Validating prognostic or predictive candidate genes in appropriately powered breast cancer cohorts are of utmost interest. Our aim was to develop an online tool to draw survival plots, which can be used to assess the relevance of the expression levels of various genes on the clinical outcome both in untreated and treated breast cancer patients. A background database was established using gene expression data and survival information of 1,809 patients downloaded from GEO (Affymetrix HGU133A and HGU133+2 microarrays). The median relapse free survival is 6.43 years, 968/1,231 patients are estrogen-receptor (ER) positive, and 190/1,369 are lymph-node positive. After quality control and normalization only probes present on both Affymetrix platforms were retained (n = 22,277). In order to analyze the prognostic value of a particular gene, the cohorts are divided into two groups according to the median (or upper/lower quartile) expression of the gene. The two groups can be compared in terms of relapse free survival, overall survival, and distant metastasis free survival. A survival curve is displayed, and the hazard ratio with 95% confidence intervals and logrank P value are calculated and displayed. Additionally, three subgroups of patients can be assessed: systematically untreated patients, endocrine-treated ER positive patients, and patients with a distribution of clinical characteristics representative of those seen in general clinical practice in the US. Web address: www.kmplot.com . We used this integrative data analysis tool to confirm the prognostic power of the proliferation-related genes TOP2A and TOP2B, MKI67, CCND2, CCND3, CCNDE2, as well as CDKN1A, and TK2. We also validated the capability of microarrays to determine estrogen receptor status in 1,231 patients. The tool is highly valuable for the preliminary assessment of biomarkers, especially for research groups with limited bioinformatic resources.

OBJECTIVE

Three genes, namely DNA methyltransferase (DNMT) 1, DNMT3A, and DNMT3B, coding for DNMTs that affect promoter methylation status are thought to play an important role in the development of cancers. Little is known of the biological and clinical significance of these genes in human breast cancer.

METHODS

We used real-time reverse transcription-PCR assays to quantify the mRNA expression of the three DNMT genes in a series of 130 breast cancer patients. We also sought relationships between mRNA levels of the DNMTs and those of 20 target genes involved in the DNMT pathway (subgroup of 46 breast tumors).

RESULTS

The DNMT3B gene showed the highest range of expression (81.8 compared with 16.6 and 14 for DNMT1 and DNMT3A, respectively). DNMT3B was overexpressed in 30% of the patients (5.4 and 3.1% for DNMT1 and DNMT3A, respectively). DNMT3B overexpression was significantly related to Scarff, Bloom, and Richardson histopathological grade III (P = 0.002), ERalpha negativity (P = 0.0015), and strong MKI67 expression (P = 3 x 10(-6)). In univariate analysis, DNMT3B overexpression was associated with poor relapse-free survival in the subgroup of patients who received adjuvant hormone therapy (with or without chemotherapy; P = 0.0064). Although the poor prognosis associated with DNMT3B overexpression was confirmed by univariate analysis in an independent series of 98 postmenopausal women exclusively treated with adjuvant tamoxifen therapy (P = 0.0036), DNMT3B expression status did not persist as an independent prognostic factor in multivariate analysis.

CONCLUSIONS

Although we failed to identify underexpression of specific target genes associated with DNMT increasing expression, the frequent overexpression of DNMT3B in this breast tumor series points to DNMT3B as a potential new therapeutic target in breast cancer.

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

BACKGROUND

Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity in patients with multiple myeloma, often requiring adjustment of treatment and affecting quality of life. We investigated the molecular profiles of early-onset (within one treatment cycle) versus late-onset (after two or three treatment cycles) bortezomib-induced peripheral neuropathy and compared them with those of vincristine-induced peripheral neuropathy during the induction phase of a prospective phase 3 trial.

METHODS

In the induction phase of the HOVON-65/GMMG-HD4 trial, patients (aged 18-65 years) with newly diagnosed Salmon and Durie stage 2 or 3 multiple myeloma were randomly assigned to three cycles of bortezomib-based or vincristine-based induction treatment. We analysed the gene expression profiles and single-nucleotide polymorphisms (SNPs) of pretreatment samples of myeloma plasma cells and peripheral blood, respectively. This study is registered, number ISRCTN64455289.

