DNA methylation aberrancies delineate clinically distinct subsets of colorectal cancer and provide novel targets for epigenetic therapies.

Colorectal cancer (CRC) is a worldwide health concern with respect to both incidence and mortality, and as a result, CRC tumorigenesis, progression and metastasis have been heavily studied, especially with respect to identifying genetic, epigenetic, transcriptomic and proteomic profiles of disease. DNA methylation alterations are hallmarks of CRC, and epigenetic driver genes have been identified that are thought to be involved in early stages of tumorigenesis. Moreover, distinct CRC patient subgroups are organized based on DNA methylation profiles. CRC tumors displaying CpG island methylator phenotypes (CIMPs), defined as DNA hypermethylation at specific CpG islands in subsets of tumors, show high concordance with specific genetic alterations, disease risk factors and patient outcome. This review details the DNA methylation alterations in CRC, the significance of CIMP status, the development of treatments based on specific molecular profiles and the application of epigenetic therapies for CRC patient treatment.

CD70-restricted specific activation of TRAILR1 or TRAILR2 using scFv-targeted TRAIL mutants.

To combine the CD27 stimulation inhibitory effect of blocking CD70 antibodies with an antibody-dependent cellular cytotoxicity (ADCC)-independent, cell death-inducing activity for targeting of CD70-expressing tumors, we evaluated here fusion proteins of the apoptosis-inducing TNF family member TRAIL and a single-chain variable fragment (scFv) derived from a high-affinity llama-derived anti-human CD70 antibody (lαhCD70). A fusion protein of scFv:lαhCD70 with TNC-TRAIL, a stabilized form of TRAIL, showed strongly enhanced apoptosis induction upon CD70 binding and furthermore efficiently interfered with CD70-CD27 interaction. Noteworthy, introduction of recently identified mutations that discriminate between TRAILR1 and TRAILR2 binding into the TRAIL part of scFv:lαhCD70-TNC-TRAIL resulted in TRAIL death receptor-specific fusion proteins with CD70-restricted activity.

CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells.

Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction.

DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status.

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

DNA methylation profiles of female steroid hormone-driven human malignancies.

Tumor DNA contains valuable clues about the origin and pathogenesis of human cancers. Alterations in DNA methylation can lead to silencing of genes associated with distinct tumorigenic pathways. These pathway-specific DNA methylation changes help define tumor-specific DNA methylation profiles that can be used to further our understanding of tumor development, as well as provide tools for molecular diagnosis and early detection of cancer. Female sex hormones have been implicated in the etiology of several of the women's cancers including breast, endometrial, ovarian, and proximal colon cancers. We have reviewed the DNA methylation profiles of these cancers to determine whether the hormonal regulation of these cancers results in specific DNA methylation alterations. Although subsets of tumors in each of these four types of cancers were found to share some DNA methylation alterations, we did not find evidence for global hormone-specific DNA methylation alterations, suggesting that female sex hormones may participate in different tumorigenic pathways that are associated with distinct DNA methylation-based molecular signatures. One such pathway may include MLH1 methylation in the context of the CpG island methylator phenotype.

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

BACKGROUND: The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. AIM: To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. MATERIALS AND METHODS: We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. RESULTS: There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, p<10(-5)) and non-CIMP MSS tumours (6.6%, p<10(-4)), respectively). CONCLUSION: CIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

Comparison of multitarget fluorescence in situ hybridization in urine with other noninvasive tests for detecting bladder cancer.

OBJECTIVES: To present a single-centre study investigating aneuploidy at chromosomes 3, 7, 17 and 9p21 (e.g. loss at 9p21) using a multitarget fluorescence in situ hybridization (FISH) system, as identifying genetic alterations in urine specimens is a promising approach for the noninvasive detection of bladder cancer. PATIENTS AND METHODS: Urine samples from 103 patients were evaluated, including those from 46 with histologically confirmed urothelial carcinoma, two with other urological malignancies, and 55 who acted as controls. The urine samples were taken before any manipulation. The validity of FISH (Urovision, Vysis, Downers Grove, Ill, USA) was compared with other noninvasive urine tests, including the BTA-Stat test, the nuclear matrix protein (NMP)-22 test, and immunocytology against 486p3/12 and LewisX. Those evaluating the tests were unaware of the clinical and histopathological data. FISH was considered positive if five or more urinary cells had gains of two or more chromosomes. The threshold for the urine tests were 10 U/mL (NMP-22), 30% positive cells (486p3/12), or 5% positive cells, respectively (LewisX). RESULTS: The sensitivity was 69% (FISH), 67% (BTA-Stat), 69% (486p3/12), 96% (LewisX) and 71% (NMP22), respectively; the respective specificity was 89%, 78%, 76%, 33% and 66%. CONCLUSION: Multitarget FISH had a better specificity than the other urine markers but because of its inadequate sensitivity it does not seem to be powerful enough to replace endoscopy. Optimizing the marker panel could provide a higher sensitivity.

A 3D high-resolution gamma camera for radiopharmaceutical studies with small animals.

The results of studies conducted with a small field of view tomographic gamma camera based on a Position Sensitive Photomultiplier Tube are reported. The system has been used for the evaluation of radiopharmaceuticals in small animals. Phantom studies have shown a spatial resolution of 2mm in planar and 2-3mm in tomographic imaging. Imaging studies in mice have been carried out both in 2D and 3D. Conventional radiopharmaceuticals have been used and the results have been compared with images from a clinically used system.

Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis.

Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.

Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations.

