ + Bev at recurrence (recurrent group). PET scans (FDG, MET, FLT, and FMISO) were performed before and 4 weeks after the start of the Bev treatment. FDG, MET, and FLT measured tumor-to-normal ratio (TNR), and FMISO measured tumor-to-blood ratio (TBR), as well as the percent change in SUVmax, TNR, TBR, and MTV before and after Bev treatment. Using the Cox proportional hazards model, we examined which of these rates of change were the prognostic factors. Results The median PFS (months) and median OS from Bev treatment were 8.87, 16.53, and 4.0 and 12.53, 22.13, and 8.0, respectively. Significant differences in the percent change in MTV for MET, FLT, and FMISO in the first-line group, the percent change in MTV for FDG and MET, the percent change in FDG TNR and FMISO TBR in the recurrent group, and the percent change in MTV for MET and FLT in the neoadjuvant group were shown in the Cox proportional hazards model. Conclusion Neoadjuvant treatment with Bev prolonged PFS and OS. The decrease in MET MTV change rate and FMISO TBR.

Background Phase II clinical trial funded by Ministry of Health, Labour and Welfare from 1995 to 2003 evaluated efficacy of pathology-based three-group treatment stratification for CNS germ cell tumors (GCTs). We here present long-term follow-up results. Methods Total 228 cases were registered. Germinoma was treated with carboplatin+etoposide (CARE) and extended-local irradiation, local irradiation was added for intermediate-prognosis-group, and poor-prognosis-group was treated with ifosfamide+cisplatin+etoposide (ICE) and whole-brain or craniospinal irradiation. Results Mean/median ages at diagnosis were 16.8/16 years and female-to-male ratio was 40-188. Registry included 123 germinomas, 76 intermediate-prognosis-group cases (including 38 germinoma with STGC), 28 poor-prognosis-group cases and 1 mature teratoma. Median 222-months follow-up was conducted, and 56 recurrences and 39 deaths were recorded. 10 and 20-year recurrence-free survival (RFS) for germinoma, intermediate and poor-prognosis-groups were 84/79%, 83/76% and 59/59%, respectively, and overall survival (OS) for each were 97/91%, 92/85% and 57/53%, respectively. Prognosis for germinoma with or without STGC was the same. Basal ganglia germinoma showed significantly shorter RFS but OS was not different from other locations. Median age at death was 24 years, and ages were significantly different depending on causes, such as disease-related (14 years on average) and complications (29 years). OS after recurrence at 5/10/20 years were 64/62/48%.Hormonal supplementation was seen in 82% for neurohypophyseal cases and antidiuretic hormone supplementation was most frequent (82%). Among available cases, 20-out-of-155 cases showed neoplastic/vascular complications, among which cavernous malformation was the most (n=9). Median period until complication presentation was 235 months, and the rate at 20 years was 11%. Conclusions Germinoma and intermediate-prognosis-group cases showed long-term survival for approximately 90%, while more intensive treatment would be necessitated for poor-prognosis-group. Long-term survivors often required hormonal supplementation, and increasing frequency of treatment-related complications was observed. There is no end of outpatient follow-up for CNS GCT patients.

Background Gd-enhanced MRI and T2WI/FLAIR images are used to determine the efficacy of bevacizumab(Bev) in glioblastoma(GBM). FMISO-PET reflects hypoxia in tumor tissue. In this study, we hypothesized that FMISO-PET could detect a series of changes in the hypoxic environment during the Bev effect and at the time of Bev recurrence, and compared images and pathological findings after initial Bev treatment and recurrence. Subjects and Methods Seven patients with first-episode GBM, IDH-wild type were included in the study. 3 patients underwent tumor resection after completion of Bev, radiation, and temozolomide treatment, and reoperation was performed at recurrence. FMISO-PET was performed at each time point. Four patients who underwent tumor resection after FMISO-PET in the absence of treatment were also included. The explanted specimens were analyzed for expression of hypoxia (CA9) and stem cell surface marker (FOXM1) by immunohistochemistry (IHC). Results All three patients treated with preoperative chemoradiotherapy showed reduced FMISO accumulation. Two of them showed recurrence and increased FMISO accumulation, and IHC showed increased CA9 and FOXM1 positive cells at the time of reoperation. There was a trend toward fewer CA9-positive cells in patients with low FMISO accumulation, including controls. Conclusion FMISO-PET was used to visualize the improvement of oxygenation in tumor tissues after preoperative chemoradiotherapy including Bev, and the increased FMISO accumulation at the time of Bev resistance suggested that FMISO-PET may be useful for monitoring the duration of Bev treatment effect by reflecting the oxygenation of tumor tissues. The results suggest that FMISO-PET may be useful for monitoring the duration of response to Bev therapy.

Background The present study tested the possible utility of fractal analysis from l-[methyl-11C]-methionine (MET) uptake in patients with newly diagnosed gliomas for differentiating glioma, especially in relation to isocitrate dehydrogenase 1 (IDH1) mutation status, and as compared with the conventional standardized uptake value (SUV) parameters. Methods Investigations of MET PET/CT were performed retrospectively in 47 patients with newly diagnosed glioma. Tumors were divided into three groups: lower grade glioma (IDH1-mutant diffuse astrocytoma and IDH1-mutant anaplastic astrocytoma), higher grade glioma (IDH1-wildtype diffuse astrocytoma and IDH1-wildtype anaplastic astrocytoma), and glioblastoma. The fractal dimension for tumor, maximum SUV (SUVmax) for tumor (T) and mean SUV for normal contralateral hemisphere (N) were calculated, and the tumor-to-normal (T/N) ratio was determined. Metabolic tumor volume (MTV) and total lesion MET uptake (TLMU) were also measured. Results There were significant differences in SUVmax (p = 0.006) and T/N ratio (p = 0.02) between lower grade glioma and glioblastoma. There were no significant differences among any of the three groups in MTV or TLMU. Significant differences were obtained in the fractal dimension between lower grade glioma and higher grade glioma (p = 0.006) and glioblastoma (p Conclusions The results of this preliminary study in a small patient population suggest that the fractal dimension using MET PET in patients with newly diagnosed gliomas is useful for differentiating glioma, especially in relation to IDH1 mutation status, which has not been possible with SUV parameters.

Abnormal function of human body enzymes and epigenetic alterations such as DNA methylation have been shown to lead to human carcinogenesis. Lysyl oxidase (LOX) enzyme has attracted attention due to its involvement in tumor progression in various cancers. The purpose of this study was to clarify the clinical importance of LOX expression and its epigenetic regulation in the pathogenesis of esophageal squamous cell carcinoma (ESCC). Using a database of 284 ESCCs, we examined LOX expression and its prognostic characteristics. The functional role of LOX was assessed by in vitro growth, migration, and invasion assays. The relationship between LOX expression, global DNA hypomethylation (ie, LINE‐1 methylation), and LOX promoter methylation was evaluated by using mRNA expression arrays and pyrosequencing technology. High LOX expression cases had a significantly shorter overall survival and cancer‐specific survival (log‐rank, P, This is the first study to provide the evidence for the relationship between lysyl oxidase (LOX) expression, poor prognosis, and aggressive phenotype in esophageal cancer. In addition, we revealed that LOX expression was regulated epigenetically by genome‐wide hypomethylation using in vitro assay and integrated gene analysis in tissues and cell lines.

