Prediction of clinically significant prostate cancer through urine metabolomic signatures: A large-scale validated study.

PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.

LMNB1, a potential marker for early prostate cancer progression.

Although prostate cancer (PC) is the most common cancer among men in the Western world, there are no good biomarkers that can reliably differentiate between potentially aggressive and indolent PC. This leads to overtreatment, even for patients who can be managed conservatively. Previous studies have suggested that nuclear lamin proteins-especially lamin B1 (LMNB1)-play important roles in PC progression. However, the results of these studies are inconsistent. Here, we transfected the LMNB1 gene into the telomerase reverse transcriptase-immortalized benign prostatic epithelial cell line, EP156T to generate a LMNB1-overexpressing EP156T (LMN-EP156T) cell line with increased cellular proliferation. However, LMN-EP156T cells could neither form colonies in soft agar, nor establish subcutaneous growth or metastasis in the xenograft NOD/SCID mouse model. In addition, immunohistochemical staining of LMNB1 in PC specimens from 143 patients showed a statistically significant trend of stronger LMNB1 staining with higher Gleason scores. A univariate analysis of the clinicopathological parameters of 85 patients with PC who underwent radical prostatectomy revealed that pathological stage, resection margin, and extracapsular extension were significant predictors for biochemical recurrence (BCR). However, LMNB1 staining showed only a non-significant trend of association with BCR (high vs. low staining: hazard ratio (HR), 1.83; 95% confidence interval (CI), 0.98-3.41; P = 0.059). In multivariate analysis, only pathological stage was a significant independent predictor of BCR (pT3 vs. pT2: HR, 2.29; 95% CI, 1.18-4.43; P = 0.014). In summary, LMNB1 may play a role in the early steps of PC progression, and additional molecular alterations may be needed to confer full malignancy potential to initiated cells.

Overexpression of Notch Signaling Induces Hyperosteogeny in Zebrafish.

Notch signaling is one of the evolutionarily conserved signaling pathways in multicellular organisms. It plays an important role in embryonic development. During skeletal development of vertebrates, it regulates bone homeostasis by manipulating both osteoblastogenesis and osteoclastogenesis through different mechanisms. However, due to the different nature of Notch signaling in mesenchymal stem cell and osteoblast, regulation of Notch signaling in bone-related diseases remains unsettled. Previous studies by cell culture and mouse models showed contradictory results regarding the role of Notch signaling in bone homeostasis. To clarify the role of Notch signaling in osteogenesis, we established a zebrafish model, in which Notch1a intracellular domain (N1aICD) was specifically expressed in the osteoblasts. We found that overexpression of N1aICD in osteoblasts caused hyperosteogeny in the column region of zebrafish with the morphology of narrowed neural/hemal canals. Moreover, increased metabolic activity of osteoblasts instead of augmenting osteoblast number led to hyperosteogeny in N1aICD-overexpressed zebrafish. In summary, we successfully established a transgenic zebrafish line overexpressing N1aICD to clarify the in-vivo function of Notch signaling during osteoblastogenesis. In the future, this fish line can serve as a valuable tool to test the therapeutic drugs for hyperosteogeny.

The Power of Fish Models to Elucidate Skin Cancer Pathogenesis and Impact the Discovery of New Therapeutic Opportunities.

Animal models play important roles in investigating the pathobiology of cancer, identifying relevant pathways, and developing novel therapeutic tools. Despite rapid progress in the understanding of disease mechanisms and technological advancement in drug discovery, negative trial outcomes are the most frequent incidences during a Phase III trial. Skin cancer is a potential life-threatening disease in humans and might be medically futile when tumors metastasize. This explains the low success rate of melanoma therapy amongst other malignancies. In the past decades, a number of skin cancer models in fish that showed a parallel development to the disease in humans have provided important insights into the fundamental biology of skin cancer and future treatment methods. With the diversity and breadth of advanced molecular genetic tools available in fish biology, fish skin cancer models will continue to be refined and expanded to keep pace with the rapid development of skin cancer research. This review begins with a brief introduction of molecular characteristics of skin cancers, followed by an overview of teleost models that have been used in the last decades in melanoma research. Next, we will detail the importance of the zebrafish (Danio rerio) animal model and other emerging fish models including platyfish (Xiphophorus sp.), and medaka (Oryzias latipes) in future cutaneous malignancy studies. The last part of this review provides the recent development and genome editing applications of skin cancer models in zebrafish and the progress in small molecule screening.