Although alterations in chromosome number have frequently been detected in human tumor cells and associated with tumor initiation and progression, the causal mechanisms are still not understood. One protein known to be involved in maintaining genetic stability is tumor suppressor p53. In mice, p53 has been implicated in the maintenance of diploidy (Cross et al., 1995) and the regulation of centrosome duplication (Fukasawa et al., 1996). Here we report on cerebral primitive neuroectodermal tumors that lacked the wild-type p53 gene (TP53) and showed multiple numerical chromosome aberrations, as detected by comparative genomic hybridization. In these tumors, the centrosome number was significantly higher than in a control tumor without a detected TP53 mutation and with few chromosomal imbalances. These findings indicate that abnormal centrosome amplification can occur in human tumors lacking wild-type TP53 and may be a mechanism by which numerical chromosome aberrations are generated.

Looking at Christmas trees in the nucleolus.

We describe novel nucleolar structures, observed by thin section electron microscopy in oocyte nuclei of the grasshopper Locusta migratoria, which we interpret, based on morphological and compositional criteria, as rDNA transcription units. Morphologically they resemble the condensed and foreshortened "Christmas trees" seen in Miller spreads of nucleolar chromatin prepared from the same biological material. They contain DNA and rRNA as shown by immunocytochemistry and in situ hybridization and are concentrated in several intranucleolar cavities. The presumptive rDNA transcription units extend throughout the interior of these nucleolar pockets or are selectively enriched at their outermost zones in close contact with the surrounding fibrillarin-positive dense component. We suggest that the nucleolar pockets of Locusta oocytes are equivalent to the fibrillar centers of somatic nucleoli and discuss possible implications for the current understanding of the functional organization of nucleoli.

A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis.

When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.

The nucleolus.

The transcriptionally active rRNA genes have the remarkable ability to organize and integrate the biochemical pathway of ribosome production into a structural framework, the nucleolus. The past year has seen numerous advances in our understanding of the relationships between nucleolar substructures, the site of ribosomal RNA (rRNA) gene transcription and the pathway of ribosome maturation. Progress has also been made both in the molecular identification of nucleolar constituents and in our understanding of the interactions between these components and their assembly into higher order structures.

Clinical features and prognosis of follicular large-cell lymphoma: a report from the Nebraska Lymphoma Study Group.

PURPOSE: Our purpose was to describe the treatment outcome of patients with follicular large-cell lymphoma (FLCL) and to identify prognostic factors that affect the treatment outcome. PATIENTS AND METHODS: Between 1980 and 1991, 107 newly diagnosed, previously untreated patients with FLCL were prospectively treated using treatment plans of the Nebraska Lymphoma Study Group (NLSG). Most stage I/II patients received two to three cycles of one of four closely related six-drug combination chemotherapy regimens (cyclophosphamide, doxorubicin or mitoxantrone, and procarbazine, plus bleomycin, vincristine, and prednisone or dexamethasone [CAP/BOP I-IV]) plus involved-field radiotherapy; 10 patients received involved-field irradiation only. Stage III/IV patients received six to eight cycles of CAP/BOP. RESULTS: Forty-four percent of patients had stage I/II disease. Stage I/II patients were older and more often female than stage III/IV patients. Cytogenetic studies were available on 35 patients: seven were normal; the most common abnormality was a translocation involving 14q32. Abnormalities of 1p or 1q were also common, often secondary to a 14q32 abnormality. The median follow-up of surviving patients is 2 years. The complete response rates observed were stage I/II, 88%; stage III/IV, 49%. Complete response rates were affected by both age and tumor bulk. Failure-free survival (FFS; time to first occurrence of progression, relapse after response, or death from any cause) at 3 years was estimated to be 61% for stage I/II patients and 34% for stage III/IV patients. Survival at 3 years was estimated to be 76% and 61%, respectively. FFS of stage III/IV patients was poorer for stage IV patients and those with composite lymphomas. Significantly poorer survival was only seen in patients older than 70 years of age. CONCLUSION: A proportion of stage I/II FLCL patients may obtain long-term disease control with combination chemotherapy plus radiotherapy. Results for patients with stage III/IV FLCL are similar to those seen for other follicular lymphomas.

Assigning functions to nucleolar structures.

Nucleoli provide the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical features of the formation and stepwise maturation of ribosomes. Localization of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-functional relationships of the nucleolus. The present review describes some recent results obtained by electron microscopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) participate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA maturation. The intranucleolar distribution of U3 snRNA is consistent with the view that it is involved in both early and late stages of pre-rRNA processing.

Pulmonary emboli in patients receiving chemotherapy for non-Hodgkin's lymphoma.

Combination chemotherapy has dramatically improved the prognosis of patients with intermediate and high grade histologic subtypes of non-Hodgkin's lymphomas. Treatment-related complications, however, are considerable, and a common problem encountered is respiratory distress or respiratory insufficiency. Usually these difficulties have been attributed to infectious etiologies or to chemotherapy-induced interstitial fibrosis, most often involving bleomycin. We describe five patients presenting with respiratory problems several weeks after the initiation of chemotherapy. These patients, who represent 3 percent of all patients treated with a single bleomycin-containing regimen for intermediate or high grade non-Hodgkin's lymphoma, were all initially thought to have chemotherapy-induced interstitial fibrosis but were found on subsequent evaluation to have pulmonary emboli. Of the three patients in whom pulmonary emboli were diagnosed antemortem, two had symptoms suggestive of pulmonary emboli and all were successfully treated and remained well and free of lymphoma for over 24 months. Two additional patients were diagnosed at autopsy. We suggest that pulmonary emboli may contribute significantly to the morbidity and mortality of patients undergoing chemotherapy for non-Hodgkin's lymphoma and recommend that patients presenting with respiratory difficulties be evaluated for pulmonary emboli.