Background Tumor hypoxia and glycolysis have been recognized as determinant factors characterizing tumor aggressiveness in malignant gliomas. To clarify in vivo hypoxia and glucose metabolism in relation to isocitrate dehydrogenase (IDH) mutation and chromosome 1p and 19q (1p/19q) codeletion status, we retrospectively analyzed hypoxia as assessed by positron emission tomography (PET) with [18F]-fluoromisonidazole (FMISO) and glucose metabolism as assessed by PET with [18F]-fluoro-2-deoxy-d-glucose (FDG) in newly diagnosed malignant gliomas. Methods In total, 87 patients with newly diagnosed supratentorial malignant (WHO grade III and IV) gliomas were enrolled in this study. They underwent PET studies with FMISO and FDG before surgery. The molecular features and histopathological diagnoses based on the 2016 WHO classification were determined using surgical specimens. Maximal tumor-to-normal ratio (TNR) was calculated for FDG PET, and maximal tumor-to-blood SUV ratio (TBR) was calculated for FMISO PET. The PET uptake values in relation to IDH mutation and 1p/19q codeletion status were statistically analyzed. Results In all tumors and malignant astrocytomas, the median FMISO TBR in IDH-wildtype tumors was significantly higher than that in IDH-mutant tumors (P P IDH-wildtype tumors tended to be higher than that in IDH-mutant tumors, but the difference was not statistically significant. In WHO grade III anaplastic astrocytomas, there were no significant differences in median FMISO TBR or FDG TNR between IDH-mutant and IDH-wildtype tumors. In IDH-mutant WHO grade III anaplastic gliomas, there were no significant differences in median FMISO TBR or FDG TNR between anaplastic astrocytomas and anaplastic oligodendrogliomas. Conclusions Tumor hypoxia as assessed by FMISO PET was informative for prediction of the IDH mutation status in newly diagnosed malignant gliomas. However, the accuracy of the discrimination was not satisfactory for clinical application. On the other hand, glucose metabolism as assessed by FDG PET could not differentiate the IDH-mutant status. Moreover, PET studies using FMISO and FDG could not predict IDH mutation and 1p/19q codeletion status in WHO grade III tumors.

Although vascular endothelial growth factor (VEGF) promotes the immunosuppressive microenvironment, the efficacy of bevacizumab (Bev) on tumor immunity has not been fully investigated. The present study used 47 glioblastoma tissues obtained at 3 different settings: tumors of initial resection (naïve Bev group), tumors resected following Bev therapy (effective Bev group), and recurrent tumors after Bev therapy (refractory Bev group). The paired samples of the initial and post‐Bev recurrent tumors from 9 patients were included. The expression of programmed cell death‐1 (PD‐1)/PD ligand‐1 (PD‐L1), CD3, CD8, Foxp3, and CD163 was analyzed by immunohistochemistry. The PD‐L1+ tumor cells significantly decreased in the effective or refractory Bev group compared with the naïve Bev group (P

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2. The enzyme 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) degrades prostaglandin and is frequently decreased in several types of cancer; however, the molecular mechanisms of 15‐PGDH suppression are unclear. The current study was carried out to elucidate the molecular mechanisms and clinical significance of 15‐PGDH suppression in PDAC. Here, we showed that interleukin‐1β (IL‐1β), a pro‐inflammatory cytokine, downregulates 15‐PGDH expression in PDAC cells, and that IL‐1β expression was inversely correlated with 15‐PGDH levels in frozen PDAC tissues. We also found that activated macrophages produced IL‐1β and reduced 15‐PGDH expression in PDAC cells. Furthermore, the number of CD163‐positive tumor‐associated macrophages was shown to be inversely correlated with 15‐PGDH levels in PDAC cells by immunohistochemical staining of 107 PDAC samples. Finally, we found that low 15‐PGDH expression was significantly associated with advanced tumors, presence of lymph node metastasis and nerve invasion, and poor prognosis in PDAC patients. Our results indicate that IL‐1β derived from TAMs suppresses 15‐PGDH expression in PDAC cells, resulting in poor prognosis of PDAC patients.

OBJECTIVE The maximum resection of Glioblastoma (GBM) is the standard therapy and is expected to improve prognosis. Image-guided surgery using a neuronavigation system is the standard technique for glioma. However, due to the brain shift during surgery, intraoperative technologies, such as 5-ALA fluorescence and intraoperative MRI (IoMRI), are employed. Radiotracers are used during positron emission tomography (PET) for metabolic imaging and assist the evaluation of glioma metabolism. We compared the effectiveness of these intraoperative technologies. METHODS Between January 2016 and May 2021, 52 patients with gliomas underwent IoMRI. 21 patients were selected for 5-ALA fluorescence-guided resection of GBM and underwent multiple PET studies (MET, FLT, and FMISO). We graded fluorescence level as strong, vague, or none. Following tumor resection, we identified the fluorescence level and evaluated the residual volume of gadolinium-enhanced T1WI (T1-Gd) on IoMRI and at each PET study. After calculating the extent of resection (EOR) for T1-Gd, we compared the residual volume on T1-Gd for IoMRI and each PET study, between EOR ≥ 93% and EOR < 93%. RESULTS We detected strong 5-ALA fluorescence during induction and before tumor resection in all 21 (100%) patients with a newly-diagnosed and histopathologically-confirmed GBM. Following tumor resection, we noted an EOR ≥ 93% for T1-Gd in 12 cases (vague, 4; none, 8) and an EOR < 93% for T1-Gd in 9 cases (vague, 5; none, 4). The compared median residual volume (mL) with no fluorescence between EOR ≥ 93% and EOR < 93% for T1-Gd were T1-Gd (0.22, 0.74), MET (0.29, 3.31), FLT (0.24, 1.77), and FMISO (0.22, 1.02). CONCLUSIONS GBM cells are difficult to distinguish in cases without 5-ALA fluorescence. For cases without 5-ALA fluorescence, we were able to maximize the resection of GBM by extracting the area of MET accumulation.

Although bevacizumab was shown to improve progression-free survival and performance status of the patients with high-grade gliomas, clinical trials consistently showed lack of benefit in terms of patients’ overall survival. The recurrent tumors are inevitably more aggressive and invasive as compared with the original tumors, and the in-situ observation in actual human specimens is essential to elucidate the mechanism of resistance. In Japan, bevacizumab was approved not only for recurrent but also for newly diagnosed cases. The safety as well as efficacy of resection following neoadjuvant bevacizumab has been reported, and a phase II study is currently ongoing (UMIN-000025579). In the present study, the expression of angiogenic factors other than VEGF (basic fibroblast growth factor (bFGF), placenral growth factor (PlGF), angiopoietin1/2 and ephrinA2) was investigated by immunohistochemistry to be compared among tumors with no previous bevacizumab treatment, those resected following bevacizumab, and those refractory to bevacizumab. Fifty-nine samples from 42 patients were included; 24 of newly diagnosed glioblastomas with no previous bevacizumab (naïve group), 16 resected following neoadjuvant bevacizumab (effective group), and 6 resected after recurrence or autopsied (refractory/autopsied group). 12 were paired samples (8 naïve and refractory, 4 effective and refractory). The expression of PlGF significantly increased in the effective group as compared with the naïve group (p=0.003). In the paired specimens, there was a trend towards increased expression of PlGF in the second specimens (refractory/autopsied group) as compared with the specimens of initial surgery (p=0.083). Angiopoietin1, angiopoietin2 and ephrinA2 were characteristically expressed in the microvessels less than 15μm. The increased expression of PlGF might be associated with the recurrence after bevacizumab.

Anti-angiogenic therapy induces the apparent normalization of vascular structure, decreases microvessel density (MVD), and improves tumor oxygenation in glioblastomas (GBMs). Six initial and recurrent tumor pairs after bevacizumab (Bev) treatment were compared with GBMs from nine patients resected under neoadjuvant Bev treatment with regard to histological characteristics; MVD; MIB-1 index; and expression of vascular endothelial growth factor (VEGF) and its receptors, hypoxia markers (hypoxia-inducible factor 1 alpha, carbonic anhydrase 9), and nestin as a marker of glioma stem-like cells. In recurrent tumors post-Bev treatment, while the MVD remained low compared with the paired initial tumors (pre-Bev tumors), the expression of hypoxic markers were increased and were even higher in expression compared with the paired pre-Bev tumors in three of the six cases. MIB-1 indices were similar among the initial GBMs, neoadjuvant group, and recurrent tumors post-Bev treatment. The nestin-positive cell ratio of the post-Bev recurrent tumors was as high as that of the pre-Bev tumors. The expression of VEGF and VEGFR1 was increased in the post-Bev recurrent tumors in three and four cases, respectively, compared with the paired pre-Bev tumors. In the majority of Bev-refractory GBMs, tumor hypoxia was present with a paradoxical decrease in MVD. These findings suggest that re-activation of tumor angiogenesis is not initially involved in the acquisition of resistance to Bev.

