Massimo Loda

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types. Two Articles in this issue add major data sets to the growing picture of the cancer genome. Bignell et al. analysed a large number of homozygous gene deletions in a collection of 746 publicly available cancer cell lines. Combined with information about hemizygous deletions of the same genes, the data suggest that many deletions found in cancer reflect the position of a gene at a fragile site in the genome, rather than as a recessive cancer gene whose loss confers a selective growth advantage. Beroukhim et al. present the largest data set to date on somatic copy-number variations across more than 3,000 specimens of human primary cancers. Many alterations are shared between multiple tumour types. Functional experiments demonstrate an oncogenic role for the apoptosis genes MCL1 and BCL2L1 that are associated with amplifications found in many cancers. One way of discovering genes with key roles in cancer development is to identify genomic regions that are frequently altered in human cancers. Here, high-resolution analyses of somatic copy-number alterations (SCNAs) in numerous cancer specimens provide an overview of regions of focal SCNA that are altered at significant frequency across several cancer types. An oncogenic function is also found for the anti-apoptosis genes MCL1 and BCL2L1, which reside in amplified genome regions in many cancers.

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%–60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients.

Prostate tumors are among the most heterogeneous of cancers, both histologically and clinically. Microarray expression analysis was used to determine whether global biological differences underlie common pathological features of prostate cancer and to identify genes that might anticipate the clinical behavior of this disease. While no expression correlates of age, serum prostate specific antigen (PSA), and measures of local invasion were found, a set of genes was identified that strongly correlated with the state of tumor differentiation as measured by Gleason score. Moreover, a model using gene expression data alone accurately predicted patient outcome following prostatectomy. These results support the notion that the clinical behavior of prostate cancer is linked to underlying gene expression differences that are detectable at the time of diagnosis.

The optimal treatment of patients with cancer depends on establishing accurate diagnoses by using a complex combination of clinical and histopathological data. In some instances, this task is difficult or impossible because of atypical clinical presentation or histopathology. To determine whether the diagnosis of multiple common adult malignancies could be achieved purely by molecular classification, we subjected 218 tumor samples, spanning 14 common tumor types, and 90 normal tissue samples to oligonucleotide microarray gene expression analysis. The expression levels of 16,063 genes and expressed sequence tags were used to evaluate the accuracy of a multiclass classifier based on a support vector machine algorithm. Overall classification accuracy was 78%, far exceeding the accuracy of random classification (9%). Poorly differentiated cancers resulted in low-confidence predictions and could not be accurately classified according to their tissue of origin, indicating that they are molecularly distinct entities with dramatically different gene expression patterns compared with their well differentiated counterparts. Taken together, these results demonstrate the feasibility of accurate, multiclass molecular cancer classification and suggest a strategy for future clinical implementation of molecular cancer diagnostics.

Coronavirus disease 2019 (COVID-19) is a novel, viral-induced respiratory disease that in ∼10-15% of patients progresses to acute respiratory distress syndrome (ARDS) triggered by a cytokine storm. In this Perspective, autopsy results and literature are presented supporting the hypothesis that a little known yet powerful function of neutrophils-the ability to form neutrophil extracellular traps (NETs)-may contribute to organ damage and mortality in COVID-19. We show lung infiltration of neutrophils in an autopsy specimen from a patient who succumbed to COVID-19. We discuss prior reports linking aberrant NET formation to pulmonary diseases, thrombosis, mucous secretions in the airways, and cytokine production. If our hypothesis is correct, targeting NETs directly and/or indirectly with existing drugs may reduce the clinical severity of COVID-19.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Heterogeneity in the genomic landscape of metastatic prostate cancer has become apparent through several comprehensive profiling efforts, but little is known about the impact of this heterogeneity on clinical outcome. Here, we report comprehensive genomic and transcriptomic analysis of 429 patients with metastatic castration-resistant prostate cancer (mCRPC) linked with longitudinal clinical outcomes, integrating findings from whole-exome, transcriptome, and histologic analysis. For 128 patients treated with a first-line next-generation androgen receptor signaling inhibitor (ARSI; abiraterone or enzalutamide), we examined the association of 18 recurrent DNA- and RNA-based genomic alterations, including androgen receptor ( AR ) variant expression, AR transcriptional output, and neuroendocrine expression signatures, with clinical outcomes. Of these, only RB1 alteration was significantly associated with poor survival, whereas alterations in RB1 , AR , and TP53 were associated with shorter time on treatment with an ARSI. This large analysis integrating mCRPC genomics with histology and clinical outcomes identifies RB1 genomic alteration as a potent predictor of poor outcome, and is a community resource for further interrogation of clinical and molecular associations.

The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.

Somatic mutations in DNA mismatch repair genes have been observed in sporadic tumors as well as cell lines and xenografts derived from such tumors implicating genetic defects of mismatch repair genes in the development of such tumors. However, the proportion of sporadic tumors in which mismatch repair genes have been inactivated has not been determined accurately. We have analyzed 66 sporadic colorectal tumors for the expression of hMLH1 by immunohistochemistry and identified 4 tumors that do not express hMLH1. These four colorectal tumors, a colon tumor cell line (SW48) and an endometrial tumor cell line (AN3CA), did not express hMLH1, despite the absence of mutations in its coding sequence. Cytosine methylation of the hMLH1 promoter region was found in these four colorectal tumors, whereas cytosine methylation of the hMLH1 promoter region was absent in adjacent normal tissue or in nine tumors that expressed hMLH1. In addition, cytosine methylation of the hMLH1 promoter region was observed in the SW48 and AN3CA cell lines that do not express hMLH1 but not in four tumor cell lines known to express hMLH1 mRNA. Our data indicate that DNA methylation is likely to be a common mode of mismatch repair gene inactivation in sporadic tumors.

Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

The CpG island methylator phenotype (CIMP), characterised by widespread promoter methylation, is associated with microsatellite instability (MSI) and BRAF mutation in colorectal cancer. The independent effect of CIMP, MSI and BRAF mutation on prognosis remains uncertain.Utilising 649 colon cancers (stage I-IV) in two independent cohort studies, we quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) as well as CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN by using MethyLight technology. We examined MSI, KRAS and BRAF status. Cox proportional hazard models computed hazard ratios (HRs) for colon cancer-specific and overall mortalities, adjusting for patient characteristics and tumoral molecular features.After adjustment for other predictors of patient survival, patients with CIMP-high cancers (126 (19%) tumours with >or=6/8 methylated CIMP-specific promoters) experienced a significantly low colon cancer-specific mortality (multivariate HR 0.44, 95% confidence interval (CI) 0.22 to 0.88), whereas the BRAF mutation was significantly associated with a high cancer-specific mortality (multivariate HR 1.97, 95% CI 1.13 to 3.42). A trend toward a low cancer-specific mortality was observed for MSI-high tumours (multivariate HR 0.70, 95% CI 0.36 to 1.37). In stratified analyses, CIMP-high tumours were associated with a significant reduction in colon cancer-specific mortality, regardless of both MSI and BRAF status. The relation between CIMP-high and lower mortality appeared to be consistent across all stages. KRAS mutation was unrelated to prognostic significance.CIMP-high appears to be an independent predictor of a low colon cancer-specific mortality, while BRAF mutation is associated with a high colon cancer-specific mortality.

On activation by receptors, the ubiquitously expressed class IA isoforms (p110alpha and p110beta) of phosphatidylinositol-3-OH kinase (PI(3)K) generate lipid second messengers, which initiate multiple signal transduction cascades. Recent studies have demonstrated specific functions for p110alpha in growth factor and insulin signalling. To probe for distinct functions of p110beta, we constructed conditional knockout mice. Here we show that ablation of p110beta in the livers of the resulting mice leads to impaired insulin sensitivity and glucose homeostasis, while having little effect on phosphorylation of Akt, suggesting the involvement of a kinase-independent role of p110beta in insulin metabolic action. Using established mouse embryonic fibroblasts, we found that removal of p110beta also had little effect on Akt phosphorylation in response to stimulation by insulin and epidermal growth factor, but resulted in retarded cell proliferation. Reconstitution of p110beta-null cells with a wild-type or kinase-dead allele of p110beta demonstrated that p110beta possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110beta was required for G-protein-coupled receptor signalling triggered by lysophosphatidic acid and had a function in oncogenic transformation. Most strikingly, in an animal model of prostate tumour formation induced by Pten loss, ablation of p110beta (also known as Pik3cb), but not that of p110alpha (also known as Pik3ca), impeded tumorigenesis with a concomitant diminution of Akt phosphorylation. Taken together, our findings demonstrate both kinase-dependent and kinase-independent functions for p110beta, and strongly indicate the kinase-dependent functions of p110beta as a promising target in cancer therapy.

The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.

The WNT/ β-catenin signalling pathway, which normally plays a pivotal part in development, is deregulated in almost all colorectal cancers. Retinoblastoma tumour suppressor protein (pRB) is a cell-cycle regulator that is mutated in many different types of cancer. Two papers in this issue show that signalling through the WNT pathway and that mediated by pRB are highly interconnected, and that a common denominator of their deregulation is colorectal cancer. Firestein et al. combined RNAi screening for genes required for colon cancer cell proliferation with genomic data from human colon cancer to identifty CDK8 as a novel human oncogene. CDK8, a general transcriptional regulator, functions in part by enhancing the activity of the Wnt signalling pathway. Morris et al. report that E2F1, a transcription factor that is a target of pRB, is a potent and specific inhibitor of β-catenin, and that its activity is negatively regulated by CDK8. They point out that the interaction between E2F1 and β-catenin explains the long-standing paradox that pRB, an important tumour suppressor in most other contexts, is preserved in colorectal carcinomas. In an accompanying News & Views, René Bernards considers how the crosstalk between E2F and β-catenin signalling can lead to colorectal cancer. Aberrant activation of the canonical WNT/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival1,2. Although dysregulated β-catenin activity drives colon tumorigenesis, further genetic perturbations are required to elaborate full malignant transformation3. To identify genes that both modulate β-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex4, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Here we show that the suppression of CDK8 expression inhibits proliferation in colon cancer cells characterized by high levels of CDK8 and β-catenin hyperactivity. CDK8 kinase activity was necessary for β-catenin-driven transformation and for expression of several β-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer a clinical benefit in β-catenin-driven malignancies.

The anemia of chronic disease is a prevalent, poorly understood condition that afflicts patients with a wide variety of diseases, including infections, malignancies, and rheumatologic disorders. It is characterized by a blunted erythropoietin response by erythroid precursors, decreased red blood cell survival, and a defect in iron absorption and macrophage iron retention, which interrupts iron delivery to erythroid precursor cells. We noted that patients with large hepatic adenomas had severe iron refractory anemia similar to that observed in anemia of chronic disease. This anemia resolved spontaneously after adenoma resection or liver transplantation. We investigated the role of the adenomas in the pathogenesis of the anemia and found that they produce inappropriately high levels of hepcidin mRNA. Hepcidin is a peptide hormone that has been implicated in controlling the release of iron from cells. We conclude that hepcidin plays a major, causative role in the anemia observed in our subgroup of patients with hepatic adenomas, and we speculate that it is important in the pathogenesis of the anemia of chronic disease in general.

The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many epithelial organs and its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. Here, we show that prostate basal cells, but not secretory or neuroendocrine cells, express p63. In addition, prostate basal cells in culture predominantly express the DeltaNp63alpha isotype. In contrast, p63 protein is not detected in human prostate adenocarcinomas. Finally, and most importantly, p63(-/-) mice do not develop the prostate. These results indicate that p63 is required for prostate development and support the hypothesis that basal cells represent and/or include prostate stem cells. Furthermore, our results show that p63 immunohistochemistry may be a valuable tool in the differential diagnosis of benign versus malignant prostatic lesions.

PTEN acts as a tumor suppressor, at least in part, by antagonizing phosphoinositide 3-kinase (PI3K)/Akt signaling. Here we show that Forkhead transcription factors FKHRL1 and FKHR, substrates of the Akt kinase, are aberrantly localized to the cytoplasm and cannot activate transcription in PTEN-deficient cells. Restoration of PTEN function restores FKHR to the nucleus and restores transcriptional activation. Expression of a constitutively active form of FKHR that cannot be phosphorylated by Akt produces the same effect as reconstitution of PTEN on PTEN-deficient tumor cells. Specifically, activated FKHR induces apoptosis in cells that undergo PTEN-mediated cell death and induces G(1) arrest in cells that undergo PTEN-mediated cell cycle arrest. Furthermore, both PTEN and constitutively active FKHR induce p27(KIP1) protein but not p21. These data suggest that Forkhead transcription factors are critical effectors of PTEN-mediated tumor suppression.

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Cyclin-dependent kinases (CDKs) are activated by CDC25 phosphatases, which remove inhibitory phosphate from tyrosine and threonine residues. In human cells, CDC25 proteins are encoded by a multigene family, consisting of CDC25A, CDC25B, and CDC25C. In rodent cells, human CDC25A or CDC25B but not CDC25C phosphatases cooperate with either Ha-RASG12V or loss of RB1 in oncogenic focus formation. Such transformants were highly aneuploid, grew in soft agar, and formed high-grade tumors in nude mice. Overexpression of CDC25B was detected in 32 percent of human primary breast cancers tested. The CDC25 phosphatases may contribute to the development of human cancer.

Whole-exome sequencing of circulating tumor cells enables accurate and powered calling of somatic point mutations. Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Fatty acid synthase (FASN) is a key enzyme involved in neoplastic lipogenesis. Overexpression of FASN is common in many cancers, and accumulating evidence suggests that it is a metabolic oncogene with an important role in tumor growth and survival, making it an attractive target for cancer therapy. Early small-molecule FASN inhibitors such as cerulenin, C75 and orlistat have been shown to induce apoptosis in several cancer cell lines and to induce tumor growth delay in several cancer xenograft models but their mechanism is still not well understood. These molecules suffer from pharmacological limitations and weight loss as a side effect that prevent their development as systemic drugs. Several potent inhibitors have recently been reported that may help to unravel and exploit the full potential of FASN as a target for cancer therapy in the near future. Furthermore, novel sources of FASN inhibitors, such as green tea and dietary soy, make both dietary manipulation and chemoprevention potential alternative modes of therapy in the future.

