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Positive controls of anti-mouse and anti-human antibodies for immunohistochemistry

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Purpose: High-fat diet (HFD) could induce prostate cancer progression. The aim of this study is to identify mechanisms of HFD-induced prostate cancer progression, focusing on inflammation.Experimental Design: We administered HFD and celecoxib to autochthonous immunocompetent Pb-Cre+;Pten(fl/fl) model mice for prostate cancer. Tumor growth was evaluated by tumor weight and Ki67 stain, and local immune cells were assessed by flow cytometry at 22 weeks of age. Cytokines which correlated with tumor growth were identified, and the changes of tumor growth and local immune cells after inhibition of the cytokine signals were evaluated in the mice. IHC analyses using prostatectomy specimens of obese patients were performed.Results: HFD accelerated tumor growth and increased the myeloid-derived suppressor cells (MDSCs) fraction and M2/M1 macrophage ratio in the model mice. Celecoxib-suppressed tumor growth, and decreased both local MDSCs and M2/M1 macrophage ratio in HFD-fed mice. HFD-induced tumor growth was associated with IL6 secreted by prostatic macrophages, as were phosphorylated STAT3 (pSTAT3)-positive tumor cells. Anti-IL6 receptor antibody administration suppressed tumor growth, and decreased local MDSCs and pSTAT3-positive cell fractions in HFD-fed mice. The tumor-infiltrating CD11b-positive cell count was significantly higher in prostatectomy specimens of obese than those of nonobese patients with prostate cancer.Conclusions: HFD increased MDSCs and accelerated prostate cancer tumor growth via IL6/pSTAT3 signaling in the mice. This mechanism could exist in obese patients with prostate cancer. IL6-mediated inflammation could be a therapeutic target for prostate cancer. Clin Cancer Res; 24(17); 4309–18. ©2018 AACR.

// Yoshiyuki Yamamoto 1 , Motohide Uemura 1, 2 , Kosuke Nakano 1 , Yujiro Hayashi 1 , Cong Wang 1 , Yu Ishizuya 1 , Toshiro Kinouchi 1 , Takuji Hayashi 1 , Kyosuke Matsuzaki 1 , Kentaro Jingushi 2 , Taigo Kato 1 , Atsunari Kawashima 1 , Takeshi Ujike 1 , Akira Nagahara 1 , Kazutoshi Fujita 1 , Ryoichi Imamura 1 and Norio Nonomura 1 1 Department of Urology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan 2 Department of Therapeutic Urologic Oncology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan Correspondence to: Motohide Uemura, email: uemura@uro.med.osaka-u.ac.jp Keywords: circulating cell-free DNA; renal cell carcinoma; plasma; level; fragment size Received: December 14, 2017 Accepted: March 11, 2018 Published: April 17, 2018 ABSTRACT Background: Reliable biomarkers for renal cell carcinoma (RCC) have yet to be found. Circulating cell-free DNA (cfDNA) is an emerging resource for the diagnosis and prognosis of various cancers. This study aims to identify novel blood biomarkers for RCC. Materials And Methods: Plasma cfDNA was extracted from RCC patients ( n = 92) and healthy controls ( n = 41). Levels of cfDNA were determined using quantitative real-time PCR of ACTB as the target gene, and cfDNA fragment size was measured using a microfluidics-based platform. Diagnostic potential was assessed using receiver operating characteristic (ROC) and logistic regression analysis, and prognostic potential was evaluated using log-rank test. Results: Median levels of cfDNA from RCC patients were significantly higher than those from healthy controls (3803 vs 2242 copies/ml, p < 0.001). Median fragment sizes of cfDNA in RCC patients were shorter than those in healthy controls (170 vs 171 bp, p = 0.052). To evaluate level of cfDNA as a diagnostic tool for RCC, ROC curve analysis revealed a sensitivity of 63.0% and a specificity of 78.1%. Multivariate analysis indicated that age, gender and the level of cfDNA were significantly associated with the presence of RCC ( p < 0.001, p = 0.013, p < 0.001, respectively). Additionally, shorter cfDNA fragment size was negatively associated with progression-free survival ( p = 0.006). Conclusions: Our study demonstrates the diagnostic and prognostic potential of plasma cfDNA as a biomarker for RCC.

Summary There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly up-regulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by Receiver Operating Characteristic curve analysis (sensitivity of 70.0% and specificity of 64.0%, AUC = 0.722, a multiple logistic regression analysis, OR, 1.07; 95% CI, 1.03–1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in peripheral blood mononuclear cells than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in peripheral blood mononuclear cells from healthy volunteers was significantly elevated following co-culture with RCC cells compared to those co-cultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC. This article is protected by copyright. All rights reserved.