RESULTS

We analysed gene expression profiles of myeloma plasma cells from 329 (39%) of 833 patients at diagnosis, and SNPs in DNA samples from 369 (44%) patients. Early-onset bortezomib-induced peripheral neuropathy was noted in 20 (8%) patients, and 63 (25%) developed the late-onset type. Early-onset and late-onset vincristine-induced peripheral neuropathy was noted in 11 (4%) and 17 (7%) patients, respectively. Significant genes in myeloma plasma cells from patients that were associated with early-onset bortezomib-induced peripheral neuropathy were the enzyme coding genes RHOBTB2 (upregulated by 1·59 times; p=4·5×10(-5)), involved in drug-induced apoptosis, CPT1C (1·44 times; p=2·9×10(-7)), involved in mitochondrial dysfunction, and SOX8 (1·68 times; p=4·28×10(-13)), involved in development of peripheral nervous system. Significant SNPs in the same patients included those located in the apoptosis gene caspase 9 (odds ratio [OR] 3·59, 95% CI 1·59-8·14; p=2·9×10(-3)), ALOX12 (3·50, 1·47-8·32; p=3·8×10(-3)), and IGF1R (0·22, 0·07-0·77; p=8·3×10(-3)). In late-onset bortezomib-induced peripheral neuropathy, the significant genes were SOD2 (upregulated by 1·18 times; p=9·6×10(-3)) and MYO5A (1·93 times; p=3·2×10(-2)), involved in development and function of the nervous system. Significant SNPs were noted in inflammatory genes MBL2 (OR 0·49, 95% CI 0·26-0·94; p=3·0×10(-2)) and PPARD (0·35, 0·15-0·83; p=9·1×10(-3)), and DNA repair genes ERCC4 (2·74, 1·56-4·84; p=1·0×10(-3)) and ERCC3 (1·26, 0·75-2·12; p=3·3×10(-3)). By contrast, early-onset vincristine-induced peripheral neuropathy was characterised by upregulation of genes involved in cell cycle and proliferation, including AURKA (3·31 times; p=1·04×10(-2)) and MKI67 (3·66 times; p=1·82×10(-3)), and the presence of SNPs in genes involved in these processes-eg, GLI1 (rs2228224 [0·13, 0·02-0·97, p=1·18×10(-2)] and rs2242578 [0·14, 0·02-1·12, p=3·00×10(-2)]). Late-onset vincristine-induced peripheral neuropathy was associated with the presence of SNPs in genes involved in absorption, distribution, metabolism, and excretion-eg, rs1413239 in DPYD (3·29, 1·47-7·37, 5·40×10(-3)) and rs3887412 in ABCC1 (3·36, 1·47-7·67, p=5·70×10(-3)).

CONCLUSIONS

Our results strongly suggest an interaction between myeloma-related factors and the patient's genetic background in the development of treatment-induced peripheral neuropathy, with different molecular pathways being implicated in bortezomib-induced and vincristine-induced peripheral neuropathy.

BACKGROUND

Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line.

RESULTS

We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported.

CONCLUSIONS

CT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.

BACKGROUND

Atopic dermatitis (AD) is classified as extrinsic and intrinsic, representing approximately 80% and 20% of patients with the disease, respectively. Although sharing a similar clinical phenotype, only extrinsic AD is characterized by high serum IgE levels. Because most patients with AD exhibit high IgE levels, an "allergic"/IgE-mediated disease pathogenesis was hypothesized. However, current models associate AD with T-cell activation, particularly TH2/TH22 polarization, and epidermal barrier defects.

OBJECTIVE

We sought to define whether both variants share a common pathogenesis.

