Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Malignant pilomatricoma in 3 dogs.

Malignant pilomatricoma, also known as pilomatrix carcinoma and calcifying epitheliocarcinoma (in the human literature), has been considered a rare neoplasm of dogs. The authors present 3 canine cases of malignant pilomatricoma (2 with distant metastasis) and compare its behavior with reported behavior. Cases include an 8-year-old spayed female Airedale Terrier, a 7-year-old male Bassett Hound, and a 12-year-old intact male Giant Schnauzer. In all cases, the histologic features included trabeculae of basaloid cells, abrupt keratinization, "ghost" or "shadow" cells, and various features of malignancy consistent with a diagnosis of malignant pilomatricoma. Metastasis, including that to bone, was confirmed in 2 cases. Four cases of the 13 canine pilomatricomas diagnosed within a 24-month period (2006-2008) at the Ohio State University (2 of which are discussed in this report) were classified as malignant, which suggests that malignant pilomatricoma is more common than previously reported.

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptide-treated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1-target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML.

Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC).

Head and neck squamous cell carcinomas (HNSCC) often metastasise to the cervical lymph nodes. It is known for HNSCC as well as other cancers that progression from normal tissue to primary tumour and finally to metastatic tumour is characterised by an accumulation of genetic mutations. DNA methylation, an epigenetic modification, can result in loss of gene function in cancer, similar to genetic mutations such as deletions and point mutations. We have investigated the DNA methylation phenotypes of both primary HNSCC and metastatic tumours from 13 patients using restriction landmark genomic scanning (RLGS). With this technique, we were able to assess the methylation status of an average of nearly 1300 CpG islands for each tumour. We observed that the number of CpG islands hypermethylated in metastatic tumours is significantly greater than what is found in the primary tumours overall, but not in every patient. Interestingly, the data also clearly show that many loci methylated in a patient's primary tumour are no longer methylated in the metastatic tumour of the same patient. Thus, even though metastatic HNSCC methylate a greater proportion of CpG islands than do the primary tumours, they do so at different subsets of loci. These data show an unanticipated variability in the methylation state of loci in primary and metastatic HNSCCs within the same patient. We discuss two possible explanations for how different epigenetic events might arise between the primary tumour and the metastatic tumour of a person.

Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon.

OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB.

Global methylation profiling of lung cancer identifies novel methylated genes.

Epigenetic changes, including DNA methylation, are a common finding in cancer. In lung cancers methylation of cytosine residues may affect tumor initiation and progression in several ways, including the silencing of tumor suppressor genes through promoter methylation and by providing the targets for adduct formation of polycyclic aromatic hydrocarbons present in combustion products of cigarette smoke. Although the importance of aberrant DNA methylation is well established, the extent of DNA methylation in lung cancers has never been determined. Restriction landmark genomic scanning (RLGS) is a highly reproducible two-dimensional gel electrophoresis that allows the determination of the methylation status of up to 2000 promoter sequences in a single gel. We selected 1184 CpG islands for RLGS analysis and determined their methylation status in 16 primary non-small cell lung cancers. Some tumors did not show methylation whereas others showed up to 5.3% methylation in all CpG islands of the profile. Cloning of 21 methylated loci identified 11 genes and 6 ESTs. We demonstrate that methylation is part of the silencing process of BMP3B in primary tumors and lung cancer cell lines.

Evaluation of microcytosis in 18 Shibas.

OBJECTIVE: To determine whether microcytosis is a typical finding in Shibas. DESIGN: Prospective study. ANIMALS: 18 Shibas. PROCEDURE: Blood and serum samples were obtained for automated hematologic analyses (18 dogs) and for determination of ferritin concentration, using ELISA (14 dogs). Blood samples from 30 clinically normal dogs of various other breeds was analyzed to establish a reference range for ferritin concentration. RESULTS: Erythrocyte mean corpuscular volume in Shibas ranged from 55.6 to 69.1 fl (mean+/-SD, 61.2+/-4.3 fl; median, 60.6 fl; reference range, 63 to 73 fl). Microcytosis was identified in 12 of 18 dogs. Males and females were affected equally. Mean corpuscular hemoglobin concentration was slightly low (range, 32.0 to 33.9%; reference range, 34 to 38%) in 6 dogs, 4 of which had microcytic RBC. Serum ferritin concentrations ranged from 61.2 to 277.0 ng/ml (mean+/-SD, 110.6+/-51.4 ng/ml; median, 106 ng/ml). Reference range for serum ferritin concentration was 50.7 to 440.0 ng/ml (mean+/-SD, 121.2+/-67.1 ng/ml; median, 111.5 ng/ml). Thrombocytopenia (range, 110,000 to 196,000 platelets; reference range, 200,000 to 450,000 platelets) was found in 7 dogs, 6 of which also had microcytic RBC. CLINICAL IMPLICATIONS: Microcytosis can be a typical finding in Shibas. Common origin of Shibas and Akitas, a breed predisposed to microcytosis, suggests a hereditary basis for this finding.

