Correction to: Phase I-II trial of imatinib mesylate (Gleevec; STI571) in treatment of recurrent oligodendroglioma and mixed oligoastrocytoma. North central cancer treatment group study N0272 (ALLIANCE/NCCTG).

The last author's first name was truncated in the initial online publication. The original article has been corrected.

Phase I-II trial of imatinib mesylate (Gleevec; STI571) in treatment of recurrent oligodendroglioma and mixed oligoastrocytoma. North central cancer treatment group study N0272 (ALLIANCE/NCCTG).

PURPOSE: To evaluate the pharmacokinetics and efficacy of imatinib in patients with recurrent oligodendroglial tumors. METHODS: Patients with progressive WHO grade II-III recurrent tumors after prior RT and chemotherapy were eligible. A phase I dose-escalation study was conducted for patients on enzyme-inducing anticonvulsants (EIAC). A phase II study for non-EIAC patients utilized a fixed dose of 600 mg/D. Primary efficacy endpoint was 6-month progression-free survival (PFS6). A 2-stage design was utilized, with 90% power to detect PFS6 increase from 25 to 45%. RESULTS: In the Phase I, maximum tolerated dose was not reached at 1200 mg/D. For phase II patients, overall PFS6 was 33% and median PFS 4.0 months (95% CI 2.1, 5.7). Median overall survival (OS) was longer in imatinib-treated patients compared with controls (16.6 vs. 8.0 months; HR = 0.64, 95% CI 0.41,1.0, p = 0.049), and longer in patients with 1p/19q-codeleted tumors (19.2 vs. 6.2 months, HR = 0.43, 95% CI 0.21,0.89, p = 0.019). Confirmed response rate was 3.9% (PR = 1; REGR = 1), with stable disease observed in 52.9%. At 600 mg/D, mean steady-state imatinib plasma concentration was 2513 ng/ml (95% CI 1831,3195). Grade 3-4 adverse events (hematologic, fatigue, GI, hypophosphatemia, or hemorrhage) occurred in 61%. CONCLUSIONS: Although adequate plasma levels were achieved, the observed PFS6 of 33% did not reach our pre-defined threshold for success. Although OS was longer in imatinib-treated patients than controls, this finding would require forward validation in a larger cohort. Imatinib might show greater activity in a population enriched for PDGF-dependent pathway activation in tumor tissue.

TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced expression in tumours may be an informative biomarker of disease progression and perhaps metastatic disease.

Analysis of Ly49 gene transcripts in mature NK cells supports a role for the Pro1 element in gene activation, not gene expression.

The variegated expression of murine Ly49 loci has been associated with the probabilistic behavior of an upstream promoter active in immature cells, the Pro1 element. However, recent data suggest that Pro1 may be active in mature natural killer (NK) cells and function as an enhancer element. To assess directly if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA-sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49(+) NK cells that contained high levels of Pro2 transcripts. Low levels of Ly49g Pro1 transcripts were found in both Ly49G(+) and Ly49G(-) populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in interleukin-2. Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on Pro2 in a mature Ly49-expressing cell line. Taken together, the results are consistent with Pro1 transcription having a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.

Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes.

The human killer cell immunoglobulin-like receptor (KIR) genes contain multiple promoters that control the process of gene activation and variegated expression of KIR on natural killer (NK) and T cells. Specific subfamilies of KIR genes have differences in the timing and tissue specificity of expression: however, previous studies of the proximal KIR promoters have not shown significant differences in activity between differentially expressed KIR gene subsets. The recent identification of an intermediate KIR promoter (ProI) associated with KIR2DL1 expression suggested a central role for this element in KIR expression. The current study identifies ProI elements in all of the KIR genes, revealing four classes of ProI that correspond with four distinct expression phenotypes of KIR subgroups: KIR2DL2/S2/L3 that are expressed early in reconstituting NK after transplant; KIR2DL4 that is expressed by CD56-bright NK in a non-variegated manner; KIR3DL3 that is not expressed by circulating NK cells; and the remaining KIR that are expressed by subsets of CD56-dim NK. The four classes of ProI are structurally diverse and display distinct functional properties. Altogether, these results indicate that KIR ProI elements contribute to the tissue/cell-type specificity of KIR transcription and cooperate with the probabilistic proximal promoter to control KIR expression.

Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression.

Members of the human KIR (killer cell immunoglobulin-like receptor) class I major histocompatibility complex receptor gene family contain multiple promoters that determine the variegated expression of KIR on natural killer cells. In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression. An individual with a single copy of the KIR2DL1 gene but a very low level of gene expression was identified. The low expression phenotype was associated with a single-nucleotide polymorphism (SNP) that created a binding site for the inhibitory ZEB1 (Zinc finger E-box-binding homeobox 1) transcription factor adjacent to a c-Myc binding site previously implicated in distal promoter activity. Individuals possessing this SNP had a substantial decrease in distal KIR2DL1 transcripts initiating from a novel intermediate promoter located 230 bp upstream of the proximal promoter start site. Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter. Reduced intermediate promoter activity revealed the existence of alternatively spliced KIR2DL1 transcripts containing premature termination codons that initiated from the proximal KIR2DL1 promoter. Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.

Identification of a KIR antisense lncRNA expressed by progenitor cells.

Human NK cells express cell surface class I MHC receptors (killer cell immunoglobulin-like receptor, KIR) in a probabilistic manner. Previous studies have shown that a distal promoter acts in conjunction with a proximal bidirectional promoter to control the selective activation of KIR genes. We report here the presence of an intron 2 promoter in several KIR genes that produce a spliced antisense transcript. This long noncoding RNA (lncRNA) transcript contains antisense sequence complementary to KIR-coding exons 1 and 2 as well as the proximal promoter region of the KIR genes. The antisense promoter contains myeloid zinc finger 1 (MZF-1)-binding sites, a transcription factor found in hematopoietic progenitors and myeloid precursors. The KIR antisense lncRNA was detected only in progenitor cells or pluripotent cell lines, suggesting a function that is specific for stem cells. Overexpression of MZF-1 in developing NK cells led to decreased KIR expression, consistent with a role for the KIR antisense lncRNA in silencing KIR gene expression early in development.

Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome.

The mouse Ly49 and human killer cell immunoglobulin-like receptors (KIR) gene clusters encode activating and inhibitory class I MHC receptors on natural killer (NK) cells. A direct correlation between the presence of multiple activating KIR and various human autoimmune diseases including diabetes has been shown. Previous studies have implicated NK cell receptors in the development of diabetes in the non-obese diabetic (NOD) inbred mouse strain. To assess the contribution of Ly49 to NOD disease acceleration the Ly49 gene cluster of these mice was sequenced. Remarkably, the NOD Ly49 haplotype encodes the largest haplotype and the most functional activating Ly49 of any known mouse strain. These activating Ly49 include three Ly49p-related and two Ly49h-related genes. The NOD cluster contains large regions highly homologous to both C57BL/6 and 129 haplotypes, suggesting unequal crossing over as a mechanism of Ly49 haplotype evolution. Interestingly, the 129-like region has duplicated in the NOD genome. Thus, the NOD Ly49 cluster is a unique mix of elements seen in previously characterized Ly49 haplotypes resulting in a disproportionately large number of functional activating Ly49 genes. Finally, the functionality of activating Ly49 in NOD mice was confirmed in cytotoxicity assays.

Autoimmunity, spontaneous tumourigenesis, and IL-15 insufficiency in mice with a targeted disruption of the tumour suppressor gene Fus1.

The Fus1 gene resides in the critical 3p21.3 human chromosomal region deleted in lung and breast cancers. Recently, the tumour suppressor properties of Fus1 were confirmed experimentally by intra-tumoural administration of Fus1 that suppressed experimental lung metastasis in mice. We generated Fus1-deficient mice that were viable, fertile, and demonstrated a complex immunological phenotype. Animals with a disrupted Fus1 gene developed signs of autoimmune disease, such as vasculitis, glomerulonephritis, anaemia, circulating autoantibodies, and showed an increased frequency of spontaneous vascular tumours. Preliminary analysis of immune cell populations revealed a consistent defect in NK cell maturation in Fus1 null mice that correlated with changes in the expression of IL-15. Injection of IL-15 into Fus1 knockout mice completely rescued the NK cell maturation defect. Based on these results, we propose the hypothesis that Fus1 deficiency affects NK cell maturation through the reduction of IL-15 production but does not directly alter their developmental capacity. Since acquired immunity was not affected in Fus1-deficient animals, we suggest a relationship between the Fus1 protein and the regulation of innate immunity via IL-15 production. The increased frequency of spontaneous cancers and the development of an autoimmune syndrome in Fus1 null mice imply that these mice could serve as a model for studying molecular mechanisms of anti-tumour immunity and autoimmunity.