(Cell 161, 1215–1228; May 21, 2015) Our paper presented an integrated sequencing analysis of clinical samples from advance prostate cancer patients. Parts of the findings were listed in Table S3, Mutations Identified in the 150 Cases. During the preparation of our table, we inadvertently mislabeled three gene names. In row 14118, 11-Sep should be SEPT11. In row 22306, 12-Sep should be SEPT12. In row 22927, 1-Mar should be MARCH1. These errors do not affect any conclusions of the paper, and the corrected version of Table S3 has been updated online. We apologize for any inconvenience this may have caused. Integrative Clinical Genomics of Advanced Prostate CancerRobinson et al.CellMay 21, 2015In BriefA multi-institutional integrative clinical sequencing analysis reveals that the majority of affected individuals with metastatic castration-resistant prostate cancer harbor clinically actionable molecular alterations, highlighting the need for genetic counseling to inform precision medicine in affected individuals with advanced prostate cancer. Full-Text PDF Open Archive

PAX8 is a paired-box gene important in embryogenesis of the thyroid, Müllerian, and renal/upper urinary tracts, and expression of PAX8 has been previously described in carcinomas from each of these sites. However, a large study including a wide variety of epithelial neoplasms from multiple organ sites other than the thyroid, kidney, or Müllerian system has not been performed. The goal of this study was to evaluate the utility of PAX8 immunostaining based on the evaluation of a wide range of epithelial tumors. PAX8 immunohistochemistry was performed on 1357 tumors (486 tumors in whole-tissue sections and 871 tumors in tissue microarrays, predominantly epithelial) from multiple organs. Only nuclear staining was scored as positive, and tumors were evaluated for the extent and intensity of staining. Western blot analysis with PAX8 was also performed on multiple tumor cell lines. Nuclear PAX8 staining was present in 91% (60 of 66) of thyroid tumors, 90% (158 of 176) of renal cell carcinomas (RCCs), 81% (13 of 16) of renal oncocytomas, 99% (164 of 165) of high-grade ovarian serous carcinomas, 71% (32 of 49) of nonserous ovarian epithelial neoplasms, 91% (10 of 11) of cervical epithelial lesions, and 98% (152 of 155) of endometrial adenocarcinomas. Of the remaining 719 evaluated tumors, only 30 cases (4%), including 12 thymic neoplasms, 3 bladder urothelial carcinomas, 4 lung squamous cell carcinomas, 2 esophageal adenocarcinomas, 1 pancreatic adenocarcinoma, 2 cholangiocarcinomas, 1 ovarian Sertoli-Leydig cell tumor, 1 ovarian sex cord stromal tumor, 3 testicular mixed germ cell tumors, and 1 acinic cell carcinoma, showed at least weak or focal PAX8 positivity. The unexpected finding was diffuse, moderate staining of PAX8 in a subset of thymomas and thymic carcinomas. The 689 remaining tumors, including but not limited to those from the prostate, colon, stomach, liver, adrenal gland, and head and neck, and small cell carcinomas from the lung, cervix, and ovary, were PAX8 negative. PAX8 specificity was confirmed by Western blot analysis, as expression was detected only in ovarian and RCC cell lines. These results show that PAX8 is a highly sensitive marker for thyroid, renal, Müllerian, and thymic tumors. Importantly, all lung adenocarcinomas, breast and adrenal neoplasms, and the majority of gastrointestinal tumors were negative for PAX8. Therefore, PAX8 is an excellent marker for confirming primary tumor site. In a subset of cases, additional markers, including but not limited to thyroid transcription factor-1, RCC, and Wilms tumor-1, may be needed to distinguish between the 3 most common PAX8-positive tumors.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. We developed quantitative real-time polymerase chain reaction (MethyLight) to measure percentage of methylated reference (PMR, ie, degree of methylation) for the <i>MGMT, MLH1</i>, and <i>CDKN2A</i> (pl6) promoters. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. To assess run-to-run variation, we repeated MethyLight five times. Bisulfìte-to-bisulfìte coefficient of variation (CV) of PMR ranged from 0.10 to 0.38 (mean, 0.21), and run-to-run CV of PMR ranged from 0.046 to 0.60 (mean, 0.31). Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. Of 272 colorectal cancers evaluated, most showed PMR either <1 or >10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression (P < 10⁻¹⁶). In conclusion, sodium bisulfite conversion and quantitative MethyLight assays have good precision and linearity and can be effectively used for high-throughput DNA methylation analysis on paraffin-embedded tissue.

The tumor suppressor gene PTEN/MMAC-1/TEP-1 (referred to hereafter as PTEN) maps to chromosome 10q23 and encodes a dual specificity phosphatase. The PTEN protein negatively regulates cell migration and cell survival and induces a G1 cell cycle block via negative regulation of the phosphatidylinositol 3'-kinase/protein kinase B/Akt signaling pathway. PTEN is frequently mutated or deleted in both prostate cancer cell lines and primary prostate cancers. A murine polyclonal antiserum was raised against a glutathione S-transferase fusion polypeptide of the COOH terninus of PTEN. Archival paraffin tissue sections from 109 cases of resected prostate cancer were immunostained with the antiserum, using DU145 and PC-3 cells as positive and negative controls, respectively. PTEN expression was seen in the secretory cells. Cases were considered positive when granular cytoplasmic staining was seen in all tumor cells, mixed when areas of both positive and negative tumor cell clones were seen, and negative when adjacent benign prostate tissue but not tumor tissue showed positive staining. Seventeen cases (15.6%) of prostate cancer were positive, 70 cases (64.2%) were mixed, and 22 cases (20.2%) were negative. Total absence of PTEN expression correlated with the Gleason score (P = 0.0081) and correlated more significantly with a Gleason score of 7 or higher (P = 0.0004) and with advanced pathological stage (American Joint Committee on Cancer stages T3b and T4; P = 0.0078). Thus, loss of PTEN protein is correlated with pathological markers of poor prognosis in prostate cancer.

Master transcription factors interact with DNA to establish cell type identity and to regulate gene expression in mammalian cells. The genome-wide map of these transcription factor binding sites has been termed the cistrome. Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13 colocalized at the reprogrammed AR binding sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR cistrome reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium toward transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis.

Metastasis is a fatal complication of prostate cancer, but its mechanisms remain largely unknown. In this report, the authors identify a signaling pathway commonly deregulated in human prostate cancer and describe how it can foster both primary growth and metastatic tumor progression. Epigenetic silencing of the RasGAP DAB2IP by EZH2 overexpression results in aberrant activation of Ras signaling, but also of NF-κB. These two events are mediated by different DAB2IP domains and have distinct roles in localized growth and distant dissemination. Metastasis is responsible for the majority of prostate cancer–related deaths; however, little is known about the molecular mechanisms that underlie this process. Here we identify an oncogene–tumor suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-κB (NF-κB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-κB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-κB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis.

Purpose Gleason grading is an important predictor of prostate cancer (PCa) outcomes. Studies using surrogate PCa end points suggest outcomes for Gleason score (GS) 7 cancers vary according to the predominance of pattern 4. These studies have influenced clinical practice, but it is unclear if rates of PCa mortality differ for 3 + 4 and 4 + 3 tumors. Using PCa mortality as the primary end point, we compared outcomes in Gleason 3 + 4 and 4 + 3 cancers, and the predictive ability of GS from a standardized review versus original scoring. Patients and Methods Three study pathologists conducted a blinded standardized review of 693 prostatectomy and 119 biopsy specimens to assign primary and secondary Gleason patterns. Tumor specimens were from PCa patients diagnosed between 1984 and 2004 from the Physicians' Health Study and Health Professionals Follow-Up Study. Lethal PCa (n = 53) was defined as development of bony metastases or PCa death. Hazard ratios (HR) were estimated according to original GS and standardized GS. We compared the discrimination of standardized and original grading with C-statistics from models of 10-year survival. Results For prostatectomy specimens, 4 + 3 cancers were associated with a three-fold increase in lethal PCa compared with 3 + 4 cancers (95% CI, 1.1 to 8.6). The discrimination of models of standardized scores from prostatectomy (C-statistic, 0.86) and biopsy (C-statistic, 0.85) were improved compared to models of original scores (prostatectomy C-statistic, 0.82; biopsy C-statistic, 0.72). Conclusion Ignoring the predominance of Gleason pattern 4 in GS 7 cancers may conceal important prognostic information. A standardized review of GS can improve prediction of PCa survival.

Overexpression of the fatty acid synthase (FASN) gene has been implicated in prostate carcinogenesis. We sought to directly assess the oncogenic potential of FASN.We used immortalized human prostate epithelial cells (iPrECs), androgen receptor-overexpressing iPrECs (AR-iPrEC), and human prostate adenocarcinoma LNCaP cells that stably overexpressed FASN for cell proliferation assays, soft agar assays, and tests of tumor formation in immunodeficient mice. Transgenic mice expressing FASN in the prostate were generated to assess the effects of FASN on prostate histology. Apoptosis was evaluated by Hoechst 33342 staining and by fluorescence-activated cell sorting in iPrEC-FASN cells treated with stimulators of the intrinsic and extrinsic pathways of apoptosis (ie, camptothecin and anti-Fas antibody, respectively) or with a small interfering RNA (siRNA) targeting FASN. FASN expression was compared with the apoptotic index assessed by the terminal deoxynucleotidyltransferase-mediated UTP end-labeling method in 745 human prostate cancer samples by using the least squares means procedure. All statistical tests were two-sided.Forced expression of FASN in iPrECs, AR-iPrECs, and LNCaP cells increased cell proliferation and soft agar growth. iPrECs that expressed both FASN and androgen receptor (AR) formed invasive adenocarcinomas in immunodeficient mice (12 of 14 mice injected formed tumors vs 0 of 14 mice injected with AR-iPrEC expressing empty vector (P < .001, Fisher exact test); however, iPrECs that expressed only FASN did not. Transgenic expression of FASN in mice resulted in prostate intraepithelial neoplasia, the incidence of which increased from 10% in 8- to 16-week-old mice to 44% in mice aged 7 months or more (P = .0028, Fisher exact test), but not in invasive tumors. In LNCaP cells, siRNA-mediated silencing of FASN resulted in apoptosis. FASN overexpression protected iPrECs from apoptosis induced by camptothecin but did not protect iPrECs from Fas receptor-induced apoptosis. In human prostate cancer specimens, FASN expression was inversely associated with the apoptotic rate (mean percentage of apoptotic cells, lowest vs highest quartile of FASN expression: 2.76 vs 1.34, difference = 1.41, 95% confidence interval = 0.45 to 2.39, Ptrend = .0046).These observations suggest that FASN can act as a prostate cancer oncogene in the presence of AR and that FASN exerts its oncogenic effect by inhibiting the intrinsic pathway of apoptosis.

With the advent of effective tools to study lipids, including mass spectrometry-based lipidomics, lipids are emerging as central players in cancer biology. Lipids function as essential building blocks for membranes, serve as fuel to drive energy-demanding processes and play a key role as signaling molecules and as regulators of numerous cellular functions. Not unexpectedly, cancer cells, as well as other cell types in the tumor microenvironment, exploit various ways to acquire lipids and extensively rewire their metabolism as part of a plastic and context-dependent metabolic reprogramming that is driven by both oncogenic and environmental cues. The resulting changes in the fate and composition of lipids help cancer cells to thrive in a changing microenvironment by supporting key oncogenic functions and cancer hallmarks, including cellular energetics, promoting feedforward oncogenic signaling, resisting oxidative and other stresses, regulating intercellular communication and immune responses. Supported by the close connection between altered lipid metabolism and the pathogenic process, specific lipid profiles are emerging as unique disease biomarkers, with diagnostic, prognostic and predictive potential. Multiple preclinical studies illustrate the translational promise of exploiting lipid metabolism in cancer, and critically, have shown context dependent actionable vulnerabilities that can be rationally targeted, particularly in combinatorial approaches. Moreover, lipids themselves can be used as membrane disrupting agents or as key components of nanocarriers of various therapeutics. With a number of preclinical compounds and strategies that are approaching clinical trials, we are at the doorstep of exploiting a hitherto underappreciated hallmark of cancer and promising target in the oncologist's strategy to combat cancer.

Cellular levels of key regulatory proteins are controlled via ubiquitination and subsequent degradation. Deubiquitinating enzymes or isopeptidases can potentially prevent targeted destruction of protein substrates through deubiquitination prior to proteasomal degradation. However, only one deubiquitinating enzyme to date has been matched to a specific substrate in mammalian cells and shown to functionally modify it. Here we show that the isopeptidase USP2a (ubiquitin-specific protease-2a) interacts with and stabilizes fatty acid synthase (FAS), which is often overexpressed in biologically aggressive human tumors. Further, USP2a is androgen-regulated and overexpressed in prostate cancer, and its functional inactivation results in decreased FAS protein and enhanced apoptosis. Thus, the isopeptidase USP2a plays a critical role in prostate cancer cell survival through FAS stabilization and represents a therapeutic target in prostate cancer.

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation is a distinct phenotype in colorectal cancer. However, a choice of markers for CIMP has been controversial. A recent extensive investigation has selected five methylation markers (<i>CACNA1G</i>, <i>IGF2</i>, <i>NEUROG1</i>, <i>RUNX3</i>, and <i>SOCS1</i>) as surrogate markers for epigenomic aberrations in tumor. The use of these markers as a CIMP-specific panel needs to be validated by an independent, large dataset. Using MethyLight assays on 920 colorectal cancers from two large prospective cohort studies, we quantified DNA methylation in eight CIMP-specific markers [the above five plus <i>CDKN2A</i> (p16), <i>CRABP1</i>, and <i>MLH1</i>]. A CIMP-high cutoff was set at ≥6/8 or ≥5/8 methylated promoters, based on tumor distribution and <i>BRAF</i>/<i>KRAS</i> mutation frequencies. All but two very specific markers [<i>MLH1</i> (98% specific) and <i>SOCS1</i> (93% specific)] demonstrated ≥85% sensitivity and ≥80% specificity, indicating overall good concordance in methylation patterns and good performance of these markers. Based on sensitivity, specificity, and false positives and negatives, the eight markers were ranked in order as: <i>RUNX3</i>, <i>CACNA1G</i>, <i>IGF2</i>, <i>MLH1</i>, <i>NEUROG1</i>, <i>CRABP1</i>, <i>SOCS1</i>, and <i>CDKN2A</i>. In conclusion, a panel of markers including at least <i>RUNX3</i>, <i>CACNA1G</i>, <i>IGF2</i>, and <i>MLH1</i> can serve as a sensitive and specific marker panel for CIMP-high.

Adam Bass, Gad Getz, Scott Carter and colleagues report the whole-exome sequences of 25 pairs of esophageal adenocarcinoma and Barrett's esophagus. They identify two pathways by which Barrett's esophagus can develop into esophageal adenocarcinoma. Barrett's esophagus is thought to progress to esophageal adenocarcinoma (EAC) through a stepwise progression with loss of CDKN2A followed by TP53 inactivation and aneuploidy. Here we present whole-exome sequencing from 25 pairs of EAC and Barrett's esophagus and from 5 patients whose Barrett's esophagus and tumor were extensively sampled. Our analysis showed that oncogene amplification typically occurred as a late event and that TP53 mutations often occurred early in Barrett's esophagus progression, including in non-dysplastic epithelium. Reanalysis of additional EAC exome data showed that the majority (62.5%) of EACs emerged following genome doubling and that tumors with genomic doubling had different patterns of genomic alterations, with more frequent oncogenic amplification and less frequent inactivation of tumor suppressors, including CDKN2A. These data suggest that many EACs emerge not through the gradual accumulation of tumor-suppressor alterations but rather through a more direct path whereby a TP53-mutant cell undergoes genome doubling, followed by the acquisition of oncogenic amplifications.

The PIK3CA gene encoding the p110alpha subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110beta, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110alpha and p110beta in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110alpha, H1047R and E545K, potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110alpha, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110beta a mutation homologous to the E545K allele of p110alpha, the resulting p110beta mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110beta with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110alpha at the corresponding sites in p110beta failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the beta isoform.

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease.Using real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6) in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18) and FFPE serous ovarian tumours (n = 22). Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry.We propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer.

Abstract Background: Whether the genomic rearrangement transmembrane protease, serine 2 (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (ERG) has prognostic value in prostate cancer is unclear. Methods: Among men with prostate cancer in the prospective Physicians' Health and Health Professionals Follow-Up Studies, we identified rearrangement status by immunohistochemical assessment of ERG protein expression. We used Cox models to examine associations of ERG overexpression with biochemical recurrence and lethal disease (distant metastases or cancer-specific mortality). In a meta-analysis including 47 additional studies, we used random-effects models to estimate associations between rearrangement status and outcomes. Results: The cohort consisted of 1,180 men treated with radical prostatectomy between 1983 and 2005. During a median follow-up of 12.6 years, 266 men experienced recurrence and 85 men developed lethal disease. We found no significant association between ERG overexpression and biochemical recurrence [hazard ratio (HR), 0.99; 95% confidence interval (CI), 0.78–1.26] or lethal disease (HR, 0.93; 95% CI, 0.61–1.43). The meta-analysis of prostatectomy series included 5,074 men followed for biochemical recurrence (1,623 events), and 2,049 men followed for lethal disease (131 events). TMPRSS2:ERG was associated with stage at diagnosis [risk ratio (RR)≥T3 vs. T2, 1.23; 95% CI, 1.16–1.30) but not with biochemical recurrence (RR, 1.00; 95% CI, 0.86–1.17) or lethal disease (RR, 0.99; 95% CI, 0.47–2.09). Conclusions: These results suggest that TMPRSS2:ERG, or ERG overexpression, is associated with tumor stage but does not strongly predict recurrence or mortality among men treated with radical prostatectomy. Impact: This is the largest prospective cohort study to examine associations of ERG overexpression and lethal prostate cancer among men treated with radical prostatectomy. Cancer Epidemiol Biomarkers Prev; 21(9); 1497–509. ©2012 AACR.

