The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

BACKGROUND

Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq).

OBJECTIVE

We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort.

METHODS

RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, ≥ 2.0; false discovery rate ≤ 0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation.

RESULTS

Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory (S100A8/A9/A12, CXCL1, and 2'-5'-oligoadenylate synthetase-like [OASL]) and barrier (MKi67, keratin 16 [K16], and claudin 8 [CLDN8]) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation.

CONCLUSIONS

This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.

Temozolomide (TMZ) was used for the treatment of glioblastoma (GBM) for over a decade, but its treatment benefits are limited by acquired resistance, a process that remains incompletely understood. Here we report that an enhancer, located between the promoters of marker of proliferation Ki67 (MKI67) and O6-methylguanine-DNA-methyltransferase (MGMT) genes, is activated in TMZ-resistant patient-derived xenograft (PDX) lines and recurrent tumor samples. Activation of the enhancer correlates with increased MGMT expression, a major known mechanism for TMZ resistance. We show that forced activation of the enhancer in cell lines with low MGMT expression results in elevated MGMT expression. Deletion of this enhancer in cell lines with high MGMT expression leads to a dramatic reduction of MGMT and a lesser extent of Ki67 expression, increased TMZ sensitivity, and impaired proliferation. Together, these studies uncover a mechanism that regulates MGMT expression, confers TMZ resistance, and potentially regulates tumor proliferation.

We examined estrogen receptor (ER) mRNA expression and molecular subtypes in stage I-III breast cancers that are progesterone receptor (PR) positive but ER and HER2 negative by immunohistochemistry (IHC) or fluorescent in situ hybridization. The ER, PR, and HER2 status was determined by IHC as part of routine clinical assessment (N = 501). Gene expression profiling was done with the Affymetrix U133A gene chip. We compared expressions of ESR1 and MKI67 mRNA, distribution of molecular subtypes by the PAM50 classifier, the sensitivity to endocrine therapy index, and the DLDA30 chemotherapy response predictor signature among ER/PR-positive (n = 223), ER-positive/PR-negative (n = 73), ER-negative/PR-positive (n = 20), and triple-negative (n = 185) cancers. All patients received neoadjuvant chemotherapy with an anthracycline and taxane and had adjuvant endocrine therapy only if ER or PR>> 10 % positive. ESR1 expression was high in 25 % of ER-negative/PR-positive, in 79 % of ER-positive/PR-negative, in 96 % of ER/PR-positive, and in 12 % of triple-negative cancers by IHC. The average MKI67 expression was significantly higher in the ER-negative/PR-positive and triple-negative cohorts. Among the ER-negative/PR-positive patients, 15 % were luminal A, 5 % were Luminal B, and 65 % were basal like. The relapse-free survival rate of ER-negative/PR-positive patients was equivalent to ER-positive cancers and better than the triple-negative cohort. Only 20-25 % of the ER-negative/PR-positive tumors show molecular features of ER-positive cancers. In this rare subset of patients (i) a second RNA-based assessment may help identifying the minority of ESR1 mRNA-positive, luminal-type cancers and (ii) the safest clinical approach may be to consider both adjuvant endocrine and chemotherapy.

BACKGROUND

Dupilumab is an IL-4 receptor α mAb inhibiting signaling of IL-4 and IL-13, key drivers of type 2-driven inflammation, as demonstrated by its efficacy in patients with atopic/allergic diseases.

OBJECTIVE

This placebo-controlled, double-blind trial (NCT01979016) evaluated the efficacy, safety, and effects of dupilumab on molecular/cellular lesional and nonlesional skin phenotypes and systemic type 2 biomarkers of patients with moderate-to-severe atopic dermatitis (AD).

METHODS

Skin biopsy specimens and blood were evaluated from 54 patients randomized 1:1 to weekly subcutaneous doses of 200 mg of dupilumab or placebo for 16 weeks.