The human microbiome Fusobacterium nucleatum, which primarily inhabits the oral cavity, causes periodontal disease and has also been implicated in the development of colorectal cancer. However, whether F. nucleatum is present in other gastroenterological cancer tissues remains to be elucidated. The present study evaluated whether quantitative polymerase chain reaction (qPCR) assays were able to detect F. nucleatum DNA and measure the quantity of F. nucleatum DNA in esophageal, gastric, pancreatic and liver cancer tissues. The accuracy of the qPCR assay was determined from a calibration curve using DNA extracted from cells from the oral cavity. Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 20 patients with gastroenterological [esophageal (squamous cell carcinoma), gastric, colorectal, pancreatic and liver] cancer and 20 matched normal tissues were evaluated for F. nucleatum DNA content. The cycle threshold values in the qPCR assay for F. nucleatum and solute carrier organic anion transporter family member 2A1 (reference sample) decreased linearly with the quantity of input DNA (r2>0.99). The F. nucleatum detection rate in esophageal, gastric and colorectal cancer tissues were 20% (4/20), 10% (2/20) and 45% (9/20), respectively. F. nucleatum was not detected in liver and pancreatic cancer tissues. The qPCR results from the frozen and FFPE tissues were consistent. Notably, F. nucleatum was detected at a higher level in superficial areas compared with the invasive areas. F. nucleatum in esophageal, gastric and colorectal cancer tissues was evaluated by qPCR using FFPE tissues. F. nucleatum may be involved in the development of esophageal, gastric and colorectal cancer.

OBJECTIVE Pediatric brain tumors are primarily diagnosed using MRI or CT examination; however, determining the correct diagnosis using only morphological MRI can sometimes be challenging. Positron emission tomography (PET) uses radiotracers for metabolic and molecular imaging. We examined the accumulation of multiple PET (FDG, MET, FLT, and FMISO) studies for diagnosing pediatric cystic brain tumors. METHODS We performed PET scans for eight pediatric patients (five pilocytic astrocytoma, one pleomorphic xanthoastrocytoma, one diffuse astrocytoma with IDH1 mutation, one ganglioglioma) from April 2010 to December 2019. The resulting studies were compared by measuring the tumor-to-normal lesion (T/N) ratio of FDG, MET, and FLT and the tumor-to-blood value (T/B) ratio of FMISO between each pediatric cystic brain tumor. RESULTS All pediatric brain tumors showed tumor uptake of FDG, MET, and FLT. We could not examine FMISO PET for one diffuse astrocytoma with IDH1 mutation. The T/N ratios of FDG, MET, and FLT and the T/B ratio of FMISO were 1.07, 2.76, 4.6, and 1.12 for pilocytic astrocytoma; 0.65, 4.6, 7.67, and 1.38 for pleomorphic xanthoastrocytoma; 0.61, 2.14, and 3.82 for diffuse astrocytoma with IDH1 mutation; and 0.79, 1.78, 5, and 1.49 for ganglioglioma, respectively. The T/N ratios of MET and FLT for pleomorphic xanthoastrocytoma were high, but the Ki-67 labeling index was 1%. In the ganglioglioma, the T/N ratio of FLT was high, but the T/N ratio of MET was low. CONCLUSION Specialized multiple PET accumulation patterns for tumors are useful for discriminating each tumor.

OBJECTIVE Neuronavigation systems with magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging (methionine [MET], fluorothymidine [FLT], and fluoromisonidazole [FMISO]) are routinely used in glioblastoma surgery. Residual tumor identified using intraoperative MRI (IoMRI) or 5-aminolevulinic acid (5-ALA) fluorescence is removed. Neoadjuvant bevacizumab therapy is offered to patients with low Karnovsky performance status (KPS) or with tumors in eloquent regions. We evaluated the usefulness of neoadjuvant bevacizumab therapy. METHODS Twelve patients with glioblastoma with low KPS or tumors in eloquent regions on multiple PET and IoMRI evaluations were enrolled between January 2016 and April 2019. Six had received neoadjuvant bevacizumab before surgery; six had not. Postsurgical 5-ALA fluorescence (strong, vague, and none) tumor extraction rate, residual volume on MRI and PET imaging, and prognosis in the patients with and without bevacizumab were compared. RESULTS In patients with bevacizumab adjuvant therapy, the KPS scores immediately prior to surgery were 90 in 3 cases, 80 in 2, and 70 in 1. The scores in patients without bevacizumab were 50 in 2 and 40 in 4. The 5-ALA fluorescence in patients with bevacizumab was vague in one and none in five. Vague fluorescence was noted in all six patients without bevacizumab. Tumor extraction rates in patients with vs. those without bevacizumab were 97.6% vs. 91.5% by T1-Gd, 95.4% vs. 99.9% by MET, 96.2% vs. 90.2% by FLT, and 97% vs. 92% by FMISO. Corresponding residual volumes (ml) were (0.6 vs. 1.7) for T1-Gd, 1.2 vs. 2.9 for MET, 1.0 vs. 2.1 for FLT, and 0.5 vs. 1.1 for. FLT. Median progression free survival (PFS) was 10.1 vs. 4.9 months; median overall survival (OS) was 15.7 vs. 13.3 months. CONCLUSIONS Neoadjuvant bevacizumab therapy improved KPS at the time of surgery, increased extraction rate, reduced residual tumor volume, and improved PFS and OS prognosis.

OBJECTIVE Use of the positron emission tomography (PET), such as 18F-fluorodeoxyglucose (FDG), 11C-Methionine (MET), 18F- Fluorothymidine (FLT), and 18F-Fluoromisonidazole (FMISO), is expected to lead the way for novel applications aimed at achieving efficient malignancy grading and treatment of gliomas. The aim of this study was to assess FDG, MET, FLT, and FMISO PET studies to evaluate the biological effects induced by bevacizumab (BEV) therapy in glioblastoma patients. METHODS Seventy-one patients with glioblastoma were treated biweekly with BEV from July 2013 to November 2020. FDG, MET, FLT, and FMISO PET scans were obtained at baseline and at follow-up (4 weeks after treatment onset). Measures of FDG, MET, FLT, and FMISO avidity were recorded; the measures were SUVmax, metabolic tumor volume (MTV; volume of tumor with SUV>42% of SUVmax), SUVmean (within the MTV), tumor-to-normal ratio (TNR), tumor-to blood ratio (TBR), and total lesion avidity (TLA; calculated as MTV x SUVmean). The prognostic analysis was performed in relation to the response assessment by multiple PET tracers using progression-free survival (PFS) and overall survival (OS). RESULTS Under the assessment of the Cox proportional hazard model, increased changes of FDG SUVmax, MET TLA at follow-up, FLT TLA at follow up, increased changes of FLT TLA, increased changes of FMISO TBR and FMISO MTV were significant prognostic factor of PFS. Increase changes of FDG TLA and FLT TLA and increased changes of FMISO TBR were significant prognostic factor of OS. CONCLUSION Increased changes in FLT TLA and FMISO-PET after BEV therapy may be a useful biomarker for predicting PFS and OS in glioblastoma.

The nucleoside analog 3′-deoxy-3′-18F-fluorothymidine (FLT) has been investigated for evaluating tumor proliferating activity in brain tumors. We evaluated FLT uptake heterogeneity using textural features from the histogram analysis in patients with newly diagnosed gliomas and examined correlation of the results with proliferative activity and patient prognosis, in comparison with the conventional PET parameters. FLT PET was investigated in 37 patients with newly diagnosed gliomas. The conventional parameters [tumor-to-contralateral normal brain tissue (T/N) ratio and metabolic tumor volume (MTV)] and textural parameters (standard deviation, skewness, kurtosis, entropy, and uniformity) were derived from FLT PET images. Linear regression analysis was used to compare PET parameters and the proliferative activity as indicated by the Ki-67 index. The associations between parameters and overall survival (OS) were tested by Cox regression analysis. Median OS was 662 days. For the conventional parameters, linear regression analysis indicated a significant correlation between T/N ratio and Ki-67 index (p = 0.02) and MTV and Ki-67 index (p = 0.02). Among textural parameters, linear regression analysis indicated a significant correlation for kurtosis (p = 0.003), entropy (p