Abstract Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or “personalized”) medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR. Significance: The study of exceptional responders represents a promising approach to better understanding the mechanisms that underlie sensitivity to targeted anticancer therapies. Here, we identify two activating mTOR mutations in a patient with exquisite sensitivity to everolimus and pazopanib, suggesting an approach to identifying patients who might benefit most from mTOR inhibitors. Cancer Discov; 4(5); 546–53. ©2014 AACR. See related commentary by Rejto and Abraham, p. 513 This article is highlighted in the In This Issue feature, p. 495

Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. Here we report the engineering of personalized tumour ecosystems that contextually conserve the tumour heterogeneity, and phenocopy the tumour microenvironment using tumour explants maintained in defined tumour grade-matched matrix support and autologous patient serum. The functional response of tumour ecosystems, engineered from 109 patients, to anticancer drugs, together with the corresponding clinical outcomes, is used to train a machine learning algorithm; the learned model is then applied to predict the clinical response in an independent validation group of 55 patients, where we achieve 100% sensitivity in predictions while keeping specificity in a desired high range. The tumour ecosystem and algorithm, together termed the CANScript technology, can emerge as a powerful platform for enabling personalized medicine.

Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

Prostate cancer (PCa) metabolism appears to be unique in comparison with other types of solid cancers. Normal prostate cells mainly rely on glucose oxidation to provide precursors for the synthesis and secretion of citrate, resulting in an incomplete Krebs cycle and minimal oxidative phosphorylation for energy production. In contrast, during transformation, PCa cells no longer secrete citrate and they reactivate the Krebs cycle as energy source. Moreover, primary PCas do not show increased aerobic glycolysis and therefore they are not efficiently detectable with 18F-FDG-PET. However, increased de novo lipid synthesis, strictly intertwined with deregulation in classical oncogenes and oncosuppressors, is an early event of the disease. Up-regulation and increased activity of lipogenic enzymes (including fatty acid synthase and choline kinase) occurs throughout PCa carcinogenesis and correlates with worse prognosis and poor survival. Thus, lipid precursors such as acetate and choline have been successfully used as alternative tracers for PET imaging. Lipid synthesis intermediates and FA catabolism also emerged as important players in PCa maintenance. Finally, epidemiologic studies suggested that systemic metabolic disorders including obesity, metabolic syndrome, and diabetes as well as hypercaloric and fat-rich diets might increase the risk of PCa. However, how metabolic disorders contribute to PCa development and whether dietary lipids and de novo lipids synthesized intra-tumor are differentially metabolized still remains unclear. In this review, we examine the switch in lipid metabolism supporting the development and progression of PCa and we discuss how we can exploit its lipogenic nature for therapeutic and diagnostic purposes. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial-stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion-positive PCa.

Purpose Cure rates for localized high-risk prostate cancers (PCa) and some intermediate-risk PCa are frequently suboptimal with local therapy. Outcomes are improved by concomitant androgen-deprivation therapy (ADT) with radiation therapy, but not by concomitant ADT with surgery. Luteinizing hormone–releasing hormone agonist (LHRHa; leuprolide acetate) does not reduce serum androgens as effectively as abiraterone acetate (AA), a prodrug of abiraterone, a CYP17 inhibitor that lowers serum testosterone (&lt; 1 ng/dL) and improves survival in metastatic PCa. The possibility that greater androgen suppression in patients with localized high-risk PCa will result in improved clinical outcomes makes paramount the reassessment of neoadjuvant ADT with more robust androgen suppression. Patients and Methods A neoadjuvant randomized phase II trial of LHRHa with AA was conducted in patients with localized high-risk PCa (N = 58). For the first 12 weeks, patients were randomly assigned to LHRHa versus LHRHa plus AA. After a research prostate biopsy, all patients received 12 additional weeks of LHRHa plus AA followed by prostatectomy. Results The levels of intraprostatic androgens from 12-week prostate biopsies, including the primary end point (dihydrotestosterone/testosterone), were significantly lower (dehydroepiandrosterone, Δ 4 -androstene-3,17-dione, dihydrotestosterone, all P &lt; .001; testosterone, P &lt; .05) with LHRHa plus AA compared with LHRHa alone. Prostatectomy pathologic staging demonstrated a low incidence of complete responses and minimal residual disease, with residual T3- or lymph node–positive disease in the majority. Conclusion LHRHa plus AA treatment suppresses tissue androgens more effectively than LHRHa alone. Intensive intratumoral androgen suppression with LHRHa plus AA before prostatectomy for localized high-risk PCa may reduce tumor burden.

Abstract Animal models, particularly mouse models, play a central role in the study of the etiology, prevention, and treatment of human prostate cancer. While tissue culture models are extremely useful in understanding the biology of prostate cancer, they cannot recapitulate the complex cellular interactions within the tumor microenvironment that play a key role in cancer initiation and progression. The National Cancer Institute (NCI) Mouse Models of Human Cancers Consortium convened a group of human and veterinary pathologists to review the current animal models of prostate cancer and make recommendations about the pathologic analysis of these models. More than 40 different models with 439 samples were reviewed, including genetically engineered mouse models, xenograft, rat, and canine models. Numerous relevant models have been developed over the past 15 years, and each approach has strengths and weaknesses. Analysis of multiple genetically engineered models has shown that reactive stroma formation is present in all the models developing invasive carcinomas. In addition, numerous models with multiple genetic alterations display aggressive phenotypes characterized by sarcomatoid carcinomas and metastases, which is presumably a histologic manifestation of epithelial–mesenchymal transition. The significant progress in development of improved models of prostate cancer has already accelerated our understanding of the complex biology of prostate cancer and promises to enhance development of new approaches to prevention, detection, and treatment of this common malignancy. Cancer Res; 73(9); 2718–36. ©2013 AACR.

Abstract Lineage plasticity has emerged as an important mechanism of treatment resistance in prostate cancer. Treatment-refractory prostate cancers are increasingly associated with loss of luminal prostate markers, and in many cases induction of developmental programs, stem cell–like phenotypes, and neuroendocrine/neuronal features. Clinically, lineage plasticity may manifest as low PSA progression, resistance to androgen receptor (AR) pathway inhibitors, and sometimes small cell/neuroendocrine pathologic features observed on metastatic biopsy. This mechanism is not restricted to prostate cancer as other malignancies also demonstrate lineage plasticity during resistance to targeted therapies. At present, there is no established therapeutic approach for patients with advanced prostate cancer developing lineage plasticity or small cell neuroendocrine prostate cancer (NEPC) due to knowledge gaps in the underlying biology. Few clinical trials address questions in this space, and the outlook for patients remains poor. To move forward, urgently needed are: (i) a fundamental understanding of how lineage plasticity occurs and how it can best be defined; (ii) the temporal contribution and cooperation of emerging drivers; (iii) preclinical models that recapitulate biology of the disease and the recognized phenotypes; (iv) identification of therapeutic targets; and (v) novel trial designs dedicated to the entity as it is defined. This Perspective represents a consensus arising from the NCI Workshop on Lineage Plasticity and Androgen Receptor-Independent Prostate Cancer. We focus on the critical questions underlying lineage plasticity and AR-independent prostate cancer, outline knowledge and resource gaps, and identify strategies to facilitate future collaborative clinical translational and basic studies in this space.

<h2>Summary</h2> Patients with cancer may be at increased risk of severe coronavirus disease 2019 (COVID-19), but the role of viral load on this risk is unknown. We measured SARS-CoV-2 viral load using cycle threshold (C_T) values from reverse-transcription polymerase chain reaction assays applied to nasopharyngeal swab specimens in 100 patients with cancer and 2,914 without cancer who were admitted to three New York City hospitals. Overall, the in-hospital mortality rate was 38.8% among patients with a high viral load, 24.1% among patients with a medium viral load, and 15.3% among patients with a low viral load (p < 0.001). Similar findings were observed in patients with cancer (high, 45.2% mortality; medium, 28.0%; low, 12.1%; p = 0.008). Patients with hematologic malignancies had higher median viral loads (C_T = 25.0) than patients without cancer (C_T = 29.2; p = 0.0039). SARS-CoV-2 viral load results may offer vital prognostic information for patients with and without cancer who are hospitalized with COVID-19.

A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119–mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7–driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

The role of lycopene in prostate cancer prevention remains controversial. We examined the associations between dietary lycopene intake and prostate cancer, paying particular attention to the influence of prostate-specific antigen screening, and evaluated tissue biomarkers in prostate cancers in relation to lycopene intake.Among 49898 male health professionals, we obtained dietary information through questionnaires and ascertained total and lethal prostate cancer cases from 1986 through January 31, 2010. Cox regression was used to estimate multivariable hazard ratios (HRs) and 95% confidence intervals (CIs). Tissue microarrays and immunohistochemistry were used to assess tumor biomarker expression in a subset of men. Two-sided χ(2) tests were used to calculate the P values.Higher lycopene intake was inversely associated with total prostate cancer and more strongly with lethal prostate cancer (top vs bottom quintile: HR = 0.72; 95% CI = 0.56 to 0.94; P(trend) = .04). In a restricted population of screened participants, the inverse associations became markedly stronger (for lethal prostate cancer: HR = 0.47; 95% CI = 0.29 to 0.75; P trend = .009). Comparing different measures of dietary lycopene, early intake, but not recent intake, was inversely associated with prostate cancer. Higher lycopene intake was associated with biomarkers in the cancer indicative of less angiogenic potential.Dietary intake of lycopene was associated with reduced risk of lethal prostate cancer and with a lesser degree of angiogenesis in the tumor. Because angiogenesis is a strong progression factor, an endpoint of lethal prostate cancer may be more relevant than an endpoint of indolent prostate cancer for lycopene in the era of highly prevalent prostate-specific antigen screening.

Abstract 5′AMP‐activated kinase (AMPK) constitutes a hub for cellular metabolic and growth control, thus representing an ideal therapeutic target for prostate cancers (PCas) characterized by increased lipogenesis and activation of mTORC 1 pathway. However, whether AMPK activation itself is sufficient to block cancer cell growth remains to be determined. A small molecule screening was performed and identified MT 63–78, a specific and potent direct AMPK activator. Here, we show that direct activation of AMPK inhibits PCa cell growth in androgen sensitive and castration resistant PCa (CRPC) models, induces mitotic arrest, and apoptosis. In vivo , AMPK activation is sufficient to reduce PCa growth, whereas the allelic loss of its catalytic subunits fosters PCa development. Importantly, despite mTORC 1 blockade, the suppression of de novo lipogenesis is the underpinning mechanism responsible for AMPK‐mediated PCa growth inhibition, suggesting AMPK as a therapeutic target especially for lipogenesis‐driven PCas. Finally, we demonstrate that MT 63–78 enhances the growth inhibitory effect of AR signaling inhibitors MDV3100 and abiraterone. This study thus provides a rationale for their combined use in CRPC treatment.

Standard imaging for assessing osseous metastases in advanced prostate cancer remains focused on altered bone metabolism and is inadequate for diagnostic, prognostic, or predictive purposes. We performed a first-in-human phase I/II study of (89)Zr-DFO-huJ591 ((89)Zr-J591) PET/CT immunoscintigraphy to assess performance characteristics for detecting metastases compared with conventional imaging modalities (CIM) and pathology.Fifty patients with progressive metastatic castration-resistant prostate cancers were injected with 5 mCi of (89)Zr-J591. Whole-body PET/CT scans were obtained, and images were analyzed for tumor visualization. Comparison was made to contemporaneously obtained bone scintigraphy and cross-sectional imaging on a lesion-by-lesion basis and with biopsies of metastatic sites.Median standardized uptake value for (89)Zr-J591-positive bone lesions (n = 491) was 8.9 and for soft-tissue lesions (n = 90), it was 4.8 (P < 0.00003). (89)Zr-J591 detected 491 osseous sites compared with 339 by MDP and 90 soft-tissue lesions compared with 124 by computed tomography (CT). Compared with all CIMs combined, (89)Zr-J591 detected an additional 99 osseous sites. Forty-six lesions (21 bone and 25 soft tissue) were biopsied in 34 patients; 18 of 19 (89)Zr-J591-positive osseous sites and 14 of 16 (89)Zr-J591-positive soft tissue sites were positive for prostate cancer. The overall accuracy of (89)Zr-J591 was 95.2% (20 of 21) for osseous lesions and 60% (15 of 25) for soft-tissue lesions.(89)Zr-J591 imaging demonstrated superior targeting of bone lesions relative to CIMs. Targeting soft-tissue lesions was less optimal, although (89)Zr-J591 had similar accuracy as individual CIMs. This study will provide benchmark data for comparing performance of proposed prostate-specific membrane antigen (PSMA) targeting agents for prostate cancer.

TP53, PTEN, and RB1 tumor suppressor genes (TSGs) are recurrently altered in treatment-resistant prostate cancer. Cooperative loss of two or more TSGs may drive more aggressive disease.To determine clinical outcomes of single and compound TSG alterations across the spectrum of prostate cancer.Massively parallel targeted sequencing using castration-sensitive prostate cancer (CSPC; localized [L] and metastatic [M1]) and castration-resistant prostate cancer (CRPC) specimens (n=285). TSG altered (TSG-alt) was any copy number loss or deleterious mutation of one or more TSGs (TP53, PTEN, and RB1).For L-CSPC, event-free survival (EFS) and time to CRPC were estimated. For M1-CSPC and M1-CRPC, overall survival (OS) was estimated. Cox regression models assessed the association between cumulative TSG hits (zero hits vs one hit vs two to three hits) and outcomes with multivariable analyses adjusted for clinicopathological factors.TSG variants increased with advanced disease (L-CSPC: 39%; M1-CSPC: 63%, M1-CRPC: 92%). TSG-alt L-CSPC had shorter EFS (median 2.6yr, hazard ratio [HR] 1.95, 95% confidence interval [CI] 1.22-3.13) and time to CRPC (median 9.5mo, HR 3.36, 95% CI 1.01-11.16). Cumulative gene hits led to an incremental risk of relapse (EFS: one gene, HR 1.69, 95% CI 0.99-2.87; two to three genes, HR 2.70, 95% CI 1.43-5.08; both versus zero genes, p=0.004). There was evidence of inferior OS with increasing TSG hits in the metastatic cohorts. Only four (8%) patients in the M1-CRPC cohort were TSG-neg, one of whom died after 5.2yr. Multivariable analyses adjusting for mutational and copy number burden did not demonstrate a significant independent association of increasing gene hits and poorer outcomes.Deleterious TSG variants are associated with an increased risk of relapse (L) and death (M1) in CSPC. Poorer outcomes are seen with compound gene hits in both early and advanced disease, and this may in part reflect increasing global genomic instability.Men with prostate tumors with compound tumor suppressor gene mutations have poorer outcomes. These findings help identify patients with aggressive features who may benefit from intensified treatment.

The precise role of 5'AMP-activated kinase (AMPK) in cancer and its potential as a therapeutic target is controversial. Although it is well established that activation of this energy sensor inhibits the main anabolic processes that sustain cancer cell proliferation and growth, AMPK activation can confer on cancer cells the plasticity to survive under metabolic stress such as hypoxia and glucose deprivation, which are commonly observed in fast growing tumors. Thus, AMPK is referred to as both a "conditional" tumor suppressor and "contextual" oncogene. To add a further layer of complexity, AMPK activation in human cancer tissues and its correlation with tumor aggressiveness and progression appears to vary in different contexts. The current review discusses the different faces of this metabolic regulator, the therapeutic implications of its modulation, and provides an overview of the most relevant data available on AMPK activation and AMPK-activating drugs in human studies.

PTEN is a tumor suppressor frequently deleted in prostate cancer that may be a useful prognostic biomarker. However, the association of PTEN loss with lethal disease has not been tested in a large, predominantly surgically treated cohort.In the Health Professionals Follow-up Study and Physicians' Health Study, we followed 1044 incident prostate cancer cases diagnosed between 1986 and 2009 for cancer-specific and all-cause mortality. A genetically validated PTEN immunohistochemistry (IHC) assay was performed on tissue microarrays (TMAs). TMPRSS2:ERG status was previously assessed in a subset of cases by a genetically validated IHC assay for ERG. Cox proportional hazards models adjusting for age and body mass index at diagnosis, Gleason grade, and clinical or pathologic TNM stage were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association with lethal disease. All statistical tests were two-sided.On average, men were followed 11.7 years, during which there were 81 lethal events. Sixteen percent of cases had complete PTEN loss in all TMA cores and 9% had heterogeneous PTEN loss across cores. After adjustment for clinical-pathologic variables, complete PTEN loss was associated with lethal progression (HR = 1.8, 95% CI = 1.2 to 2.9). The association of PTEN loss (complete or heterogeneous) with lethal progression was only among men with ERG-negative (HR = 3.1, 95% CI = 1.7 to 5.7) but not ERG-positive (HR = 1.2, 95% CI = 0.7 to 2.2) tumors.PTEN loss is independently associated with increased risk of lethal progression, particularly in the ERG fusion-negative subgroup. These validated and inexpensive IHC assays may be useful for risk stratification in prostate cancer.