Identification of bidirectional promoters in the human KIR genes.

Although the class I MHC receptors expressed by human and mouse natural killer (NK) cells have distinct molecular origins, they are functional analogues that are expressed in a variegated pattern. The murine Ly49 class I receptors contain bidirectional promoters that have been proposed to control the probabilistic expression of these genes. Whether similar elements are present in the human killer Ig-like receptor (KIR) genes is a fundamental question. A detailed analysis of the 2 kb intergenic region separating the KIR2DL4 gene and the adjacent KIR3DL1 gene revealed that additional promoter elements exist in the human KIR genes. Remarkably, the previously characterized KIR3DL1 proximal promoter possesses bidirectional promoter activity that maps to an 88 bp DNA fragment containing CREB, AML, Sp1 and Ets transcription factor binding sites. Individual KIR genes and alleles possess bidirectional promoters with distinct properties. Analysis of KIR(+)and KIR(-) NK cells and NK precursors indicates that reverse transcripts from the bidirectional promoter are found in cells that lack KIR protein expression, but are not present in mature KIR-expressing NK cells, suggesting that reverse transcription from the proximal promoter blocks gene activation in immature NK and precursor cells.

Identification of distal KIR promoters and transcripts.

A more complete understanding of the transcriptional control of the human and murine class I MHC receptors will help to shed light on the mechanism of selective, stochastic, gene activation that operates in these gene families. Studies of the murine Ly49 class I MHC receptor genes have revealed an important role for distal transcripts originating upstream of the proximal promoter. To date, there have been no reports of distal promoters within the functionally analogous human KIR family of class I MHC receptors. In the current study, reverse transcriptase-polymerase chain reaction (RT-PCR) and RNase protection assays were used to reveal the presence of distal KIR transcripts initiating upstream of the previously characterized proximal KIR promoter. The intergenic promoter elements detected were associated with repetitive elements of the Alu and L1 families. Unlike the proximal KIR promoter, the distal promoter regions were not NK cell-specific. KIR genes expressed in a variegated manner produced a low level of distal transcripts containing a large 5' untranslated region. In contrast, the highly expressed KIR2DL4 gene possessed a higher level of spliced distal transcripts that were capable of producing KIR2DL4 protein. The identification of distal KIR promoter elements suggests that intergenic transcripts may influence the expression of KIR genes.

Transcriptional regulation of NK cell receptors.

The stochastic expression of individual members of NK cell receptor gene families on subsets of NK cells has attracted considerable interest in the transcriptional regulation of these genes. Each receptor gene can contain up to three separate promoters with distinct properties. The recent discovery that an upstream promoter can function as a probabilistic switch element in the Ly49 gene family has revealed a novel mechanism of variegated gene expression. An important question to be answered is whether or not the other NK cell receptor gene families contain probabilistic switches. The promoter elements currently identified in the Ly49, NKR-P1, CD94, NKG2A, and KIR gene families are described.

A single-gene biomarker identifies breast cancers associated with immature cell type and short duration of prior breastfeeding.

The pathogenesis of breast cancers that do not express estrogen receptors or Her-2/neu receptors (ER-/HER2- phenotype) is incompletely understood. We had observed markedly elevated gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunit pi (GABApi, GABRP) in some breast cancers with ER-/HER2- phenotype. In this study, transcriptional profiles (TxPs) were obtained from 82 primary invasive breast cancers by oligonucleotide microarrays. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to measure GABApi gene expression in a separate cohort of 121 invasive breast cancers. GABApi gene expression values from TxP and RT-PCR were standardized and compared with clinicopathologic characteristics in the 203 patients. GABApi gene expression was increased in 16% of breast cancers (13/82 TxP, 20/ 121 RT-PCR), particularly in breast cancers with ER-/HER2- phenotype (60%), and breast cancers with basal-like genomic profile (60%). The profile of genes coexpressed with GABApi in these tumors was consistent with an immature cell type. In multivariate linear regression analysis, the level of GABApi gene expression was associated with ER-/HER2- phenotype (P < 0.0001), younger age at diagnosis (P = 0.0003), and shorter lifetime duration of breastfeeding (< or = 6 months) in all women (P = 0.017) and specifically in parous women (P = 0.013). GABApi gene expression was also associated with combinations of high grade with ER-/HER2- phenotype (P = 0.002), and with Hispanic ethnicity (P = 0.036). GABApi gene expression is increased in breast cancers of immature (undifferentiated) cell type and is significantly associated with shorter lifetime history of breastfeeding and with high-grade breast cancer in Hispanic women.