// Ryota Tamura 1 , Toshihide Tanaka 2 , Keisuke Miyake 3 , Yusuke Tabei 4 , Kentaro Ohara 5 , Oltea Sampetrean 6 , Maya Kono 1 , Katsuhiro Mizutani 1 , Yohei Yamamoto 2 , Yuichi Murayama 7 , Takashi Tamiya 3 , Kazunari Yoshida 1 and Hikaru Sasaki 1 1 Department of Neurosurgery, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan 2 Department of Neurosurgery, Jikei University Kashiwa Hospital, Kashiwashita, Kashiwa-shi, Chiba, Japan 3 Department of Neurosurgery, Kagawa University Hospital, Ikedo, Mituki-cho, Kita-gun, Kagawa, Japan 4 Department of Neurosurgery, Japan Red Cross Medical Center, Hiroo, Shibuya-ku, Tokyo, Japan 5 Division of Diagnostic Pathology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan 6 Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan 7 Department of Neurosurgery, Jikei University Hospital, Nishishinbashi, Minato-ku, Tokyo, Japan Correspondence to: Hikaru Sasaki, email: // Keywords : bevacizumab; neoadjuvant; vascular normalization; hypoxia; glioma stem cell Received : February 08, 2016 Accepted : May 09, 2016 Published : May 17, 2016 Abstract To date, no clinical observations have been reported for histopathological changes in human gliomas under antiangiogenic treatment. We collected six glioblastomas resected under bevacizumab treatment. Histopathological investigation was performed by hematoxilyn-eosin staining and immunohistochemistry for CD34, VEGF, VEGFR1/2, HIF-1α, CA9, and nestin as compared to eleven control glioblastomas to assess the differences in histological features, microvessel density, expression of VEGF and its receptors, tumor oxygenation, and status of glioma stem-like cells. In the six tumors resected under bevacizumab, microvascular proliferation was absent, and microvessel density had significantly decreased compared with that of the controls. The expressions of VEGF and its receptors were downregulated in two cases of partial response. HIF-1α or CA9 expression was decreased in five of the six tumors, whereas the decreased expression of these markers was noted in only one of the 11 control glioblastomas. The expression of nestin significantly decreased in the six tumors compared with that of the controls, with the remaining nestin-positive cells being relatively concentrated around vessels. We provide the first clinicopathological evidence that antiangiogenic therapy induces the apparent normalization of vascular structure, decrease of microvessel density, and improvement of tumor oxygenation in glioblastomas. These in situ observations will help to optimize therapy.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to α-ketoglutarate. Somatic point mutations in IDH1/2 confer a gain-of-function in cancer cells, resulting in overproduction of an oncometabolite, 2-hydroxyglutarate (2HG). 2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. Given that IDH1 and IDH2 are attracting attention as promising therapeutic targets, better evaluation of the incidence of IDH1 and IDH2 mutations and 2HG level in human cancers is clinically important. This is the first study to assess their incidence in esophageal squamous cell carcinomas (ESCCs). First, we established pyrosequencing assays for IDH1 and IDH2 mutations and revealed that these mutations were absent in 10 ESCC cell lines and 96 ESCC tissues. Second, utilizing IDH1 and IDH2 overexpression vectors, we demonstrated that LC-MS/MS assays can accurately evaluate 2HG level and found that some ESCC cases presented a high level of 2HG. In conclusion, IDH1 or IDH2 mutations play a limited role in the development of ESCC. 2HG is potentially synthesized to high levels in the absence of IDH1 and IDH2 mutations, and this may correlate with progression of ESCCs.

OBJECTIVE: Image-guided surgery, such as neuronavigation systems with magnetic resonance imaging (MRI) have become standard techniques for glioma surgeries. However, MRI is limited by correct metabolism of glioma cells. Positron-emission tomography (PET) uses radiotracers to achieve metabolic and molecular imaging, but can only be evaluated preoperatively. We compared the utility of intraoperative real-time MRI (IoMRI) and multiple PET studies (methionine [MET], fluorothymidine [FLT], and fluoromisonidazole [FMISO]) for malignant glioma surgery. METHODS: Between January 2016 and May 2018, 39 patients with gliomas underwent IoMRI, whereas 19 (excluding biopsy cases) underwent multiple PET studies for tumor removal. Of these 19 patients, 12 had recurrence and 7 did not. Excision rate and residual region by IoMRI and PET studies were compared. RESULTS: The respective excision rates in the recurrent and nonrecurrent groups were 91.9% (80.499) and 97.7% (92.7100) with IoMRI, 92.3% (79.297.1) and 99.4% (98.3100) with MET, 94.5% (84.597.5) and 99.2% (97.5100) with FLT, and 95.9% (90.298.3) and 98.7% (93.5100) with FMISO. In the recurrent group, the residual volume of each PET tracer was significantly larger than that of IoMRI. All recurrences wherein accumulation areas of each PET tracer remained in the IoMRI excision areas were detected. Recurrence was observed from these sites, particularly the residual region overlapping residual accumulations of MET and FLT. CONCLUSIONS: Fusion of IoMRI images with preoperative multiple PET images during navigation remains challenging. When accumulation areas of more than two PET tracers, particularly MET and FLT, overlap with the residual IoMRI region, considering tumor removal becomes important.

// Ryuma Tokunaga 1, * , Yu Imamura 1, 2, * , Kenichi Nakamura 1 , Takatsugu Ishimoto 1 , Shigeki Nakagawa 1 , Keisuke Miyake 1 , Yu Nakaji 2, 3 , Yasuo Tsuda 2 , Masaaki Iwatsuki 1 , Yoshifumi Baba 1 , Yasuo Sakamoto 1 , Yuji Miyamoto 1 , Hiroshi Saeki 2 , Naoya Yoshida 1 , Eiji Oki 2 , Masayuki Watanabe 1, 2 , Yoshinao Oda 3 , Adam J. Bass 4, 5, 6 , Yoshihiko Maehara 2 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 2 Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 3 Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 4 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA 5 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 6 Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA * These authors contributed equally to this work Correspondence to: Hideo Baba, e-mail: hdobaba@kumamoto-u.ac.jp Keywords: esophagogastric junction adenocarcinoma, esophageal adenocarcinoma, FGFR2, targeting therapy, amplification Received: November 02, 2015 Accepted: January 31, 2016 Published: February 27, 2016 ABSTRACT Background: Fibroblast growth factor receptor 2 (FGFR2) genetic alterations lead to tumor cell proliferation in various types of cancer. We hypothesized that FGFR2 amplification is associated with FGFR2 expression, resulting in tumor growth and poorer outcome in esophagogastric junction (EGJ) adenocarcinoma. Patients and Methods: A total of 176 consecutive chemo-naive patients with EGJ adenocarcinoma were enrolled from two academic institutions. FGFR2 amplification was examined by real-time PCR ( N = 140) and FGFR2 expression with immunohistochemical staining ( N = 176), and compared against clinicopathological factors and patient outcomes. The effects of FGFR2 inhibition or overexpression on cell proliferation, cell cycle, and apoptosis assays were investigated in EGJ adenocarcinoma cell lines. Downstream FGFR2, AKT and ERK were also examined. Results: Based on the correlation between FGFR2 levels and FGFR2 overexpression in vitro , FGFR2 amplification was defined as copy number > 3.0. In clinical samples, FGFR2 amplification and FGFR2 IHC expression were 15% and 61%, respectively. Although these two statuses were significantly correlated ( P < 0.05), only FGFR2 IHC expression was significantly associated with tumor depth (multivariate P < 0.001) and overall survival of patients (univariate P = 0.007). Supporting these findings, FGFR2 overexpression was associated with tumor cell proliferation, cell cycle progression, and anti-apoptosis. Selective inhibition of FGFR2 sufficiently suppressed tumor cell proliferation through de-phosphorylation of AKT and ERK. Conclusions: FGFR2 amplification was significantly associated with FGFR2 expression. FGFR2 expression (but not FGFR2 amplification) was associated with tumor growth and patient outcomes. Our findings support FGFR2 as a novel therapeutic target for EGJ adenocarcinoma.