Abstract Purpose: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. Experimental Design: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish “favorable” versus “nonfavorable” pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). Results: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of &amp;gt;0.80 (possible range between 0 and 1) were defined on “false-negative” and “false-positive” rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores &amp;gt;0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P &amp;lt; 0.0001; OR, 20.9) and GS-6 versus non–GS-6 pathology (AUC, 0.65; P &amp;lt; 0.0001; OR, 12.95). Conclusions: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy. Clin Cancer Res; 21(11); 2591–600. ©2015 AACR.

The CYP17A1 inhibitor abiraterone markedly reduces androgen precursors and is thereby effective in castration-resistant prostate cancer (CRPC). However, abiraterone increases progesterone, which can activate certain mutant androgen receptors (AR) identified previously in flutamide-resistant tumors. Therefore, we sought to determine if CYP17A1 inhibitor treatment selects for progesterone-activated mutant ARs.AR was examined by targeted sequencing in metastatic tumor biopsies from 18 patients with CRPC who were progressing on a CYP17A1 inhibitor (17 on abiraterone, 1 on ketoconazole), alone or in combination with dutasteride, and by whole-exome sequencing in residual tumor in one patient treated with neoadjuvant leuprolide plus abiraterone.The progesterone-activated T878A-mutant AR was present at high allele frequency in 3 of the 18 CRPC cases. It was also present in one focus of resistant tumor in the neoadjuvant-treated patient, but not in a second clonally related resistant focus that instead had lost one copy of PTEN and both copies of CHD1. The T878A mutation appeared to be less common in the subset of patients with CRPC treated with abiraterone plus dutasteride, and transfection studies showed that dutasteride was a more potent direct antagonist of the T878A versus the wild-type AR.These findings indicate that selection for tumor cells expressing progesterone-activated mutant ARs is a mechanism of resistance to CYP17A1 inhibition.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Prostate cancers are considered to be immunologically 'cold' tumors given the very few patients who respond to checkpoint inhibitor (CPI) therapy. Recently, enrichment of interferon-stimulated genes (ISGs) predicted a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is overexpressed in prostate cancer and known to negatively regulate ISGs. In the present study, we demonstrate that EZH2 inhibition in prostate cancer models activates a double-stranded RNA-STING-ISG stress response upregulating genes involved in antigen presentation, Th1 chemokine signaling and interferon response, including programmed cell death protein 1 (PD-L1) that is dependent on STING activation. EZH2 inhibition substantially increased intratumoral trafficking of activated CD8+ T cells and increased M1 tumor-associated macrophages, overall reversing resistance to PD-1 CPI. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. These data suggest EZH2 inhibition as a therapeutic direction to enhance prostate cancer response to PD-1 CPI.

Abstract c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Analyses of clinical specimens reveal that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq uncover an increase in RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.

Cediranib, a pan-vascular endothelial growth factor receptor inhibitor, suppresses expression of homologous recombination repair (HRR) genes and increases sensitivity to poly-(ADP-ribose) polymerase inhibition in preclinical models. We investigated whether cediranib combined with olaparib improves the clinical outcomes of patients with prostate cancer.Patients with progressive metastatic castration-resistant prostate cancer (mCRPC) were randomly assigned 1:1 to arm A: cediranib 30 mg once daily plus olaparib 200 mg twice daily or arm B: olaparib 300 mg twice daily alone. The primary end point was radiographic progression-free survival (rPFS) in the intention-to-treat patients. The secondary end points were rPFS in patients with HRR-deficient and HRR-proficient mCRPC.In the intention-to-treat set of 90 patients, median rPFS was 8.5 (95% CI, 5.4 to 12.0) and 4.0 (95% CI, 3.2 to 8.5) months in arms A and B, respectively. Cediranib/olaparib significantly improved rPFS versus olaparib alone (hazard ratio [HR], 0.617; 95% CI, 0.392 to 0.969; P = .0359). Descriptive analyses showed a median rPFS of 10.6 (95% CI, 5.9 to not assessed [NA]) and 3.8 (95% CI, 2.33 to NA) months (HR, 0.64; 95% CI, 0.272 to 1.504) among patients with HRR-deficient mCRPC, and 13.8 (95% CI, 3.3 to NA) and 11.3 (95% CI, 3.8 to NA) months (HR, 0.98; 95% CI, 0.321 to 2.988) among patients with BRCA2-mutated mCRPC in arms A and B, respectively. The incidence of grades 3-4 adverse events was 61% and 18% in arms A and B, respectively.Cediranib combined with olaparib improved rPFS compared with olaparib alone in men with mCRPC. This combination was associated with an increased incidence of grades 3-4 adverse events. BRCA2-mutated subgroups treated with olaparib with or without cediranib were associated with a numerically longer median rPFS.

Allelic loss of chromosome 18q predicts a poor outcome in patients with stage II colorectal cancer. Although the specific gene inactivated by this allelic loss has not been elucidated, the DCC (deleted in colorectal cancer) gene is a candidate. We investigated whether the expression of the DCC protein in tumor cells is a prognostic marker in colorectal carcinoma.

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer. However, no study has comprehensively examined features of colorectal cancer with less extensive promoter methylation (designated as “CIMP-low”). Using real-time polymerase chain reaction (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1] in 840 relatively unbiased, population-based colorectal cancer samples, obtained from two large prospective cohort studies. CIMP-low (defined as 1/5 to 3/5 methylated promoters) colorectal cancers were significantly more common among men (38 versus 30% in women, P = 0.01) and among KRAS-mutated tumors (44 versus 30% in KRAS/BRAF wild-type tumors, P = 0.0003; 19% in BRAF-mutated tumors, P < 0.0001). In addition, KRAS mutations were significantly more common in CIMP-low tumors (47%) than in CIMP-high tumors (with ≥4/5 methylated promoters, 12%, P < 0.0001) and CIMP-0 tumors (with 0/5 methylated promoters, 37%, P = 0.007). The associations of CIMP-low tumors with male sex and KRAS mutations still existed after tumors were stratified by microsatellite instability status. In conclusion, CIMP-low colorectal cancer is associated with male sex and KRAS mutations. The hypothesis that CIMP-low tumors are different from CIMP-high and CIMP-0 tumors needs to be tested further. The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer. However, no study has comprehensively examined features of colorectal cancer with less extensive promoter methylation (designated as “CIMP-low”). Using real-time polymerase chain reaction (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1] in 840 relatively unbiased, population-based colorectal cancer samples, obtained from two large prospective cohort studies. CIMP-low (defined as 1/5 to 3/5 methylated promoters) colorectal cancers were significantly more common among men (38 versus 30% in women, P = 0.01) and among KRAS-mutated tumors (44 versus 30% in KRAS/BRAF wild-type tumors, P = 0.0003; 19% in BRAF-mutated tumors, P < 0.0001). In addition, KRAS mutations were significantly more common in CIMP-low tumors (47%) than in CIMP-high tumors (with ≥4/5 methylated promoters, 12%, P < 0.0001) and CIMP-0 tumors (with 0/5 methylated promoters, 37%, P = 0.007). The associations of CIMP-low tumors with male sex and KRAS mutations still existed after tumors were stratified by microsatellite instability status. In conclusion, CIMP-low colorectal cancer is associated with male sex and KRAS mutations. The hypothesis that CIMP-low tumors are different from CIMP-high and CIMP-0 tumors needs to be tested further. Transcriptional inactivation by cytosine methylation at promoter CpG islands of tumor suppressor genes is thought to be an important mechanism in human carcinogenesis.1Laird PW Cancer epigenetics.Hum Mol Genet. 2005; : R65-R76Crossref PubMed Scopus (437) Google Scholar A number of tumor suppressor genes, such as CDKN2A (the p16/INK4a gene), MGMT, and MLH1, have been shown to be silenced by promoter methylation in colorectal cancers.1Laird PW Cancer epigenetics.Hum Mol Genet. 2005; : R65-R76Crossref PubMed Scopus (437) Google Scholar2Issa JP CpG island methylator phenotype in cancer.Nat Rev Cancer. 2004; 4: 988-993Crossref PubMed Scopus (865) Google Scholar3Rashid A Issa JP CpG island methylation in gastroenterologic neoplasia: a maturing field.Gastroenterology. 2004; 127: 1578-1588Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar In fact, a subset of colorectal cancers have been shown to exhibit promoter methylation in multiple genes, which is referred to as the CpG island methylator phenotype (CIMP).2Issa JP CpG island methylator phenotype in cancer.Nat Rev Cancer. 2004; 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In this study using quantitative DNA methylation analysis (MethyLight) and a large number of relatively unbiased, population-based colorectal cancer samples,11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar we have examined molecular features of CIMP-low tumors (defined as the presence of methylation in 1/5 to 3/5 promoters) compared with those of CIMP-0 tumors (with 0/5 methylated promoters) and CIMP-high tumors (with ≥4/5 methylated promoters). MethyLight assays can reliably distinguish high from low levels of DNA methylation, the latter of which likely have little or no biological significance.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar,19Coleman WB Rivenbark AG Quantitative DNA methylation analysis: the promise of high-throughput epigenomic diagnostic testing in human neoplastic disease.J Mol Diagn. 2006; 8: 152-156Abstract Full Text Full Text PDF PubMed Scopus (26) Google ScholarMaterials and MethodsStudy GroupTo recruit patients into this study, we used the databases of two large prospective cohort studies; the Nurses' Health Study (N = 121,700 women followed since 1976)20Colditz GA Hankinson SE The Nurses' Health Study: lifestyle and health among women.Nat Rev Cancer. 2005; 5: 388-396Crossref PubMed Scopus (451) Google Scholar and the Health Professional Follow-up Study (N = 51,500 men followed since 1986).21Wei EK Giovannucci E Fuchs CS Willett WC Mantzoros CS Low plasma adiponectin levels and risk of colorectal cancer in men: a prospective study.J Natl Cancer Inst. 2005; 97: 1688-1694Crossref PubMed Scopus (405) Google Scholar Informed consent was obtained from all participants before inclusion in the cohorts. All cohort participants were free of cancer (except for non-melanoma skin cancer) at the time of study entry. A subset of the cohort participants developed colorectal cancers during prospective follow-up. Thus, these colorectal cancers represented population-based, relatively unbiased samples. Based on availability of tissue samples and results at the time of the study, a total of 840 colorectal cancer cases (362 from the men's cohort and 478 from the women's cohort) were included, among which 460 cases were examined in our previous study.11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar Tissue collection and analyses were approved by the Dana-Farber Cancer Institute and Brigham and Women's Hospital Institutional Review Boards.Genomic DNA ExtractionAfter tumor areas were marked on a hematoxylin and eosin (H&E)-stained section with a pen, tumor tissue was dissected manually from additional tissue sections by a sterile needle. Normal colonic tissue for microsatellite analysis was obtained from the margins of the resection specimens. The dissected tissue was placed in buffered proteinase K solution at 56°C for 3 hours. Genomic DNA was then extracted using QIAmp DNA Mini Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions.Real-Time Polymerase Chain Reaction (PCR) (MethyLight) for Quantitative DNA Methylation AnalysisSodium bisulfite treatment on genomic DNA was performed as previously described.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar For DNA methylation analysis, we typically used one to two tissue sections (10-μm thick) when large tumor sections were available. Real-time PCR to measure DNA methylation (MethyLight) was performed as previously described.22Eads CA Danenberg KD Kawakami K Saltz LB Danenberg PV Laird PW CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.Cancer Res. 1999; 59: 2302-2306PubMed Google Scholar23Eads CA Danenberg KD Kawakami K Saltz LB Blake C Shibata D Danenberg PV Laird PW MethyLight: a high-throughput assay to measure DNA methylation.Nucleic Acids Res. 2000; 28: E32Crossref PubMed Scopus (1200) Google Scholar24Widschwendter M Siegmund KD Muller HM Fiegl H Marth C Muller-Holzner E Jones PA Laird PW Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.Cancer Res. 2004; 64: 3807-3813Crossref PubMed Scopus (279) Google Scholar We used ABI 7300 (Applied Biosystems, Foster City, CA) for quantitative real-time PCR. Using five sets of primers and probes, we amplified five CIMP-specific promoters [calcium channel, voltage-dependent, T type alpha-1G subunit (CACNA1G); cyclin-dependent kinase inhibitor 2A (CDKN2A) (p16/INK4A); cellular retinoic acid binding protein 1 (CRABP1); MLH1; and neurogenin 1 (NEUROG1)].11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar COL2A1 (the collagen 2A1 gene) was used to normalize for the amount of input bisulfite-converted DNA.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar,24Widschwendter M Siegmund KD Muller HM Fiegl H Marth C Muller-Holzner E Jones PA Laird PW Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.Cancer Res. 2004; 64: 3807-3813Crossref PubMed Scopus (279) Google Scholar Primers and probes were previously described for the following genes: CACNA1G, CRABP1, and NEUROG1;11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar CDKN2A and COL2A1;24Widschwendter M Siegmund KD Muller HM Fiegl H Marth C Muller-Holzner E Jones PA Laird PW Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.Cancer Res. 2004; 64: 3807-3813Crossref PubMed Scopus (279) Google Scholar and MLH1.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar The percentage of methylated reference (PMR, ie, degree of methylation) at a specific locus was calculated by dividing the GENE/COL2A1 ratio of the amounts of templates in a sample by the GENE/COL2A1 ratio in M. SssI-treated human genomic DNA (presumably fully methylated) and multiplying this value by 100.25Eads CA Lord RV Wickramasinghe K Long TI Kurumboor SK Bernstein L Peters JH DeMeester SR DeMeester TR Skinner KA Laird PW Epigenetic patterns in the progression of esophageal adenocarcinoma.Cancer Res. 2001; 61: 3410-3418PubMed Google Scholar A PMR cutoff value of 4 was based on previously validated data.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar,22Eads CA Danenberg KD Kawakami K Saltz LB Danenberg PV Laird PW CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.Cancer Res. 1999; 59: 2302-2306PubMed Google Scholar23Eads CA Danenberg KD Kawakami K Saltz LB Blake C Shibata D Danenberg PV Laird PW MethyLight: a high-throughput assay to measure DNA methylation.Nucleic Acids Res. 2000; 28: E32Crossref PubMed Scopus (1200) Google Scholar24Widschwendter M Siegmund KD Muller HM Fiegl H Marth C Muller-Holzner E Jones PA Laird PW Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.Cancer Res. 2004; 64: 3807-3813Crossref PubMed Scopus (279) Google Scholar25Eads CA Lord RV Wickramasinghe K Long TI Kurumboor SK Bernstein L Peters JH DeMeester SR DeMeester TR Skinner KA Laird PW Epigenetic patterns in the progression of esophageal adenocarcinoma.Cancer Res. 2001; 61: 3410-3418PubMed Google Scholar26Eads CA Lord RV Kurumboor SK Wickramasinghe K Skinner ML Long TI Peters JH DeMeester TR Danenberg KD Danenberg PV Laird PW Skinner KA Fields of aberrant CpG island hypermethylation in Barrett's esophagus and associated adenocarcinoma.Cancer Res. 2000; 60: 5021-5026PubMed Google Scholar Based on the distribution of PMR values at the CRABP1 locus,11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar we raised PMR cutoff to 6 for CRABP1, which improved specificity of CRABP1 for the prediction of overall CIMP status (data not shown). Precision and performance characteristics of bisulfite conversion and subsequent MethyLight assays have been previously evaluated, and the assays have been validated.18Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs C Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google ScholarMicrosatellite Instability (MSI) AnalysisFor MSI analysis, whole genome amplification of genomic DNA was performed by PCR using random 15-mer primers.27Ogino S Kawasaki T Brahmandam M Yan L Cantor M Namgyal C Mino-Kenudson M Lauwers GY Loda M Fuchs CS Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.J Mol Diagn. 2005; 7: 413-421Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar Methods to determine MSI status have been previously described.28Ogino S Brahmandam M Cantor M Namgyal C Kawasaki T Kirkner G Meyerhardt JA Loda M Fuchs CS Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.Mod Pathol. 2006; 19: 59-68Crossref PubMed Scopus (203) Google Scholar In addition to the recommended MSI panel consisting of D2S123, D5S346, D17S250, BAT25, and BAT26,29Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar we also used BAT40, D18S55, D18S56, D18S67, and D18S487 (ie, 10-marker panel).28Ogino S Brahmandam M Cantor M Namgyal C Kawasaki T Kirkner G Meyerhardt JA Loda M Fuchs CS Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.Mod Pathol. 2006; 19: 59-68Crossref PubMed Scopus (203) Google Scholar A “high degree of MSI” (MSI-H) was defined as the presence of instability in ≥30% of the markers in the 10-marker panel. A low degree of MSI (MSI-L) was defined as the presence of instability in <30% of the markers, and “microsatellite-stable” (MSS) tumors were defined as tumors without an unstable marker.Sequencing of KRAS and BRAFMethods of PCR and sequencing targeted for KRAS codons 12 and 13 and BRAF codon 600 have been previously described.28Ogino S Brahmandam M Cantor M Namgyal C Kawasaki T Kirkner G Meyerhardt JA Loda M Fuchs CS Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.Mod Pathol. 2006; 19: 59-68Crossref PubMed Scopus (203) Google Scholar,30Ogino S Meyerhardt JA Cantor M Brahmandam M Clark JW Namgyal C Kawasaki T Kinsella K Michelini AL Enzinger PC Kulke MH Ryan DP Loda M Fuchs CS Molecular alterations in tumors and response to combination chemotherapy with gefitinib for advanced colorectal cancer.Clin Cancer Res. 2005; 11: 6650-6656Crossref PubMed Scopus (130) Google Scholar All forward sequencing results were confirmed by reverse sequencing. Pyrosequencing methods for KRAS and BRAF sequencing were implemented since the study began and performed on a subset of cases. Methods of KRAS pyrosequencing have been validated as described.27Ogino S Kawasaki T Brahmandam M Yan L Cantor M Namgyal C Mino-Kenudson M Lauwers GY Loda M Fuchs CS Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.J Mol Diagn. 2005; 7: 413-421Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar BRAF pyrosequecing was performed using the PSQ96 HS System (Biotage AB and Biosystems, Uppsala, Sweden) according to the manufacturer's instructions. For BRAF pyrosequencing, PCR primers were 5′-CAGTAAAAATAGGTGATTTTG-3′ (forward) and biotin-5′-CAACTGTTCAAACTGATGGG-3′ (reverse), pyrosequencing primer was 5′-TGATTTTGGTCTAGCTACA-3′, and the dispensation order was TGAGTCAGTCAGTCAGTCAGTCAGTC.Tissue Microarray (TMA) Construction and Immunohistochemistry for p53TMAs were constructed as previously described31Ogino S Brahmandam M Kawasaki T Kirkner GJ Loda M Fuchs CS Combined analysis of COX-2 and p53 expressions reveals synergistic inverse correlations with microsatellite instability and CpG island methylator phenotype in colorectal cancer.Neoplasia. 2006; 8: 458-464Abstract Full Text PDF PubMed Scopus (74) Google Scholar using the Automated Arrayer (Beecher Instruments, Sun Prairie, WI). We analyzed whole tissue sections for cases in which there was not enough tumor tissue for TMAs or no definitive results by TMA immunohistochemistry. Methods for p53 immunohistochemistry were previously described.28Ogino S Brahmandam M Cantor M Namgyal C Kawasaki T Kirkner G Meyerhardt JA Loda M Fuchs CS Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.Mod Pathol. 2006; 19: 59-68Crossref PubMed Scopus (203) Google Scholar Only strong and unequivocal nuclear staining in 50% or more of tumor cells was interpreted as positive. Appropriate positive and negative controls were included in each run of immunohistochemistry. All slides were interpreted by a pathologist (S.O.) blinded from any other laboratory data.Statistical AnalysisFor statistical analysis, the χ2 test (or Fisher's exact test for categories with an N value of less than 10) was performed on categorical data using the SAS program (version 9.1; SAS Institute, Cary, NC). All P values were two-sided, and statistical significance was set at P values of ≤0.05.ResultsCriteria for CIMP-High, CIMP-Low, and CIMP-0We obtained 840 colorectal cancer specimens and quantified DNA methylation in the five CIMP-specific gene promoters (CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1) by MethyLight technology. We have previously validated the selection and use of these five loci for the determination of CIMP-high in colorectal cancer.11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar We also examined normal mucosa from resected segments of colon in a subset of cases and shown infrequent, at most low-levels, of DNA methylation in these loci (data not shown). Distributions of PMR values were bimodal, and only rare cases showed PMR within the range of PMR cutoff ±1 (data on the first 460 cases11Ogino S Cantor M Kawasaki T Brahmandam M Kirkner G Weisenberger DJ Campan M Laird PW Loda M Fuchs CS CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.Gut. 2006; 55: 1000-1006Crossref PubMed Scopus (296) Google Scholar and data not shown for all 840 cases). Thus, the methylation status (positive or negative) at each locus could be unequivocally determined for a vast majority of the cases.Table 1 shows the distributions of the number of methylated promoters (from 0 to five) in all 840 colorectal cancers composed of 362 from the men's cohort and 478 from the women's cohort. As in Table 1, 122 MSI-H tumors in this study showed a striking bimodal distribution with only one tumor (0.8%), exhibiting 3/5 methylated promoters. Based on this bimodal distribution, CIMP-high was defined as the presence of ≥4/5 methylated promoters. Overall, 130 (15%) of all 840 tumors were CIMP-high. The proportion of CIMP-high cases increased progressively from MSS (6.0%) to MSI-L (11%) and MSI-H (69%) tumor status (MSI-H versus MSI-L or MSS, P < 0.0001). CIMP-low tumors, which were defined as tumors with 1/5 to 3/5 methylated promoters, constituted 33% (279/840) of all tumors. CIMP-0 tumors, defined as tumors with no methylated promoter, constituted 51% (431/840) of all tumors.Table 1Distribution of Colorectal Cancers According to the Number of Methylated PromotersNumber of methylated promoters0 (CIMP-0)12345CIMP-low 1 to 3CIMP-high≥4All cases (N = 840)431 (51%)14390465476279 (33%)130 (15%)Men (N = 362)189 (52%)7343211521137 (38%); P = 0.0136 (9.9%); P = 0.0001Women (N = 478)242 (51%)7047253955142 (30%); P = 0.0194 (20%); P = 0.0001MSI-H (N = 122)22 (18%)951166915 (13%)85 (70%)MSI-L (N = 72)36 (50%)11985328 (39%)8 (11%)MSS (N = 621)356 (57%)1207236334228 (37%)37 (6.0%)P values are based on comparisons of CIMP-low and CIMP-high frequencies between men and women. Open table in a new tab Characteristics of CIMP-High, CIMP-Low, and CIMP-0We examined relations of CIMP-0, CIMP-low, and CIMP-high with sex. Whereas CIMP-high tumors were more common in women (20%) than men (9.9%, P = 0.0001), CIMP-low tumors were significantly more common in men (38%) than in women (30%, P = 0.01) among all 840 tumors (Table 1). No significant difference was observed in the frequencies of CIMP-0 tumors between men and women. Next, we stratified tumors according to MSI status. CIMP-high tumors were still significantly more common in women than men among MSI-H tumors (77% in women versus 53% in men, P = 0.007) and MSS tumors (8.1% in women versus 3.3% in men, P = 0.02). In contrast, CIMP-low tumors were more common in men than women among MSS tumors (42% in men versus 32% in women, P = 0.01) and MSI-H tumors (19% in men versus 8.9% in women, although statistical significance was not reached).We also examined relations between CIMP status and KRAS and BRAF mutations. We subclassified tumors into KRAS-mutated tumors (with wild-type BRAF) (N = 277), BRAF-mutated tumors (with wild-type KRAS) (N = 103), and tumors with both wild-type KRAS and BRAF (N = 387). The results of tumors with mutations in both KRAS and BRAF are not shown because of the small number of such tumors (N = 5). Figure 1 shows the frequencies of CIMP-0, CIMP-low, and CIMP-high among KRAS/BRAF wild-type tumors, KRAS-mutated tumors, and BRAF-mutated tumors. In contrast to CIMP-0, which was more common in KRAS/BRAF wild-type tumors (61%) than in KRAS-mutated tumors (52%, P = 0.02), CIMP-low was more common in KRAS-mutated tumors (44%) than in KRAS/BRAF wild-type tumors (30%, P = 0.0003). BRAF-mutated tumors showed a very high frequency of CIMP-high (71%) compared with KRAS/BRAF wild-type tumors (9.0%, P < 0.0001) and KRAS-mutated tumors (4.7%, P < 0.0001). We also examined the frequencies of KRAS mutations among CIMP-high, CIMP-low, and CIMP-0 tumors. CIMP-low tumors showed a significantly higher KRAS mutation rate (47%) than CIMP-high tumors (12%, P < 0.0001) and CIMP-0 tumors (37%, P = 0.007) (Figure 2).Figure 2Frequencies of KRAS mutat

