Genomic characterization of HER2-positive breast cancer and response to neoadjuvant trastuzumab and chemotherapy-results from the ACOSOG Z1041 (Alliance) trial.

Background: HER2 (ERBB2) gene amplification and its corresponding overexpression are present in 15-30% of invasive breast cancers. While HER2-targeted agents are effective treatments, resistance remains a major cause of death. The American College of Surgeons Oncology Group Z1041 trial (NCT00513292) was designed to compare the pathologic complete response (pCR) rate of distinct regimens of neoadjuvant chemotherapy and trastuzumab, but ultimately identified no difference. Patients and methods: In supplement to tissues from 37 Z1041 cases, 11 similarly treated cases were obtained from a single institution study (NCT00353483). We have extracted genomic DNA from both pre-treatment tumor biopsies and blood of these 48 cases, and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have generated RNA-seq profiles from 42 of the tumor biopsies. Among patients in this cohort, 24 (50%) achieved a pCR. Results: We have characterized the genomic landscape of HER2-positive breast cancer and investigated associations between genomic features and pCR. Cases assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR compared to the luminal, basal-like, or normal-like subtypes (19/27 versus 3/15; P = 0.0032). Mutational events led to the generation of putatively active neoantigens, but were overall not associated with pCR. ERBB2 and GRB7 were the genes most commonly observed in fusion events, and genomic copy number analysis of the ERBB2 locus indicated that cases with either no observable or low-level ERBB2 amplification were less likely to achieve a pCR (7/8 versus 17/40; P = 0.048). Moreover, among cases that achieved a pCR, tumors consistently expressed immune signatures that may contribute to therapeutic response. Conclusion: The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those HER2-positive breast cancer patients who will not respond to treatment with chemotherapy and trastuzumab. ClinicalTrials.gov identifiers: NCT00513292, NCT00353483.

Cloning and expression of a developmentally regulated protein that induces mitogenic and neurite outgrowth activity.

A heparin binding mitogenic protein isolated from bovine uterus shares NH2-terminal amino acid sequence with a protein isolated from newborn rat brain. The cDNA's of the bovine, human, and rat genes have been isolated and encode extraordinarily conserved proteins unrelated to known growth or neurotrophic factors, although identity of nearly 50 percent has been found with the predicted sequence of a retinoic acid induced transcript in differentiating mouse embryonal carcinoma cells. Lysates of COS-7 cells transiently expressing this protein were mitogenic for NRK cells and initiated neurite outgrowth from mixed cultures of embryonic rat brain cells. RNA transcripts encoding this protein were widely distributed in tissues and were developmentally regulated. This protein, previously designated as heparin binding growth factor (HBGF)-8, is now renamed pleiotrophin (PTN) to reflect its diverse activities. PTN may be the first member of a family of developmentally regulated cytokines.

DNA transposition: jumping gene machine, some assembly required.

Transposition of the mobile DNA element Mu is stringently controlled by the assembly of an elaborate jumping gene machine, which is inactive until all the pieces are in place.

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.

The cytochrome P-450 monooxygenase 3A4 (CYP3A4) is responsible for the oxidative metabolism of a wide variety of xenobiotics including an estimated 60% of all clinically used drugs. Although expression of the CYP3A4 gene is known to be induced in response to a variety of compounds, the mechanism underlying this induction, which represents a basis for drug interactions in patients, has remained unclear. We report the identification of a human (h) orphan nuclear receptor, termed the pregnane X receptor (PXR), that binds to a response element in the CYP3A4 promoter and is activated by a range of drugs known to induce CYP3A4 expression. Comparison of hPXR with the recently cloned mouse PXR reveals marked differences in their activation by certain drugs, which may account in part for the species-specific effects of compounds on CYP3A gene expression. These findings provide a molecular explanation for the ability of disparate chemicals to induce CYP3A4 levels and, furthermore, provide a basis for developing in vitro assays to aid in predicting whether drugs will interact in humans.

The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction.