Immunoglobulin crystals in reactive plasma cells in a dog.

Intracellular crystalline deposits of immunoglobulin are occasionally seen in human B-cell lymphoproliferative disorders such as multiple myeloma, chronic lymphocytic leukemia, and various forms of lymphoma. Even more uncommon is the occurrence of immunoglobulin crystals in benign plasma cells or reactive lymphocytes. Here we describe the histologic, immunohistochemical, and ultrastructural features of intracellular immunoglobulin crystals in nonneoplastic plasma cells in a chronic inflammatory lesion in a dog. Microscopically, the intracellular, nonbirefringent eosinophilic crystals were square to rectangular, 2-20 microm long, and caused nuclear displacement to the periphery. The crystal-containing cells, as well as some of the crystals themselves, were positive for lambda light chain. Ultrastructural findings were consistent with a lattice network of protein-molecule alignment. The cause and significance of the crystals is unknown.

Quantification of plasma DNA as a prognostic indicator in canine lymphoid neoplasia.

Dogs have a similar incidence of spontaneous cancers as people, and a noninvasive test to monitor disease status in dogs would be of great value. Humans with cancer often have increased levels of cell-free circulating DNA in their plasma, which has shown promise for diagnosis, prognosis and detection of residual disease. We hypothesized that dogs with cancer have increased circulating DNA compared with healthy dogs or dogs with non-neoplastic diseases. Plasma DNA was measured in 40 healthy dogs, 20 dogs with non-neoplastic diseases and 80 dogs with cancer. The reference interval for plasma DNA in healthy dogs was 1-15 ng mL(-1). Dogs with lymphoma and lymphoid leukaemia had significantly higher concentrations (range: 0-91 ng mL(-1), P < 0.0001). Antigen receptor rearrangement assays suggest that plasma DNA had the same clonality as the primary lymphoid tumours. Dogs with lymphoid neoplasia and plasma DNA >25 ng mL(-1) had shorter remission times than those with < 25 ng mL(-1) (P = 0.0116). In contrast to humans, where increased plasma DNA is seen in many diseases, dogs with nonlymphoid malignancies and non-neoplastic diseases had plasma DNA concentrations similar to healthy dogs. This study shows that a portion of dogs with lymphoid neoplasia have increased tumour-derived plasma DNA, which serves as a negative prognostic indicator.

Aberrant hypermethylation of the major breakpoint cluster region in 17p11.2 in medulloblastomas but not supratentorial PNETs.

Deletions of 17p have been consistently reported in up to 50% of medulloblastomas (MBs), and the major breakpoint interval has been localized to chromosome segment 17p11.2. Based on several reports linking aberrant DNA methylation and chromosomal disruption, we examined the methylation pattern in this region by employing restriction landmark genomic scanning (RLGS). Several CpG islands located in the major breakpoint cluster region were identified using a bacterial artificial chromosome (BAC) contig of the breakpoint region. A long-range methylation map was established for 20 MBs and 5 supratentorial primitive neuroectodermal tumors (stPNETs). Selected CpG islands were examined using Southern and bisulfite sequencing analysis. Aberrantly hypermethylated CpG islands in 17p11. 2 were found in 33% of MBs. Interestingly, one CpG island was methylated in MBs, but not in any of the examined stPNETs. A BAC clone covering three of the methylated CpG islands was partially sequenced in the search for a potential tumor suppressor gene. None of the expressed sequence tag sequences and full-length mouse/human cDNAs that were associated with aberrant methylation showed a change in expression levels due to methylation. The potential link between chromosomal instability in 17p11.2 and hypermethylation in this region is discussed.

Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies.

Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.

Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci.

Aberrant DNA methylation is believed to be important in tumorigenesis by causing either transcriptional inactivation of genes or chromosomal instability. Several laboratories have identified promoter hypermethylation of tumor suppressor genes in acute myeloid leukemia (AML). However, these studies do not provide a global assessment of overall methylation changes and do not allow the identification of novel methylated sequences. Previously, nonrandom CpG island methylation was reported in 17 adult de novo AML diagnostic samples when compared with the corresponding remission samples by means of restriction landmark genomic scanning (RLGS). That study has been expanded on by an analysis of a larger set of CpG islands (1740 vs 1184), which now provides details of 33 cloned methylated loci, including 21 known genes or expressed sequence tags. Five of these cloned loci appear to be methylated only in AML and not in the 6 solid tumors studied in this study (more than 98 samples analyzed). Chromosomal location was available for 30 of the 33 loci, and 5 of these 30 (17%) are localized to chromosome 11, suggesting a trend toward overrepresentation of methylation events on this chromosome. These results provide evidence for widespread aberrant methylation in AML, with identification of novel methylation targets, epigenetic changes that appear unique to AML, and apparent preferential methylation on chromosome 11.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia.

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.