To determine whether Akt activation was sufficient for the transformation of normal prostate epithelial cells, m urine p rostate restricted A kt k inase activity was generated in t ransgenic mice (MPAKT mice). Akt expression led to p70 S6K activation, prostatic intraepithelial neoplasia (PIN), and bladder obstruction. mRNA expression profiles from MPAKT ventral prostate revealed similarities to human cancer and an angiogenic signature that included three angiogenin family members, one of which was found elevated in the plasma of men with prostate cancer. Thus, the MPAKT model may be useful in studying the role of Akt in prostate epithelial cell transformation and in the discovery of molecular markers relevant to human disease.

In 1920, Warburg suggested that tumors consistently rely on anaerobic pathways to convert glucose to ATP even in the presence of abundant oxygen [Warberg, 1956] despite the fact that it is less efficient for energy supply than aerobic glycolysis. The reasons for this remain obscure to date. More often than not, the microenvironment of solid tumors contains regions of poor oxygenation and high acidity. In this context hypoxia can act in an epigenetic fashion, inducing changes in gene expression and in metabolism for survival. It is reasonable to assume that only the tumor cells capable of developing an unusual tolerance to limiting oxygen availability and to the acidosis resulting from excessive lactate production, can survive. In addition to the striking changes that occur in glucose metabolism, studies in human cancer patients suggest that there is often also an increase in free fatty acid turnover, oxidation and clearance [Legaspi et al., 1987; Hyltander et al., 1991]. For instance, a lipid mobilizing factor produced by tumor cells appears to be responsible for the increase in whole body fatty acid oxidation [Russell and Tisdale, 2002]. Fatty acids synthesis in tumor tissues also occurs at very high rates, as first demonstrated more than half a century ago [Medes et al., 1953]. Importantly, (14)C glucose studies have shown that in tumor cells almost all fatty acids derive from de novo synthesis despite adequate nutritional supply [Sabine and Abraham, 1967; Ookhtens et al., 1984; Weiss et al., 1986]. In addition, tumors overexpressing fatty acid synthase (FAS), the enzyme responsible for de novo synthesis of fatty acids, display aggressive biologic behavior compared to those tumors with normal FAS levels, suggesting that FAS overexpression confers a selective growth advantage. Here, we will review the roles that FAS plays in important cellular processes such as apoptosis and proliferation. In addition, speculations on the putative role of FAS in the altered metabolic pathways of prostate cancer cells will be explored. Because of the frequent overexpression of this enzyme prostate cancer, FAS constitutes a therapeutic target in this disease.

Abstract Purpose: High-level expression of the telomerase reverse transcriptase (hTERT) in &amp;gt;85% of human cancers, in contrast with its restricted expression in normal adult tissues, points to hTERT as a broadly applicable molecular target for anticancer immunotherapy. CTLs recognize peptides derived from hTERT and kill hTERT+ tumor cells of multiple histologies in vitro. Moreover, because survival of hTERT+ tumor cells requires functionally active telomerase, hTERT mutation or loss as a means of escape may be incompatible with sustained tumor growth. Experimental Design: A Phase I clinical trial was performed to evaluate the clinical and immunological impact of vaccinating advanced cancer patients with the HLA-A2-restricted hTERT I540 peptide presented with keyhole limpet hemocyanin by ex vivo generated autologous dendritic cells. Results: As measured by peptide/MHC tetramer, enzyme-linked immunospot, and cytotoxicity assays, hTERT-specific T lymphocytes were induced in 4 of 7 patients with advanced breast or prostate carcinoma after vaccination with dendritic cells pulsed with hTERT peptide. Tetramer-guided high-speed sorting and polyclonal expansion achieved highly enriched populations of hTERT-specific cells that killed tumor cells in an MHC- restricted fashion. Despite concerns of telomerase activity in rare normal cells, no significant toxicity was observed. Partial tumor regression in 1 patient was associated with the induction of CD8+ tumor infiltrating lymphocytes. Conclusions: These results demonstrate the immunological feasibility of vaccinating patients against telomerase and provide rationale for targeting self-antigens with critical roles in oncogenesis.

Despite its potential role as a tumor suppressor, p27 gene, a member of the Cip/Kip family of cyclin-dependent kinase inhibitor genes, has never been found mutated in human tumors. We investigated p27 protein expression in a series of 108 non-small cell lung cancers (57.4% stage 1, 16.7% stage 2, and 25.9% stage 3) to determine whether the lack or altered expression of this protein correlates with neoplastic transformation and/or progression. We performed immunohistochemistry and Western blot analysis of each specimen. We found that tumors expressing low to undetectable levels of p27 contained high p27 degradation activity. When we evaluated the outcome of the patients in relationship to p27 expression, we found p27 to be a prognostic factor correlating with the overall survival times (P = 0.0012). The possibility of a simple assay, such as the immunohistochemical analysis of p27 expression on routinely formalin-fixed, paraffin-embedded specimens, has considerable value for the prognosis of patients who undergo surgical resection. In addition, confirmation of the involvement of the proteasome-mediated proteolysis in p27 degradation should stimulate new strategies of nonsurgical treatments of non-small cell lung cancer.