Cooperation of MLL/AF10(OM-LZ) with PTPN11 activating mutation induced monocytic leukemia with a shorter latency in a mouse bone marrow transplantation model.

PTPN11 mutation, a RAS signaling pathway mutation, is associated with MLL translocations in acute leukemia. A girl with MLL/AF10 AML was found to carry PTPN11G503A . To study the impact of PTPN11G503A cooperating with MLL/AF10 on leukemogenesis, we established a retroviral transduction/transplantation mouse model. Compared to the MLL/AF10(OM-LZ) leukemia cells harboring PTPN11wt , the cells harboring PTPN11G503A were hypersensitive to GM-CSF and IL3, and more resistant to death upon treatment with daunorubicin but sensitive to cytarabine. The cells harboring PTPN11G503A autonomously differentiated into macrophages (1.8%) in the medium containing IL3. Further studies showed that the cells had an elevated (∼2.9-fold) Csf1 transcription level and secreted more (∼4.5-fold) M-CSF to the medium which can stimulate monocyte/macrophage differentiation of BM cells. Mice transplanted with the cells harboring PTPN11G503A had a higher concentration of M-CSF in plasma. When mixed with the MLL/AF10(OM-LZ) leukemia cells harboring PTPN11wt , the cells harboring PTPN11G503A had an increased competitive engraftment and clonal expansion in the BM and spleen of recipient mice, although no competitive growth advantage was observed in the in vitro co-culturing assays. The mice transplanted with the MLL/AF10(OM-LZ) cells harboring PTPN11wt developed myelomonocytic leukemia, while those transplanted with the cells harboring PTPN11G503A -induced monocytic leukemia in a shorter latency. Our results demonstrated that addition of PTPN11G503A to MLL/AF10 affected cell proliferation, chemo-resistance, differentiation, in vivo BM recruitment/clonal expansion and accelerated disease progression.

High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

Biological Activities of RUNX1 Mutants Predict Secondary Acute Leukemia Transformation from Chronic Myelomonocytic Leukemia and Myelodysplastic Syndromes.

PURPOSE: Transcription factor RUNX1 is essential for normal hematopoiesis. High mutation frequencies of RUNX1 gene in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) have been described, whereas the biologic significances of the mutations were not investigated. Here, we aimed to correlate the biologic activities of the RUNX1 mutants with the clinical outcomes of patients. EXPERIMENTAL DESIGN: We examined the mutational status of RUNX1 in 143 MDS and 84 CMML patients. Then, we studied the DNA and CBFß binding abilities of all the RUNX1 mutants identified by using electrophoretic mobility shift assay and co-immunoprecipitation assay, and also determined their activities on target C-FMS gene induction by Western blotting and luciferase reporter assay. Using luciferase reporter assay, the relative biologic activities of each RUNX1 mutant could be quantified and correlated with the patient outcomes by statistical analyses. RESULTS: We observed that most RUNX1 mutants had reduced abilities in DNA binding, CBFß heterodimerization, and C-FMS gene induction. The relative biologic activities of RUNX1 mutants were grouped into high- and low-activity mutations. Correlation of the activities of RUNX1 mutants with the clinical outcomes revealed that patients harboring lower activities of RUNX1 mutants had a higher risk and shorter time to secondary acute myeloid leukemia transformation in MDS and CMML. In multivariate analysis, low RUNX1 activity remained an independent predictor for secondary acute myeloid leukemia-free survival in MDS patients. CONCLUSIONS: The biologic activity rather than the mutational status of RUNX1 might be an indicator in predicting outcome of patients with MDS and CMML.

Frequencies of ETV6-RUNX1 fusion and hyperdiploidy in pediatric acute lymphoblastic leukemia are lower in far east than west.