Jumonji-domain containing 3 (JMJD3) affects transcriptional regulation by demethylating lysine 27 residue of histone 3. We investigated its function and prognostic significance in colorectal cancer (CRC). The influence of JMJD3 on cell proliferation was assessed using quantitative RT-PCR and western blot on the downstream target gene of JMJD3, in knock-down (KD) experiments and clinical samples from 151 CRC patients. Cells with KD JMJD3 significantly increased proliferation through cell cycle progression and apoptosis suppression. Expression of P15INK4B was remarkably decreased in KD JMJD3 cells; and JMJD3 expression strongly correlated with p15INK4B expression in clinical CRC samples (P

// Asuka Murata 1, * , Yoshifumi Baba 1, * , Takatsugu Ishimoto 1 , Keisuke Miyake 1 , Keisuke Kosumi 1 , Kazuto Harada 1 , Junji Kurashige 1 , Shiro Iwagami 1 , Yasuo Sakamoto 1 , Yuji Miyamoto 1 , Naoya Yoshida 1 , Manabu Yamamoto 2 , Shinya Oda 3 , Masayuki Watanabe 4 , Mitsuyoshi Nakao 5 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Japan 2 Department of Surgery, National Hospital Organization Kyushu Cancer Center, Japan 3 Department of Cancer Biology, National Kyushu Cancer Center Clinical Research Institute, Japan 4 Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Japan 5 Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan * These authors have contributed equally to this work Correspondence to: Yoshifumi Baba, e-mail: y-baba@kumamoto-u.ac.jp Keywords: epigenetics, markers, demethylation, TET Received: April 17, 2015 Accepted: May 26, 2015 Published: June 08, 2015 ABSTRACT Mammalian DNA is epigenetically marked by 5′-cytosine methylation (5-methylcytosine [5-mC]). The Ten-eleven translocation (TET) enzymes (TET1, TET2, and TET3) are implicated in DNA demethylation, through dioxygenase activity that converts 5-mC to 5-hydroxymethylcytosine (5-hmC). Although decreased TET is reportedly associated with decreased 5-hmC levels in various cancers, functions of 5-hmC and TET expression in esophageal squamous cell carcinoma (ESCC) are unclear. We used ELISA and immunohistochemistry tests to analyze 5-hmC status in ESCC tissues, RT-qPCR to analyze TET family mRNA expression in normal and tumor tissues, and pyrosequencing to quantify LINE-1 (i.e., global DNA methylation) levels. ELISA and immunohistochemical testing showed 5-hmC levels were significantly lower in ESCC than in paired normal tissues ( P < 0.0001). TET2 expression was significantly lower in ESCCs than paired normal tissues ( P < 0.0001), and significantly associated with 5-hmC levels in ESCCs ( P = 0.003, r = 0.33). 5-hmC levels were also significantly associated with LINE-1 methylation level ( P = 0.0002, r = 0.39). Patients with low 5-hmC levels had shorter overall survival than those with higher levels, although not significantly so ( P = 0.084). In conclusion, 5-hmC expression was decreased in ESCC tissues, and was associated with TET2 expression level. TET2 reduction and subsequent 5-hmC loss might affect ESCC development.

Although current treatment advances prolong patient survival, treatment for glioblastoma (GBM) in the elderly has become an emerging issue. The definition of "elderly" differs across articles; GBM predominantly occurs at an age ≥65 years, and the prognosis worsens with increasing age. Regarding molecular markers, isocitrate dehydrogenase (IDH) mutations are less common in the elderly with GBM. Meanwhile, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation has been identified in approximately half of patients with GBM. Surgery should be considered as the first-line treatment even for elderly patients, and maximum safe resection is recommended if feasible. Concurrently, radiotherapy is the standard adjuvant therapy. Hypofractionated radiotherapy (e.g., 40 Gy/15 Fr) is suitable for elderly patients. Studies also supported the concurrent use of temozolomide (TMZ) with radiotherapy. In cases wherein elderly patients cannot tolerate chemoradiation, TMZ monotherapy is an effective option when MGMT promoter methylation is verified. Conversely, tumors with MGMT unmethylated promoter may be treated with radiotherapy alone to reduce the possible toxicity of TMZ. Meanwhile, the efficacy of bevacizumab (BEV) in elderly patients remains unclear. Similarly, further studies on the efficacy of carmustine wafers are needed. Based on current knowledge, we propose a treatment diagram for GBM in the elderly.

Introduction: Diagnostic imaging such as magnetic resonance imaging (MRI) is the most commonly used morphological method for determination of size and location of brain tumors. However, it is difficult to evaluate the effect of bevacizumab for malignant gliomas by using only MRI. In contrast, positron emission tomography (PET) uses radiotracers to achieve metabolic and molecular imaging, and this approach can improve diagnoses. Notably, 11C-Methionine (MET) and 18F-Fluoromisonidazole (FMISO) are expected to lead ways for novel applications aimed at achieving efficient malignancy grading and treatment of gliomas. Because of difficulty evaluating the effect of bevacizumab, aim of this study was to assess MET and FMISO PET studies to evaluate pathological examinations induced by bevacizumab in malignant glioma patients. METHODS: Thirty-three patients with malignant gliomas were treated biweekly with bevacizumab at a dose of 10 mg/kg from July 2013 to July 2016. MRI, as well as MET and FMISO PET scans were obtained. Five glioblastoma patients out of thirty-three malignant glioma patients had been treated by resection after bevacizumab therapy. Based on MET and FMISO accumulation, intraoperative biopsies were selected by surgeon and investigated for histological characteristics, Ki-67 proliferative indices, and expression levels of VEGF and HIF-1 alpha. We determined that MET accumulation with a tumor to normal (T/N) ratio cutoff of > 3 and FMISO accumulation with a tumor to blood (T/B) ratio cutoff of > 1.6 were related to metabolically active tumor cells. Sensitivities, specificities, positive predictive values (PPVs) and negative predictive values (NPVs) for evaluation between presence of tumor and accumulation of PET radiotracers were calculated. Results: MET accumulation correlated with presence of tumor for sensitivity of 88.5%, specificity of 39.5%, PPV of 50% and NPV of 83.3% (p-value 0.022). FMISO accumulation correlated with presence of tumor for sensitivity of 91.9%, specificity of 55.6%, PPV of 73.9% and NPV of 83.3% (p-value 3) and FMISO (T/B > 1.6), we could detect high microvessel density and high expression levels of VEGF and HIF-1 alpha. Ki-67 labeling index was more than 50%. In the region with a little high MET T/N ratio and low FMISO T/B ratio, we detected the metabolically non-active tumor and necrosis areas. Conclusions: We suspect that high accumulation areas of MET and FMISO are related to the resistance of bevacizumab therapy, and MET and FMISO may be useful biomarkers for the evaluation of pathological examination of malignant gliomas during bevacizumab therapy.

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2 (PGE2). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) participates in the degradation of PGE2 and has attracted attention in recent years. Although inhibition of 15-PGDH is reported to lead to PGE2 accumulation and expand tissue stem cell fraction, the role for pancreatic tumor progression is still unknown. The aim of this study is to elucidate the regulatory mechanism and functional role of 15-PGDH for cancer stem-like cell expansion during tumor progression in pancreas. We found that 15-PGDH expression is frequently down-regulated in PDAC cells and significantly correlated with the number of infiltrating macrophages in human PDAC tissues. Moreover, direct co-culture assay revealed that macrophage derived interleukin-1 beta down-regulates 15-PGDH expression in PDAC cells. Furthermore, pharmacological blockade of 15-PGDH led to PGE2 accumulation, and promoted growth and sphere formation through the expansion of ALDH1-positive cells. We also elucidated the molecular mechanism that PGE2 accumulation by 15-PGDH inhibition increases CYP26A1 expression and subsequently depletes all-trans retinoic acid, and results in ALDH1 up-regulation. Finally, genetic ablation of 15-Pgdh promoted tumorigenesis in KrasLSL-G12D; Ptf1aCre/+ mice through the expansion of ALDH1-positive cells. Our findings highlight the role and significance of PGE2 degradation pathway for PDAC tumor progression. Note: This abstract was not presented at the meeting. Citation Format: Takatsugu Ishimoto, Kota Arima, Tomoyuki Uchihara, Keisuke Miyake, Atsuko Yonemura, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. Inhibition of 15-PGDH causes Kras-driven tumor expansion through prostaglandin E2-ALDH1 signaling in the pancreas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4677.