RESULTS

Dupilumab (vs placebo) significantly improved clinical signs and symptoms of AD, was well tolerated, and progressively shifted the lesional transcriptome toward a nonlesional phenotype (weeks 4-16). Mean improvements in a meta-analysis-derived AD transcriptome (genes differentially expressed between lesional and nonlesional skin) were 68.8% and 110.8% with dupilumab and -10.5% and 55.0% with placebo (weeks 4 and 16, respectively; P < .001). Dupilumab significantly reduced expression of genes involved in type 2 inflammation (IL13, IL31, CCL17, CCL18, and CCL26), epidermal hyperplasia (keratin 16 [K16] and MKi67), T cells, dendritic cells (ICOS, CD11c, and CTLA4), and TH17/TH22 activity (IL17A, IL-22, and S100As) and concurrently increased expression of epidermal differentiation, barrier, and lipid metabolism genes (filaggrin [FLG], loricrin [LOR], claudins, and ELOVL3). Dupilumab reduced lesional epidermal thickness versus placebo (week 4, P = .001; week 16, P = .0002). Improvements in clinical and histologic measures correlated significantly with modulation of gene expression. Dupilumab also significantly suppressed type 2 serum biomarkers, including CCL17, CCL18, periostin, and total and allergen-specific IgEs.

CONCLUSIONS

Dupilumab-mediated inhibition of IL-4/IL-13 signaling through IL-4 receptor α blockade significantly and progressively improved disease activity, suppressed cellular/molecular cutaneous markers of inflammation and systemic measures of type 2 inflammation, and reversed AD-associated epidermal abnormalities.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3' untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention.

BACKGROUND

Dyskerin encoded by the DKC1 gene is a predominantly nucleolar protein essential for the formation of pseudouridine in RNA and the telomerase RNA subunit hTR. Inherited mutations inactivating dyskerin cause dyskeratosis congenita, a syndrome with progeroid features characterised by skin defects and haematopoiesis failure, as well as cancer susceptibility. In this study, we report DKC1 overexpression in prostate cancers.

METHODS

Expression of DKC1 was measured by quantitative RT-PCR in prostate cancer tissues in relation to hTR and the proliferation marker MKI67. Effects of dyskerin downregulation on proliferation, apoptosis and senescence of prostate cancer cell lines were determined.

RESULTS

DKC1 was significantly overexpressed in prostate cancers, particularly in high-stage and recurring cases, correlating moderately with hTR and MKI67. Dyskerin downregulation in prostate carcinoma cell lines by siRNA diminished cell proliferation, but elicited neither apoptosis nor senescence. Apoptosis induction by TNF-alpha or tunicamycin was not enhanced. Long-term downregulation led predominantly to cell shrinking and loss of adhesion.

CONCLUSIONS

DKC1 upregulation in prostate cancers is common and likely to be necessary for extensive tumour growth. The phenotype of prostate carcinoma cell lines after dyskerin downregulation suggests that its most critical function is sustaining protein biosynthesis. Intriguingly, compromised function and overexpression of dyskerin can both contribute to cancer development.

Identification and characterization of crucial gene target(s) that will allow focused therapeutics development remains a challenge. We have interrogated the putative therapeutic targets associated with the transcription factor Grainy head-like 2 (GRHL2), a critical epithelial regulatory factor. We demonstrate the possibility to define the molecular functions of critical genes in terms of their personalized expression profiles, allowing appropriate functional conclusions to be derived. A novel methodology, relative expression analysis with gene-set pairs (RXA-GSP), is designed to explore the potential clinical utility of cancer-biology discovery. Observing that Grhl2-overexpression leads to increased metastatic potential in vitro, we established a model assuming Grhl2-induced or -inhibited genes confer poor or favorable prognosis respectively for cancer metastasis. Training on public gene expression profiles of 995 breast cancer patients, this method prioritized one gene-set pair (GRHL2, CDH2, FN1, CITED2, MKI67 versus CTNNB1 and CTNNA3) from all 2717 possible gene-set pairs (GSPs). The identified GSP significantly dichotomized 295 independent patients for metastasis-free survival (log-rank tested p = 0.002; severe empirical p = 0.035). It also showed evidence of clinical prognostication in another independent 388 patients collected from three studies (log-rank tested p = 3.3e-6). This GSP is independent of most traditional prognostic indicators, and is only significantly associated with the histological grade of breast cancer (p = 0.0017), a GRHL2-associated clinical character (p = 6.8e-6, Spearman correlation), suggesting that this GSP is reflective of GRHL2-mediated events. Furthermore, a literature review indicates the therapeutic potential of the identified genes. This research demonstrates a novel strategy to integrate both biological experiments and clinical gene expression profiles for extracting and elucidating the genomic impact of a novel factor, GRHL2, and its associated gene-sets on the breast cancer prognosis. Importantly, the RXA-GSP method helps to individualize breast cancer treatment. It also has the potential to contribute considerably to basic biological investigation, clinical tools, and potential therapeutic targets.

