Data sets for the qualification of volumetric CT as a quantitative imaging biomarker in lung cancer.

The drug development industry is faced with increasing costs and decreasing success rates. New ways to understand biology as well as the increasing interest in personalized treatments for smaller patient segments requires new capabilities for the rapid assessment of treatment responses. Deployment of qualified imaging biomarkers lags apparent technology capabilities. The lack of consensus methods and qualification evidence needed for large-scale multi-center trials, as well as the standardization that allows them, are widely acknowledged to be the limiting factors. The current fragmentation in imaging vendor offerings, coupled with the independent activities of individual biopharmaceutical companies and their contract research organizations (CROs), may stand in the way of the greater opportunity were these efforts to be drawn together. A preliminary report, "Volumetric CT: a potential biomarker of response," of the Quantitative Imaging Biomarkers Alliance (QIBA) activity was presented at the Medical Imaging Continuum: Path Forward for Advancing the Uses of Medical Imaging in the Development of New Biopharmaceutical Products meeting of the Extended Pharmaceutical Research and Manufacturers of America (PhRMA) Imaging Group sponsored by the Drug Information Agency (DIA) in October 2008. The clinical context in Lung Cancer and a methodology for approaching the qualification of volumetric CT as a biomarker has since been reported [Acad. Radiol. 17, 100-106, 107-115 (2010)]. This report reviews the effort to collect and utilize publicly available data sets to provide a transparent environment in which to pursue the qualification activities in such a way as to allow independent peer review and verification of results. This article focuses specifically on our role as stewards of image sets for developing new tools.

Change in lung tumor volume as a biomarker of treatment response: a critical review of the evidence.

SPECIFIC AIM: To review the evidence indicating that volumetric image analysis of computed tomography scans meets specifications for qualification as a biomarker in clinical trials or the management of individual patients with lung cancer. METHODS: Claims of value were broken down into questions about technical feasibility, accuracy, the precision of measurement, sensitivity, the correlations with health outcomes, and the risks of producing misleading information. For each claim, the pertinent literature was reviewed. RESULTS: Technical feasibility has now been shown, but only in limited contexts. Accuracy has been demonstrated, but only for tumors with favorable anatomical features. Measurement error still makes the assessment of change in small nodules precarious in diagnostic settings unless rigorous image acquisition and analysis procedures are followed. Precision is sufficient in some larger masses to make volumetrics a sensitive biomarker. In a few trials, correlations with clinical outcomes have been higher for volumetric-based measures than for unidimensional or bidimensional diameters. Value in clinical practice settings and clinical trials has been suggested, but not proven. CONCLUSION: The weight of the evidence indicates there are circumstances in which volumetric image analysis adds value to clinical trial science and the practice of medicine.

hREC2, a RAD51-like gene, is disrupted by t(12;14) (q15;q24.1) in a uterine leiomyoma.

A balanced translocation between chromosomes 12 and 14 is commonly seen in uterine leiomyoma (UL). We have previously cloned and characterized a 2 Mb segment of human chromosomal subband 14q24.1, and have shown that the t(12;14)(q15;q24.1) breakpoints from several ULs map within this region. Exon trapping of DNA clones spanning one such breakpoint revealed coding sequences from hREC2, a gene that shows significant amino acid sequence identity to the double-strand break repair enzyme RAD51. We report here that this breakpoint is located within a 19 kb intron of the hREC2 gene and that the translocation results in the premature truncation of the major hREC2 transcript. Mapping and sequence analyses show that alternative transcripts of the hREC2 gene, including novel isoforms identified in testis and uterus, are not interrupted by the translocation.

Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome.

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein-Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. The XLP gene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions. We describe here the presence of small deletions and intragenic mutations that specifically disrupt a gene named DSHP in 6 of 10 unrelated patients with XLP. This gene encodes a predicted protein of 128 amino acids composing a single SH2 domain with extensive homology to the SH2 domain of SHIP, an inositol polyphosphate 5-phosphatase that functions as a negative regulator of lymphocyte activation. DSHP is expressed in transformed T cell lines and is induced following in vitro activation of peripheral blood T lymphocytes. Expression of DSHP is restricted in vivo to lymphoid tissues, and RNA in situ hybridization demonstrates DSHP expression in activated T and B cell regions of reactive lymph nodes and in both T and B cell neoplasms. These observations confirm the identity of DSHP as the gene responsible for XLP, and suggest a role in the regulation of lymphocyte activation and proliferation. Induction of DSHP may sustain the immune response by interfering with SHIP-mediated inhibition of lymphocyte activation, while its inactivation in XLP patients results in a selective immunodeficiency to EBV.

The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein.