Background: Cancer-associated fibroblasts (CAFs) enhance tumor progression through secretion of soluble factors. Although we demonstrated that direct-interaction between CAFs and cancer cells confer on gastric cancer (GC) cells the ability to invade extracellular matrix (ECM), the molecular mechanism underlying the drug resistance by soluble factors from CAFs has not been determined. Methods: We have established primary fibroblasts from more than 100 GC patients and collected conditioned medium (CM) of CAFs. Human GC cell lines cultured in normal medium or CAF-CM were examined the drug resistance with or without extracellular matrix (ECM). We isolated extracellular vesicles (EVs) from CAF-CM by ultracentrifugal separation. We performed RNA sequencing of GC cell lines and mass analysis of GC-EVs and CAF-EVs. Results: GC cells treated with CAF-CM or CAF-EVs did not show morphological change on normal coated plates. Notably, we found that GC cells with CAF-CM or CAF-EVs on ECM-coated plates showed unique network formation and cisplatin resistance. Given that the morphological change appeared within 1-3 hours, we predicted that the stimulation by CAF-EV had an impact on the expression pattern of membrane proteins in GC cells. Mass analysis using isolated CAF-EVs was conducted and revealed that CAF-EVs contained high amount of annexin family protein members. Moreover, we showed that annexin in CAF-EVs is critical for network formation and drug resistance of GC cells. Finally, we identified integrinβ1/FAK signaling was activated in GC cells treated with CAF-EVs during network formation. Conclusions: These findings highlight the underlying mechanism whereby CAF-EVs in tumor-microenvironment confer GC cells on drug resistance. Citation Format: Tomoyuki Uchihara, Atsuko Yonemura, Keisuke Miyake, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. Extracellular vesicles from cancer associated fibroblasts induce drug resistance via integrinβ1/FAK signaling in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 107.

Background: LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs. Methods: Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined. Results: The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1–90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI. Conclusion: Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

To investigate the relationship between the long interspersed nucleotide element-1 (L1/LINE-1) methylation level and the disease-free survival and cancer-specific survival in patients with esophageal squamous cell carcinoma (ESCC).Cancer cells exhibit 2 types of deoxyribonucleic acid (DNA) methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the LINE-1 repetitive element is a good indicator of the global DNA methylation level. Although the LINE-1 methylation level is attracting interest as a useful marker for predicting cancer prognosis, the prognostic significance of LINE-1 hypomethylaiton in ESCC remains unclear.Using 217 curatively resected ESCC specimens, we quantified the LINE-1 methylation by utilizing the bisulfite pyrosequencing technology. Promoter methylation levels of MGMT and MLH1 were also evaluated by pyrosequencing.ESCC showed significantly lower LINE-1 methylation levels in comparison with matched normal esophageal mucosa (P0.0001; N = 50). LINE-1 hypomethylation was significantly associated with disease-free survival [log-rank P = 0.0008; univariate hazard ratio (HR): 2.32, 95% confidence interval (CI): 1.38-3.84, P = 0.0017; multivariate HR: 1.81, 95% CI: 1.06-3.05, P = 0.031] and cancer-specific survival (log-rank P = 0.0020; univariate HR: 2.21, 95% CI: 1.33-3.60, P = 0.0026; multivariate HR: 1.87, 95% CI: 1.12-3.08, P = 0.018]. MGMT and MLH1 hypermethylation were not associated with patient prognosis.LINE-1 hypomethylation in ESCC is associated with a shorter survival, thus suggesting that it has potential for use as a prognostic biomarker.

Vascular endothelial growth factor (VEGF) is an attractive target of antiangiogenic therapy in glioblastomas. Bevacizumab (Bev), a humanized anti-VEGF antibody, is associated with the improvement of progression-free survival and performance status in patients with glioblastoma. However, randomized trials uniformly suggest that these favorable clinical effects of Bev do not translate into an overall survival benefit. The mechanisms of action of Bev appear to include the inhibition of tumor angiogenesis, as well as indirect effects such as the depletion of niches for glioma stem cells and stimulation of antitumor immunity. Although several molecules/pathways have been reported to mediate adaptation and resistance to Bev, including the activation of alternative pro-angiogenic pathways, the resistance mechanisms have not been fully elucidated; for example, the mechanism that reinduces tumor hypoxia remains unclarified. The identification of imaging characteristics or biomarkers predicting the response to Bev, as well as the better understanding of the mechanisms of action and resistance, is crucial to improve the overall clinical outcome and optimize individual therapy. In this article, the authors review the results of important clinical trials/studies, the current understanding of the mechanisms of action and resistance, and the knowledge of imaging characteristics and biomarkers predicting the response to Bev.

The authors report on a case of concurrent herpes simplex encephalitis (HSE) and malignant glioma. The co-occurrence of HSE and malignant glioma is very rare, but it can occur during glioma treatment. Both radiotherapy and chemoradiation with temozolomide can induce viral reactivation, leading to HSE relapse. Careful observation for HSE is necessary when administering chemoradiation to patients with a history of HSE. Antiviral therapy for HSE must be initiated immediately, and the chemoradiation for glioma should be stopped; however, it is not clear what antitumor therapy is optimal when HSE co-occurs during the treatment of glioma.

Purpose: Fusobacterium nucleatum (F. nucleatum) is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. Although there are several reports of the relationship between F. nucleatum and the clinical outcome in human cancers, its prognostic significance in esophageal cancer remains unclear. Experimental Design: We quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. Significant pathways in F. nucleatum–positive esophageal cancer tissues were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using microarray data. Results: Esophageal cancer tissues contained significantly more F. nucleatum DNA than matched normal esophageal mucosa (P = 0.021; n = 60). F. nucleatum DNA was detected in 74 of 325 cases (23%). F. nucleatum DNA positivity was significantly associated with tumor stage, but not with sex, age, performance status, tobacco use, alcohol use, histology, tumor location, or preoperative treatment. F. nucleatum DNA positivity was also significantly associated with cancer-specific survival [log-rank P = 0.0039; univariate HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.031]. The top-ranked KEGG pathway in F. nucleatum–positive tissues was “cytokine–cytokine receptor interaction.” A significant relationship between F. nucleatum and the chemokine CCL20 was validated by IHC. Conclusions: F. nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. F. nucleatum might also contribute to aggressive tumor behavior through activation of chemokines, such as CCL20. Clin Cancer Res; 22(22); 5574–81. ©2016 AACR.

// Kenichi Nakamura 1 , Yoshifumi Baba 1 , Keisuke Kosumi 1 , Kazuto Harada 1 , Hironobu Shigaki 1 , Keisuke Miyake 1 , Yuki Kiyozumi 1 , Mayuko Ohuchi 1 , Junji Kurashige 1 , Takatsugu Ishimoto 1 , Masaaki Iwatsuki 1 , Yasuo Sakamoto 1 , Naoya Yoshida 1 , Masayuki Watanabe 2 , Mitsuyoshi Nakao 3 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan 2 Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan 3 Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Correspondence to: Hideo Baba, email: hdobaba@kumamoto-u.ac.jp Keywords: LINE-1, methylation, esophageal cancer, prognosis, UHRF1 Received: March 24, 2016 Accepted: July 19, 2016 Published: August 05, 2016 ABSTRACT Background: Global DNA hypomethylation contributes to oncogenesis through various mechanisms. The level of long interspersed nucleotide element-1 (LINE- 1) methylation is considered a surrogate marker of global DNA methylation, and is attracting interest as a good predictor of cancer prognosis. However, the mechanism how LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully elucidated. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays a crucial role in DNA methylation. UHRF1 is overexpressed in many cancers, and UHRF1 overexpression may be a mechanism underlying DNA hypomethylation in cancer cells. Nonetheless, the relationship between UHRF1, LINE-1 methylation level, and clinical outcome in esophageal squamous cell carcinoma (ESCC) remains unclear. Results: In ESCC cell lines, vector-mediated UHRF1 overexpression caused global DNA (LINE-1) hypomethylation and, conversely, UHRF1 knockdown using siRNA increased the global DNA methylation level. In ESCC tissues, UHRF1 expression was significantly associated with LINE-1 methylation levels. Furthermore, UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients. Materials and Methods: The relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) were investigated using ESCC tissues and cell lines. In addition, we examined the correlation between UHRF1 expression, LINE-1 methylation, and clinical outcome in patients with ESCC. Conclusions: Our results suggest that UHRF1 is a key epigenetic regulator of DNA methylation and might be a potential target for cancer treatment.