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be determined. Circulating tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. The present study aims to clarify the clinical utility of ctDNA for RCC. Fifty-three patients histologically diagnosed with clear cell RCC were enrolled. Targeted sequencing was carried out using plasma cell-free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR (ddPCR) to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics-based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50-166 bp) to large (167-250 bp) cfDNA fragments. Association of mutant allele frequency of ctDNA with clinical course was analyzed. Prognostic potential was evaluated using log-rank test. A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6) and VHL (n = 5), and median mutant allele frequency of ctDNA was 10%. We designed specific ddPCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. Positive ctDNA was significantly associated with a higher proportion of cfDNA fragments (P = .033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer-specific survival (P < .001, P = .011). In conclusion, our study shows the clinical utility of ctDNA status and cfDNA fragment size as biomarkers for prognosis and disease monitoring in RCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, responsible for approximately 90‑95% of cases. We previously reported a novel method that enables direct extraction of extracellular vesicles (EVs) from surgically resected viable tissues, yielding what we term tissue‑exudative extracellular vesicles (Te‑EVs). Quantitative LC/MS analysis identified 3,871 proteins in Te‑EVs, among which leukocyte‑associated immunoglobulin‑like receptor 1 (LAIR1) was highly enriched in tumor Te‑EVs. In the present study, we found that LAIR1 was significantly upregulated in clinical specimens of human RCC tumor tissues compared to that noted in adjacent non‑cancerous renal tissues as determined by quantitative PCR analysis. LAIR1 overexpression resulted in accelerated cell proliferation and tumor growth in RCC cells. Moreover, knockdown of LAIR1 using siRNA significantly inhibited cell proliferation in RCC cells. Mechanistically, LAIR1 upregulated the phosphorylation status of Akt, which in turn increased cell proliferation in RCC cells. In clinical RCC specimens, RCC patients with high LAIR1 mRNA expression showed poor progression‑free survival compared to those with low LAIR1 expression. These findings indicate that LAIR1 promotes tumorigenesis in RCC.

Purpose: High-fat diet (HFD) could induce prostate cancer progression. The aim of this study is to identify mechanisms of HFD-induced prostate cancer progression, focusing on inflammation. Experimental Design: We administered HFD and celecoxib to autochthonous immunocompetent Pb-Cre+;Pten(fl/fl) model mice for prostate cancer. Tumor growth was evaluated by tumor weight and Ki67 stain, and local immune cells were assessed by flow cytometry at 22 weeks of age. Cytokines which correlated with tumor growth were identified, and the changes of tumor growth and local immune cells after inhibition of the cytokine signals were evaluated in the mice. IHC analyses using prostatectomy specimens of obese patients were performed. Results: HFD accelerated tumor growth and increased the myeloid-derived suppressor cells (MDSCs) fraction and M2/M1 macrophage ratio in the model mice. Celecoxib-suppressed tumor growth, and decreased both local MDSCs and M2/M1 macrophage ratio in HFD-fed mice. HFD-induced tumor growth was associated with IL6 secreted by prostatic macrophages, as were phosphorylated STAT3 (pSTAT3)-positive tumor cells. Anti-IL6 receptor antibody administration suppressed tumor growth, and decreased local MDSCs and pSTAT3-positive cell fractions in HFD-fed mice. The tumor-infiltrating CD11b-positive cell count was significantly higher in prostatectomy specimens of obese than those of nonobese patients with prostate cancer. Conclusions: HFD increased MDSCs and accelerated prostate cancer tumor growth via IL6/pSTAT3 signaling in the mice. This mechanism could exist in obese patients with prostate cancer. IL6-mediated inflammation could be a therapeutic target for prostate cancer. Clin Cancer Res; 24(17); 4309–18. ©2018 AACR.

Background Tumor-infiltrating macrophages, which are thought to be derived from blood monocytes, interact with tumor cells to promote cancer progression. The aim of this study was to assess the association of peripheral blood monocyte count with pathological findings and local tumor-infiltrating macrophages in prostatectomy specimens. Methods Preoperative peripheral blood monocyte counts were retrospectively assessed for their associations with pathological findings (pathological T stage, Gleason Score, extraprostatic extension, seminal vesicle invasion, and surgical margin) and biochemical recurrence of 248 patients who underwent radical prostatectomy. Local tumor-infiltrating macrophages were also evaluated immunohistochemically for their association with peripheral monocyte counts. Results The peripheral monocyte counts of the patients with extraprostatic extension, seminal vesicle invasion, or primary Gleason ≥4 were significantly higher than those of the patients without each of these pathological findings (P