No AccessJournal of UrologyClinical Urology: Original Articles1 Mar 1998LOW P27 EXPRESSION PREDICTS POOR DISEASE-FREE SURVIVAL IN PATIENTS WITH PROSTATE CANCER RONALD M. YANG, JOHN NAITOH, MICHAEL MURPHY, HE-JING WANG, JULIA PHILLIPSON, JEAN B. DEKERNION, MASSIMO LODA, and ROBERT E. REITER RONALD M. YANGRONALD M. YANG More articles by this author , JOHN NAITOHJOHN NAITOH More articles by this author , MICHAEL MURPHYMICHAEL MURPHY More articles by this author , HE-JING WANGHE-JING WANG More articles by this author , JULIA PHILLIPSONJULIA PHILLIPSON More articles by this author , JEAN B. DEKERNIONJEAN B. DEKERNION More articles by this author , MASSIMO LODAMASSIMO LODA More articles by this author , and ROBERT E. REITERROBERT E. REITER More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(01)63776-5AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: p27 is an inhibitor of the cell cycle with potential tumor suppressor function. Decreased levels of p27 protein expression have been correlated with poor prognosis in patients with breast and colorectal carcinomas. Although as many as a third of patients with clinically localized prostate cancer will have relapse after radical prostatectomy, predicting who will have recurrence remains enigmatic. We examined the ability of p27 protein levels to predict outcome in patients with clinically localized disease who underwent radical prostatectomy. Materials and Methods: p27 protein expression was evaluated in 86 patients with clinical stage T1-2 prostate cancer who were treated with radical prostatectomy. Archived paraffin embedded specimens were sectioned and immunostained with p27 antibody, and scored by 2 independent observers in a blinded fashion. The absence or presence of p27 protein was then correlated with biochemical relapse in univariate and multivariate analyses. Results: In a multivariate analysis that included age, preoperative prostate specific antigen, Gleason score and pathological stage p27 was a strong independent predictor of disease-free survival (p = 0.0184, risk ratio 3.04), second only to pathological stage (p = 0.0001, risk ratio 6.73). Even more strikingly, multivariate analysis demonstrated that p27 was the strongest predictor of biochemical recurrence (p = 0.0081, risk ratio 4.99) among factors studied in patients with pathological T2a-T3b disease. Conclusions: Absent or low levels of p27 protein expression appear to be an adverse prognostic factor in patients with clinically organ confined disease treated by radical prostatectomy. 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Google Scholar From the Departments of Urology, Pathology and Biomathematics, UCLA School of Medicine, Los Angeles, California, and Department of Pathology, Beth Israel Deaconess Medical Center, West Campus, Harvard Medical School, Boston, Massachusetts.© 1998 by American Urological Association, Inc.FiguresReferencesRelatedDetailsCited byFEBBO P and SELLERS W (2018) Use of Expression Analysis to Predict Outcome After Radical ProstatectomyJournal of Urology, VOL. 170, NO. 6S, (S11-S20), Online publication date: 1-Dec-2003.BEN-IZHAK O, LAHAV-BARATZ S, MERETYK S, BEN-ELIEZER S, SABO E, DIRNFELD M, COHEN S and CIECHANOVER A (2018) Inverse Relationship Between Skp2 Ubiquitin Ligase and the Cyclin Dependent Kinase Inhibitor p27Kip1 in Prostate CancerJournal of Urology, VOL. 170, NO. 1, (241-245), Online publication date: 1-Jul-2003.FREEDLAND S, deGREGORIO F, SACOOLIDGE J, ELSHIMALI Y, CSATHY G, DOREY F, REITER R and ARONSON W (2018) Preoperative p27 Status is an Independent Predictor of Prostate Specific Antigen Failure Following Radical ProstatectomyJournal of Urology, VOL. 169, NO. 4, (1325-1330), Online publication date: 1-Apr-2003.VIS A, NOORDZIJ M, FITOZ K, WILDHAGEN M, SCHRÖDER F and van der KWAST T (2018) PROGNOSTIC VALUE OF CELL CYCLE PROTEINS p27kip1 AND MIB-1, AND THE CELL ADHESION PROTEIN CD44s IN SURGICALLY TREATED PATIENTS WITH PROSTATE CANCERJournal of Urology, VOL. 164, NO. 6, (2156-2161), Online publication date: 1-Dec-2000.Taneja S (2018) EDITORIAL: MOLECULAR MARKERS OF CANCER PROGRESSION. READY OR NOT, HERE THEY COMEJournal of Urology, VOL. 164, NO. 6, (1996-1997), Online publication date: 1-Dec-2000.THOMAS G, SCHRAGE M, ROSENFELT L, KIM J, SALUR G, deKERNION J, DOREY F, SAID J and REITER R (2018) PREOPERATIVE PROSTATE NEEDLE BIOPSY p27 CORRELATES WITH SUBSEQUENT RADICAL PROSTATECTOMY p27, GLEASON GRADE AND PATHOLOGICAL STAGEJournal of Urology, VOL. 164, NO. 6, (1987-1991), Online publication date: 1-Dec-2000.KIBEL A, FAITH D, BOVA G and ISAACS W (2018) LOSS OF HETEROZYGOSITY AT 12P12–13 IN PRIMARY AND METASTATIC PROSTATE ADENOCARCINOMAJournal of Urology, VOL. 164, NO. 1, (192-196), Online publication date: 1-Jul-2000.D'AMICO A, WHITTINGTON R, MALKOWICZ S, FONDURULIA J, CHEN M, TOMASZEWSKI J and WEIN A (2018) THE COMBINATION OF PREOPERATIVE PROSTATE SPECIFIC ANTIGEN AND POSTOPERATIVE PATHOLOGICAL FINDINGS TO PREDICT PROSTATE SPECIFIC ANTIGEN OUTCOME IN CLINICALLY LOCALIZED PROSTATE CANCERJournal of Urology, VOL. 160, NO. 6 Part 1, (2096-2101), Online publication date: 1-Dec-1998.DE MARZO A, NELSON W, MEEKER A and COFFEY D (2018) STEM CELL FEATURES OF BENIGN AND MALIGNANT PROSTATE EPITHELIAL CELLSJournal of Urology, VOL. 160, NO. 6 Part 2, (2381-2392), Online publication date: 1-Dec-1998. Volume 159Issue 3March 1998Page: 941-945 Advertisement Copyright & Permissions© 1998 by American Urological Association, Inc.Metrics Author Information RONALD M. YANG More articles by this author JOHN NAITOH More articles by this author MICHAEL MURPHY More articles by this author HE-JING WANG More articles by this author JULIA PHILLIPSON More articles by this author JEAN B. DEKERNION More articles by this author MASSIMO LODA More articles by this author ROBERT E. REITER More articles by this author Expand All Advertisement PDF downloadLoading ...

Although most oncogenic phenotypes of PTEN loss are attributed to AKT activation, AKT alone is not sufficient to induce all of the biological activities associated with PTEN inactivation. We searched for additional PTEN-regulated pathways through gene set enrichment analysis (GSEA) and identified genes associated with JNK activation. PTEN null cells exhibit higher JNK activity, and genetic studies demonstrate that JNK functions parallel to and independently of AKT. Furthermore, PTEN deficiency sensitizes cells to JNK inhibition and negative feedback regulation of PI3K was impaired in PTEN null cells. Akt and JNK activation are highly correlated in human prostate cancer. These findings implicate JNK in PI3K-driven cancers and demonstrate the utility of GSEA to identify functional pathways using genetically defined systems.

Signet ring cell carcinoma and mucinous carcinoma are distinct subtypes of colorectal adenocarcinoma. The morphologic and molecular spectra of colorectal carcinomas with various signet ring cell components and colorectal carcinomas with various mucinous components, compared to non-mucinous adenocarcinomas, have not been examined. The study groups consisted of 39 carcinomas with various signet ring cell components ('the signet group'), 167 carcinomas with various mucinous components ('the mucinous group'), and 457 nonmucinous adenocarcinoma. We visually estimated the amounts of signet ring cell and mucinous components in tumors, and subclassified the signet and mucinous groups according to the amount of each component (< or = 19, 20-49, and > or = 50%). We sequenced BRAF and KRAS, analyzed for microsatellite instability (MSI) and 18q loss of heterozygosity (LOH), and performed immunohistochemistry for TP53, cyclooxygenase-2 (COX2), MLH1, O-6-methylguanine DNA methyltransferase (MGMT), p16 (CDKN2A), and fatty acid synthase (FASN). Signet ring cell carcinoma (> or = 50% signet ring cell tumors) and < or = 49% signet ring cell tumors showed similar molecular features. Except for MSI and MGMT, > or = 50% mucinous tumors and < or = 49% mucinous tumors also showed similar molecular features. BRAF mutations, MSI, and MLH1 loss were more frequent in both the signet and mucinous groups than nonmucinous carcinoma. More frequent KRAS mutations and less frequent p16 loss and TP53 positivity were observed in the mucinous group than nonmucinous carcinoma. 18q LOH and COX2 overexpression were less common in the signet group than nonmucinous carcinoma. FASN levels were highest in the mucinous group, followed by nonmucinous carcinoma, and lowest in the signet group. In conclusion, a minor (< or = 49%) signet ring cell or mucinous component in colorectal carcinoma suggests molecular features similar to > or = 50% signet ring cell or mucinous carcinoma, respectively. Signet ring cell carcinoma and mucinous carcinoma are related subtypes of colorectal adenocarcinoma, but have molecular features distinct from each other.

Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry.We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing.These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing.This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.