Background Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing. Methods The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay. Results The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs. Conclusions These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

Cystine/glutamic acid transporter (xCT) plays a role in tumor progression by regulating the redox status in several types of cancers. To demonstrate the importance of xCT expression for predicting the prognosis of hepatocellular carcinoma (HCC), we analyzed xCT gene expression in 130 paired HCC and non-cancerous tissues. xCT protein expression was confirmed using 7 HCC cell lines and samples from human subjects. xCT mRNA expression was detected in 34 (26%) tumor tissues. Expression of xCT was higher in HCC tissues compared to the corresponding normal tissues according to quantitative reverse transcriptase-polymerase chain reaction findings (P

Background Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, LINE-1 methylation level is attracting interest as a predictive marker for patient prognosis. However, the prognostic significance of LINE-1 hypomethylation in gastric cancer remains unclear. Methods Using 203 resected gastric cancer specimens, we quantified LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical and pathological variables. Results Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p 0.25). Conclusions LINE-1 hypomethylation in gastric cancer is associated with shorter survival, suggesting that it has potential for use as a prognostic biomarker.

Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Selected gene promoter hypermethylation in normal esophageal mucosae has attracted attention as a surrogate marker for the epigenetic field defect induced by smoking and/or drinking in esophageal squamous cell carcinoma (ESCC). However, the significance of global DNA hypomethylation for field cancerization remains unclear.By using histologically normal esophageal mucosa samples from 109 ESCC cases and 20 autopsy cases without ESCCs, we measured long interspersed nucleotide element 1 (LINE-1) methylation levels by pyrosequencing, which correlates with global DNA methylation level.LINE-1 methylation levels in normal esophageal mucosae of ESCC patients were significantly lower than those of autopsy individuals (P = 0.017). LINE-1 methylation of noncancerous mucosae ranged 68.3-93.0 on a 0-100 scale (mean 81.7, median 82.2, standard deviation 5.9). LINE-1 hypomethylation was significantly associated with smoking history (P = 0.014 for smoking duration; P = 0.0017 for number of cigarettes per day; P = 0.0002 for tobacco pack-year). LINE-1 methylation was not associated with alcohol drinking or age at diagnosis.A history of tobacco use was significantly associated with LINE-1 hypomethylation in noncancerous esophageal mucosae of ESCC patients. These results support the potential of LINE-1 methylation level as an indicator of epigenetic field for ESCC cancerization, particularly when caused by tobacco smoking.

Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element 1 L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Nonetheless, no study has examined LINE-1 methylation in esophageal squamous cell carcinoma (ESCC). The aim of this study is to assess the precision of sodium bisulfite conversion and polymerase chain reaction (PCR) pyrosequencing assay for evaluating LINE-1 methylation in ESCC. To measure assay precision, we performed bisulfite conversion on 5 different DNA specimen aliquots (bisulfite-to-bisulfite) and repeated PCR pyrosequencing five times (run to run). Second, to assess heterogeneity of LINE-1 methylation levels within tumor, we made 5 different tissue sections from one tumor and examined LINE-1 methylation level of each section (section to section). Third, to evaluate LINE-1 methylation status in ESCC, we applied this assay to 30 ESCCs and 30 matched normal esophageal mucosa. Bisulfite-to-bisulfite standard deviation (SD) ranged from 1.44 to 2.90 (median 2.32) in ESCCs; and 0.57 to 4.02 (median 1.23) in normal esophagus. Run-to-run SD ranged from 0.63 to 3.25 (median 1.54) in ESCCs. Section-to-section SD ranged from 1.37 to 3.31 (median 1.94). ESCC tissues showed significantly lower levels of LINE-1 methylation than matched normal mucosa (P

BACKGROUND: Comorbidity is a common problem in the treatment of elderly patients, but there is no standard method of the evaluation in glioblastoma patients. Gagne comorbidity index (GCI) has been proposed to be an integrated prognostic index of the Charlson comorbidity index and Elixhauser index, associating with the short-term survival of various diseases. We examined the relationship between GCI and prognosis of elderly patients with glioblastoma. METHODS: 31 patients over 70 years treated at our hospital from Dec.2006 to Aug.2017. 16 males and 15 females. Mean age 76.8 years (70–86 years old). Relationship to overall survival (OS) was examined focusing on the age, GCI at hospitalization(low:≤1, intermediate:2–4, high:≥5), Karnofsky Performance Status (KPS), surgery, radiotherapy (standard and hypofractionated), and temozolomide-based chemotherapy. RESULT: Median OS of entire cohort was 13.0 months. There was no significant correlation between age and GCI, but a negative correlation between KPS and GCI (r=-0.427). Median OS was longer in the resection group (resection; 13.5m vs non-resection; 8.6m), and in chemo-radiotherapy (standard radiotherapy+TMZ: 13.5m and hypofractionated radiotherapy+TMZ: 10.1m), though, GCI has no relationship to such treatment option. Nevertheless, high GCI indicated poor prognosis with a median OS in the group of low GCI:13.0m, intermediate GCI:14.5m and high GCI:5.3m (p=0.002). DISCUSSION: The presence of comorbidities is one of the factors related to prognosis, and glioblastoma affecting the brain is in a disadvantageous condition due to cognitive dysfunction and paralysis. Since surgical excision and radiation chemotherapy lead to improvement in prognosis even in the elderly, it is important to complete the treatment paying maximum attention to the management of comorbidities. GCI would be useful to evaluate comorbidities. Further examination is needed in large cohort.

Background: Tumor microenvironment consists of various types of cells. Although cancer associated fibroblasts (CAFs) have been reported to enhance tumor progression and anticancer drug resistance through secretion of soluble factors so far, the crucial mechanism is not fully understood. On the other hand, advanced gastric cancer (GC) is still poor prognosis due to metastasis and recurrence despite the progression of diagnosis and treatment. The aim of this study is to identify the molecular mechanism underlying anticancer drug resistance mediated by CAFs in GC. Methods: We have isolated CAFs from surgically resected GC tissues and collected conditioned medium (CM) of CAFs. Human GC cell lines with or without CAF-CM were examined the drug sensitivity and analyzed expression of specific genes using quantitative reverse transcription PCR. We isolated extracellular vesicles (EVs) from CAF-CM by ExoQuick-TC ® and ultracentrifugal separation. We observed GC cells with confocal microscopy after the solution of stained EVs treated. We performed EVs from CAF-CM and GC-CM to mass analysis. Results: GC cells with CAF-CM showed drug resistance on normal coated plates. Moreover, we found that GC cells with CAF-CM on extracellular matrix (ECM)-coated plates showed remarkable morphological change and much higher drug resistance than those without CAF-CM. In contrast, GC cells with GC cell line-CM on ECM- coated plates did not show morphological change. We also found that EVs isolated from CAF-CM caused morphological change and drug resistance of GC cells. Moreover, we identified five proteins included in CAF-EVs as possible candidates involving in GC drug resistance by mass analysis. Gene expression analysis revealed that SNAI2, which is one of the Epithelial Mesenchymal Transition (EMT)-related gene, was significantly up-regulated in GC cells cultured with CAF-CM or EVs from CAF-CM on ECM-coated plates. Conclusion and future perspective: These findings in current study suggest that EVs from CAFs cause characteristic gene expression change and enhance drug resistance along with morphological change of GC cells. We are trying to identify the critical factor in CAF-EVs for drug resistance of GC cells. Citation Format: Tomoyuki Uchihara, Keisuke Miyake, Eri Oda, Yuki Koga, Taisuke Yagi, Daisuke Kuroda, Tsugio Eto, Yuki Kiyozumi, Kota Arima, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. To identify the critical factors in extracellular vesicles derived from cancer-associated fibroblasts for drug resistance of gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5086.

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2 (PGE2). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) participates in the degradation of PGE2 and has attracted attention in recent years. Although inhibition of 15-PGDH is reported to lead to PGE2 accumulation and expand tissue stem cell fraction, the role for pancreatic tumor progression is still unknown. The aim of this study is to elucidate the regulatory mechanism and functional role of 15-PGDH for cancer stem-like cell expansion during tumor progression in pancreas. We found that 15-PGDH expression is frequently down-regulated in PDAC cells and significantly correlated with the number of infiltrating macrophages in human PDAC tissues. Moreover, direct co-culture assay revealed that macrophage derived interleukin-1 beta down-regulates 15-PGDH expression in PDAC cells. Furthermore, pharmacological blockade of 15-PGDH led to PGE2 accumulation, and promoted growth and sphere formation through the expansion of ALDH1-positive cells. We also elucidated the molecular mechanism that PGE2 accumulation by 15-PGDH inhibition increases CYP26A1 expression and subsequently depletes all-trans retinoic acid, and results in ALDH1 up-regulation. Finally, genetic ablation of 15-Pgdh promoted tumorigenesis in KrasLSL-G12D; Ptf1aCre/+ mice through the expansion of ALDH1-positive cells. Our findings highlight the role and significance of PGE2 degradation pathway for PDAC tumor progression. Citation Format: Takatsugu Ishimoto, Kota Arima, Luke Bu, Tomoyuki Uchihara, Keisuke Miyake, Tsugio Eto, Rumi Itoyama, Hideo Baba. Expansion of pancreatic cancer stem-like cells through PGE2 accumulation in inflammatory environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1987.