METHODS

We stratified 51 patients with severe AD into extrinsic AD (n = 42) and intrinsic AD (n = 9) groups (with similar mean disease activity/SCORAD scores) and analyzed the molecular and cellular skin pathology of lesional and nonlesional intrinsic AD and extrinsic AD by using gene expression (real-time PCR) and immunohistochemistry.

RESULTS

A significant correlation between IgE levels and SCORAD scores (r = 0.76, P < 10(-5)) was found only in patients with extrinsic AD. Marked infiltrates of T cells and dendritic cells and corresponding epidermal alterations (keratin 16, Mki67, and S100A7/A8/A9) defined lesional skin of patients with both variants. However, higher activation of all inflammatory axes (including TH2) was detected in patients with intrinsic AD, particularly TH17 and TH22 cytokines. Positive correlations between TH17-related molecules and SCORAD scores were only found in patients with intrinsic AD, whereas only patients with extrinsic AD showed positive correlations between SCORAD scores and TH2 cytokine (IL-4 and IL-5) levels and negative correlations with differentiation products (loricrin and periplakin).

CONCLUSIONS

Although differences in TH17 and TH22 activation exist between patients with intrinsic AD and those with extrinsic AD, we identified common disease-defining features of T-cell activation, production of polarized cytokines, and keratinocyte responses to immune products. Our data indicate that a TH2 bias is not the sole cause of high IgE levels in patients with extrinsic AD, with important implications for similar therapeutic interventions.

The molecular mechanisms of acute hepatitis C virus (HCV) infection, end-stage hepatitis (cirrhosis), and hepatocellular carcinoma have been extensively studied, but little is known of the changes in liver gene expression during the early stages of liver fibrosis associated with chronic HCV infection, that is, the transition from normal liver (NL) of uninfected patients to the first stage of liver fibrosis (F1-CH-C). To obtain insight into the molecular pathogenesis of F1-CH-C, we used real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to study the mRNA expression of 240 selected genes in liver tissue with F1-CH-C, in comparison with NL. The expression of 54 (22.5%) of the 240 genes was significantly different between F1-CH-C and NL; 46 genes were upregulated and 8 were downregulated in F1-CH-C. The most noteworthy changes in gene expression mainly affected the transcriptional network regulated by interferons (IFNs), including both IFN-alpha/beta-inducible genes (STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2, MX1) and IFN-gamma-inducible genes (CXCL9, CXCL10, CXCL11). Interesting, upregulation of IFN-alpha/beta-inducible genes (but not IFN-gamma-inducible genes) was independent of histological scores (grade and stage of fibrosis) and HCV characteristics (hepatic HCV mRNA levels and the HCV genotype), and was specific to HCV (as compared to hepatitis B virus (HBV)). Other genes dysregulated in F1-CH-C, albeit less markedly than IFN-alpha/beta- and IFN-gamma-inducible genes, were mainly involved in the activation of lymphocytes infiltrating the liver (IFNG, TNF, CXCL6, IL6, CCL8, CXCR3, CXCR4, CCR2), cell proliferation (p16/CDKN2A, MKI67, p14/ARF), extracellular matrix remodeling (MMP9, ITGA2), lymphangiogenesis (XLKD1/LYVE), oxidative stress (CYP2E1), and cytoskeleton microtubule organization (STMN2/SCG10). Thus, a limited number of signaling pathways, and particularly the transcriptional network regulated by interferons, are dysregulated in the first stage of HCV-induced liver fibrosis. Some of the genes identified here could form the basis for new approaches aimed at refining IFN-based therapies for chronic HCV infection.