Du et al. describe a bead-based method for high-throughput detection of phosphorylated tyrosine kinases and use it to profile 130 human cancer lines. They show that the tyrosine kinase SRC is frequently activated in glioblastoma cells and that a SRC inhibitor blocks the growth of glioblastoma tumors. The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase–activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome–wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16INK4a. In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27Kip1, by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27Kip1 is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27Kip1 is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27Kip1. Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27Kip1 levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27Kip1down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27Kip1, even when PI3K was inhibited by LY-294002. The mechanism of p27Kip1 regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27Kip1 levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27Kip1protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo. Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16INK4a. In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27Kip1, by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27Kip1 is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27Kip1 is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27Kip1. Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27Kip1 levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27Kip1down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27Kip1, even when PI3K was inhibited by LY-294002. The mechanism of p27Kip1 regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27Kip1 levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27Kip1protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo. chronic myelogenous leukemia acute lymphoblastic leukemia interleukin cyclin-dependent kinase cyclin-dependent kinase inhibitor phosphatidylinositol 3-kinase hemagglutinin phosphate-buffered saline polyvinylidene difluoride Tris-buffered saline TBS with 0.5% Tween polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase polyacrylamide gel electrophoresis green fluorescent protein wild type Chronic myelogenous leukemia (CML)1 is a myeloproliferative disorder associated with expression of the Philadelphia chromosome (1Nowell P.C. Hungerford D.A. J. Natl. Cancer Inst. 1960; 25: 85-109PubMed Google Scholar), a translocation between chromosomes 9 and 22 that fuses the Bcr and Abl genes (2Rowley J.D. Nature. 1973; 243: 290-293Crossref PubMed Scopus (3396) Google Scholar, 3Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1124) Google Scholar, 4McWhirter J.R. Wang J.Y. Mol. Cell. Biol. 1991; 11: 1553-1565Crossref PubMed Google Scholar). Unlike many other leukemia oncogenes, BCR/ABL does not appear to alter differentiation of granulocyte lineage cells. In contrast, recent studies have suggested that the major cellular effects of BCR/ABL are related to increased mitogenic activity (5Puil L. Liu J. Gish G. Mbamalu G. Bowtell D. Pelicci T.G. Arlinghaus R. Pawson T. EMBO J. 1994; 13: 764-773Crossref PubMed Scopus (401) Google Scholar), reduced sensitivity to apoptosis (6Bedi A. Zehnbauer B.A. Barber J.P. Sharkis S.J. Jones R.J. Blood. 1994; 83: 2038-2044Crossref PubMed Google Scholar), and altered adhesion and homing of CML progenitor cells (7Gordon M.Y. Dowding C.R. Riley G.P. Goldman J.M. Greaves M.F. Nature. 1987; 328: 342-344Crossref PubMed Scopus (396) Google Scholar). The BCR/ABL oncogene is associated with both myeloproliferative disease and acute leukemias in human and in murine models. There are three known breakpoints in the gene, resulting in three different protein products, p190, p210, and p230, which vary in the length of Bcr present in the fusion protein (8Quackenbush R.C. Reuther G.W. Miller J.P. Courtney K.D. Pear W.S. Pendergast A.M. Blood. 2000; 95: 2913-2921Crossref PubMed Google Scholar). Interestingly, the three proteins tend to be associated with different leukemias: ALL, CML, and chronic neutrophilic leukemia, respectively, for p190, p210, and p230BCR/ABL. Each of the BCR/ABL proteins have elevated Abl tyrosine kinase activity (9Konopka J.B. Witte O.N. Mol. Cell. Biol. 1985; 5: 3116-3123Crossref PubMed Scopus (193) Google Scholar), and this increased kinase activity is necessary for transformation (3Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1124) Google Scholar). Although a number of substrates of the BCR/ABL tyrosine kinase have been identified, including CBL (10Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar), CrkL (11Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar), Dok (12Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), STAT5 (13Shuai K. Halpern J. ten Hoeve J. Rao X. Sawyers C.L. Oncogene. 1996; 13: 247-254PubMed Google Scholar, 14Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (434) Google Scholar), SHP-2 (15Tauchi T. Feng G.S. Marshall M.S. Shen R. Mantel C. Pawson T. Broxmeyer H.E. J. Biol. Chem. 1994; 269: 25206-25211Abstract Full Text PDF PubMed Google Scholar), Shc (16Pelicci G. Lanfrancone L. Salcini A.E. Romano A. Mele S. Grazia Borrello M. Segatto O. Di Fiore P.P. Pelicci P.G. Oncogene. 1995; 11: 899-907PubMed Google Scholar), and Fak (17Salgia R. Sattler M. Pisick E. Li J.L. Griffin J.D. Exp. Hematol. 1996; 24: 310-313PubMed Google Scholar), the signaling pathways that result in dysregulated growth, viability, and adhesion are not yet well defined. The mitogenic effects of BCR/ABL are likely to be important in the pathogenesis of CML. BCR/ABL reduces growth factor requirements of primary hematopoietic stem cells (18Sanchez-Garcia I. Grutz G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5287-5291Crossref PubMed Scopus (197) Google Scholar), converts IL-3-dependent murine hematopoietic cell lines to growth factor independence (19Mandanas R.A. Boswell H.S. Lu L. Leibowitz D. Leukemia ( Baltimore ). 1992; 6: 796-800PubMed Google Scholar), and is mitogenic in fibroblasts (20Sawyers C.L. McLaughlin J. Witte O.N. J. Exp. Med. 1995; 181: 307-313Crossref PubMed Scopus (248) Google Scholar). When compared with normal progenitor cells, CML progenitor cells are more likely to be in S phase, both in the marrow and blood, and the fraction of cells in G0 is reduced (21Eaves A.C. Cashman J.D. Gaboury L.A. Kalousek D.K. Eaves C.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5306-5310Crossref PubMed Scopus (176) Google Scholar). Thus, BCR/ABL is likely to deregulate checkpoints at one or more sites within the cell cycle. Previous studies have shown that several immediate-early genes are induced by BCR/ABL, including myc (22Sawyers C.L. Callahan W. Witte O.N. Cell. 1992; 70: 901-910Abstract Full Text PDF PubMed Scopus (356) Google Scholar), fos, andjun (23Mandanas R.A. Leibowitz D.S. Gharehbaghi K. Tauchi T. Burgess G.S. Miyazawa K. Jayaram H.N. Boswell H.S. Blood. 1993; 82: 1838-1847Crossref PubMed Google Scholar). The rapid induction of these genes correlates with an enhanced rate of transition from G0 to G1. The increased fraction of cells in S phase suggests that G1/S transition checkpoints are also suppressed. A number of molecules play a key role in regulating cell cycle progression from G1 to S, including the G1cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs). CKIs can be grouped in two categories based on similarities of sequence and actions: the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the CIP/KIP family (p21WAF1/CIP1, p27Kip1, and p57Kip2), reviewed in Sherr and Roberts (24Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5155) Google Scholar). INK4 family members specifically inhibit the activity of cdk4 and 6, whereas the CIP/KIP family members have a broader action. Overexpression of each of these CKI have been shown to induce a G1 arrest. p21CIP1 has recently been directly shown to be important for regulating hematopoiesis in vivo in mice (25Cheng T. Rodrigues N. Shen H. Yang Y. Dombkowski D. Sykes M. Scadden D.T. Science. 2000; 287: 1804-1808Crossref PubMed Scopus (1085) Google Scholar, 26Mantel C. Braun S.E. Reid S. Henegariu O. Liu L. Hangoc G. Broxmeyer H.E. Blood. 1999; 93: 1390-1398Crossref PubMed Google Scholar). Although PI3K and AKT have previously been reported to play essential roles in BCR/ABL transformation (27Skorski T. Bellacosa A. Nieborowska-Skorska M. Majewski M. Martinez R. Choi J.K. Trotta R. Wlodarski P. Perrotti D. Chan T.O. Wasik M.A. Tsichlis P.N. Calabretta B. EMBO J. 1997; 16: 6151-6161Crossref PubMed Scopus (558) Google Scholar), the mechanisms and downstream signaling targets have been unclear. PI3K and AKT have been linked to enhanced cell survival through the phosphorylation and subsequent inhibition of the pro-apoptotic molecule Bad (28Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (165) Google Scholar). However, it has been difficult to demonstrate phosphorylation of Bad in some cell types transformed by BCR/ABL, so identification of other downstream targets is of interest. In the present study we demonstrate that BCR/ABL regulates the expression of p27Kip1 in a proteasome-dependent manner and through activation of PI3K and AKT. Anti-Abl monoclonal antibody 3F12 was a gift from R. Salgia (Dana Farber Cancer Institute). Monoclonal antibodies against p27Kip1 (K25020) and Rb (14001A) were purchased from Transduction Laboratories (Pharmingen/Transduction Laboratories, San Diego, CA). Anti-p85 antiserum (06–195) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-HA monoclonal was purchased from Babco (Richmond, CA). AKT constructs, inserted in a pCDNA3.1 backbone, were described previously (29Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). RNase A, lactacystine, andN-acetyl-leucyl-leucine norleucinal (LLnL) were purchased from Sigma. E64 and calpain inhibitors were purchased from Calbiochem. The IL-3-dependent Ba/F3 cell line was maintained in RPMI 1640 (Mediatech Cellgro, Herndon, VA) supplemented with 10% fetal calf serum, 1 mg/ml l-glutamine, penicillin-streptomycin, and 10% WEHI-3B conditioned medium (WEHI-3B-CM) as a source of IL-3. Ba/F3 is commonly used as a model for BCR/ABL signaling because it is non-leukemic and factor-dependent in the absence of BCR/ABL-transformation but becomes leukemic in syngeneic mice and factor-independent after transformation by BCR/ABL. p210BCR/ABL-transformed Ba/F3 cells (Ba/F3-p210) are maintained in culture in the medium described above, except without IL-3. All cells were maintained at 37 °C in a 5% CO2 humidified incubator. Ba/F3 cells expressing the reverse tet-transactivator pUHD172–1 (Ton.B.1) and Ton.B.210 cells in which p210BCR/ABL expression is induced by the addition of doxycycline were obtained from G. Daley (Whitehead institute, Cambridge, MA) and grown as described previously (30Gesbert F. Griffin J. Blood. 2000; 96: 2269-2276Crossref PubMed Google Scholar). In experiments using transiently transfected cells, 1 × 107 cells were transfected by electroporation (Gene-Pulser Bio-Rad, 960 microfarads, 350V). 40 μg of the indicated plasmids were cotransfected with 10 μg of a pEGFP plasmid (CLONTECH, Palo Alto, CA). 24-h post-transfection, the green fluorescent protein-expressing cells were sorted on a high speed cell sorter (Coulter Electronics, Miami, FL). After sorting, cells were pelleted, resuspended in culture medium, and kept in culture for 24 h with or without treatment as indicated. For protein analysis, cells were harvested, washed in PBS and lysed at 5 × 107 cells/ml in cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 10 mm NaF, 1 mm EDTA, 1 mmEGTA, 1 mm phenylmethylsulfonyl fluoride, 1 mmNaVO3, 1 μg/ml each leupeptin and aprotinin) for 30 min. Lysates were clarified by centrifugation at 15,000 ×g for 20 min at 4 °C. The protein concentration was determined by Bradford assay, and equivalent amounts of proteins were separated by gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). Filters were blocked for 2 h at room temperature with either 5% nonfat dry milk or 3% bovine serum albumin in Tris-buffered saline (TBS), 0.5% Tween (TBS-T). Filters were washed three times in TBS-T and incubated for 1 h with optimal concentrations of primary antibodies diluted in TBS, 0.1% Tween. After four additional washes in TBS-T, filters were further incubated 45 min with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Visualization was performed using PerkinElmer Life Sciences, Renaissance system and Kodak X-Omat blue film (Eastman Kodak Co.). Ton.B.210 cells were either left untreated or treated with 1 μg/ml doxycycline for at least 24 h before cycloheximide treatment in RPMI 1640 medium supplemented with 10% fetal calf serum and 10% WEHI-CM. 8 h before treatment, the cells were harvested, washed twice in 1× PBS, and resuspended in RPMI 1640 supplemented with 1% bovine serum albumin at a cell density of 1 × 106cells/ml with or without doxycycline. After 8 h of IL-3 deprivation, 10 μm cycloheximide was added to the culture, and an aliquot of the cells was harvested at the indicated times. Cells were lysed as described above. For cell cycle analysis, cells were treated as specified. 1–2 × 106 cells were harvested, washed once in 4 ml of PBS, and fixed in 1 ml of 70% ethanol solution. Fixed cells were kept at −20 °C and stained just before analysis. For staining, fixed cells were pelleted, washed once in PBS, and resuspended in 1 ml of propidium iodide-staining solution (PBS, 0.1% Triton X-100, 20 μg/ml propidium iodide, and 100 units/ml RNase A added extemporaneously). Cells were left in propidium iodide staining solution for 30 min at room temperature and analyzed immediately. DNA content and hence the cell cycle distribution was determined by flow cytometry. Repartition of the cells in the various stages of cell cycle was determined with cell cycle analysis software. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed in a 20 μl of reaction mixture containing 250 μm each dNTP, 20 units of RNase inhibitor, 50 units of murine leukemia virus reverse transcriptase, 2.5 μm random hexamers, and 1× buffer (1.5 mmMgCl2) (all reagents were purchased from PE Applied Biosystems, Foster City, CA). The reaction mix was incubated at 42 °C for 45 min and then denatured at 99 °C for 5 min. For each sample, a control reaction not containing the reverse transcriptase enzyme was also performed. Specific primers and probe for p27 (forward: 5′-GGTGGACCAAATGCCTGACT-3′; reverse: 5′-GCCCTTTTGTTTTGCGAAGA-3′; probe: 5′ AATCTTCTGCCGCAGGTCGCTTCC-3′) were designed from sequences in the GenBankTM data base using the Primer Express 1.0 Software (PE Applied Biosystems). The hybridization probe spanned an intron to exclude annealing to genomic DNA. The gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control to standardize the amount of RNA in each reaction (Taqman Rodent GAPDH control reagents). All primers and probes were synthesized by PE Applied Biosystems. PCR was performed on the cDNA samples using an ABI PRISM 7700 sequence detector (PE Applied Biosystems). The Taqman® PCR Core reagent kit (PE Applied Biosystems) was used according to the manufacturer's protocol with the modification that dUTP was replaced by dTTP, and incubation with AmpErase was omitted. For each sample tested, PCR reaction was carried out in a 50-μl volume containing 1 μl of cDNA reaction (equivalent to 50 ng of template RNA) and 2.5 units of AmpliTaq Gold. Oligonucleotide primers and fluorogenic probe were added to a final concentration of 100 nm each. The amplification step consisted of 60 cycles of 94 °C for 45 s, 58 °C for 45 s, and 65 °C for 1 min. In each experiment, additional reactions with 7 serial 2-fold dilutions of Ton.B.210 cDNA, prepared from cells induced or not with doxycycline, as template were performed with each set of primers and probes on the same 96-well plate to generate standard curves, which related the threshold cycle (CT) to the log input amount of template. All samples were amplified in triplicate. The relative amount of p27 transcripts in each sample was determined by using the standard curve method and by normalizing for GAPDH mRNA expression levels, as described previously (ABI PRISM sequence detection system user bulletin No. 2 (PE Applied Biosystems and Ref. 31Fink L. Seeger W. Ermert L. Hanze J. Stahl U. Grimminger F. Kummer W. Bohle R.M. Nat. Med. 1998; 4: 1329-1333Crossref PubMed Scopus (524) Google Scholar). The ability of BCR/ABL to promote survival and proliferation in the absence of growth factors has been well documented in certain hematopoietic-derived cell lines. The Ba/F3 cell line used in our studies is a pre-B cell fully dependent on the presence of IL-3 for survival and proliferation. IL-3 withdrawal for 16 h induces a partial G1 arrest in parental Ba/F3 cells but not in Ba/F3 cells transformed by p210BCR/ABL (Fig. 1, upper panels). Similar results were obtained with the previously described Ton.B.210 cell line (Fig. 1, lower panels). This cell line, derived from Ba/F3 cells, expresses p210BCR/ABL in response to the addition of doxycycline in the culture medium. After withdrawal of IL-3 for 16 h, non-induced Ton.B.210 cells arrest at G0/G1, whereas BCR/ABL-expressing Ton.B.210 cells progress through G1 to S phase (Fig. 1, lower panels). These results demonstrate that BCR/ABL expression regulates cell cycle progression in hematopoietic cells in a manner similar to cytokine stimulation. Based on these results, we sought to identify cell cycle-related proteins that might be regulated by BCR/ABL activity. The Ton.B.210 cell line was used to assure that changes were specifically due to BCR/ABL and not due to unrelated mutations in the cultured cell lines. Cells were left untreated or stimulated with doxycycline for 24 h and IL-3-deprived for 16 h. Immunoblotting of p27 on lysates from non-treated or doxycycline-treated Ton.B.210 cells demonstrated that resting, non-induced cells expressed a high amount of the CKI p27Kip1, whereas BCR/ABL-expressing cells displayed a very low amount of p27Kip1(Fig. 2 A). In addition, p21Cip1 expression levels were higher in proliferating cells.(Fig. 2 A, lower panel). Discordant expression levels of p21Cip1 and p27Kip1 has also been described in other proliferating cells (32Li Y. Jenkins C.W. Nichols M.A. Xiong Y. Oncogene. 1994; 9: 2261-2268PubMed Google Scholar). In some circumstances, CKIs can promote rather than inhibit the formation of active cyclin D-cdk4 complexes (33Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (974) Google Scholar, 34LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1222) Google Scholar). Our data would be consistent with a model in which the increased level of p21Cip1expression is enough to participate in the activation of cyclin D-cdk4 but is not high enough to inhibit cyclinE-cdk2. In contrast to p27Kip1, there was no variation of expression of cyclins A, E, D1, or D3 (data not shown). These results suggest that regulation of p27Kip1 expression by BCR/ABL might be an important mechanism in BCR/ABL-mediated proliferative signaling. To confirm that p27Kip1 down-regulation was directly due to BCR/ABL activity, doxycycline-induced Ton.B.210 cells were treated for 14 h with increasing concentrations of the small molecule Abl tyrosine kinase inhibitor STI571 (Fig. 2 B). At an optimal concentration of 1 × 10−6mSTI571, BCR/ABL tyrosine kinase activity was inhibited, and this was accompanied by a substantial increase in p27Kip1expression. In these experiments, the level of expression of the 85-kDa subunit of PI3K was used as a control for equal loading of gel lanes. The results described above indicated that p27Kip1 might be an important target for BCR/ABL in deregulating cell cycle control mechanisms, and therefore, we next sought to identify the signaling pathway responsible for p27Kip1 expression. As an initial screen, three drugs that inhibit different signaling pathways were studied: PD98059, LY-294002, and rapamycin, known to specifically inhibit mitogen-activated protein kinase, PI3K, and p70S6K pathways, respectively. Treatment of p210BCR/ABL-expressing Ba/F3 cells with carrier alone (Me2SO) or with PD98059 had no effect on cell cycle progression (Fig. 3 A, upper panels), whereas treatment for 14 h with either LY-294002 or rapamycin induced a G0/G1 arrest, without any significant effect on cell viability (note the absence of a sub-G1population in Fig. 3 A, lower panels). The effects of these three drugs on BCR/ABL-induced expression of p27Kip1 was then investigated (Fig. 3 B). Treatment of Ba/F3p210BCR/ABL with LY-294002 induced a dramatic increase of p27Kip1 expression, whereas a treatment with rapamycin and PD98059 had no effect. Since rapamycin effectively induces a G1 arrest in these cells, it is unlikely that the p27Kip1 up-regulation induced by LY-294002 is simply a consequence of cell cycle arrest. As p27Kip1 is a known inhibitor of the pRb kinase cdk2, we also sought to determine if pRb was found in a hyperphosphorylated form in BCR/ABL-expressing cells and if this phosphorylation was regulated by PI3K. As shown in Fig. 3 C, hyper- (ppRb) and hypo- (pRb) phosphorylated forms of Rb can be distinguished by an electrophoretic shift on a 6.5% SDS-PAGE. Ba/F3 p210 cells displayed almost exclusively a hyperphosphorylated form of Rb. Although PD98052, rapamycin, or Me2SO had no or little effect on Rb phosphorylation, treatment of Ba/F3 p210 cells with the PI3K inhibitor LY-294002 had a dramatic effect, with Rb reverting to an almost exclusively hypophosphorylated state. These results demonstrate that the PI3K pathway regulates p27Kip1 expression and Rb phosphorylation, most likely through the well known ability of p27Kip1 to regulate cdk2 activity. The serine/threonine kinase AKT/protein kinase B is a downstream mediator of PI3K activity. In an effort to determine if AKT activity was sufficient to mediate down-regulation of p27Kip1, an AKT mutant rendered constitutively active by membrane targeting through the fusion of a CAAX box (HA-AKT-CAAX), was transiently expressed in the BCR/ABL-inducible cell line, Ton-B-210. The Ton-B-210 cells were co-transfected with a plasmid encoding green fluorescent protein (GFP) and either a wild-type AKT construct (HA-AKT-WT), as control, or HA-AKT-CAAX construct. Twenty-four hours after transfection, GFP-positive cells were isolated by flow cytometry and then maintained in culture for an additional 24 h either in the absence or presence of doxycycline in order to induce BCR/ABL expression. The cells were then IL-3-deprived for 18 h and lysed. As shown in Fig. 4 A, IL-3 deprivation of cells without BCR/ABL leads to a dramatic increase of p27Kip1expression in cells transfected either with an empty vector or with a vector encoding for a wild-type form of AKT. Expression of an activated form of AKT or BCR/ABL, however, led to a significant and equivalent decrease of p27Kip1 expression. These results suggest that activation of AKT, a known consequence of BCR/ABL signaling, is sufficient in these cells to regulate p27Kip1expression. To determine if AKT functions downstream of PI3K in the regulation of p27Kip1 expression in Ba/F3 cells, activated AKT was expressed in BCR/ABL-transformed cells in which PI3K had been inhibited by LY-294002 (Fig. 4 B). Ba/F3-p210 cells were co-transfected as above with plasmids encoding GFP and either wild type or the activated form of AKT. After isolation of GFP-positive cells by flow sorting, the positive cells were allowed to recover for an additional 18 h and then either left untreated or were treated with LY-294002 or rapamycin. The expression of the constitutively active form of AKT (HA-AKT-CAAX) completely inhibited the increase of p27Kip1 expression induced by LY-294002, whereas the expression of a wild type form (HA-AKT-WT) had no detectable effect. These results indicate that AKT can regulate p27Kip1expression in hematopoietic cells and that it is likely downstream of PI3K activity, because an activated AKT mutant can override the effect of PI3K inhibition on p27Kip1 expression. In other cell systems, p27Kip1 regulation of expression has been shown to occur at both transcriptional and postranscriptional levels (35Servant M.J. Coulombe P. Turgeon B. Meloche S. J. Cell Biol. 2000; 148: 543-556Crossref PubMed Scopus (124) Google Scholar). In some cells, p27Kip1 has been shown to be ubiquitinated and thereby targeted for proteasome-mediated degradation (36Podust V.N. Brownell J.E. Gladysheva T.B. Luo R.S. Wang C. Coggins M.B. Pierce J.W. Lightcap E.S. Chau V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4579-4584Crossref PubMed Scopus (224) Google Scholar, 37Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1735) Google Scholar, 38Alessandrini A. Chiaur D.S. Pagano M. Leukemia ( Baltimore ). 1997; 11: 342-345Crossref PubMed Scopus (145) Google Scholar). To determine if BCR/ABL-induced p27Kip1 down-regulation is mediated by a protein degradation pathway, we studied several protease inhibitors and two different proteasome inhibitors, lactacystine andN-acetyl-leucyl-leucine norleucinal. As shown in Fig. 5 A, after a 5-h treatment, both proteasome inhibitors specifically induced an increase of p27Kip1 expression in Ba/F3-p210 cells, whereas the protease inhibitors had little or no effect. To further investigate possible degradation of p27Kip1, Ton.B.210 cells, induced or not with doxycycline, were treated with 10 μmribosomal complex inhibitor cycloheximide. By blocking translation with cycloheximide, the role of post-translational events such as degradation in regulating protein levels can be specifically evaluated. Lysates of the cells were prepared at different time points of treatment. Equivalent amounts of protein were loaded on SDS-PAGE and probed with an anti- p27Kip1 antibody. As shown in Fig. 5 B, we clearly show that the disappearance of p27Kip1 protein is faster in BCR/ABL-expressing cells (Fig. 5 B, lower panel) than in non-induced cells (Fig. 5 B, upper panel), suggesting a faster degradation of p27Kip1 in BCR/ABL-expressing cells. After densitometry analysis of the bands, the half-life of p27Kip1 was estimated to be longer than 8 h in non-induced cells and 2 h in BCR/ABL- expressing cells. These results suggest that p27Kip1 protein down-regulation in BCR/ABL-transformed cells is likely to be due predominantly to proteasome-dependent degradation and that this process is regulated by PI3-kinase and AKT. In other cells, p27Kip1 has also been shown to be regulated at the level of transcription (35Servant M.J. Coulombe P. Turgeon B. Meloche S. J. Cell Biol. 2000; 148: 543-556Crossref PubMed Scopus (124) Google Scholar, 39Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar). Therefore, p27Kip1RNA levels were compared before and after induction of BCR/ABL in Ton.B.210 cells using semiquantitative real time PCR, as described under “Materials and Methods.” Treatment of Ton.B.210 cells with doxycycline in presence or in absence of LY-294002 resulted in a les

BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist.The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein.Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases.Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein.We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells.Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens.We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER-and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option.