Endothelial progenitor cells (EPCs) play an important role in accelerating endothelial repair after vascular injury. The proliferation and migration of EPCs is a critical first step in restoring endothelial. However, mechanisms for modulating EPC proliferation and migration are still being elucidated. Our previous study found that transient receptor potential canonical-1 (TRPC1) is involved in regulating store-operated Ca(2+) entry in EPCs through stromal interaction molecule 1. Therefore, in the present study, we sought to further investigate the regulation of proliferation and migration of EPCs by TRPC1. We found that the silencing of TRPC1 by 2 different RNA interference methods suppressed the proliferation and migration of EPCs. In addition, knockdown of TRPC1 significantly reduced of the amplitude of store-operated Ca(2+) entry and caused arrest of the EPC cell cycle in G1 phase. Analysis of the expression of 84 cell cycle genes by microarray showed that 9 genes were upregulated and 4 were downregulated by >2-fold in EPCs following TRPC1 silencing. The genes with expression changes were Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, Prm1, Bcl2, Mki67, Pmp22, and Ppp2r3a. Finally, we found that a Schlafen 1-blocking peptide partially reversed the abnormal cell cycle distribution and proliferation induced by TRPC1 knockdown, suggesting that Schlafen 1 is downstream of TRPC1 silencing in regulating EPC proliferation. In summary, these findings provide a new mechanism for modulating the biological properties of EPCs and suggest that TRPC1 may be a new target for inducing vascular repair by EPCs.

OBJECTIVE

We studied the ethnicity-specific expression of prostate cancer (PC) -associated biomarkers to evaluate whether genetic/biologic factors affect ethnic disparities in PC pathogenesis and disease progression.

METHODS

A total of 154 African American (AA) and 243 European American (EA) patients from four medical centers were matched according to the Cancer of the Prostate Risk Assessment postsurgical score within each institution. The distribution of mRNA expression levels of 20 validated biomarkers reported to be associated with PC initiation and progression was compared with ethnicity using false discovery rate, adjusted Wilcoxon-Mann-Whitney, and logistic regression models. A conditional logistic regression model was used to evaluate the interaction between ethnicity and biomarkers for predicting clinicopathologic outcomes.

RESULTS

Of the 20 biomarkers examined, six showed statistically significant differential expression in AA compared with EA men in one or more statistical models. These include ERG (P < .001), AMACR (P < .001), SPINK1 (P = .001), NKX3-1 (P = .03), GOLM1 (P = .03), and androgen receptor (P = .04). Dysregulation of AMACR (P = .036), ERG (P = .036), FOXP1 (P = .041), and GSTP1 (P = .049) as well as loss-of-function mutations for tumor suppressors NKX3-1 (P = .025) and RB1 (P = .037) predicted risk of pathologic T3 disease in an ethnicity-dependent manner. Dysregulation of GOLM1 (P = .037), SRD5A2 (P = .023), and MKi67 (P = .023) predicted clinical outcomes, including 3-year biochemical recurrence and metastasis at 5 years. A greater proportion of AA men than EA men had triple-negative (ERG-negative/ETS-negative/SPINK1-negative) disease (51% v 35%; P = .002).

CONCLUSIONS

We have identified a subset of PC biomarkers that predict the risk of clinicopathologic outcomes in an ethnicity-dependent manner. These biomarkers may explain in part the biologic contribution to ethnic disparity in PC outcomes between EA and AA men.

The objective of this study was to examine the association between the immunohistochemical Ki67 labeling index (IHC Ki67), Ki67 mRNA expression level, and first-generation gene signatures in a cohort of breast cancer patients. We assessed associations between IHC Ki67 and first-generation gene signatures in a panel of 39 tumor samples, using an oligonucleotide microarray. Gene expression analyses included Ki67 alone (MKi67), 21-gene signature, mitosis kinome score signature, and genomic grade index. Correlation coefficients were calculated by Spearman's rank correlation test. In all cases, IHC Ki67, MKi67, and three genetic markers were highly correlated (ρ, 0.71-0.97). Estrogen receptor (ER)-positive cases showed strong correlations between IHC Ki67 and other signatures (ρ, 0.79-0.83). The ER-negative cases showed slightly lower correlations (ρ, 0.58-0.73). In ER-positive cases, the low IHC Ki67 group showed significantly longer relapse-free survival than the high IHC Ki67 group (P = 0.007). This difference was confirmed by multivariate analysis. Our data indicate that IHC Ki67 shows similar predictive power for proliferation in ER-positive cancers as genomic markers. Further study of IHC Ki67 is needed to define prognostic factors and predictive factors for chemotherapy using central laboratory assessment.