In pregnancy, the decidua is infiltrated by leukocytes promoting fetal development without causing immunological rejection. Murine regulatory T (Treg) cells are known to be important immune regulators at this site. The aim of the study was to characterize the phenotype and origin of Treg cells and determine the quantitative relationship between Treg, T-helper type 1 (T(H)1), T(H)2, and T(H)17 cells in first-trimester human decidua. Blood and decidual CD4(+) T cells from 18 healthy first-trimester pregnant women were analyzed for expression of Treg-cell markers (CD25, FOXP3, CD127, CTLA4, and human leukocyte antigen-DR [HLA-DR]), chemokine receptors (CCR4, CCR6, and CXCR3), and the proliferation antigen MKI67 by six-color flow cytometry. Treg cells were significantly enriched in decidua and displayed a more homogenous suppressive phenotype with more frequent expression of FOXP3, HLA-DR, and CTLA4 than in blood. More decidual Treg cells expressed MKI67, possibly explaining their enrichment at the fetal-maternal interface. Using chemokine receptor expression profiles of CCR4, CCR6, and CXCR3 as markers for T(H)1, T(H)2, and T(H)17 cells, we showed that T(H)17 cells were nearly absent in decidua, whereas T(H)2-cell frequencies were similar in blood and decidua. CCR6(+) T(H)1 cells, reported to secrete high levels of interferon gamma (IFNG), were fewer, whereas the moderately IFNG-secreting CCR6(-) T(H)1 cells were more frequent in decidua compared with blood. Our results point toward local expansion of Treg cells and low occurrence of T(H)17 cells. Furthermore, local, moderate T(H)1 activity seems to be a part of normal early pregnancy, consistent with a mild inflammatory environment controlled by Treg cells.

OBJECTIVE

Kruppel-like factor (KLF5) is a cell growth mediator in various epithelial cells. Higher KLF5 increases cell growth rate and leads to transformed phenotypes. Because tumor cell proliferation is tightly associated with tumor progression, and consequently, with survival of cancer patients, we wanted to examine the prognostic value of KLF5 gene expression for patients with breast cancer.

METHODS

The gene expression levels of KLF5, ER, PR, HER2, and MKI67 were quantified in the tumor tissues of 90 patients with breast cancer and correlated with disease-free survival and overall survival of the patients. The correlations of gene expression between KLF5 and ER, PR, HER2, and MKI67 were analyzed. In addition, KLF5 expression was also compared with clinical data and age of patients.

RESULTS

Statistically significant correlations were found between gene expression of KLF5 and both disease-free survival (univariate analysis) and overall survival (univariate and multivariate analysis). Patients with higher KLF5 expression had shorter disease-free survival and overall survival time, whereas patients with lower KLF5 expression had better survival. Moreover, KLF5 was also found to be positively correlated with HER2 and MKI67, and negatively correlated with age of the patients at diagnosis.

CONCLUSIONS

The gene expression of KLF5 is directly correlated with cell proliferation in vivo and is a prognostic factor for patients with breast cancer. Patients with higher KLF5 expression have shorter disease-free survival and overall survival than patients with lower KLF5 expression. In addition, KLF5 has higher expression in patients ages </=50 years old than in patients >50 years old.

This prospective study aimed to develop reproducible diagnostic criteria for sporadic Burkitt lymphoma (BL), applicable to routine practice, and to evaluate the efficacy of dose-modified (dm) CODOX-M/IVAC in patients diagnosed using these criteria. The study was open to patients with an aggressive B-cell lymphoma with an MKI67 fraction approaching 100%. Immunophenotype and fluorescent in situ hybridization (FISH) were used to separate BL from other aggressive B-cell lymphomas. BL was characterized by the presence of a cMYC rearrangement as a sole cytogenetic abnormality occurring in patients with a germinal center phenotype with absence of BCL-2 expression and abnormal TP53 expression. A total of 128 patients were eligible for the study, of whom 58 were considered to have BL and 70 to have diffuse large B-cell lymphoma (DLBCL). There were 110 clinically fit patients who received dmCODOX-M (methotrexate, dose 3 g/m(2)) with or without IVAC according to risk group. The 2-year progression-free survival was 64% (95% confidence interval [CI] 51%-77%) for BL, 55% (95% CI 42%-66%) for DLBCL, 85% (95% CI 73%-97%) for low risk, and 49% (95% CI 38%-60%) for high-risk patients. The observed differences in outcome and other clinical features validate the proposed diagnostic criteria. Compared with the previous trial LY06 with full-dose methotrexate (6.7 g/m(2)), there was a reduction in toxicity with comparable outcomes. This study was registered at www.clinicaltrials.gov as NCT00040690.

BACKGROUND

To identify markers and mechanisms of resistance to adalimumab therapy, we studied global gene expression profiles in synovial tissue specimens obtained from severe rheumatoid arthritis (RA) patients before and after initiation of treatment.

METHODS

Paired synovial biopsies were obtained from the affected knee of 25 DMARD (disease-modifying antirheumatic drug)-resistant RA patients at baseline (T0) and 12 weeks (T12) after initiation of adalimumab therapy. DAS28-CRP (disease activity score using 28 joint counts-C-reactive protein) scores were computed at the same time points, and patients were categorized as good, moderate, or poor responders according to European League Against Rheumatism criteria. Global gene expression profiles were performed in a subset of patients by means of GeneChip Human Genome U133 Plus 2.0 Arrays, and confirmatory immunohistochemistry experiments were performed on the entire cohort.

RESULTS

Gene expression studies performed at baseline identified 439 genes associated with poor response to therapy. The majority (n = 411) of these genes were upregulated in poor responders and clustered into two specific pathways: cell division and regulation of immune responses (in particular, cytokines, chemokines, and their receptors). Immunohistochemistry experiments confirmed that high baseline synovial expression of interleukin-7 receptor alpha chain (IL-7R), chemokine (C-X-C motif) ligand 11 (CXCL11), IL-18, IL-18 receptor accessory (IL-18rap), and MKI67 is associated with poor response to adalimumab therapy. In vitro experiments indicated that genes overexpressed in poor responders could be induced in fibroblast-like synoviocytes (FLS) cultures by the addition of tumor necrosis factor-alpha (TNF-alpha) alone, IL-1beta alone, the combination of TNF-alpha and IL-17, and the combination of TNF-alpha and IL-1beta.

CONCLUSIONS

Gene expression studies of the RA synovium may be useful in the identification of early markers of response to TNF blockade. Genes significantly overexpressed at baseline in poor responders are induced by several cytokines in FLSs, thereby suggesting a role for these cytokines in the resistance to TNF blockade in RA.

BACKGROUND

Severe atopic dermatitis (AD) has a high unmet need for effective and safe therapeutics. In early-phase trials, dupilumab, a fully human mAb targeting IL-4 receptor α, markedly improved disease activity, but the effect of IL-4/IL-13 blockade on AD at the molecular level has not been characterized.

OBJECTIVE

We sought to evaluate dupilumab modulation of the AD molecular signature.

METHODS

We performed transcriptomic analyses of pretreatment and posttreatment skin biopsy specimens from patients with moderate-to-severe AD treated weekly with 150 or 300 mg of dupilumab or placebo.

RESULTS

Exacerbation of the AD transcriptome was observed in placebo-treated patients. Dupilumab improved the AD signature in a dose-dependent manner. Expression of genes upregulated in AD lesions decreased in patients treated with dupilumab by 26% (95% CI, 21% to 32%) and 65% (95% CI, 60% to 71%) for treatment with 150 and 300 mg, respectively. Genes downregulated in AD lesions increased by 21% (95% CI, 16% to 27%) and 32% (95% CI, 26% to 37%) with dupilumab (150 and 300 mg, respectively). The molecular changes paralleled improvements in clinical scores. A dupilumab treatment signature of 821 probes (>2-fold change, P < .05) significantly modulated in the 300-mg dupilumab group at week 4 compared with baseline was identified in this sample set. Significant (P < .05) decreases in mRNA expression of genes related to hyperplasia (K16 and MKI67), T cells, and dendritic cells (CD1b and CD1c) and potent inhibition of TH2-associated chemokines (CCL17, CCL18, CCL22, and CCL26) were noted without significant modulation of TH1-associated genes (IFNG).

CONCLUSIONS

This is the first report showing rapid improvement of the AD molecular signature with targeted anti-IL-4 receptor α therapy. These data suggest that IL-4 and IL-13 drive a complex, TH2-centered inflammatory axis in patients with AD.

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was>> 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was>> 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of>> 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred>> 2) versus PEPI>> 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI>> 0 (recurrence hazard ratio [PEPI = 0 v PEPI>> 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Hepatocarcinogenesis is a stepwise process. It involves several genetic and epigenetic alterations, e.g., loss of tumor suppressor gene expression (TP53, PTEN, RB) as well as activation of oncogenes (c-MYC, MET, BRAF, RAS). However, the role of RNA-binding proteins (RBPs), which regulate tumor suppressor and oncogene expression at the posttranscriptional level, are not well understood in hepatocellular carcinoma (HCC). Here we analyzed RBPs induced in human liver cancer, revealing 116 RBPs with a significant and more than 2-fold higher expression in HCC compared to normal liver tissue. We focused our subsequent analyses on the Insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 1 (IGF2BP1) representing the most strongly up-regulated RBP in HCC in our cohort. Depletion of IGF2BP1 from multiple liver cancer cell lines inhibits proliferation and induces apoptosis in vitro. Accordingly, murine xenograft assays after stable depletion of IGF2BP1 reveal that tumor growth, but not tumor initiation, strongly depends on IGF2BP1 in vivo. At the molecular level, IGF2BP1 binds to and stabilizes the c-MYC and MKI67 mRNAs and increases c-Myc and Ki-67 protein expression, two potent regulators of cell proliferation and apoptosis. These substrates likely mediate the impact of IGF2BP1 in human liver cancer, but certainly additional target genes contribute to its function.

CONCLUSIONS

The RNA-binding protein IGF2BP1 is an important protumorigenic factor in liver carcinogenesis. Hence, therapeutic targeting of IGF2BP1 may offer options for intervention in human HCC.

The 21-gene Recurrence Score (RS) assay is a validated prognostic/predictive tool in early hormone receptor-positive breast cancer (BC); however, only a few prospective outcome results have been available so far. In the phase III PlanB trial, RS was prospectively used to define a subset of patients who received only endocrine therapy. We present 3-year outcome data and concordance analysis (among biomarkers/RS).

Central tumor bank was established prospectively from PlanB (intermediate and high-risk, locally human epidermal growth factor receptor 2-negative BC). After an early amendment, HR-positive, pN0-1 patients with RS ≤ 11 were recommended to omit chemotherapy.

From 2009 to 2011, PlanB enrolled 3,198 patients with a median age of 56 years; 41.1% had node-positive and 32.5% grade 3 disease. In 348 patients (15.3%), chemotherapy was omitted based on RS ≤ 11. After 35 months median follow-up, 3-year disease-free survival in patients with RS ≤ 11 and endocrine therapy alone was 98% versus 92% and 98% in RS>> 25 and RS 12 to 25 in chemotherapy-treated patients, respectively. Nodal status, central and local grade, the Ki-67 protein encoded by the MKI67 gene, estrogen receptor, progesterone receptor, tumor size, and RS were univariate prognostic factors for disease-free survival; only nodal status, both central and local grade, and RS were independent multivariate factors. Histologic grade was discordant between central and local laboratories in 44%. RS was positively but moderately correlated with the Ki-67 protein encoded by the MKI67 gene and grade and negatively correlated with progesterone receptor and estrogen receptor.

In this prospective trial, patients with enhanced clinical risk and omitted chemotherapy on the basis of RS ≤ 11 had excellent 3-year survival. The substantial discordance observed between traditional prognostic markers and RS emphasizes the need for standardized assessment and supports the potential integration of standardized, well-validated genomic assays such as RS with clinicopathologic prognostic factors for chemotherapy indication in early hormone receptor-positive BC.

Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. These tumors are three to four times more prevalent in African American women, who also have a 10 times higher incidence of hypovitaminosis D than white women. Recent studies have demonstrated the antitumor effects of 1,25-dihydroxyvitamin D3 on several cancers, but its effects on uterine leiomyomas are still unknown. To determine the antitumor and therapeutic effects of 1,25-dihydroxyvitamin D3 on uterine leiomyomas, female Eker rats (14-16 mo old) harboring uterine leiomyomas were randomized into control and experimental groups and were given vehicle versus 1,25-dihydroxyvitamin D3 (0.5 μg/kg per day) subcutaneously for 3 wk, respectively. At the end of the experiment, the rats were euthanized, and the leiomyoma tumors were analyzed. Treatment with 1,25-dihydroxyvitamin D3 significantly reduced leiomyoma tumor size in Eker rats. It also reduced leiomyoma size by suppressing cell growth and proliferation-related genes (Pcna, cyclin D1 [Ccnd1], Myc, Cdk1, Cdk2, and Cdk4), antiapoptotic genes (Bcl2 and Bcl2l1 [Bcl-x]), and estrogen and progesterone receptors. Additionally, immunohistochemistry revealed decreased expression of PCNA and MKI67 (a marker of proliferation) and increased expression of caspase 3 in 1,25-dihydroxyvitamin D3-treated Eker rat leiomyomas. Toxicity analyses using serum samples showed similar levels of SGOT, SGPT, calcium, and total bilirubin in 1,25-dihydroxyvitamin D3-treated and vehicle-treated control Eker rats. These results support that 1,25-dihydroxyvitamin D3 is an antitumor agent that may be a potential safe, nonsurgical therapeutic option for the treatment of uterine leiomyomas.

Gene co-expression network analysis is an effective method for predicting gene functions and disease biomarkers. However, few studies have systematically identified co-expressed genes involved in the molecular origin and development of various types of tumors. In this study, we used a network mining algorithm to identify tightly connected gene co-expression networks that are frequently present in microarray datasets from 33 types of cancer which were derived from 16 organs/tissues. We compared the results with networks found in multiple normal tissue types and discovered 18 tightly connected frequent networks in cancers, with highly enriched functions on cancer-related activities. Most networks identified also formed physically interacting networks. In contrast, only 6 networks were found in normal tissues, which were highly enriched for housekeeping functions. The largest cancer network contained many genes with genome stability maintenance functions. We tested 13 selected genes from this network for their involvement in genome maintenance using two cell-based assays. Among them, 10 were shown to be involved in either homology-directed DNA repair or centrosome duplication control including the well-known cancer marker MKI67. Our results suggest that the commonly recognized characteristics of cancers are supported by highly coordinated transcriptomic activities. This study also demonstrated that the co-expression network directed approach provides a powerful tool for understanding cancer physiology, predicting new gene functions, as well as providing new target candidates for cancer therapeutics.

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (β-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by β-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the translation of target mRNA transcripts. In this study, we demonstrated that miR-519d was downregulated in human HCC and could suppress growth of the human HCC cell line QGY-7703. Bioinformatics analysis indicated that MKi67 was a putative target of miR-519d. In an EGFP reporter system, we confirmed that MKi67 was a direct target gene of miR-519d. Furthermore, knockdown of MKi67 inhibited QGY-7703 cell growth. These findings indicate that miR-519d targets the MKi67 transcript and suppresses HCC cell growth, suggesting that miR-519d has a tumor suppressive role in human HCC pathogenesis.

BACKGROUND

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex range of clinical symptoms. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors (MPNSTs), and the underlying molecular mechanisms are largely unknown.

RESULTS

To obtain further insight into the molecular pathogenesis of MPNSTs, we used real-time quantitative RT-PCR to quantify the mRNA expression of 489 selected genes in MPNSTs, in comparison with plexiform neurofibromas. The expression of 28 (5.7%) of the 489 genes was significantly different between MPNSTs and plexiform neurofibromas; 16 genes were upregulated and 12 were downregulated in MPNSTs. The altered genes were mainly involved in cell proliferation (MKI67, TOP2A, CCNE2), senescence (TERT, TERC), apoptosis (BIRC5/Survivin, TP73) and extracellular matrix remodeling (MMP13, MMP9, TIMP4, ITGB4). More interestingly, other genes were involved in the Ras signaling pathway (RASSF2, HMMR/RHAMM) and the Hedgehog-Gli signaling pathway (DHH, PTCH2). Several of the down-regulated genes were Schwann cell-specific (L1CAM, MPZ, S100B, SOX10, ERBB3) or mast cell-specific (CMA1, TPSB), pointing to a depletion and/or dedifferentiation of Schwann cells and mast cells during malignant transformation of plexiform neurofibromas.

CONCLUSIONS

These data suggest that a limited number of signaling pathways, and particularly the Hedgehog-Gli signaling pathway, may be involved in malignant transformation of plexiform neurofibromas. Some of the relevant genes or their products warrant further investigation as potential therapeutic targets in NF1.

OBJECTIVE

Clinical studies have implicated the mechanistic target of rapamycin (serine/threonine kinase; MTOR) pathway in the regulation of neuroendocrine tumor (NET) growth. We explored whether expression of MTOR pathway components has prognostic significance in NET patients.

METHODS

We evaluated immunohistochemical expression of MTOR and phospho (p) -MTOR; its downstream targets RPS6KB1, RPS6, and EIF4EBP1; and its upstream regulators, in a cohort of 195 archival neuroendocrine tumors. We correlated expression levels with clinical outcomes, after adjusting for other prognostic variables.

RESULTS

We observed anticipated correlations between expression of upstream components of the MTOR pathway and their downstream targets. Expression of PIK3CA, MTOR, or p-EIF4EBP1 was associated with high MKI67 (Ki-67) labeling index. We failed to identify clinical correlations associated with expression of the upstream regulators TSC1, TSC2, AKT, p-AKT, PDPK1, PTEN, PIK3R1, or PIK3CA. In contrast, high expression of MTOR or its activated downstream targets p-RPS6KB1, p-RPS6, or p-EIF4EBP1 was associated with adverse clinical outcomes.

CONCLUSIONS

Our observations suggest that expression of MTOR or its downstream targets may be adverse prognostic factors in neuroendocrine tumors.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma, where survival has been remarkably improved by use of protocols including high dose cytarabine, rituximab and autologous stem cell transplantation, such as the Nordic MCL2/3 protocols. In 2008, a MCL international prognostic index (MIPI) was created to enable stratification of the clinical diverse MCL patients into three risk groups. So far, use of the MIPI in clinical routine has been limited, as it has been shown that it inadequately separates low and intermediate risk group patients. To improve outcome and minimize treatment-related morbidity, additional parameters need to be evaluated to enable risk-adapted treatment selection. We have investigated the individual prognostic role of the MIPI and molecular markers including SOX11, TP53 (p53), MKI67 (Ki-67) and CCND1 (cyclin D1). Furthermore, we explored the possibility of creating an improved prognostic tool by combining the MIPI with information on molecular markers. SOX11 was shown to significantly add prognostic information to the MIPI, but in multivariate analysis TP53 was the only significant independent molecular marker. Based on these findings, we propose that TP53 and SOX11 should routinely be assessed and that a combined TP53/MIPI score may be used to guide treatment decisions.

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.

The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction with DNA methyltransferases. EZH2 expression increases during prostate cancer progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2 expression, DNA methylation alterations escalate in higher stage prostate cancers, raising the question whether these epigenetic changes are related. Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC, ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation. SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless, cancers with higher DNMT1 and BMI-1 expression had worse clinical characteristics, as did those with elevated EZH2. In particular, above median DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were well correlated with increased MKI67. Immunohistochemistry confirmed limited EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1 by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically among all factors investigated, was associated with more frequent hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1 hypomethylation. Our data reveal complex changes in the composition of polycomb repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and slightly increased EZH2 may characterize less malignant cancers, whereas more aggressive cases express both at higher levels. SIRT1 appears to be generally increased in prostate cancers. Intriguingly, our data suggest a direct influence of increased EZH2 on altered DNA methylation patterns in prostate cancer.