Abstract Purpose: More than 1,300,000 prostate needle biopsies are done annually in the United States with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer on subsequent needle biopsies in prostate-specific antigen–screened populations. In contemporary series, prostate cancer is detected in ∼20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histologic subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in prostate cancer has also been shown to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive clinical course. Experimental Design: In this study, we assessed a series of HGPIN lesions and paired prostate cancer for the presence of TMPRSS2-ERG gene fusion. Results: Fusion-positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances, the matching cancer shared the same fusion pattern. Sixty percent of TMPRSS2-ERG fusion prostate cancer had fusion-negative HGPIN. Conclusions: Given the more aggressive nature of TMPRSS2-ERG prostate cancer, the findings of this study raise the possibility that gene fusion-positive HGPIN lesions are harbingers of more aggressive disease. To date, pathologic, molecular, and clinical variables do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion prostate cancer in subsequent biopsies.

The phosphatase Cdc25A plays an important role in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases, and it has been shown to transform diploid murine fibroblasts in cooperation with activated Ras. Here we show that Cdc25A is overexpressed in primary breast tumors and that such overexpression is correlated with higher levels of cyclin-dependent kinase 2 (Cdk2) enzymatic activity in vivo. Furthermore, in the breast cancer cell line MCF-7, Cdc25A activity is necessary for both the activation of Cdk2 and the subsequent induction of S-phase entry. Finally, in a series of small (< 1 cm) breast carcinomas, overexpression of Cdc25A was found in 47% of patients and was associated with poor survival. These data suggest that overexpression of Cdc25A contributes to the biological behavior of primary breast tumors and that both Cdc25A and Cdk2 are suitable therapeutic targets in early-stage breast cancer.

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.

Lung metastases from colorectal carcinomas (CRC) can be resected with improved survival. The distinction between primary lung adenocarcinomas and metastases from CRC may sometimes be difficult, especially on cytologic specimens or small bronchoscopic biopsies. Immunohistochemistry may be of help in this setting: available markers include TTF-1 and SP-A, which are markers of lung origin, whereas there are no good markers of intestinal origin, besides cytokeratin 7 and 20 coexpression pattern, which is not very specific. The nuclear CDX-2 transcription factor, which is the product of a homeobox gene necessary for intestinal organogenesis, is expressed in normal colonic epithelia and most colorectal adenocarcinomas, and could potentially be of diagnostic usefulness. Our aim was to investigate CDX-2 immunohistochemical expression using a new monoclonal antibody and to verify if CDX-2 can be a reliable marker to identify the colorectal origin of lung metastases. CDX-2 expression was evaluated in formalin-fixed, paraffin-embedded samples of normal adult human tissues (50 samples) and in 299 surgically resected carcinomas of different origins, including 125 non-lung adenocarcinomas, 117 primary lung tumors, 5 mesotheliomas, and 52 adenocarcinomas metastatic to the lung. CDX-2 was also evaluated on a series of 20 bioptic and 10 cytologic specimens (5 cases of colorectal metastases to the lung, 5 cases of metastases from other organs, and 10 primary lung adenocarcinomas). In normal tissues CDX-2 immunoreactivity was observed only in ileal and colorectal epithelia. CDX-2 was expressed in almost all primary and metastatic CRC (88 of 90) and was never observed in primary lung tumors. CDX-2 was also expressed in a limited group of adenocarcinomas of other sites (gastric, biliopancreatic, and mucinous ovarian adenocarcinomas). CDX-2 could be easily detected in all bioptic and cytologic samples of CRC metastases. CDX-2 is a reliable, specific, and sensitive immunohistochemical marker of normal and neoplastic intestinal epithelium. CDX-2 can be easily applied to routine histologic and cytologic material and is therefore a useful marker in the differential diagnosis of primary versus metastatic adenocarcinomas in the lung, and among metastases from an unknown primary, supports intestinal origin.

CDX2 is a homeobox domain-containing transcription factor that is important in the development and differentiation of the intestines. Based on recent studies, CDX2 expression is immunohistochemically detectable in normal colonic enterocytes and is retained in most, but not all, colorectal adenocarcinomas. CDX2 expression has also been documented in a subset of adenocarcinomas arising in the stomach, esophagus and ovary. In this study, we examined CDX2 expression in a series of large tissue microarrays representing 4652 samples of normal and neoplastic tissues. Strong nuclear staining for CDX2 was observed in 97.9% of 140 colonic adenomas, 85.7% of 1109 colonic adenocarcinomas overall and 81.8% of 55 mucinous variants. There was no significant difference in the staining of well-differentiated (96%) and moderately differentiated tumors (90.8%, P=0.18), but poorly differentiated tumors showed reduced overall expression (56.0%, P<0.000001). Correspondingly, there was an inverse correlation between CDX2 expression and tumor stage, with a significant decrease in staining between pT2 and pT3 tumors (95.8 vs 89.0%, P<0.012), and between pT3 and pT4 tumors (89.0 vs 79.8%, P<0.016). Analysis of 140 locally advanced, CDX2-positive colorectal adenocarcinomas coarrayed with their matching lymph node metastases revealed that expression of this marker was retained in 82.1% of the metastases. Consistent with previous reports, CDX2 staining was observed in gastric adenocarcinomas (n=71), more commonly in the intestinal-type than the diffuse-type (28.9 vs 11.5%, P<0.05). Occasional ovarian carcinomas were positive for CDX2, including mucinous (10.5%), endometrioid (9.3%) and serous variants (2%), but expression was either very rare or absent in primary carcinomas of the lung, breast, thyroid, pancreas, liver, gallbladder, kidney, endometrium and urinary bladder. A low frequency of CDX2 expression in pancreatic and biliary carcinomas observed on the microarrays was pursued further by comparing these tumors with ampullary adenocarcinomas on conventional sections. Ampullary adenocarcinomas were more commonly positive for CDX2 (19/24, 79%) than cholangiocarcinomas (1/11, 9%) and pancreatic carcinomas (3/20, 15%). In summary, CDX2 is a sensitive and specific marker for colorectal adenocarcinoma, although its expression is decreased among higher grade and stage tumors, and it is not invariably present in metastases from positive primaries. CDX2 may also be helpful in distinguishing adenocarcinomas of the ampulla from those arising in the pancreas and biliary tree.

Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients with low p27 expression had a median survival of 139 months (17% mortality) versus 174 months (9% mortality) in the group with high p27 expression (P = 0.0233). Lack of p27 was associated with poor prognosis when node-positive patients were excluded (P = 0.0252). Nodal status and low p27 were found to be the only independent prognostic parameters by both univariate and multivariate analysis, with relative risks of dying of disease of 4.9 (P = 0.001) and 3.4 (P = 0.0306), respectively. Assessment of p27, which yields prognostic information in node-negative patients, could be useful to identify patients with small, invasive breast carcinomas who might benefit from adjuvant therapy.

Abstract Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in diverse human tumors and may play a direct role in malignant transformation. However, the full complement of target genes that STAT3 regulates to promote oncogenesis is not known. We created a system to express a constitutively active form of STAT3, STAT3-C, in mouse fibroblasts and used it to identify STAT3 targets. We showed that a subset of these targets, which include transcription factors regulating cell growth, survival, and differentiation, are coexpressed in a range of human tumors. Using immunohistochemical staining of tissue microarrays, we showed that these targets are enriched in breast and prostate tumors harboring activated STAT3. Finally, we showed that STAT3 is required for the expression of these genes in a breast cancer cell line. Taken together, these results identify a cohort of STAT3 targets that may mediate its role in oncogenesis.

Aside from the well-established roles of c-Myc in the regulation of cell cycle, differentiation, and apoptosis, a recent picture is beginning to emerge linking c-Myc to the regulation of metabolic pathways. Here, we define a further function for c-Myc in determining cellular redox balance, identifying glutathione (GSH) as the leading molecule mediating this process. The link between c-Myc and GSH is gamma-glutamyl-cysteine synthetase (gamma-GCS), the rate-limiting enzyme catalyzing GSH biosynthesis. Indeed, c-Myc transcriptionally regulates gamma-GCS by binding and activating the promoters of both gamma-GCS heavy and light subunits. Exposure to H2O2 enhances c-Myc recruitment to gamma-GCS regulatory regions through ERK-dependent phosphorylation. Phosphorylation at Ser-62 is required for c-Myc recruitment to gamma-GCS promoters and determines the cellular response to oxidative stress induced by different stimuli. Thus, the c-Myc phosphorylation-dependent activation of the GSH-directed survival pathway can contribute to oxidative stress resistance in tumor cells, which generally exhibit deregulated c-Myc expression.

Abstract Purpose: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and determined the occurrence and clinical significance of these events in a series of bladder carcinomas. Experimental Design: Eight hundred and twenty-five tumor samples from 572 bladder cancer patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray were subjected to antigen retrieval and processed by immunohistochemistry for the expression of E-cadherin, plakoglobin, β-catenin, N-cadherin, and vimentin. Results: Pathologic expression of E-cadherin, β-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly influenced overall survival (P = 0.02) in which the median time to death was 2 years compared with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for cancer-specific survival, low levels of both plakoglobin (P = 0.02) and β-catenin (P = 0.02) significantly influenced survival. Conclusion: The putative markers of EMT defined within a panel of bladder carcinoma cell lines were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations associated with plakoglobin were identified as significant prognostic features in these tumors.

Abstract Purpose: To determine the clinical, pathologic, and molecular effects of neoadjuvant docetaxel chemotherapy in high-risk localized prostate cancer. Experimental Design: Patients with biopsy Gleason scores of 8 to 10, serum prostate-specific antigen levels &amp;gt;20 ng/mL, and/or clinical stage T3 disease received weekly docetaxel (36 mg/m2) for 6 months, followed by radical prostatectomy, and were monitored with weekly visits, serum prostate-specific antigen measurements, and endorectal magnetic resonance imaging (MRI). Frozen tumor specimens were collected for microarray analysis. Results: The 19 patients enrolled received 82% of the planned chemotherapy. Toxicity was mild to moderate; fatigue and taste disturbance were common. Prostate-specific antigen declines of &amp;gt;50% were seen in 11 of 19 patients (58%; 95% confidence interval, 33-80%) and endorectal MRI showed maximum tumor volume reduction of at least 25% in 13 of 19 patients (68%; 95% confidence interval, 47-85%) and at least 50% in 4 patients (21%; 95% confidence interval, 6-46%). Sixteen patients completed chemotherapy and had radical prostatectomy; none achieved pathologic complete response. Microarray analysis identified coordinate up-regulation of genes involved in androgen metabolism associated with docetaxel therapy. Specifically, RNA expression for genes that decrease cellular levels of bioactive androgens was coordinately increased in response to chemotherapy. Conclusions: Neoadjuvant docetaxel administered for 6 months before radical prostatectomy is feasible, well tolerated, and often results in prostate-specific antigen declines of &amp;gt;50% and decreased tumor volume on endorectal MRI. No pathologic complete responses were observed. Altered androgen metabolism may partially account for the noted declines in prostate-specific antigen and be a mechanism for chemotherapy resistance.

Abstract VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with ≥4N DNA content, followed by cell death. Here, we define the role of p53 and p21Waf1/Cip1 in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21Waf1/Cip1 and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21Waf1/Cip1 within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21Waf1/Cip1 is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21Waf1/Cip1 expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21Waf1/Cip1 in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21Waf1/Cip1–dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis. (Cancer Res 2006; 66(15): 7668-77)

Energy balance seems to be important in the pathogenesis of colon cancer. Fatty acid synthase (FASN) is physiologically regulated by energy balance and is often upregulated in colorectal cancer. Nonetheless, the influence of FASN expression on patient outcome is uncertain.Using the database of 647 patients with colon cancer in two independent cohort studies, FASN overexpression was detected in 84 tumors (13%) by immunohistochemistry. Cox proportional hazards models calculated hazard ratios (HRs) of colon cancer-specific and overall mortalities, adjusted for patient characteristics and related tumoral features, including KRAS, BRAF, p53, microsatellite instability and the CpG island methylation phenotype.There were 279 deaths, including 160 colon cancer-specific deaths. FASN overexpression was associated with a significant reduction in colon cancer-specific mortality by both univariate and multivariate analyses (adjusted HR, 0.41; 95% CI, 0.19 to 0.89) and an insignificant trend toward improved overall mortality (adjusted HR, 0.75; 95% CI, 0.50 to 1.13). Notably, the effect of FASN expression on mortality might be different according to body mass index (BMI; P(interaction) = .019); the adjusted HR of overall mortality for FASN overexpression was 0.63 (95% CI, 0.39 to 1.02) among patients with BMI less than 27.5 kg/m(2) and 2.91 (95% CI, 1.19 to 7.12) among those with BMI >or= 27.5 kg/m(2). Moreover, the adverse effect of moderate overweight/obesity on overall survival was limited to FASN-positive tumors (adjusted HR, 4.10; 95% CI, 1.14 to 14.8; BMI >or= 27.5 kg/m(2) v < 27.5 kg/m(2)). CONCLUSION Among nonobese patients with colon cancer, tumoral FASN overexpression is associated with improved survival, whereas among moderately overweight or obese patients (BMI >or= 27.5 kg/m(2)), FASN overexpression may predict a worse outcome.

Human kidney injury molecule-1 (hKIM-1) is a type 1 transmembrane protein that is not detectable in normal kidney tissue but is expressed at high levels in human and rodent kidneys with dedifferentiated proximal tubule epithelial cells after ischemic or toxic injury. Therefore, it was hypothesized that renal tumors express hKIM-1 and release this protein into the urine. Forty renal cell carcinoma (RCC) and 484 nonrenal tumors were analyzed by immunohistochemistry for expression of hKIM-1 (group 1). Urine samples before nephrectomy and nephrectomy tissue samples were collected from an additional 42 patients with renal tumors, from 30 normal control subjects, and also from 10 patients with prostate carcinoma (group 2). In five additional patients with RCC, urine was collected before and after nephrectomy (group 3). Tissue was examined for expression of hKIM-1, and cell-free urine supernatants were analyzed for hKIM-1 by ELISA. Urinary hKIM-1 was normalized to the urinary creatinine concentration (U(Cr)). Expression of hKIM-1 was present in 32 tissue sections (91%) of 35 clear cell RCC (group 1). In group 2, the normalized urinary hKIM-1 levels were significantly higher in patients with clear cell RCC (0.39 +/- 0.08 ng/mg U(Cr); n = 21), compared with levels in patients with prostate carcinoma (0.12 +/- 0.03 ng/mg U(Cr); P < 0.02; n = 10), or normal control subjects (0.05 +/- 0.01 ng/mg U(Cr); P < 0.005; n = 30). Tissue sections from 28 (82%) of 34 primary RCC stained positively for the expression of hKIM-1. In all patients with a detectable prenephrectomy urinary hKIM-1 level, there was either complete disappearance or marked reduction after nephrectomy (group 3). In conclusion, the cleaved ectodomain of hKIM-1 can be detected in the urine of patients with RCC and may serve as a new biomarker for early detection of RCC.

Distinguishing aggressive from indolent disease and developing effective therapy for advanced disease are the major challenges in prostate cancer research. Chromosomal rearrangements involving ETS transcription factors, such as ERG and ETV1, occur frequently in prostate cancer. How they contribute to tumorigenesis and whether they play similar or distinct in vivo roles remain elusive. Here we show that in mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factor cooperated with loss of a single copy of Pten , leading to localized cancer, but only ETV1 appeared to support development of invasive adenocarcinoma under the background of full Pten loss. Mechanistic studies demonstrated that ERG and ETV1 control a common transcriptional network but largely in an opposing fashion. In particular, while ERG negatively regulates the androgen receptor (AR) transcriptional program, ETV1 cooperates with AR signaling by favoring activation of the AR transcriptional program. Furthermore, we found that ETV1 expression, but not that of ERG, promotes autonomous testosterone production. Last, we confirmed the association of an ETV1 expression signature with aggressive disease and poorer outcome in patient data. The distinct biology of ETV1-associated prostate cancer suggests that this disease class may require new therapies directed to underlying programs controlled by ETV1.

Abstract Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.