BACKGROUND

Retrospective studies demonstrated that cell cycle-related and proliferation biomarkers add information to standard pathologic tumor features after radical cystectomy (RC). There are no prospective studies validating the clinical utility of markers in bladder cancer.

OBJECTIVE

To prospectively determine whether a panel of biomarkers could identify patients with urothelial carcinoma of the bladder (UCB) who were likely to experience disease recurrence or mortality.

METHODS

Between January 2007 and January 2012, every patient with high-grade bladder cancer, including 216 patients treated with RC and lymphadenectomy, underwent immunohistochemical staining for tumor protein p53 (Tp53); cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A); cyclin-dependent kinase inhibitor 1B (p27, Kip1); antigen identified by monoclonal antibody Ki-67 (MKI67); and cyclin E1.

METHODS

Every patient underwent RC and lymphadenectomy, and marker staining.

METHODS

Cox regression analyses tested the ability of the number of altered biomarkers to predict recurrence or cancer-specific mortality (CSM).

CONCLUSIONS

Pathologic stage among the study population was pT0 (5%), pT1 (35%), pT2 (19%), pT3 (29%), and pT4 (13%); lymphovascular invasion (LVI) was seen in 34%. The median number of removed lymph nodes was 23, and 60 patients had lymph node involvement (LNI). Median follow-up was 20 mo. Expression of p53, p21, p27, cyclin E1, and Ki-67 were altered in 54%, 26%, 46%, 15%, and 75% patients, respectively. In univariable analyses, pT stage, LNI, LVI, perioperative chemotherapy (CTx), margin status, and number of altered biomarkers predicted disease recurrence. In a multivariable model adjusting for pathologic stage, margins, LNI, and adjuvant CTx, only LVI and number of altered biomarkers were independent predictors of recurrence and CSM. The concordance index of a baseline model predicting CSM (including pathologic stage, margins, LVI, LNI, and adjuvant CTx) was 80% and improved to 83% with addition of the number of altered markers.

CONCLUSIONS

Molecular markers improve the prediction of recurrence and CSM after RC. They may identify patients who might benefit from additional treatments and closer surveillance after cystectomy.

OBJECTIVE

To examine the molecular profile of fetal human retinal progenitor cells (hRPCs) expanded in vitro and those grown in a co-culture system with mouse retina through the analysis of protein and gene expression and neurotransmitter-stimulated calcium dynamics.

METHODS

hRPCS were isolated from human retina of 14 to 18 weeks gestational age (GA) and expanded in vitro. Immunoblot, microarray, and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for 2 weeks. Cell function was assessed by using calcium imaging.

RESULTS

The ICC results showed a gradual decrease in the percentages of KI67-, SOX2-, and vimentin-positive cells from passages (P) 1 to P6, whereas a sustained expression of nestin and PAX6 was observed through P6. Microarray analysis of P1 hRPCs showed the expression of early retinal developmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). At P6, hRPCs continued to express VIM, KI67, NES, PAX6, SOX2, GNL3, and SIX6. On co-culture, there was a significant increase in the expression of MKI67, PAX6, SOX2, GNL3, SIX3, and RHO (rhodopsin). Calcium imaging showed a functional response to excitatory neurotransmitters.

CONCLUSIONS

Fetal-derived hRPCs show molecular characteristics indicative of a retinal progenitor state up to P6 (latest passage studied). They show a progressive decrease in the expression of immature markers as they reach P6. These cells are functional, respond to excitatory neurotransmitters, and exhibit changes in expression patterns in response to co-culture with mouse retina.

BACKGROUND

Until now, almost nothing is known about the tumorigenesis of atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). Our hypothesis is that AFX is the non-infiltrating precursor lesion of PDS.

METHODS

We performed the world-wide most comprehensive immunohistochemical and mutational analysis in well-defined AFX (n=5) and PDS (n=5).