BACKGROUND: Both ETV6-RUNX1 (TEL-AML1) fusion and hyperdiploidy (>50 chromosomes) in transformed lymphoblasts are favorable genetic features in childhood acute lymphoblastic leukemia (ALL). PROCEDURE: Among 433 Taiwanese children with ALL diagnosed at our hospitals between 1997 and 2007, the ETV6-RUNX1 fusion was found in 15.8%, and hyperdiploidy (>50 chromosomes) in 14.1% of the patients. These frequencies were lower than those reported in the West, leading us to conduct a meta-analysis of ETV6-RUNX1 fusion and hyperdiploidy frequencies in childhood ALL based on published reports. RESULTS: The frequency of ETV6-RUNX1 fusion in the Far East (Japan, Korea, China, Hong Kong, Chinese in Singapore, and Taiwan) was 13.4% (177/1,321, range: 9-23%, median 13%), significantly lower than the 22.8% (1,664/7,291, range: 19-26%, median 23%) in the West (West Europe and the United States) (P < 0.001, odds ratio = 2.0, 95% CI: 1.7-2.4). Similarly, the frequency of hyperdiploidy in Japan and Taiwan was 14.3% (333/2,334, range: 12-20%, median 16%), significantly lower than the 25.2% in the West (5,173/20,510, range: 18-34%, median 23.5%; P < 0.001, odds ratio = 2.0, 95% CI: 1.8-2.3). CONCLUSIONS: This meta-analysis demonstrates lower frequencies of ETV6-RUNX1 fusion and hyperdiploidy among leukemia patients in the Far East compared with the West. The integral relationship of these genetic features with a favorable outcome in childhood ALL warrants further study of potentially important epidemiologic factors, including placental exposure to leukemogenic agents, and host pharmacogenetics.

In vitro antiangiogenic activity in ex vivo expanded human limbocorneal epithelial cells cultivated on human amniotic membrane.

PURPOSE: To compare the in vitro antiangiogenic activities of ex vivo expanded human limbocorneal epithelial (HLE) cells cultivated on preserved human amniotic membrane (AM) and to identify factors responsible for the activities. METHODS: The antiangiogenic effects were compared of culture media conditioned by AM, HLE cells, or HLE cells cultivated on intact AM (HLE/IAM), on denuded AM (HLE/DAM), or on DAM cocultured with 3T3 fibroblasts (HLE/DAM/3T3). A monolayer culture of human umbilical vein endothelial cells (ECs) was used in a proliferation and migration assay. ECs suspended in type I collagen gel were used to assess capillary tube formation. Quantitative analyses of tissue inhibitor of metalloproteinase (TIMP)-1, thrombospondin (TSP)-1, pigment epithelium-derived factor (PEDF), and endostatin (proteolytic fragment of collagen XVIII) were performed by ELISA. Immunoconfocal microscopy was performed to localize the site of endostatin expression in HLE cells and AM. RESULTS: HLE cell- but not AM-conditioned medium (CM) inhibited the proliferation and migration of ECs, and coculture of HLE cells, but not of AM, with ECs inhibited capillary tube formation. Although some data from HLE cells alone are not significantly different from the control, increased inhibitory activity was expressed by HLE/IAM and HLE/DAM and was most significantly expressed by HLE/DAM/3T3. Quantitation of TIMP-1, TSP-1, PEDF, and endostatin revealed that only the level of endostatin showed an increased expression by HLE cells cultivated on AM. Neutralizing antibody to endostatin substantially abrogated the inhibitory effect on EC proliferation and migration, but was less effective on EC differentiation. Endostatin signal was more prominent in the basement membrane zone of HLE cells cultivated on denuded AM than in those cultivated on intact AM. CONCLUSIONS: The antiangiogenic effect of HLE cells was enhanced when they were cultivated on AM and cocultured with 3T3 fibroblasts, and endostatin-related antiangiogenic factor may play a major role. This highlights the significance of cell-matrix and cell-cell interaction in the regulation of antiangiogenic factor secretion by HLE cells.

Functional analysis of EBV in nasopharyngeal carcinoma cells.

A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBV+), cells demonstrated different degrees of elevated gene expression. In our animal model, EBV+ tumors grew faster and larger than EBV-free, or EBV-negative (EBV-), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBV+ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBV- tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV.