Transcriptomic analysis of hepatocellular carcinoma reveals molecular features of disease progression and tumor immune biology.

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial-mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-ß, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-ß neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies.

CHANGES IN DIABETIC RETINOPATHY SEVERITY WHEN TREATING DIABETIC MACULAR EDEMA WITH RANIBIZUMAB: DRCR.net Protocol I 5-Year Report.

PURPOSE: To explore 5-year changes from baseline in diabetic retinopathy severity among eyes treated with ranibizumab for diabetic macular edema. METHODS: Diabetic retinopathy severity was assessed from study visits and annual fundus photographs among participants in Protocol I (DRCR.net). The proportion of eyes that improved at annual examinations and the cumulative probability of worsening through 5 years were estimated. RESULTS: Among 235 participants with nonproliferative diabetic retinopathy at baseline, there were 29%, 28%, and 32% of eyes with retinopathy improvement at 1, 3, and 5 years, respectively. Among 111 participants with proliferative diabetic retinopathy, corresponding improvement percentages were 38%, 35%, and 23%. The 5-year cumulative probability of worsening was 18% (95% CI: 14%-25%) among nonproliferative diabetic retinopathy eyes and 31% (95% CI: 23%-42%) among proliferative diabetic retinopathy eyes (P = 0.01). In Years 1, 3, and 5, the mean (SD) number of ranibizumab injections was 8.1 (2.5), 2.2 (2.6), and 1.8 (2.6) for nonproliferative diabetic retinopathy eyes, and 9.0 (2.8), 2.3 (2.9), and 1.7 (2.6) for proliferative diabetic retinopathy eyes, respectively. Proportions with improvement or rates of worsening did not change with time. CONCLUSION: Individuals receiving ranibizumab therapy for diabetic macular edema may have favorable changes in DR severity throughout a 5-year period concomitant with sequential reduction in anti-vascular endothelial growth factor therapy.

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma-carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.

A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach.

Aflibercept, Bevacizumab, or Ranibizumab for Diabetic Macular Edema: Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial.

PURPOSE: To provide 2-year results comparing anti-vascular endothelial growth factor (VEGF) agents for center-involved diabetic macular edema (DME) using a standardized follow-up and retreatment regimen. DESIGN: Randomized clinical trial. PARTICIPANTS: Six hundred sixty participants with visual acuity (VA) impairment from DME. METHODS: Randomization to 2.0-mg aflibercept, 1.25-mg repackaged (compounded) bevacizumab, or 0.3-mg ranibizumab intravitreous injections performed up to monthly using a protocol-specific follow-up and retreatment regimen. Focal/grid laser photocoagulation was added after 6 months if DME persisted. Visits occurred every 4 weeks during year 1 and were extended up to every 4 months thereafter when VA and macular thickness were stable. MAIN OUTCOME MEASURES: Change in VA, adverse events, and retreatment frequency. RESULTS: Median numbers of injections were 5, 6, and 6 in year 2 and 15, 16, and 15 over 2 years in the aflibercept, bevacizumab, and ranibizumab groups, respectively (global P = 0.08). Focal/grid laser photocoagulation was administered in 41%, 64%, and 52%, respectively (aflibercept vs. bevacizumab, P < 0.001; aflibercept vs. ranibizumab, P = 0.04; bevacizumab vs. ranibizumab, P = 0.01). At 2 years, mean VA improved by 12.8, 10.0, and 12.3 letters, respectively. Treatment group differences varied by baseline VA (P = 0.02 for interaction). With worse baseline VA (20/50 to 20/320), mean improvement was 18.1, 13.3, and 16.1 letters, respectively (aflibercept vs. bevacizumab, P = 0.02; aflibercept vs. ranibizumab, P = 0.18; ranibizumab vs. bevacizumab, P = 0.18). With better baseline VA (20/32 to 20/40), mean improvement was 7.8, 6.8, and 8.6 letters, respectively (P > 0.10, for pairwise comparisons). Anti-Platelet Trialists' Collaboration (APTC) events occurred in 5% with aflibercept, 8% with bevacizumab, and 12% with ranibizumab (global P = 0.047; aflibercept vs. bevacizumab, P = 0.34; aflibercept vs. ranibizumab, P = 0.047; ranibizumab vs. bevacizumab, P = 0.20; global P = 0.09 adjusted for potential confounders). CONCLUSIONS: All 3 anti-VEGF groups showed VA improvement from baseline to 2 years with a decreased number of injections in year 2. Visual acuity outcomes were similar for eyes with better baseline VA. Among eyes with worse baseline VA, aflibercept had superior 2-year VA outcomes compared with bevacizumab, but superiority of aflibercept over ranibizumab, noted at 1 year, was no longer identified. Higher APTC event rates with ranibizumab over 2 years warrants continued evaluation in future trials.

Acquired PIK3CA amplification causes resistance to selective phosphoinositide 3-kinase inhibitors in breast cancer.

Agents targeting the PI3K/mTOR signaling axis have shown promise in early-phase clinical trials and are currently being studied in later stages of clinical development in multiple indications. Experience with other targeted agents suggests that clinical responses may be short-lived because of acquired resistance to therapy. Here, we report preclinical modeling of acquired resistance in a HER2-positive, PIK3CA mutant breast cancer cell line, KPL-4. We identified a heretofore-unreported mechanism of resistance, specifically high-level amplification of the mutant allele of PIK3CA, which resulted in a marked upregulation of PI3K signaling, enabling resistant cells to regain proliferative capacity at clinically relevant concentrations of the PI3K inhibitor, GDC-0941. We show that knockdown of the amplified PIK3CA mutant allele in these cells by small interfering RNA restored pathway signaling and sensitivity to PI3K inhibition at levels comparable to parental cells. These novel preclinical findings suggest that, in addition to assessment of other previously reported mechanisms of resistance, evaluation of PI3K copy number variation should be integrated into the exploratory analysis of biopsies obtained at disease progression.

Dexamethasone synergizes with lenalidomide to inhibit multiple myeloma tumor growth, but reduces lenalidomide-induced immunomodulation of T and NK cell function.

To determine the effect of dexamethasone on the antimyeloma effects of lenalidomide, we tested in vitro proliferation, tumor suppressor gene expression, caspase activity, cell cycling, and apoptosis levels in a series of multiple myeloma (MM) and plasma cell leukemia cell lines treated with lenalidomide and dexamethasone, alone or in combination. The effect of dexamethasone on the immunomodulatory activities of lenalidomide such as T cell and natural killer (NK) cell activation was measured via interleukin [IL]-2 production, and interferon-gamma and granzyme B production respectively. Lenalidomide inhibited proliferation in most cell lines tested, and this effect was enhanced by dexamethasone. This effect was observed in MM cells containing the high-risk cytogenetic abnormalities t(4;14), t(14;16), del17p, del13, and hypodiploidy. Mechanistically, lenalidomide plus dexamethasone synergistically induced expression of the tumor suppressor genes Egr1, Egr2, Egr3, p15, p21, and p27 in MM cell lines and MM patient cells. The combination activated caspases 3, 8, and 9; and induced cell cycle arrest and apoptosis. Lenalidomide alone increased T cell production of IL-2, and NK cell production of interferon-gamma and granzyme B. Notably, dexamethasone antagonized these immunostimulatory effects of lenalidomide in a dose-dependent manner. These data further elucidate the mechanism of action of lenalidomide and dexamethasone in MM, and suggest that use of low-dose dexamethasone with lenalidomide may retain the antiproliferative effect of lenalidomide while permitting greater immunomodulatory effects of this combination regimen.

Overlapping gene expression profiles of cell migration and tumor invasion in human bladder cancer identify metallothionein 1E and nicotinamide N-methyltransferase as novel regulators of cell migration.

Cell migration is essential to cancer invasion and metastasis and is spatially and temporally integrated through transcriptionally dependent and independent mechanisms. As cell migration is studied in vitro, it is important to identify genes that both drive cell migration and are biologically relevant in promoting invasion and metastasis in patients with cancer. Here, gene expression profiling and a high-throughput cell migration system answers this question in human bladder cancer. In vitro migration rates of 40 microarray-profiled human bladder cancer cell lines were measured by radial migration assay. Genes whose expression was either directly or inversely associated with cell migration rate were identified and subsequently evaluated for their association with cancer stage in 61 patients. This analysis identified genes known to be associated with cell invasion such as versican, and novel ones, including metallothionein 1E (MT1E) and nicotinamide N-methyltransferase (NNMT), whose expression correlated positively with cancer cell migration and tumor stage. Using loss of function analysis, we show that MT1E and NNMT are necessary for cancer cell migration. These studies provide a general approach to identify the clinically relevant genes in cancer cell migration and mechanistically implicate two novel genes in this process in human bladder cancer.

Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector.

Although the monomeric GTPases RalA and RalB have been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities in transcriptional programs regulated by RalA compared to RalB. Further, the association of these transcriptional pathways to human carcinogenesis and progression remains unclear. Here, we studied the role of RalA and/or RalB in transcriptional regulation by combining short interfering RNA depletion of Ral with gene expression profiling via microarray in the human bladder cancer cell line, UMUC-3. A large number of genes were found to be similarly modulated in cells with RalA and RalB depletion, suggesting that RalA and RalB impinge on overlapping transcriptional signaling pathways. However, smaller sets of genes were modulated by depletion of RalA or RalB, indicating that these closely related proteins also regulate nonoverlapping transcriptional pathways. Computational analysis of upstream sequences of genes modulated by Ral depletion identified Ras-responsive element-binding protein (RREB)-1, as a putative Ral transcriptional target, which we verified experimentally. Importantly, as a group, Ral-regulated probe sets identified here were disproportionally represented among those differentially expressed as a function of human bladder transformation. Taken together, these data strongly suggest that Ral family members mediate both common and specific transcriptional programs that are associated with human cancer and identify RREB-1 as a novel transcriptional effector of Ral.

A novel method for gene expression mapping of metastatic competence in human bladder cancer.

Expression profiling by DNA microarray analysis has provided insights into molecular alterations that underpin cancer progression and metastasis. Although differential expression of microarray-defined probes can be related to numerical or structural chromosomal alterations, it is unclear if such changes are also clustered in distinct chromosomes or genomic regions and whether chromosomal alterations always reflect changes in gene expression. Here we apply the dChip algorithm and a novel technique to test the hypothesis that expression changes occurring as a function of tumor progression and metastasis are nonrandomly distributed. Expression profiling of a human xenograft model of lung metastasis phenotype indicates that chromosomes 2, 11, and 20 contain higher percentages of differentially expressed genes (P < .05). Furthermore, we show that a number of differentially expressed probes mapped to chromosome 17q, defining the existence of an expression "hot spot" corresponding to an area of gain determined by comparative genomic hybridization (CGH). Interestingly, other areas of gains detected by CGH were not associated with expression hot spots. In summary, we show that gene expression changes during bladder cancer lung metastasis occur nonrandomly in specific chromosomes and intrachromosomal locations.

Overexpression of CEBPbeta correlates with decreased TFF1 in gastric cancer.

CCAAT element binding protein beta (C/EBPbeta) is an important regulator of cell growth, differentiation and in promoting tumor invasiveness. C/EBPbeta is located on chromosome 20q, which is amplified in many solid tumors including gastric cancers (GC). We sought to characterize the status of C/EBPbeta expression in GCs, which was recently found to repres TFF1 gene. Microarray analysis revealed overexpression of C/EBPbeta in 25 of 27 (93%) GC when compared to 12 normal gastric tissue samples. RT-PCR analysis confirmed the overexpression of C/EBPbeta transcripts in 54 of 59 (91%) GC. In total, 15 of 18 gastric tumors exhibited at least fivefold higher C/EBPbeta transcript levels compared to their corresponding adjacent normal gastric tissue samples. Moreover, immunohistochemistry analysis demonstrated increased nuclear staining of C/EBPbeta in 10 of 13 GC and at least fourfold overexpression of C/EBPbeta in three primary GC compared to adjacent normal gastric tissue. Furthermore, a striking correlation of decreased TFF1 expression with increased C/EBPbeta was observed in the gastric tumors studied. Microarray analysis demonstrated a loss of TFF1 expression in all 27 GC cases examined, of which 25 exhibited high C/EBPbeta expression compared to normal gastric tissue. RT-PCR analysis revealed loss of TFF1 expression in 56 of 59 gastric tumors in which 54 of these tumors exhibited overexpression of C/EBPbeta. Immunohistochemical analysis revealed overexpression of C/EBPbeta in 10 of 13 gastric tumors that exhibited low expression of TFF1 at the protein level. Thus, overexpression of the transcription factor C/EBPbeta in the majority of GCs is a novel finding.

Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer.

PURPOSE: RhoGDI2 was recently shown to be a metastasis suppressor gene in models of bladder cancer. We sought to further understand its importance in human cancer by determining the level of its expression and the distribution of its encoded protein in normal human tissues and cell lines and to evaluate whether its protein expression is a determinant of human bladder cancer progression. EXPERIMENTAL DESIGN: RhoGDI2 mRNA and protein expression was evaluated in cell lines and human tissues using Affymetrix and tissue microarrays, respectively. Tissue microarrays represented most human normal adult tissues and material from 51 patients that had undergone radical cystectomy for bladder cancer. In these 51 patients, the chi(2) test was used to test for associations between RhoGDI2 and stage, grade of urothelial carcinoma, histological type, and disease-specific survival status. Cox proportional hazards regression analyses were used to estimate the effect of RhoGDI2 expression level on time to development of metastatic disease and disease-specific survival time, adjusting for grade, stage, and histological type. RESULTS: In normal tissues, there was strong RhoGDI2 protein expression in WBCs, endothelial cells, and transitional epithelium. RhoGDI2 mRNA expression was inversely related to the invasive and metastatic phenotype in human bladder cancer cell lines. In patients with bladder cancer, univariate analysis indicated that reduced tumor RhoGDI2 protein expression was associated with a lower actuarial 5-year disease-free and disease-specific survival (P = 0.01). In addition, patients with tumors that had low or absent RhoGDI2 had a shorter time to disease-specific death (P

An integrated view of aromatase and its inhibition.

Aromatase inhibition has become a major treatment strategy for postmenopausal women with oestrogen-dependent breast cancer. Its optimal application is, however, dependent upon (i) the accurate identification of cancers which are ultimately dependent upon the activity of the aromatase enzyme, (ii) the use of the best method/inhibitor by which to blockade aromatase activity. The single best predictor of response to aromatase inhibitors is the presence of tumour oestrogen receptors; receptor-negative cancers rarely respond whereas those with high levels seem particularly likely to benefit. However, there is a need for additional discriminatory markers. The use of microarray technology coupled with neoadjuvant therapy is likely to yield promising candidate genes. The finding that, amongst peripheral tissues, the tumour itself may have high activity has led to the suggestion that the tumour aromatase measurements may be predictive; however, in situ studies and the lack of robust assays for tumour aromatase suggest that tumour aromatase may not be an influential marker. Whilst drugs such as anastrozole, exemestane, formestane and letrozole are all effective and specific inhibitors of aromatase, they differ in structure, potency and mechanism of action. Thus, differential sensitivity of tissues/tumours and non-cross resistance mean inhibitors are not equivalent and individual agents may have differing roles according to the setting in which they will be used. Aromatase inhibitors have evolved as key endocrine agents in the treatment of breast cancer. They offer the promise of rational treatment management based on the accurate identification of individual cohorts of tumours responsive to specific drugs.

Molecular classification of human carcinomas by use of gene expression signatures.

Classification of human tumors according to their primary anatomical site of origin is fundamental for the optimal treatment of patients with cancer. Here we describe the use of large-scale RNA profiling and supervised machine learning algorithms to construct a first-generation molecular classification scheme for carcinomas of the prostate, breast, lung, ovary, colorectum, kidney, liver, pancreas, bladder/ureter, and gastroesophagus, which collectively account for approximately 70% of all cancer-related deaths in the United States. The classification scheme was based on identifying gene subsets whose expression typifies each cancer class, and we quantified the extent to which these genes are characteristic of a specific tumor type by accurately and confidently predicting the anatomical site of tumor origin for 90% of 175 carcinomas, including 9 of 12 metastatic lesions. The predictor gene subsets include those whose expression is typical of specific types of normal epithelial differentiation, as well as other genes whose expression is elevated in cancer. This study demonstrates the feasibility of predicting the tissue origin of a carcinoma in the context of multiple cancer classes.

Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.

Detection, treatment, and prediction of outcome for men with prostate cancer increasingly depend on a molecular understanding of tumor development and behavior. We characterized primary prostate cancer by monitoring expression levels of more than 8900 genes in normal and malignant tissues. Patterns of gene expression across tissues revealed a precise distinction between normal and tumor samples, and revealed a striking group of about 400 genes that were overexpressed in tumor tissues. We ranked these genes according to their differential expression in normal and cancer tissues by selecting for highly and specifically overexpressed genes in the majority of cancers with correspondingly low or absent expression in normal tissues. Several such genes were identified that act within a variety of biochemical pathways and encode secreted molecules with diagnostic potential, such as the secreted macrophage inhibitory cytokine, MIC-1. Other genes, such as fatty acid synthase, encode enzymes known as drug targets in other contexts, which suggests new therapeutic approaches.

Arrays of arrays for high-throughput gene expression profiling.

Gene expression profiling using DNA arrays is rapidly becoming an essential tool for research and drug discovery and may soon play a central role in disease diagnosis. Although it is possible to make significant discoveries on the basis of a relatively small number of expression profiles, the full potential of this technology is best realized through more extensive collections of expression measurements. The generation of large numbers of expression profiles can be a time-consuming and labor-intensive process with current one-at-a-time technology. We have developed the ability to obtain expression profiles in a highly parallel yet straightforward format using glass wafers that contain 49 individual high-density oligonucleotide arrays. This arrays of arrays concept is generalizable and can be adapted readily to other types of arrays, including spotted cDNA microarrays. It is also scalable for use with hundreds and even thousands of smaller arrays on a single piece of glass. Using the arrays of arrays approach and parallel preparation of hybridization samples in 96-well plates, we were able to determine the patterns of gene expression in 27 ovarian carcinomas and 4 normal ovarian tissue samples, along with a number of control samples, in a single experiment. This new approach significantly increases the ease, efficiency, and throughput of microarray-based experiments and makes possible new applications of expression profiling that are currently impractical.

Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer, in part because of the lack of effective early detection methods. Although alterations of several genes, such as c-erb-B2, c-myc, and p53, have been identified in a significant fraction of ovarian cancers, none of these mutations are diagnostic of malignancy or predictive of tumor behavior over time. Here, we used oligonucleotide microarrays with probe sets complementary to >6,000 human genes to identify genes whose expression correlated with epithelial ovarian cancer. We extended current microarray technology by simultaneously hybridizing ovarian RNA samples in a highly parallel manner to a single glass wafer containing 49 individual oligonucleotide arrays separated by gaskets within a custom-built chamber (termed "array-of-arrays"). Hierarchical clustering of the expression data revealed distinct groups of samples. Normal tissues were readily distinguished from tumor tissues, and tumors could be further subdivided into major groupings that correlated both to histological and clinical observations, as well as cell type-specific gene expression. A metric was devised to identify genes whose expression could be considered ideal for molecular determination of epithelial ovarian malignancies. The list of genes generated by this method was highly enriched for known markers of several epithelial malignancies, including ovarian cancer. This study demonstrates the rapidity with which large amounts of expression data can be generated. The results highlight important molecular features of human ovarian cancer and identify new genes as candidate molecular markers.

Microsatellite instability in cervical carcinoma.

OBJECTIVE: To investigate the incidence of microsatellite instability (MI) in cervical carcinoma and its relationship with clinicopathological characteristics. STUDY DESIGN: A retrospective study of 100 cases of cervical carcinoma. RESULTS: MI, defined as tumor-associated alterations in at least one of five dinucleotide microsatellite markers examined, was detected in 25% of the cervical carcinomas which were observed. There was a nonsignificant trend towards MI occurrence in higher grade tumors, more advanced stage and cases with poor clinical outcome. CONCLUSION: The results suggest that microsatellite instability is present in a subset of cervical carcinoma and may be an independent prognostic factor. Further research with more samples is required.

Transcriptional regulation and function during the human cell cycle.

We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle-dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle-regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model CWR22-R1.

Treatment of metastatic prostate cancer with androgen-ablation often elicits dramatic tumor regressions, but the response is rarely complete, making clinical recurrence inevitable with time. To gain insight into therapy-related progression, changes in gene expression that occurred following androgen-deprivation of an androgen-dependent prostate tumor xenograft, CWR22, and the emergence of an androgen-independent tumor, CWR22-R, were monitored using microarray analysis. Androgen-deprivation resulted in growth arrest of CWR22 cells, as evidenced by decreased expression of genes encoding cell cycle components and basal cell metabolism, respiration and transcription, and the induced expression of putative negative regulatory genes that may act to sustain cells in a nonproliferative state. Evolution of androgen-independent growth and proliferation, represented by CWR22-R, was associated with a reentry into active cell cycle and the up-regulation of several genes that were expressed at low levels or absent in the androgen-dependent tumor. Androgen repletion to mice bearing androgen-independent CWR22-R tumors induced, augmented, or repressed the expression of a number of genes. Expression of two of these genes, the calcium-binding protein S100P and the FK-506-binding protein FKBP51, was decreased following androgen-deprivation, subsequently reexpressed in CWR22-R at levels comparable with CWR22, and elevated further upon treatment with androgens. The dysregulated behavior of these genes is analogous to other androgen-dependent genes, e.g., prostate-specific antigen and human kallikrein 2, which are commonly reexpressed in androgen-independent disease in the absence of androgens. Other androgen-responsive genes whose expression decreased during androgen-deprivation and whose expression remained decreased in CWR22 were also identified in CWR22-R. These results imply that evolution to androgen-independence is due, in part, to reactivation of the androgen-response pathway in the absence of androgens, but that this reactivation is probably incomplete.