The NGFI-B cDNA was previously isolated by virtue of its induction by nerve growth factor (NGF) in PC12 cells. It encodes a 61-kilodalton protein that has two regions of extensive homology with members of the steroid/thyroid hormone receptor gene family. The rat NGFI-B gene is approximately 7.6 kilobases long and is interrupted by six introns. Although the exon-intron structure of the gene is similar to those of several other members of the steroid/thyroid hormone receptor gene family, there is a novel splice site within the DNA-binding domain which suggests that NGFI-B constitutes yet another evolutionary digression from a postulated common ancestral receptor gene. Primer extension and S1 nuclease protection assays were used to determine the transcription initiation site, which displayed the heterogeneity typical of genes that lack a TATA box. Sequence analysis of the 5' flanking region revealed several GC boxes but no identifiable TATA box. Four potential AP1 binding sites were identified at nucleotides -49, -78, -222, and -242. Neither the serum response element nor the CArG box element, two sequences found in other growth factor-inducible genes, was detected in this region of the growth factor-inducible NGFI-B gene. Nevertheless, results of nuclear runoff experiments demonstrated that the NGFI-B gene was transcriptionally activated by nerve growth factor in PC12 cells. In vivo, a rapid, dramatic increase in NGFI-B mRNA was observed in the cerebral cortex, midbrain, and cerebellum of animals that experienced a convulsant-induced seizure.

Determination of the pressure required to cause mitral valve failure.

A method has been developed for applying water pressure to a closed mitral valve on the side corresponding to the heart's left ventricle. The pressure is increased until fluid flows through the valve, i.e. until it fails. A specific dissection technique has been developed to produce a specimen with two annular rings, mitral annulus and papillary muscle annulus. Since the valve is maintained intact, with its leaflets attached to papillary muscles by the chordae tendineae, this method allows the effects of ruptured chordae and their surgical repair or replacement to be assessed in vitro. The chamber that holds the valve supports both the mitral annulus and papillary muscle annulus of the specimen. The mitral annulus is sutured onto rubber sheeting held in the chamber. The papillary muscle annulus is held in place by a Perspex support. The main part of the apparatus consists of a water pump connected through flexible tubing to the chamber that holds the valve in place. The pressure at failure is measured using a pressure transducer. Preliminary experiments demonstrate that anterior leaflet marginal chordae, but not strut chordae, are vital to valve function. Posterior leaflet chordae have been found to be important for valve competence.

Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer.

In this report, we describe a novel cDNA isolated from a primary human breast adenocarcinoma and differentially expressed in several breast carcinoma cell lines. The protein encoded by this cDNA, which we have named mammaglobin, is homologous to a family of secreted proteins that includes rat prostatic steroid-binding protein subunit C3, human Clara cell 10-kilodalton protein, and rabbit uteroglobin. Expression of the mammaglobin gene is restricted to the adult mammary gland. More significantly, in an analysis of 35 breast tumor biopsies, mammaglobin mRNA levels were increased at least 10-fold relative to normal breast tissue in 23% of cases. The breast-specific expression of this potentially secreted protein and its frequent overexpression in primary human breast tumors suggest that mammaglobin may be a novel marker for the management of breast cancer.

Isolation of differentially expressed sequence tags from human breast cancer.

Identification of quantitative changes in gene expression that occur in the malignant mammary gland, if sufficiently characterized, may yield novel molecular markers which may be useful in the diagnosis and treatment of human breast cancer. Using modifications of a previously documented technique, the differential display polymerase chain reaction, we describe the isolation of differentially expressed sequence tags, short complementary DNA fragments corresponding to mRNAs that are differentially expressed in breast cancer biopsies, as compared to normal breast tissue controls. Direct sequencing and expression analysis of two sequence tags demonstrate that they represent sequences which are overexpressed in a number of breast carcinoma cell lines. A paradigm for generating a catalogue of these sequence tags is discussed.

Gene expression profiling with oligonucleotide microarrays distinguishes World Health Organization grade of oligodendrogliomas.