PURPOSE In this retrospective study we reported the results of salvage external beam radiotherapy for patients with biochemical recurrence after radical prostatectomy. MATERIALS AND METHODS A total of 28 patients with biochemical recurrence after radical prostatectomy underwent salvage radiotherapy with (n=16) or without (n=12) hormonal therapy. Median radiation dose was 60 Gy. Biochemical recurrence after radiotherapy was defined as a single prostate-specific antigen (PSA) of at least 0.1 ng/ml. Potential risk factors were evaluated for significant associations with biochemical recurrence. RESULTS The median follow-up period after salvage radiotherapy was 42 months. The actuarial biochemical recurrence free survival rate at 3 and 5 years was 81% and 74%, respectively. Addition of hormonal therapy to salvage radiotherapy did not alter biochemical recurrence rate (P = 0.56). Univariate analysis revealed that Gleason score of 8 to 10 (P = 0.026) and PSA before salvage therapy greater than 0.24 ng/ml (P = 0.0016) were significant risk factors for biochemical recurrence. On multivariate analysis, PSA before salvage therapy greater than 0.24 ng/ml (P = 0.017) maintained statistical significance. Of 28 patients 3 (11%) experienced late grade 3 toxicity of hematuria. CONCLUSION Our data suggest that early use of salvage radiotherapy is beneficial for patients with biochemical recurrence after radical prostatectomy.

We retrospectively reviewed 182 patients who underwent radical prostatectomy in our hospital between April, 2009 to December, 2012, and who had not received any prior hormonal therapy. We also excluded the patients who couldn't followed up more than 6 months after surgery and pN1 patients. Positive surgical margins were observed in 65 cases. We determined what were the significant factors associated with the margin status. The another aim of present study is to evaluate the risk factor which might have significance for biochemical recurrence. BMI ≥ 25.0, prostate volume < 40 cm3, and biopsy positive core ≥ 25% were significant predictors of positive surgical margin. PSA nadir ≥ 0.02 ng/ml and pT3 were the significant factors which associated with biochemical recurrence of those patients with positive margin status.

INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression and influence tumor development and progression. We previously identified miRNAs upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched-pair normal kidney using microarray analysis. However, such cancer-specific miRNAs are not always associated with patient survival and/or recurrence. This study aims to identify miRNAs that are upregulated in ccRCC and that are also associated with survival and/or recurrence. METHODS: We obtained 183 ccRCC clinical specimens from patients who underwent nephrectomy at our hospital between 2000 and 2012. We selected 24 frozen samples, of which 11 patients had evidence of disease or died from cancer (Group 1) and 13 patients had no evidence of disease (Group 2) within two years after surgery. Quantitative real time PCR (qRT-PCR) was performed on the samples to expression of 22 miRNAs that our microarray results indicated as upregulated in ccRCC, and hits were further validated using another 159 frozen samples. Functional analysis of the miRNA was performed using siRNA-mediated knockdown in RCC cell lines. RESULTS: Five miRNAs (Table.1) were upregulated in Group 1 compared to Group 2, and we validated these results using 159 samples of ccRCC. High miR-21-3p (p

The patient was a 60 year-old male who first visited a doctor because of back pain on the right side in May 2003. As a result of thorough examination, he was diagnosed with right renal pelvic cancer (cT4, N2, M1), and was referred to our department for treatment. In spite of systemic chemotherapy and radiation therapy in combination with cisplatinum on the primary tumor were performed from May 2003 to December 2005, the number and size of hepatic metastases increased. Consequently, considering hepatic metastasis as the specific prognosis factor, the patient was given a total of 14 cycles of hepatic arterial infusion chemotherapy (HAIC) from January to October 2006. As a result, the hepatic metastases completely disappeared. Then HAIC was tentatively discontinued and the patient was followed up. However, as new lung metastases were found by CT in March 2007, radiation therapy was performed on the lung metastases. As hepatic metastasis was recognized again by CT in April 2007, HAIC was resumed and the patient was given a total of 6 cycles starting from May 2007. During that period, two transurethral resection of bladder tumor were performed against the recurrence within the bladder while transarterial embolization was performed against the bleeding in the right kidney. The patient was regarded as a long-term survivor surviving for about five years after his initial consultation.

Emphysematous cystitis is a rare lower urinary tract infection. A case of emphysematous cystitis with diabetes mellitus and transverse colon cancer is reported. The patient was an 81-year-old woman complaining of nausea and vomiting. Urinalysis showed hematopyuria. Plain abdominal film and CT scan showed gaseous shadow in the bladder wall. Urine culture contained Escherichia coli. A urethral catheterization and administration of antibiotics resulted in the marked improvement in the clinical course. To our knowledge, 53 cases of emphysematous cystitis have been reported in the Japanese literature including this case and the clinical features are reviewed.

We report a case of primary malignant lymphoma of the prostate. An 84-year-old man was referred to our hospital with a chief complaint of urinary retention. Magnetic resonance imaging showed a large mass below the bladder and in front of the rectum. Histological and immunocytochemical studies of transperineal biopsy of the prostate showed diffuse large B-cell non-Hogkin's lymphoma. Radiological assessment of the disease confirmed stage IV according to the Ann Arbor classification. Although the tumor was markedly reduced in size after four cycles of combination chemotherapy with cyclophosphamide, adriamycin, vincristine, and prednisolone, he died with brain metastasis 4 months after the diagnosis.

PURPOSE Since 2003, screening with prostate specific antigen (PSA) has been conducted to detect prostate cancer. We investigated the results between 2003 and 2007. PATIENTS AND METHODS Screening with PSA alone was performed for males aged over 50 years who desired prostate cancer screening. We used a PSA cutoff value of 4.00 ng per milliliter. RESULTS In 2003, there were 18,161 males aged over 50 years in Ikeda City. 3,738, 3,905, 4,129, 4,410, and 4,515 of the males underwent PSA screening in 2003, 2004, 2005, 2006, and 2007. The rate of elevated PSA levels was 7.9%-9.8% (median 9.1%). 161, 81, 70, 75 and 60 of the males visited us for secondary screening, and prostate biopsy was performed in 130 (80.7%), 57 (70.4%), 45 (64.3%), 38 (50.7%), and 42 (70.0%). Prostate cancer was detected in 91, 33, 29, 20 and 25 males, respectively. These values corresponded to 2.43%, 0.85%, 0.70%, 0.45% and 0.55% of the males who underwent primary screening. The incidence of prostate cancer was 0.96% during the 5 years. Clinical stage was B in 137 (69.2%), C in 52 (26.3%), D in 7 (3.5%), and unknown in 2. Surgery was performed in 87 (43.9%), endocrine therapy in 61 (30.8%), irradiation in 37 (18.7%), and follow up without treatment in 7 (3.5%). Treatment for 6 (3.0%) is unknown because they desired treatment at another hospital. CONCLUSIONS 198 males were diagnosed with prostate cancer between 2003 and 2007. The clinical stage B was present in 137 (69.2%), and the early treatment was achieved. This may lead to a future decrease in the mortality rate.

We present the case of a patient with renal cell carcinoma treated preoperatively with sorafenib. Complete resection of the left renal mass measuring 7.2 x 6.6 cm seemed to be difficult at diagnosis because of large renal hilar lymph nodes. With a short period of sorafenib administration, marked shrinkage of the renal mass and lymphadenopathy was observed after the patient experienced fulminant hepatic failure and a severe hand-foot skin reaction. Two-dimensional computed tomography revealed 60%, 78% and 84% reduction in the primary renal tumor, lung metastatic nodules and lymph nodes, respectively. Tumor shrinkage allowed for complete resection of the left kidney and the lymphadenopathy. Pathological findings revealed that over 90% of the renal tumor was substituted by necrotic fibrotic tissue and that the residual neoplastic component was diagnosed as clear cell carcinoma. The lymph nodes that were resected were negative for malignancy. At 6 months after radical nephrectomy, a new computed tomography scan revealed no evidence of disease with the disappearance of lung nodules.

Cases 1 and 2 were a 84-year-old, 64-year-old female relatively. Case 2 had a history of uncontrolled diabetes mellitus. Both cases were referred to our hospital with a chief complaint of high fever. Initial diagnosis was acute pyelonephritis based on the findings of pyuria and right costovertebral angle knock pain. Abdominal computed tomography (CT) scan revealed a gas shadow in the right renal pelvis and calyx with right ureteral stone. The definitive diagnosis was emphysematous pyelonephritis (EPN). We selected transureteral catheterization into the right ureter immediately. Escherichia coli was identified from urine culture. Conservative therapy with antibiotics was also effective and general condition improved. Herein we discussed the etiology, symptomatology, choice of treatment and prognosis of emphysematous pyelonephritis. Recently CT is an effective imaging method for diagnosis at an early stage. Antibiotics therapy combined with transureteral drainage of gas-forming urolithiasis is effective as the initial conservative therapy.

We herein report four cases of sarcomatoid renal cell carcinoma. These cases comprised 4.1% of the 98 patients with renal cell carcinoma treated in our department during the past 13 years. It is confirmed that renal cell carcinoma with a sarcomatoid component often shows local invasion, distant metastasis, rapid growth and poor prognosis. In Mian's series, the percentage of sarcomatoid component (25% vsor = 25%) was associated with decreased survival time, but was independent of stage. The pathological stage was pT3b N0 M0 in case 1, pT1b N0 M1 in case 2, pT3b N0 M1 in case 3 and pT1a N0 M1 in case 4. The pT1 sarcomatoid renal cell carcinoma in case 2 and case 4, who developed poor prognosis, was composed of 60 and 80% sarcomatoid change, respectively. However in case 3 with a pathological stage of pT3, the patient is alive 35 months after resection, because the extent of sarcomatoid component was 25%. The prognosis of patients with sarcomatoid renal cell carcinoma depends on not only disease stage and tumor grade but also the pathological extent of sarcomatoid component.