After the introduction of new chemotherapeutic agents,including oxaliplatin and irinotecan, chemotherapy can induce tumor down-staging in patients with initially unresectable colorectal liver metastases (CRLM), which thus allows for hepatic resection. Recently, the application of thermal ablation,especially radiofrequency ablation (RFA),has been extended from hepatocellular carcinoma (HCC)to CRLM. However,randomized trials show little evidence to support the clinical efficacy of RFA for CRLM. We herein evaluate the clinical efficacy and the role of thermal ablation for CRLM in this era of effective chemotherapy. Non-randomized retrospective studies have demonstrated the effects of RFA alone for CRLM without new chemotherapeutic agents. The three-and five-year survival rates of RFA alone for CRLM ranged from 42% to 57%,and 21% to 37%, respectively. The local recurrence rate was between 9% and 15%. In contrast, the five-year survival rates after RFA with perioperative chemotherapy of irinotecan-or oxaliplatin-based regimens ranged from 34% to 54%. The local recurrence at the site of RFA ranged from 29.7% to 42.5%. After the introduction of new chemotherapeutic agents, RFA with perioperative chemotherapy of irinotecan-or oxaliplatin-based regimens may improve the survival in patients with CRLM compared to RFA alone. However,the rate of local recurrence of RFA with perioperative chemotherapy was similar to that after RFA alone. The three-and five-year survival rates of RFA in combination with hepatic resection for CRLM ranged from 38% to 47%,and 47% to 68%,respectively. The rate of local recurrence at the site of RFA was between 5 and 17.4%,respectively. In our study,hepatic resection combined with RFA after the FOLFOX regimen resulted in excellent local control rates(2.5% recurrence per tumor). In this era of new and more effective chemotherapy,combining RFA with chemotherapy or hepatic resection for CRLM can extend the indications of surgical resection and further improve patient survival.

The oncogenic hallmarks of pancreatic cancer (PC), such as the KRAS, BRAF, and PIK3CA mutations, have been widely investigated. However, almost all of the previous studies were limited by small sample sizes. In addition, previous data on the KRAS mutation and clinical outcomes in PC remain inconclusive. To clarify these data, we examined the mutation status of 126 PC patients and its relationship to clinical outcome. The frequencies of KRAS, BRAF, and PIK3CA mutations were determined from a non-biased database of 126 resected PCs and a high-throughput pyrosequencing assay. KRAS mutations were detected in 109 (86.5 %) of the 126 cases; the most common mutation was c.34G > T (p.G12C), which was present in 80 tumors, followed by c.35G > T (p.G12V) in 52 tumors. The KRAS mutation was not associated with any clinical or pathological features (p > 0.05 in all cases). In addition, the KRAS mutation was unrelated to overall survival (log rank p = 0.21) and cancer-specific survival (log rank p = 0.27). Importantly, the influence of KRAS mutation on patient outcome was not modified by any of the clinical or pathological variables (p for all interactions >0.05). Only one PIK3CA mutation (0.8 %) was detected on exon 9 RS3 (c.1633G > A, p.E545K). The BRAF mutation was not detected in PC. KRAS mutations appear to be unrelated to clinical outcome in PC. BRAF and PIK3CA mutations were extremely rare in PC, suggesting that they play a limited role in PC development.

There has been no clinical observation about histopathological changes in human gliomas under control of the anti-angiogenic treatment, and the tissue-based observation is likely crucial to understand mechanism of action of these treatment. We collected 4 glioblastomas resected under control of neoadjuvant bevacizumab (Bev). Histopathological investigation was performed by hematoxilyn- eosin staining and immunohistochemistry for CD34, VEGF, VEGFR1/2, HIF-1α, and Nestin as compared to 6 control glioblastomas to assess changes in histological features, microvessel density, expression of VEGF and its receptors, tumor hypoxic condition, and cancer stem cells. Tumor resection under control of Bev was uneventful in all of the 4 cases. Intraoperatively, the tumors were clearly less vasculized than usual. Histopathologically, vascular morphology was quite different from usual glioblastomas with universal disappearance of microvascular proliferation. Microvessel density was significantly decreased in the 4 tumors as compared to control tumors. The expression of VEGF and its receptors was decreased in 2 cases of partial response, suggesting possible correlation with radiologic response. The expression of HIF-1α was similar or somewhat decreased in the 4 tumors. The expression of Nestin was significantly decreased in the 4 tumors as compared to control tumors, but there still remained numerous Nestin-positive cells especially around vessels. We provide the first clinicopathological evidence that the anti-angiogenic therapy induces vascular normalization and decrease of microvessel density in glioblastomas. These in situ observation will help to elucidate the mechanism of resistance to Bev and to optimize therapy.

OBJECT: Neuroendocrine carcinoma (NEC) is a malignant tumor that usually arises in the lung, pancreas and gastrointestinal tract. Brain metastasis of NEC is merely reported. Herein, we report two cases of NEC in the brain mimicking glioblastoma (GBM). CASES: Both patients were in their 60s, cystic lesions with peritumoral edema affected the temporal lobe. Cyst walls were thin but hyperintense in diffusion weighted images implying increased cellularity. Positron emission tomography (PET) revealed malignant nature by strong uptake of methionine (MET), fluorothymidine (FLT) and fluoromisonidazole likewise GBM. However, fluorodeoxyglucose (FDG) indicated relatively weak uptakes compared as GBM. Both tumors were resected under the preoperative diagnosis of GBM, however, pathological diagnoses were NEC by immunohistochemical finding of negative GFAP and positive markers of neuroendocrine differentiation. Systemic examination failed to reveal the origin. Both cases underwent radiotherapy. One case was lost by spinal dissemination 11 months after the operation. DISCUSSION: FDG-PET of neuroendocrine tumor in the extracranial lesion does not always demonstrate strong uptake. FDG-PET for NEC in the brain could be less accumulated than GBM, while it should be evaluated keeping in mind that the background level of FDG would be high in the brain tissue. In case of malignant tumors endorsed by other tracer such as MET and FLT, inconsistent FDG-PET finding might be useful for the diagnosis of NEC in the brain.

Although cancer-associated fibroblasts (CAFs) mainly produce CXCL12 and stimulate CXCL12/CXCR4 signaling in cancer cells, the significance of this interaction in adenocarcinoma of the esophagogastric junction (AEG) was unclear. This study investigated the functional characteristics of CAF-derived CXCL12 in AEG. Immunohistochemical staining for CXCL12 was performed on sections from 123 AEG patients and analyzed against clinicopathological data. Newly isolated CAFs and normal fibroblasts were examined for phenotype. An invasion assay was performed with AEG cells co-cultured with CAFs isolated from AEG. CXCL12 expression was significantly associated with age, depth of invasion, lymphatic invasion, and lymph node metastases. High CXCL12 expression significantly correlated with poor prognosis. Isolated CAFs had abundant α-smooth muscle actin expression and showed various CXCL12 expression patterns. Notably, AEG cells co-cultured with CXCL(high)-expressing CAFs invaded more than when co-cultured with CXCL(low)-expressing CAFs; these invasive properties were impeded by CXCR4 antagonist AMD3100. We demonstrated that AEG cells co-cultured with CXCL12(high) CAFs were significantly more invasive than those co-cultured with CXCL12(low) CAFs and that high CXCL12 expression correlates with poor prognosis in AEG patients. CXCL12 derived from CAFs in tumor microenvironment stimulates CXCL12/CXCR4 signaling in AEG cells and promotes their invasive ability, resulting in tumor progression.