Naoki Izawa

HERC2 Interacts with Claspin and Regulates DNA Origin Firing and Replication Fork Progression

DNA replication, recombination, and repair are highly interconnected processes the disruption of which must be coordinated in cancer. HERC2, a large HECT protein required for homologous recombination repair, is an E3 ubiquitin ligase that targets breast cancer suppressor BRCA1 for degradation. Here, we show that HERC2 is a component of the DNA replication fork complex that plays a critical role in DNA elongation and origin firing. In the presence of BRCA1, endogenous HERC2 interacts with Claspin, a protein essential for G(2)-M checkpoint activation and replication fork stability. Claspin depletion slowed S-phase progression and additional HERC2 depletion reduced the effect of Claspin depletion. In addition, HERC2 interacts with replication fork complex proteins. Depletion of HERC2 alleviated the slow replication fork progression in Claspin-deficient cells, suppressed enhanced origin firing, and led to a decrease in MCM2 phosphorylation. In a HERC2-dependent manner, treatment of cells with replication inhibitor aphidicolin enhanced MCM2 phosphorylation. Taken together, our results suggest that HERC2 regulates DNA replication progression and origin firing by facilitating MCM2 phosphorylation. These findings establish HERC2 as a critical function in DNA repair, checkpoint activation, and DNA replication.

Gastrointestinal stromal tumor presenting with prominent calcification

We present a rare case of a gastrointestinal stromal tumor (GIST) in the stomach with prominent calcification at presentation. A 61-year-old woman visited our hospital because of epigastric discomfort. A spherical calcified lesion with a diameter of about 30 mm was incidentally shown in the left upper quadrant on an abdominal X-ray. Computed tomography demonstrated that the tumor was growing from the upper gastric body, with calcification in the peripheral ring area. A laparoscopic partial gastrectomy was performed, and the resected specimen revealed a well-circumscribed tumor with exophytic growth from the gastric muscularis propria. Microscopic examination revealed spindle-shaped tumor cells with calcification and hemorrhage. Additionally, positive immunoreactivity of the tumor to KIT and CD34 and a low mitotic index resulted in the diagnosis of very low risk GIST. There are a few case reports of heavily calcified GIST, although solitary or punctate calcification of primary GIST has been reported in several case series. Dystrophic calcification of necrotic or degenerative tissue is the supposed cause of primary calcified GISTs. In contrast, appearance of calcification after administration of imatinib mesylate, which may be one indicator of disease response, is possibly caused by a different mechanism.

Early morphological change for predicting outcome in metastatic colorectal cancer after regorafenib

Background and Objective It is unclear whether early morphological change (EMC) is a predictive marker for regorafenib in metastatic colorectal cancer (mCRC). Therefore, the present study investigated whether EMC can predict the outcome of mCRC patients receiving regorafenib. Results This study evaluated 68 patients. Among 52 patients with lung metastasis, 16 (31%) had cavity formation (CF). The median progression-free survival (PFS) and overall survival (OS) in patients with/without CF were 4.2/2.4 months (p<0.01) and 9.2/6.5 months (p=0.09), respectively. Among 45 patients with liver metastasis, 14 (31%) had active morphological response (MR). The median PFS and OS in patients with/without active MR were 5.3/2.4 months (p<0.01) and 13.6/6.9 months (p=0.02), respectively. Overall, 25 patients (37%) had EMC. The median PFS and OS in patients with/without EMC were 5.3/2.1 months (p<0.01) and 13.3/6.1 months (p<0.01), respectively. Materials and Methods This retrospective study included mCRC patients with lung and/or liver metastases receiving regorafenib. CF in lung metastasis and MR in liver metastasis were evaluated at the first post-treatment computed tomography scan. EMC was determined as CF and/or active MR. We compared PFS and OS between patients with and those without EMC. Conclusions EMC could be a useful predictive marker for regorafenib in mCRC.

Profile of trifluridine/tipiracil hydrochloride in the treatment of metastatic colorectal cancer: efficacy, safety, and place in therapy

TAS-102, with its robust survival efficacy and feasible toxicity, is one of the standard salvage-line treatments for patients with metastatic colorectal cancer (mCRC). No definitive data are available to determine which drug should be administered first during salvage-line treatment. Therefore, it is imperative that we establish the sequence of administration by considering drug toxicity profiles based on patient characteristics, such as age, performance status, comorbidities, tolerability to previous treatments, and patient preferences. The identification of predictive biomarkers in response to TAS-102 or its toxicity is urgently needed for better patient selection. Moreover, to strengthen efficacy or relieve toxicity, combinations with other agents, which could potentially emerge as standard treatment regimens, have been investigated and compared to existing active regimens for mCRC.

Pneumobilia: A Rare Complication or a Common Phenomenon of Double-Balloon Enteroscopy?

genes, were absent in the two biopsied specimens (6) . Th erefore, Crohn ’ s disease was suspected. Considering the possibility of intestinal tuberculosis, ELISPOT assay of blood specimen of the patient was carried out and showed positive results 2 days later. Four weeks later, culture of the second biopsied tissue (the fi rst biopsied specimen was not sent for cultures) grew M. tuberculosis . Th e patient received anti-tuberculosis treatment and recovery was uneventful. It is a great challenge to distinguish Crohn ’ s disease from ileocecal tuberculosis in clinical practice because the two diseases share confusingly similar clinical, endoscopic, and pathological manifestations (1,6) . Without the aid of a modern diagnostic method, this case would typically be diagnosed and treated initially as Crohn ’ s disease due to the lack of caseous necrosis in the granulomas, negative acid-fast stain, and absence of mycobacterial DNA. Th is case illustrates the potential advantages of measuring mycobacterial-specifi c immune response with ELISPOT assay for rapid diagnosis, particularly when there is no evidence of mycobacterial infection in the granulomatous intestinal lesions and when the microbiological culture results are still pending.

Identification of a FGFR3-TACC3 fusion in esophageal cancer

treatment effects for mortality from routinely collected data and subsequent randomized trials: meta-epidemiological survey. Br Med J 2016; 352: i493. 4. Chavez-MacGregor M, Giordano SH. Randomized clinical trials and observational studies: is there a battle? J Clin Oncol 2016; 34(8): 772–773. 5. Miller K, Wang M, Gralow J et al. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med 2007; 357: 2666–2676. 6. Miles DW, Chan A, Dirix LY et al. Phase III study of bevacizumab plus docetaxel compared with placebo plus docetaxel for the first-line treatment of human epidermal growth factor receptor 2-negative metastatic breast cancer. J Clin Oncol 2010; 28: 3239–3247. 7. Robert NJ, Diéras V, Glaspy J et al. RIBBON-1: randomized, doubleblind, placebo-controlled, phase III trial of chemotherapy with or without bevacizumab for first-line treatment of human epidermal growth factor receptor 2-negative, locally recurrent or metastatic breast cancer. J Clin Oncol 2011; 29: 1252–1260.

Cause of Lower Limb Edema and Hypoproteinemia Diagnosed by Double-Balloon Endoscopy

C uestion: A 67-year-old man was admitted to the hosital because of aggravated leg edema. Three years before dmission, he was evaluated for hypoproteinemia, from hich he had suffered during the last 8 years. Blood tests, omputed tomography, upper gastrointestinal endosopy, and colonoscopy were performed; however, no auses of hypoproteinemia were detected. On physical xamination, he presented a lower limb edema. Laboraory examination revealed total protein, 4.7 g/dL; albuin, 3.1 g/dL; low immunoglobulin (Ig)G (424 mg/dL); ormal IgA (150 mg/dL); and low IgM (26 mg/dL). Fat roplets were detected in stool by Sudan III staining, and igh 24-hour stool 1-antitrypsin clearance (169 ml/d) as shown. Colonoscopy and esophagogastroduodenosopy were performed, showing normal mucosa. Contrastnhanced computed tomography showed a thickening of he intestinal wall (Figure A). Therefore, oral double-balloon ndoscopy (DBE) was performed, indicating scattered white pots and white nodules, surrounded by diffuse white villi, long the enlarged mucosal folds in the ileum (Figure B). iopsy specimens of the ileum for histologic examination ere obtained. What is the diagnosis? See the GASTROENTEROLOGY web site (www. astrojournal.org) for more information on submiting your favorite image to Clinical Challenges and mages in GI. NAOKI IZAWA, MD TAKESHI SAWADA, MD FUMIO ITOH, MD Division of Gastroenterology and Hepatology St. Marianna University School of Medicine Kawasaki, Japan

Exploration of time points and cut-off values for early tumour shrinkage to predict survival outcomes of patients with metastatic colorectal cancer treated with first-line chemotherapy using a biexponential model for change in tumour size