RESULTS

In NGS-based mutation analyses of selected regions by a 17 hotspot gene panel of 102 amplicons we could detect TP53 mutations in all PDS as well as in the only analyzed AFX and PDS of the same patient. Besides, we detected mutations in the CDKN2A, HRAS, KNSTRN and PIK3CA genes.Performing immunohistochemistry for CTNNB1, KIT, CDK4, c-MYC, CTLA-4, CCND1, EGFR, EPCAM, ERBB2, IMP3, INI-1, MKI67, MDM2, MET, p40, TP53, PD-L1 and SOX2 overexpression of TP53, CCND1 and CDK4 was seen in AFX as well as in PDS. IMP3 was upregulated in 2 AFX (weak staining) and 4 PDS (strong staining).FISH analyses for the genes FGFR1, FGFR2 and FGFR3 revealed negative results in all tumors.

CONCLUSIONS

UV-induced TP53 mutations as well as CCND1/CDK4 changes seem to play essential roles in tumorigenesis of PDS. Furthermore, we found some more interesting mutated genes in other oncogene pathways (activating mutations of HRAS and PIK3CA). All AFX and PDS investigated immunohistochemically presented with similar oncogene expression profiles (TP53, CCND1, CDK4 overexpression) and the single case with an AFX and PDS showed complete identical TP53 and PIK3CA mutation profiles in both tumors. This reinforces our hypothesis that AFX is the non-infiltrating precursor lesion of PDS.

BACKGROUND

Tamoxifen is the most widely prescribed anti-estrogen treatment for patients with estrogen receptor (ER)-positive breast cancer. However, there is still a need for biomarkers that reliably predict endocrine sensitivity in breast cancers and these may well be expressed in a dynamic manner.

METHODS

In this study we assessed gene expression changes at multiple time points (days 1, 2, 4, 7, 14) after tamoxifen treatment in the ER-positive ZR-75-1 xenograft model that displays significant changes in apoptosis, proliferation and angiogenesis within 2 days of therapy.

RESULTS

Hierarchical clustering identified six time-related gene expression patterns, which separated into three groups: two with early/transient responses, two with continuous/late responses and two with variable response patterns. The early/transient response represented reductions in many genes that are involved in cell cycle and proliferation (e.g. BUB1B, CCNA2, CDKN3, MKI67, UBE2C), whereas the continuous/late changed genes represented the more classical estrogen response genes (e.g. TFF1, TFF3, IGFBP5). Genes and the proteins they encode were confirmed to have similar temporal patterns of expression in vitro and in vivo and correlated with reduction in tumour volume in primary breast cancer. The profiles of genes that were most differentially expressed on days 2, 4 and 7 following treatment were able to predict prognosis, whereas those most changed on days 1 and 14 were not, in four tamoxifen treated datasets representing a total of 404 patients.

CONCLUSIONS

Both early/transient/proliferation response genes and continuous/late/estrogen-response genes are able to predict prognosis of primary breast tumours in a dynamic manner. Temporal expression of therapy-response genes is clearly an important factor in characterising the response to endocrine therapy in breast tumours which has significant implications for the timing of biopsies in neoadjuvant biomarker studies.

Prostate cancer exhibits tremendous variability in clinical behavior, ranging from indolent to lethal disease. Better prognostic markers are needed to stratify patients for appropriately aggressive therapy. By expression profiling, we can identify a proliferation signature variably expressed in prostate cancers. Here, we asked whether one or more tissue biomarkers might capture that information, and provide prognostic utility. We assayed three proliferation signature genes: MKI67 (Ki-67; also a classic proliferation biomarker), TOP2A (DNA topoisomerase II, alpha), and E2F1 (E2F transcription factor 1). Immunohistochemical staining was evaluable on 139 radical prostatectomy cases (in tissue microarray format), with a median clinical follow-up of eight years. Each of the three proliferation markers was by itself prognostic. Notably, combining the three markers together as a "proliferation index" (0 or 1, vs. 2 or 3 positive markers) provided superior prognostic performance (hazard ratio = 2.6 (95% CI: 1.4-4.9); P = 0.001). In a multivariate analysis that included preoperative serum prostate specific antigen (PSA) levels, Gleason grade and pathologic tumor stage, the composite proliferation index remained a significant predictor (P = 0.005). Analysis of receiver-operating characteristic (ROC) curves confirmed the improved prognostication afforded by incorporating the proliferation index (compared to the clinicopathologic data alone). Our findings highlight the potential value of a multi-gene signature-based diagnostic, and define a tri-marker proliferation index with possible utility for improved prognostication and treatment stratification in prostate cancer.