Oligodendrogliomas are the second most common type of glial neoplasm with distinct prognostic and therapeutic implications. Although refinements have led to improved clinical stratification, current grading schemes are still limited by subjective histopathological criteria. In this report, we have used oligonucleotide array technology to perform expression profiling in morphologically classic oligodendrogliomas. Expression information from approximately 1100 genes divided tumors into two molecularly distinct groups that corresponded exactly to their previously assigned histological grades. Subsequent gene clustering identified a subset of 196 transcripts showing a common, differential expression pattern between tumor grades. A number of these genes have been associated with the maintenance of cytoarchitecture, cellular differentiation and maturation, immunogenicity, and chemotherapeutic resistance. These results demonstrate the utility of gene expression profiling as an objective, ancillary tool for grading oligodendrogliomas and a potential approach for classifying diffuse gliomas where histological assessment may be difficult or ambiguous.

Mammaglobin expression in primary, metastatic, and occult breast cancer.

The mammaglobin gene encodes a novel, breast cancer-associated glycoprotein. In this study, we have evaluated the frequency with which mammaglobin expression can be detected in primary and metastatic breast tumors and in breast tumor cells present in the peripheral circulation. Of 100 primary human breast tumors examined, 81 were strongly immunopositive for mammaglobin protein. Staining was independent of tumor grade and histological type. Ten of 11 lymph nodes from patients with metastatic breast cancer contained detectable mammaglobin mRNA, whereas mammaglobin expression in uninvolved lymph nodes was undetectable. Using a nested reverse transcription-PCR assay, mammaglobin mRNA was also detected in 9 of 15 products (60%) used for autologous stem cell transplant. These results suggest that larger clinical studies are warranted to investigate the full clinical utility of mammaglobin as a tool for breast cancer patient management.

Expression profiling reveals hepsin overexpression in prostate cancer.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in men. Despite this fact, many of the genetic changes that coincide with prostate cancer progression remain enigmatic. We have addressed this problem by characterizing the expression profiles of several benign and malignant human prostate samples, and we have identified several genes that are differentially expressed between benign and malignant glands. One gene that was overexpressed encodes the serine protease hepsin. We used an independent sample set to confirm that hepsin is overexpressed in prostate tumors, and in situ hybridization demonstrates that hepsin is specifically overexpressed in the carcinoma cells themselves. These facts, together with the molecular properties of hepsin, make it an ideal target for prostate cancer therapy.

An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer.

Mounting evidence suggests that catechol metabolites of estradiol may contribute to the development of estrogen-induced cancers. O-Methylation, catalyzed by catechol-O-methyltransferase (COMT), inactivates catechol estrogens. COMT is polymorphic in the human population, with 25% of Caucasians being homozygous for a low activity allele of the enzyme (COMT(LL)). We hypothesized that low activity COMT may be a risk factor for human breast cancer and designed a PCR-based RFLP assay to determine COMT genotype in a cohort of 112 matched, nested case-control samples. In the total study population, the odds ratios for the association of breast cancer risk with COMT(HL) and COMT(LL) genotypes were 1.30 [confidence interval (CI), 0.66-2.58] and 1.45 (CI, 0.69-3.07), respectively. Postmenopausal COMT(LL) women had a greater than 2-fold increased risk of developing breast cancer [odds ratio (OR), 2.18; CI, 0.93-5.11]. The association of COMT(LL) with the development of postmenopausal breast cancer was stronger and statistically significant in those women with a body mass index >24.47 kg/m2 (OR, 3.58; CI, 1.07-11.98). When COMT(LL) was combined with either glutathione S-transferase (GST) M1 null or with GSTP1 Ile-105-Val/Val-105-Val (intermediate/low activity, respectively) genotypes, the risk for developing postmenopausal breast cancer was also significantly increased. Our findings suggest that the allele encoding low activity COMT may be an important contributor to the postmenopausal development of breast cancer in certain women.

Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13.

The mammaglobin gene encodes a novel secreted protein whose corresponding mRNA is frequently up-regulated in human breast cancer. In non-malignant tissues, expression is also strictly limited to the mammary epithelium. To better understand the mechanisms controlling these patterns of expression, we have isolated the human mammaglobin gene and performed an initial assessment of its promoter activity. Mammaglobin gene architecture is very similar to that of a family of related genes that includes uteroglobin and rat prostatein subunits C1, C2, and C3. However, the mammaglobin gene itself is not well conserved phylogenetically. The human mammaglobin gene is localized by fluorescent in situ hybridization to chromosome 11 band q13, a genomic region frequently amplified in breast neoplasia. The sequence of proximal 1 kb of mammaglobin promoter contains several potential transcriptional control elements and directs high-level expression of a transfected reporter construct in human breast tumor cell lines. However, comparable levels of reporter gene expression are also seen in non-mammary human cell lines. These data suggest that, unlike related gene family members, the striking breast-specific expression and tumor-associated overexpression of mammaglobin is mediated by complex transcriptional control at more distal sequence elements.