Early-onset torsion dystonia is a movement disorder, characterized by twisting muscle contractures, that begins in childhood. Symptoms are believed to result from altered neuronal communication in the basal ganglia. This study identifies the DYT1 gene on human chromosome 9q34 as being responsible for this dominant disease. Almost all cases of early-onset dystonia have a unique 3-bp deletion that appears to have arisen idependently in different ethnic populations. This deletion results in loss of one of a pair of glutamic-acid residues in a conserved region of a novel ATP-binding protein, termed torsinA. This protein has homologues in nematode, rat, mouse and humans, with some resemblance to the family of heat-shock proteins and Clp proteases.

Fine localization of the torsion dystonia gene (DYT1) on human chromosome 9q34: YAC map and linkage disequilibrium.

The DYT1 gene, which maps to chromosome 9q34, appears to be responsible for most cases of early-onset torsion dystonia in both Ashkenazic Jewish (AJ) and non-Jewish families. This disease is inherited in an autosomal dominant mode with reduced penetrance (30%-40%). The abnormal involuntary movements associated with this disease are believed to be caused by unbalanced neural transmission in the basal ganglia. Previous linkage disequilibrium studies in the AJ population placed the DYT1 gene in a 2-cM region between the loci D9S62a and ASS. A YAC contig has now been created spanning 600 kb of this region including D9S62a. The location of the DYT1 gene has been refined within this contig using several new polymorphic loci to expand the linkage disequilibrium analysis of the AJ founder mutation. The most likely location of the DYT1 gene is within a 150 kb region between the loci D9S2161 and D9S63.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

A translocation at 12q2 refines the interval containing the Holt-Oram syndrome 1 gene.

A gene for Holt-Oram syndrome (HOS) has been previously mapped to chromosome 12q2 and designated HOS1. We have identified a HOS patient with a de novo chromosomal rearrangement involving 12q. Detailed cytogenetic analysis of this case reveals three breaks on 12q, and two of these are within the HOS1 interval. By using a combination of chromosome painting and FISH with YACs and cosmids, it has been possible to map these breakpoints within the critical HOS1 interval and thus provide a focus for HOS gene-identification efforts.

p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta-repeat domains characteristic of the beta-subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid-rich regions, a putative leucine zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy-terminal RCC1-like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1-like motif located at the amino-terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R-Ras2/TC21. These observations suggest that p619 is a Brefeldin A-sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL).

An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that it may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders.

An expression-independent catalog of genes from human chromosome 22.

To accomplish large-scale identification of genes from a single human chromosome, exon amplification was applied to large pools of clones from a flow-sorted human chromosome 22 cosmid library. Sequence analysis of more than one-third of the 6400 cloned products identified 35% of the known genes previously localized to this chromosome, as well as several unmapped genes and randomly sequenced cDNAs. Among the more interesting sequence similarities are those that represent novel human genes that are related to others with known or putative functions, such as one exon from a gene that may represent the human homolog of Drosophila Polycomb. It is anticipated that sequences from at least half of the genes residing on chromosome 22 are contained within this exon library. This approach is expected to facilitate fine-structure physical and transcription mapping of human chromosomes, and accelerate the process of disease gene identification.

Cloning of a highly conserved human protein serine-threonine phosphatase gene from the glioma candidate region on chromosome 19q13.3.

Allelic loss studies have suggested that a glioma tumor suppressor gene resides in a 425-kb region of chromosome 19q, telomeric to D19S219 and centromeric to D19S112. Exon amplification of a cosmid contig spanning this region yielded four exons with high homology to a rat protein serine-threonine phosphatase from a cosmid approximately 100 kb telomeric to D19S219. Isolation of a near full-length cDNA from a human fetal brain cDNA library revealed a protein serine-threonine phosphatase with a tetratricopeptide motif, almost identical to human PPP5C (PP5) and highly homologous to rat PPT. Northern blotting demonstrated expression in most tissues, including brain. Primary and cultured gliomas were studied for genetic alterations in this gene using pulsed-field gel electrophoresis, routine Southern blots, and genomic DNA-and RNA-based single-strand conformation polymorphism analysis. Genomic alterations were were not detected in any of the gliomas, and all studied gliomas expressed the gene, suggesting that this phosphatase is not the putative chromosome 19q glioma tumor suppressor gene.

Isolation of genes from the Batten candidate region using exon amplification. Batten Disease Consortium.

In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes.

Role of Rel-related factors in control of c-myc gene transcription in receptor-mediated apoptosis of the murine B cell WEHI 231 line.