The aim of this study is to set up single molecular secreted phosphoprotein 1 (SPP1) upstream invasive network of lung adenocarcinoma. This paper proposed an integrated method based on linear programming and a decomposition procedure with integrated analysis of the significant function cluster using Kappa statistics and fuzzy heuristic clustering. Our study proved that only modules appearing in lung adenocarcinoma include cytokine module (CXCL13, GREM1_2 inhibition), cell adhesion module (COL11A1_2 activation; CDH3 inhibition), and receptor binding module (NMU activation; CXCL13, GREM1_2 inhibition), which increase the invasion of cancer cell. We compared skeletal development, signal, biological regulation, sequence variant modules between human normal adjacent tissues and lung adenocarcinoma. SPP1 skeletal development module appears in human normal adjacent tissues (COL11A1_1 activation; COL10A1 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2 activation); signal module appears in human normal adjacent tissues (COL11A1_1, CXCL13, MMP11, SPINK1 activation; COL10A1, COL3A1 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2, MMP12 activation; CDH3, CXCL13, GREM1_2, MMP11, SPINK1 inhibition); biological regulation module appears in human normal adjacent tissues (CXCL13, MKI67, PYCR1 activation; NEK2, SPDEF, TOP2A_2, TOX3_1 inhibition), whereas in lung adenocarcinoma (HMGB3, MKI67, NMU, PYCR1, TOX3_2 activation; CXCL13, SPDEF, TOP2A_2 inhibition); sequence variant module appears in human normal adjacent tissues (COL11A1_1, MKI67, MMP11 activation; ASPM, COL10A1, COL3A1, NEK2, TMPRSS4, TOP2A_2 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2, HMMR, MKI67, MMP12 activation; ABCC3, ASPM, CDH3, MMP11, TOP2A_2 inhibition). It can be deduced that modules above in human normal adjacent tissues reflect the invasive inhibition of normal cells, whereas in lung adenocarcinoma increase the invasion of cancer cell. Our study of SPP1 upstream invasive network may be useful to identify novel and potentially targets for prognosis and therapy of lung adenocarcinoma.

BACKGROUND

Follicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.

RESULTS

Using a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.

CONCLUSIONS

Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.

The PEA3/E1AF/ETV4 gene encodes an Ets-related transcription factor that is expressed in the epithelial cells of the mammary gland. Previous reports have shown that PEA3 can up-regulate promoter activities of many genes associated with tumorigenesis. A significant fraction of those encode matrix metalloproteinases (MMP genes) required for degradation of the extracellular matrix. To better obtain a molecular characterization of PEA3 expression in sporadic breast cancer, we quantified PEA3 mRNA by means of real-time reverse transcriptase-polymerase chain reaction assay in a large series of human primary breast tumors. PEA3 expression showed wide variations in tumor tissues, being under-expressed in 30 of 130 (23.1%) and over-expressed in 18 of 130 (13.8%) compared with normal breast tissues. High PEA3 mRNA levels correlated significantly with Scarff-Bloom-Richardson histopathological grade III (P = 0.018) but not with poor prognosis, suggesting that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. We also observed positive links between the expression of PEA3 and those of MKI67 and ERBB2 (P = 0.034 and P = 0.045, respectively) and an inverse relationship with ERalpha (P = 0.0016). Our results do not support recent findings suggesting that PEA3 could be a tumor-suppressor gene that can act therapeutically in ERBB2 over-expressed tumors. Our results also suggest major roles of the MMP2, NRG1 and CGB genes (which encode type I gelatinase, heregulin and human chorionic gonadotropin beta subunit, respectively) in the PEA3 pathway dysregulation observed in breast cancer. Taken together, the data confirm the role of the PEA3 gene in breast tumorigenesis, and suggest the existence of numerous other still unknown genes transactivated by the PEA3 transcription factor.

BACKGROUND

Ki67 is a protein associated with cell cycle activity and shows a good correlation with the growth fraction, which has been proposed as a prognostic or predictive marker in breast cancer. In this study, we aimed to analyze the expression levels of Ki67 (MKI67) messenger RNA (mRNA) derived from formalin-fixed paraffin-embedded (FFPE) tissues for comparison with the immunohistochemical Ki67 labeling index, and investigate the correlation coefficients with clinical outcomes.

METHODS

We analyzed the data of Ki67 mRNA from FFPE and matched fresh-frozen (FF) tissues based on a real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay system in 203 cases of primary invasive breast cancer.

