Jun-ichiro Kuroda

MicroRNA Expression Profiling of Exfoliated Colonocytes Isolated from Feces for Colorectal Cancer Screening

To reduce the colorectal cancer (CRC) mortality rate, we have reported several CRC screening methods using colonocytes isolated from feces. Expression analysis of oncogenic microRNA (miRNA) in peripheral blood was recently reported for CRC detection. In the present study, we conducted miRNA expression analysis of exfoliated colonocytes isolated from feces for CRC screening. Two hundred six CRC patients and 134 healthy volunteers were enrolled in the study. miRNA expression of the miR-17-92 cluster, miR-21, and miR-135 in colonocytes isolated from feces as well as frozen tissues was analyzed by quantitative real-time PCR. The expression of the miR-17-92 cluster, miR-21, and miR-135 was significantly higher in CRC tissues compared with normal tissues. The exfoliated colonocytes of 197 CRC patients and 119 healthy volunteers were analyzed because of the presence of sufficient miRNA concentration. miR-21 expression did not differ significantly between CRC patients and healthy volunteers (P = 0.6). The expression of miR-17-92 cluster and miR-135 was significantly higher in CRC patients than in healthy volunteers (P < 0.0001). The overall sensitivity and specificity by using miRNA expression was 74.1% (146/197; 95% confidence interval, 67.4-80.1) and 79.0% (94/119; 95% confidence interval, 70.6-85.9), respectively. Sensitivity was dependent only on tumor location (P = 0.0001). miRNA was relatively well conserved in exfoliated colonocytes from feces both of CRC patients and healthy volunteers. miRNA expression analysis of the isolated colonocytes may be a useful method for CRC screening. Furthermore, oncogenic miRNA highly expressed in CRC should be investigated for CRC screening tests in the future. Cancer Prev Res; 3(11); 1435–42. ©2010 AACR.

Enhanced distribution of NK012, a polymeric micelle-encapsulated SN-38, and sustained release of SN-38 within tumors can beat a hypovascular tumor

Human pancreatic cancer is generally hypovascular in nature and rich in interstitium. These pathological barriers may contribute to the intractable nature of pancreatic cancer by binding the penetration of anticancer agents throughout the tumor tissue. The aim of the present study was to determine whether NK012 is an appropriate formulation for the treatment of hypovascular tumors. Among pancreatic tumor xenografts, PSN1 appeared to have the richest tumor vasculature and the least number of stromal cells and matrix. In contrast, Capan1 had the poorest tumor vasculature and most abundant stromal tissue. Fluorescence microscopy and high‐performance liquid chromatography analysis demonstrated that although NK012 accumulated and continued to be distributed for more than 48 h throughout the entire body of both tumors, CPT‐11 disappeared almost entirely from both tumors within 6 h. In addition, efficient sustained release of SN‐38 was maintained for more than 96 h in both tumors following administration of NK012. Following the administration of CPT‐11, SN‐38 was no longer detectable after 24 h in the Capan1 tumor or after 48 h in the PSN1 tumor. All tumors were eradicated in the mice treated with NK012 but not in those treated with CPT‐11. Because the antitumor activity of SN‐38 is time dependent, NK012, which combines enhanced distribution with sustained release of SN‐38 within tumors, may be ideal for the treatment of hypovascular tumors, such as pancreatic cancer. (Cancer Sci 2008; 99: 1258–1264)

Pituitary Apoplexy Associated with Endocrine Stimulation Test: Endocrine Stimulation Test, Treatment, and Outcome

Pituitary apoplexy is a rare clinical syndrome attributable to hemorrhage or hemorrhagic infarction of pituitary tumors or pituitary glands. The features of pituitary apoplexy associated with the endocrine stimulation test remain to be elucidated and the importance of surgical treatment has not been discussed enough. We report two rare patients who were treated successfully by endoscopic endonasal transsphenoidal surgery within several hours after onset of pituitary apoplexy associated with the endocrine stimulation test. Their postoperative course was uneventful. We reviewed earlier reports on this clinical entity, document its features especially as related to the endocrine stimulation test, discuss the significance of immediate surgical treatment, and present our treatment outcomes. Performing only conservative treatment is not recommended. We suggest that the necessity of endocrine stimulation test should be assessed on a case-by-case basis and in patients subjected to the test, and neurosurgical support should be sought.

Giant tumefactive perivascular spaces that expanded and became symptomatic 14 years after initial surgery.

Background: Perivascular spaces (PVSs) or Virchow–Robin spaces in the brain are pial-lined interstitial fluid (ISF)-filled structures surrounding the penetrating arteries and arterioles. These spaces appear as 1- to 2-mm in diameter, round, oval, or curvilinear smooth-walled structures on magnetic resonance imaging (MRI). Typical PVSs are asymptomatic. Occasionally, they become enlarged and cause specific clinical manifestations that depend on location and the degree of tissue compression. In this case, they are referred to as giant tumefactive PVSs. To our knowledge, there have been no reported cases in which giant PVSs increased remarkably in number and size during both the natural course and postoperative course. We describe a rare progression of giant tumefactive PVSs 14 years after initial surgery. Case Description: On first admission at age 17, endoscopic ventriculocystostomy and third ventriculostomy were performed to relieve hydrocephalus caused by cysts compressing the cerebral aqueduct. Fourteen years later, the multicystic lesion reappeared with an increase in both cyst number and size. The patient showed no hydrocephalus but presented with oculomotor and trochlear nerve palsies, which were caused by a mass effect on the midbrain. Endoscopic ventriculocystostomy was performed and symptoms improved. Conclusion: This is the first case report in which giant PVSs increased significantly in number and size.

Abstract 1819: Fecal RNA test using microRNA expressions of exfoliated colonocytes for colorectal cancer screening

Background & Aims: To reduce the mortality rate of several cancers, the development of a screening test is needed. We have been developing new screening methods for colorectal cancer (CRC) by applying molecular biological tools to exfoliated colonocytes. In the last few years especially, we have reported the fecal RNA test including the CRC-related gene expression analysis. Here, we investigated the potential of a fecal miRNA test for the early detection of CRC. Methods: CRC patients (n = 299) and healthy volunteers (n = 116) with no abnormalities detected by screening colonoscopy were enrolled in this case control study. The miRNA expressions in the colonocytes of CRC patients (n = 47) and healthy volunteers (n = 35) were analyzed in the training study and the miRNA expressions in the colonocytes of CRC patients (n = 252) and healthy volunteers (n = 81) were validated in the validation study. First, CRC related oncogenic miRNAs for CRC detection were selected by TaqMan MicroRNA Array v3.0 (corresponded to miRBase release 14). Next, the expressions of the most suitable oncogenic miRNAs were analyzed by quantitative real-time PCR in the training study. Lastly, the sensitivity and specificity of fecal miRNA test was validated by quantitative real-time PCR in the validation study. Results: According to the results of miRNA array, 20 miRNAs were selected as candidate of miRNA analysis. In the training study, significant differences in the relative expression level of miR-17, -18a, -19a, -19b, -20a, -92a, -106a, -135a, -135b, and -146a were observed between the CRC patients and the healthy volunteers (P 0.1) AUC of ROC curve using miR-17, -18a, -19a, -19b, -20a, -92a, -106a, -135b, and -146a were more than 0.7. The overall sensitivity and specificity in the training study using these miRNAs were 70.2% (33/47) and 74.3% (26/35), respectively. The overall sensitivity and specificity in the validation study were 67.5% (170/252) and 75.3% (61/81), respectively. Conclusions: We selected the suitable miRNAs for CRC detection in exfoliated colonocytes. Fecal miRNA test may be useful for CRC screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4448. doi:1538-7445.AM2012-4448

Improvement of radiation‐induced healing delay by etanercept treatment in rat arteries

Surgical treatment often causes difficulty in the irradiated field because of delayed wound healing, which is mainly due to vascular dysfunction. To overcome this difficulty, we attempted to accelerate the recovery from clamp injury in irradiated superficial epigastric arteries of rats as a model. Etanercept, a soluble receptor of tumor necrosis factor‐α, was administered four times to rats with irradiated arteries before and after clamp injury. Loss of endothelial cells and necrosis of the media in the irradiated arteries continued for more than 1 week after the injury; however, in the rats treated with etanercept, the endothelial cells recovered in the intima, and α‐smooth muscle actin‐positive smooth muscle cells recovered in the injured and irradiated arteries. After clamp injury of common carotid arteries that had previously been irradiated, the blood flow in these arteries was visualized by magnetic resonance (MR) angiography. The time‐of‐flight signal was weakened in the injured and irradiated arteries. This time‐of‐flight signal was recovered by the etanercept treatment. These findings suggest that etanercept improves the radiation‐impaired healing of arteries in rats. (Cancer Sci 2009)

BCL2 expression is associated with a poor prognosis independent of cellular origin in primary central nervous system diffuse large B-cell lymphoma

PurposePrimary central nervous system diffuse large B-cell lymphoma (CNS-DLBCL) is a distinct clinicopathological entity with a poor prognosis. Concurrent MYC and BCL2 overexpression predicts inferior prognosis in systemic DLBCL, although their prognostic significance remains unclear in primary CNS-DLBCL.MethodsPretreatment diagnostic biopsy samples were retrospectively evaluated for 79 patients with primary CNS-DLBCL who were treated between January 2001 and December 2017. Histological and immunohistochemical testing were performed to evaluate the patients’ statuses for various markers, which were also evaluated for associations with survival outcomes.ResultsAccording to the Hans criteria, 26 patients (32.9%) had the germinal center B-cell subtype and 53 patients (67.1%) had the activated B-cell subtype. Forty-one cases (51.9%) were positive for MYC (expression of ≥ 40%), 33 cases (41.8%) were positive for BCL2 (expression of ≥ 70%), 22 patients (27.8%) were positive for both MYC and BCL2, and 27 patients (34.2%) were negative for both MYC and BCL2. There were no significant differences in survival between the germinal center and activated B-cell subtypes. Furthermore, MYC positivity was not associated with overall survival (p = 0.369) or progression-free survival (PFS) (p = 0.253). However, BCL2 positivity was significantly associated with poor overall survival (p = 0.039) and PFS (p = 0.036). Co-expression of MYC and BCL2 was not associated with survival.ConclusionOur data suggest that evaluating BCL2 expression may help predict the prognosis in cases of primary CNS-DLBCL.

Abstract 4849: Implications of cancer induced blood coagulation in cancer diagnosis and therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A large body of clinical evidence indicates that abnormal coagulation occurs in a variety of cancers, especially invasive cancers. If cancer clusters erode adjacent normal or tumor vessels, hemorrhage may occur, and fibrin clots immediately form in situ to stop the bleeding. The fibrin clots are subsequently replaced by collagen, similar to the case in the normal wound healing process. Although there are many points in common between cancer-induced stroma and normal wound healing, the critical difference between the two is that the pathophysiological condition in cancer lasts for as long as cancer cells survive in the body (Adv Drug Deliv Rev 2012). First, we successfully developed a mAb that reacts only with human insoluble fibrin, not human fibrinogen (Cancer Sci 2011). In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognizes an unexplored hole that is uncovered only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ-chain that interacted closely with a counterpart region on the γ-chain in the soluble state. New anti-Bβ and anti-γ mAbs specific for peptides lining the discovered hole appeared to bind exclusively to fibrin clots. Analysis of the kinetics of fibrin clot deposition using 102-10 in several disease models indicated that fibrin clot formation occurs only in the acute phase of the disease process, thrombosis, inflammation or trauma, and that these clots virtually disappear within a few weeks to be substituted by collagen. Then, we developed a new PET probe using 102-10. The radiolabelled mAb was injected into mice bearing chemically induced spontaneous tumors, and the probe showed clear and specific accumulation in the tumors. We named this detection strategy cancer stromal targeting (CAST) diagnois. Immunohistochemistry using this mAb revealed higher fibrin deposition in WHO grade 4 gliomas than in lower-grade gliomas. Because erosive tumors are highly likely to show micro-hemorrhages, even early asymptomatic tumors detected with radiolabeled 102-10 mAb may be aggressively malignant (Sci. Rep. 2013). In conclusion, CAST diagnosis and therapy based on anti-insoluble fibrin mAb may be a useful new strategy in the field of oncology. Citation Format: Yohei Hisada, Masahiro Yasunaga, Shingo Hanaoka, Shinji Saijou, Takashi Sugino, Atsushi Tsuji, Tsuneo Saga, Kouhei Tsumoto, Shino Manabe, Jun-ichiro Kuroda, Jun-ichi Kuratsu, Yasuhiro Matsumura. Implications of cancer induced blood coagulation in cancer diagnosis and therapy. [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 4849. doi:10.1158/1538-7445.AM2014-4849

Abstract LB-433: A new method of colorectal cancer screening using microRNA expression profiles of colonocytes isolated from feces

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background and Aims: Early detection of colorectal cancer (CRC) is desirable because this cancer can be cured surgically if it is diagnosed at an early stage. To reduce the mortality rate of CRC, the development of a screening test by which the cancer can be diagnosed early is needed. To date, we have reported several CRC screening methods using colonocytes isolated from feces. Recently, expression analysis of oncogenic microRNA (miRNA) in serum was reported for CRC detection. Here, we conducted miRNA expression analysis in exfoliated colonocytes isolated from feces for CRC screening. Methods: First, oncogenic miRNA expression profiles were analyzed in CRC tissue and normal tissue. Next, 206 patients with CRC and 134 healthy volunteers were enrolled in the study, and oncogenic miRNA expression in colonocytes isolated from feces was analyzed by quantitative real-time PCR. Results: In the CRC tissue, oncogenic miRNA expression was significantly higher than in normal tissue. There was sufficient miRNA to analyze the exfoliated colonocytes of 197 CRC patients and 119 healthy volunteers. The overall sensitivity of miRNA expression analysis was 74.1% (146/197, 95% CI; 67.4-80.1) and the specificity was 79.0% (94/119, 95% CI; 70.6-85.9). The sensitivity was significantly higher for cancers in the left side of the colon ( P= 0.0001). Conclusions: miRNA was well conserved in exfoliated colonocytes. miRNA expression analysis of colonocytes isolated from feces may be useful for CRC screening. Further investigation of oncogenic miRNA highly expressed in CRC is needed for future CRC screening test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-433.