Treatment of immature murine B lymphocytes with an antiserum against their surface immunoglobulin (sIg)M results in cell death via apoptosis. The WEHI 231 B cell line (IgM, kappa) has been used extensively as a model for this anti-Ig receptor-mediated apoptosis. Anti-sIg treatment of WEHI 231 cells causes an early, transient increase in the levels of c-myc messenger RNA and gene transcription, followed by a rapid decline below control values. Given the evidence for a role of the c-myc gene in promoting apoptosis, we have characterized the nature and kinetics of changes in the binding of Rel-related factors, which modulate c-myc promoter activity. In exponentially growing WEHI 231 cells, multiple Rel-related binding activities were detectable. The major binding species was identified as p50/c-Rel heterodimers; only minor amounts of nuclear factor kappa B (NF-kappa B) (p50/p65) were detectable. Cotransfection of an inhibitor of NF-kappa B (I kappa B)-alpha expression vector reduced c-myc-promoter/upstream/exon1-CAT reporter construct activity, indicating the role of Rel factor binding in c-myc basal expression in these cells. Treatment with anti-sIg resulted in a rapid transient increase in the rate of c-myc gene transcription and in the binding of Rel factors. At later times, formation of p50 homodimer complexes occurred. In cotransfection analysis, p65 and c-Rel expression potently and modestly transactivated the c-myc promoter, respectively, whereas, overexpression of the p50 subunit caused a significant drop in its activity. The role of activation of Rel-family binding was demonstrated directly upon addition of the antioxidant pyrrolidinedithiocarbamate, which inhibited the anti-sIg-mediated activation of the endogenous c-myc gene. Similarly, induction after anti-sIg treatment of a transfected c-myc promoter was abrogated upon cotransfection of an I kappa B-alpha expression vector. These results implicate the Rel-family in Ig receptor-mediated signals controlling the activation of c-myc gene transcription in WEHI 231 cells, and suggest a role for this family in apoptosis of this line, which is mediated through a c-myc signaling pathway.

p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma.

To determine whether p16 is altered in human malignant mesothelioma (MM), molecular analysis of multiple 9p loci was performed on 40 cell lines and 23 primary tumors from 42 MM patients. We identified homozygous deletions of p16 in 34 (85%) cell lines and a point mutation in 1 line. Down-regulation of p16 was observed in 4 of the remaining cell lines, 1 of which displayed a DNA rearrangement of p16. Homozygous deletions of p16 were identified in 5 of 23 (22%) primary tumors; no mutations or rearrangements were found in these specimens. Four cell lines displayed a single homozygous deletion proximal to or distal to p16; 4 others had 2 nonoverlapping deletions, one involving p16 and the other involving a region proximal to this locus. These data indicate that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors. Furthermore, deletions of 9p21-p22 outside of the p16 locus may reflect the involvement of other putative tumor suppressor genes that could also contribute to the pathogenesis of some MMs.

MTS1/CDKN2 gene mutations are rare in primary human astrocytomas with allelic loss of chromosome 9p.

Human astrocytomas frequently have allelic losses of chromosome 9p, suggesting the presence of a 9p astrocytoma tumor suppressor gene. The MTS1 (or CDKN2) gene on chromosome 9p encodes a cell-cycle regulator and is deleted in approximately 80% of astrocytoma cell lines. To determine whether MTS1 is the tumor suppressor gene involved in human astrocytoma formation in vivo, we have analyzed chromosome 9p allelic loss and the MTS1 gene in 30 primary astrocytomas. Deletion mapping demonstrated 15 cases with allelic loss of chromosome 9p, with all losses either flanking or involving the MTS1 gene. Direct analysis of the MTS1 gene, however, revealed only a single missense mutation in a high-grade tumor that had lost the second allele. The low frequency of MTS1 mutations in primary astrocytomas with allelic 9p loss suggests that MTS1 may be more important for in vitro than in vivo astrocytoma growth, and that another 9p tumor suppressor gene may be involved in astrocytoma formation in vivo. Analysis of the MTS1 gene also demonstrated two intragenic polymorphisms, one in exon 2 and one in the 3' untranslated region, that can be used to assay allelic loss directly at MTS1.

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas.

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.

Isolation of conserved sequences from yeast artificial chromosomes by exon amplification.

Exon amplification is a technique designed to solve a central problem in mammalian molecular genetics--specifically, the isolation of genes from large regions of genomic DNA. This technique allows exons to be isolated from genomic DNA following the selective removal of introns by the eukaryotic splicing mechanism. It is a relatively rapid procedure and can theoretically be applied to test the coding potential of very long stretches of genomic DNA. In this paper we describe the application of exon amplification to a mouse yeast artificial chromosome (YAC) 175 kb in length. A number of potential coding sequences were amplified, and two sequences were shown to be conserved across a wide variety of species representing potential genes.