RESULTS

The correlation between Ki67 mRNA expression of either FFPE or FF specimens and Ki67 labeling index was positive, as was the correlation between the FFPE and FF results (P < 0.0001). Ki67 mRNA expression of FFPE specimens was significantly associated with clinicopathological characteristics: tumor size, lymph node status, nuclear grade, hormone receptors, human epidermal growth factor receptor 2 (Her2) status, and tumor subtype. In prognostic results, Ki67 gene expression in the FFPE specimens revealed almost similar patterns of significance in Kaplan-Meier curves and univariate and multivariate relapse-free survival results as the Ki67 labeling index.

CONCLUSIONS

Gene expression analysis of Ki67 of FFPE specimens could be successfully performed using RT-qPCR, closely resembling the significant clinical characteristics of Ki67 labeling index. These results confirm that Ki67 gene expression of FFPE specimens has potential for evaluation of cell cycle activity of breast cancer specimens.

The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes.

BACKGROUND

Prognostic multibiomarker signatures in prostate cancer (PCa) may improve patient management and provide a bridge for developing novel therapeutics and imaging methods. Our objective was to evaluate the association between expression of 33 candidate protein biomarkers and time to biochemical failure (BF) after prostatectomy.

METHODS

PCa tissue microarrays were constructed representing 160 patients for whom clinicopathologic features and follow-up data after surgery were available. Immunohistochemistry for each of 33 proteins was quantified using automated digital pathology techniques. Relationships between clinicopathologic features, staining intensity, and time to BF were assessed. Predictive modeling using multiple imputed datasets was performed to identify the top biomarker candidates.

RESULTS

In univariate analyses, lymph node positivity, surgical margin positivity, non-localized tumor, age at prostatectomy, and biomarkers CCND1, HMMR, IGF1, MKI67, SIAH2, and SMAD4 in malignant epithelium were significantly associated with time to BF. HMMR, IGF1, and SMAD4 remained significantly associated with BF after adjusting for clinicopathologic features while additional associations were observed for HOXC6 and MAP4K4 following adjustment. In multibiomarker predictive models, 3 proteins including HMMR, SIAH2, and SMAD4 were consistently represented among the top 2, 3, 4, and 5 most predictive biomarkers, and a signature comprised of these proteins best predicted BF at 3 and 5 years.

CONCLUSIONS

This study provides rationale for investigation of HMMR, HOXC6, IGF1, MAP4K4, SIAH2, and SMAD4 as biomarkers of PCa aggressiveness in larger cohorts.

BACKGROUND

Dyskerin (encoded by the DKC1 gene) is an essential nucleolar protein involved in cell proliferation, where it is required for the pseudo-uridylation of ribosomal RNA (rRNA) molecules and the stabilization of the telomerase RNA component. Dyskerin expression has been reported to predict poor survival in some cancer patients. The aim of the present study was to analyze the expression of dyskerin in hepatocellular carcinoma (HCC) and to determine its correlation with clinicopathologic features, including the survival of patients with HCC.

RESULTS

Dyskerin protein expression was detected by immunohistochemistry in paraffin sections of 252 HCC cases and 80 noncancerous liver tissues. The correlation was analyzed between dyskerin expression levels and clinicopathologic variables and prognosis. Dyskerin protein was significantly overexpressed in HCC tissues when compared to noncancerous liver tissue. Dyskerin overexpression was positively correlated with the hepatitis B surface antigen status, serum alpha-fetoprotein, and advanced clinical stage in HCC patients. A survival analysis indicated that HCC patients with higher dyskerin expression had a significantly shorter overall survival and 5-year survival time when compared to those with low expression. A multivariate analysis suggested that dyskerin overexpression was an independent factor for prognosis (hazard risk, 2.912; P = 0.007). Expression of DKC1 mRNA was measured by quantitative RT-PCR in 80 HCC and 50 non-cancerous tissues. The relationship between DKC1, TERT, MKI67, and MYC mRNA expression in HCC tissues was also evaluated. DKC1 mRNA was significantly overexpressed in HCC tissues and showed a significant correlation with MKI67 and MYC mRNA but a weak correlation with TERT mRNA.

CONCLUSIONS

Dyskerin overexpression in HCC patients was correlated with MYC and MKI67 expression and showed a possible involvement in the tumorigenic process. Dyskerin overexpression may be an unfavorable prognostic factor in patients with HCC.