Complete elucidation of a minimal class I MHC natural killer cell receptor haplotype.

The BALB/c inbred mouse is widely used in models of infectious disease, transplantation, and cancer. The differences in the immune responses of BALB/c compared to C57BL/6 mice are especially valuable for the identification of immune regulation genes. One striking immune variance between these mice is in the function of natural killer (NK) cells, and there is strong evidence implicating differential expression of Ly49 genes. In this study, the complete BALB/c Ly49 gene cluster has been sequenced and found to contain six functional genes and two pseudogenes. Compared to C57BL/6 mice, there is a 200 kb region absent in the BALB/c cluster including a complete lack of Ly49h-related genes, which explains the increased susceptibility of BALB/c to cytomegalovirus infection. In addition, there is no BALB/c Ly49d allele, explaining the inability of BALB/c NK cells to kill certain tumor cells. The Ly49 region has now been sequenced in three different inbred mouse strains, and comparisons indicate that the evolution of each haplotype is not straightforward and has involved large-scale deletions/insertions, gene recombination, and unequal crossing over between divergent haplotypes. This study confirms that relatively small murine class I MHC receptor haplotypes exist, analogous to observations made of human killer cell Ig-like receptor gene haplotypes.

Direct sequence comparison of two divergent class I MHC natural killer cell receptor haplotypes.

The murine Ly49 gene family encoding natural killer cell receptors for class I MHC is an example of a rapidly evolving cluster of immune response genes. Determining the genomic sequence of the 129S6/SvEvTac (129S6) Ly49 cluster and comparing it to the known sequence of the C57BL/6 (B6) region provided insight into the mechanisms of Ly49 gene evolution. 129S6 contains 20 Ly49, many of which are pseudogenes and 40% of the genes have no counterpart in the B6 genome. The difference in gene content between these two strains is primarily the result of distinct patterns of gene duplication. Phylogenetic analyses of individual exons showed that Ly49 genes form distinct sub-families and an ancestral haplotype can be surmised. Dotplot analysis supports limited allelism in the two haplotypes; however, large regions of variation punctuate these islands of co-linearity. These variable regions contain a high concentration of repetitive elements that are predicted to contribute to the dynamic evolution of this cluster. The extreme variation in Ly49 haplotype content between mouse strains provides a genetic explanation for the documented differences in natural killer cell phenotype, and also indicates that differences in natural killer cell function observed between B6 and 129-derived gene-targeted mice should be interpreted with caution.

The ever-expanding Ly49 gene family: repertoire and signaling.

The mouse lectin-related Ly49 family and the human killer cell Ig-like receptor (KIR) family represent structurally distinct, yet functionally analogous, class I MHC receptors that are expressed on natural killer cells and some T cells. The functional similarity of these two families has been borne out by the demonstration of identical signal transduction pathways associated with each receptor family. The Ly49 family therefore provides a useful model system to study the role of this dass of receptors in the regulation of the immune system. Recent data relating to the Ly49 repertoire in several mouse strains has revealed an additional evolutionary parallel between KIR and Ly49 receptor families. There is now an appreciation of the variation in the number and type of Ly49s expressed in different mouse strains, similar to the previously demonstrated differences in the number of KIR genes found in humans. This review summarizes the current members of the Ly49 gene family, their MHC class I recognition and associated signal transduction pathways.

Class I MHC-binding characteristics of the 129/J Ly49 repertoire.

The Ly49 family of NK cell receptors and its MHC-binding characteristics have only been well characterized in C57BL/6 (B6) mice. Previous studies have shown that 129/J mice express unique Ly49 genes that are not found in the B6 strain. Screening of a 129/J cDNA library led to the discovery of 10 distinct full-length Ly49-related coding sequences (Ly49e, g, i, o, p, r, s, t, u, and v). Although 129/J mice share identical class I MHC (K(b) and D(b)) transcripts with B6 mice, only one Ly49 is identical in the two strains (Ly49E). In addition to the previously characterized Ly49P, two new activating Ly49 proteins were discovered, Ly49R and U. The MHC specificity of the total 129/J Ly49 repertoire was evaluated with soluble class I MHC tetramers and found to be distinct compared with the B6 Ly49 repertoire. Ly49V bound to many types of class I MHC, suggesting that Ly49V(+) NK cells may monitor host cells for a global down-regulation in MHC levels. An activating receptor, Ly49R, was shown to bind soluble class I molecules to a moderate degree, a result not previously observed for other activating Ly49 proteins. Furthermore, tetramer-binding results were confirmed functionally with cytotoxicity assays using sorted 129/J NK cells. This study shows that the Ly49 repertoire and its MHC-binding characteristics can be very different among inbred mouse strains. Ly49 divergence should be considered when using 129-derived embryonic stem cells for the production of gene-targeted mice, especially when an immune or NK-derived phenotype is under scrutiny.

The murine Ly49 family: form and function.

The activity of natural killer (NK) cells is regulated by surface receptors that recognize class I MHC. Murine NK cells express a large family of lectin-related receptors (Ly49s) to perform this function, while human NK cells utilize a separate group of proteins containing Ig-related domains (KIRs). Although these receptor families are not structurally related, the Ly49 family appears to be the functional equivalent of human KIRs, since it uses similar signal transduction pathways for either activation or inhibition of NK cell function. Therefore, lessons learned from the study of the murine MHC class I receptor system may be relevant to human NK function. This review summarizes the current state of knowledge of the Ly49 family.

Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium.

BACKGROUND: The human lymphocyte antigen (HLA) class II proteins (DR, DQ, and DP) and DM, a protein involved in loading antigenic peptide onto the class II molecules, have a coordinate regulation that facilitates antigen presentation to CD4+ T cells. CIITA is a specific transcription factor responsible for the coordinate regulation of these genes. DR expression in the kidney was described to be constitutive on renal microvascular endothelium in the early 1980s, but expression of other genes involved in class II antigen presentation (DQ, DP, DM, and CIITA) has not been characterized. METHODS: Expression of the HLA class II proteins, DM, and CIITA in normal human kidney cortex was evaluated by immunofluorescence microscopy, Northern blots, and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The endothelium of glomerular and peritubular capillaries constitutively express DR, as indicated by colocalization of DR and CD31 antibodies. However, the endothelium of larger renal blood vessels is devoid of class II proteins. Capillaries that express DR do not have detectable DQ, DP, or DM by immunofluorescence. Northern blots identified DR, DP, and DM mRNAs but not DQ mRNA. CIITA was amplified by RT-PCR at a level that could account for the class II expressed by the microvascular endothelium. CONCLUSION: The renal microvascular endothelium constitutively expresses DR without the other class II proteins or DM. This discoordinate expression of HLA class II genes is unusual and may contribute to the kidney's ability to control CD4+ T-cell responses.

Identification of the Ly49L protein: evidence for activating counterparts to inhibitory Ly49 proteins.

Previous studies have indicated that NK cells from different strains of inbred mice may express distinct Ly49 repertoires. Screening of NK cells from the CBA/J mouse for inhibitory and activating Ly49s revealed a novel DAP12-associated receptor that was immunoprecipitated with the Ly49G-specific mAb 4D11. Degenerate primers were designed to amplify and clone Ly49 cDNAs from CBA/J NK cells. A novel activating Ly49 cDNA was identified, which bears strong homology to the partially sequenced Ly49l gene found in C57BL/6 mice. Transfection of Ly49l into a DAP12+ cell line and subsequent immunoprecipitation experiments showed that Ly49L is likely the activating Ly49 detected by the 4DD11 antibody in CBA/J NK cells. Antibody-mediated cross-linking of Ly49L induced DAP12 phosphorylation, providing evidence that Ly49L is a functional activating receptor. Comparison of the extracellular domains of Ly49 family members indicates that all known activating members have an inhibitory counterpart with a highly related extracellular region.