Background Several studies reported that early tumour shrinkage (ETS) was associated with overall survival in patients with metastatic colorectal cancer (mCRC) treated with first-line chemotherapy. However, appropriate time point and cut-off value for ETS remain unclear because these varied in previous studies. Patients and methods We investigated patients with mCRC who received FOLFOX or FOLFIRI with/without molecular-targeted agents as first-line treatment between 2005 and 2014. Using a biexponential model for change in tumour size, a relative change in the sum of the longest diameters of target lesions from baseline was estimated at a certain time point in each individual patient. Associations of survival outcomes with ETS at various time points based on various cut-off values were evaluated by Cox regression analysis with a landmark approach. Results Among the 67 patients reviewed, the objective response rate was 73.1% (95% CI 62.5% to 83.7%), the median progression-free survival was 10.9 months (95% CI 8.7 to 13.0 months) and the median overall survival was 25.6 months (95% CI 20.1 to 27.3 months). The model for change in tumour size agreed with the actual measured sizes well. Multivariate Cox regression analysis, including performance status, number of metastatic sites and use of targeted agents, showed that ETS at 8 weeks based on a cut-off value of 20% was most significantly associated with overall survival (HR: 0.404, 95% CI 0.231 to 0.707, P=0.0015). Conclusion It is suggested that a time point of 8 weeks and a cut-off value of 20% may be optimal criteria for defining ETS.

Abstract 4248: Predictive signature of response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer cells by tyrosine kinase activity profiling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: There is an urgent need to develop companion diagnostics for response prediction to increase the success rate of anticancer drug development. Constitutively activated protein kinases are major targets for cancer treatment. However, blockade of target molecules may not necessarily lead to treatment response. Thus we attempted to investigate the consequences of target kinase inhibition by comprehensive tyrosine kinases activity profiling, which can be applied to establish a response prediction signature. Here we employed clinically validated EGFR-targeted inhibition as in vitro model utilizing PamChip® peptide micro-arrays. Methods: Eight lung cancer cell lines harboring either wild-type EGFR (A549, H1781 and H441) or mutant EGFR (HCC4006, PC9, HCC827, H1650 and H1975) were used. Chemosensitivity to EGFR inhibitors (gefitinib, erlotinib and afatinib) was evaluated by MTT assay and IC50 values were obtained. For assessment of protein tyrosine kinase activity, cell lysates were prepared, aliquoted and stored at -80°C until use. The tyrosine kinase activity of the lysates was measured with or without EGFR TKIs on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Data analysis was carried out on both basal and inhibition profiles. Inhibition profiles were calculated for each EGFR TKI by taking the log-ratio of peptide phosphorylation measured with or without EGFR TKIs using BioNavigator6 software. Peptides that did not show increase of signal in time (indicative of kinase activity) were excluded from the analysis. Results: Basal tyrosine kinase activity profiles showed a clear segregation between EGFR driven (HCC4006, PC9, and HCC827) and EGFR independent (H1650, H441 and A549) cells. Additionally, overall tyrosine kinase activities in EGFR mutant cells were higher than those in EGFR wild-type cells. Based on IC50 values for three EGFR TKIs, the cell lines were divided into responders (PC9, HCC4006 and HCC827 with IC50 values ≦0.1 μmol/L), non-responders (H1650, H441 and A549 with IC50 values >1 μmol/L) and intermediate responders. Basal phosphorylation signals on a number of peptides differed between responders and non-responders. The responders showed phosphorylation on sites responsible for activating RAF, ERK1/2 and MAPK7 and on sites for inactivating the phosphatases PP2AB and PTN11. The phosphorylation was absent in the non-responders, possibly as a consequence of constitutive activation of the RAS/RAF pathway. This peptide set was suggested to serve as a predictive signature of response to EGFR TKIs. Conclusions: Kinase activity profiles reflect the biochemical consequences of EGFR mutant non-small-cell lung cancer. Tyrosine kinase activity profiling may serve as companion diagnostic in drug development and this should be further explored. Citation Format: Naoki Izawa, Masakuni Serizawa, Faris Naji, Rik de Wijn, Riet Hilhorst, Rob Ruijtenbeek, Hirofumi Yasui, Takako Nakajima, Narikazu Boku, Yasuhiro Koh. Predictive signature of response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer cells by tyrosine kinase activity profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2014-4248

Identification of DNA methylation changes in esophageal cancer before/after chemoradiation therapy using a MCA-microarray and bisulfate pyrosequencing.

10550 Background: Systemic chemo- and chemoradiation therapies for esophageal squamous cell carcinoma (ESCC) patients are widely accepted. However we sometimes experience the resistance of ESCC to these therapies, and multicentric occurrence of ESCC after the successful treatment. Although there are studies, which have shown the involvements of promoter methylation of tumor suppressor genes in esophageal carcinogenesis, it remains unclear that the epigenetic changes related to treatments against ESCC. Our aim is to identify how DNA methylation status changes in tumor tissue (T) and also adjacent normal tissue (ADJ) before/after chemoradiation therapy (CRT). Methods: Tumor and adjacent normal biopsy specimens were obtained before/after treatment from 34 patients (124 samples) treated with a uniform CRT protocol. We analyzed genomewide DNA methylation of 12 non-treated test samples (T and matched ADJ: 6 each) by methylated CpG island amplification and microarray (MCAM) method (4×44K, Agilent custom array), ...