Alternative geometries of DNA looping: an analysis using the SfiI endonuclease.

Many processes are governed by proteins that bind to separate sites in DNA and loop out the intervening DNA, but the geometries of the loops have seldom been determined. The SfiI endonuclease cleaves DNA after interacting with two recognition sites, and is a favourable system for the analysis of DNA looping. A gel-shift assay was used here to examine the binding of SfiI to a series of linear DNA molecules containing two SfiI sites separated by 109-170 base-pairs. The complexes in which SfiI trapped a loop by binding to two sites in the same DNA were separated from the complexes containing SfiI bound to separate DNA molecules. Step-wise changes in the inter-site spacing generated two forms of the looped complex with different electrophoretic mobilities. The yields of each looped complex and the complexes from intermolecular synapses all varied cyclically with the inter-site spacing, with similar periodicities ( approximately 10.5 base-pairs) but with different phases. One looped complex predominated whenever the DNA between the sites needed to be underwound in order to produce the correct helical orientation of the binding sites. The other looped complex predominated whenever the intervening DNA needed to be overwound. We conclude that the former has trapped a right-handed loop with a negative node and the latter a left-handed loop with a positive node.

Rate and selectively of synapsis of res recombination sites by Tn3 resolvase.

Site-specific recombination catalysed by Tn3 resolvase requires the formation of an intermediate synaptic complex containing two res recombination sites and several resolvase subunits. Synaptic complexes were observed directly by chemical crosslinking of resolvase subunits followed by agarose gel electrophoresis. The highest yield of synaptic complex was from a "standard" substrate, a supercoiled plasmid with res sites in direct repeat, but complexes were also made between sites in inverted repeat, or in nicked or linear molecules, or in separate molecules. The substrate selectivity for synapsis is less stringent than for recombination; thus recombination selectivity is dependent on steps after synapsis. The stability of the synapse after its formation might be a key factor, since unproductive synapses are less stable than productive ones. In a standard substrate, synapsis is fast relative to the rate of recombination. Crosslinking in active reaction mixtures yields synaptic complexes derived from both the substrate and the catenane recombination product. Although catalysis of strand exchange is at binding site I of res, a pair of isolated site 1's do not synapse, whereas a synaptic complex is formed from a plasmid carrying two copies of res binding sites II and III. Our data are consistent with a model in which the formation of the synaptic intermediate is driven, and its structure defined, by the initial interaction of these accessory sites.

Specificity from the synapsis of DNA elements by the Sfi I endonuclease.

The synapsis of DNA sites is a prerequisite for the reactions of many proteins that act at specific DNA sequences. The requirement for synapsis was investigated by analysing the reactions of Sfi I, a tetrameric restriction enzyme that cleaves DNA only after interacting with two recognition sites. In the presence of Mg2+, oligonucleotide duplexes with the cognate recognition sequence were cleaved rapidly, with cooperative kinetics, while non-cognate duplexes were not cleaved. In the absence of Mg2+, the primary complex formed by Sfi I with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated Sfi I concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to Sfi I. Pairs of non-cognate duplexes, or one cognate and one non-cognate duplex, generally failed to form synaptic complexes. On adding Mg2+to complexes with cognate DNA, cleavage occurred much more rapidly in the synaptic complex than in the secondary complex. DNA synapsis thus acts to enhance the specificity of Sfi I for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex.

Civil commitment in the psychiatric emergency room. I. The assessment of dangerousness by emergency room clinicians.

Critics of the dangerousness standard for civil commitment contend that there is no professional standard for the evaluation of dangerousness. We used Three Ratings of Involuntary Admissibility, a reliable index of behavioral indicators of danger to self, danger to others, and grave disability, and found that when combined into weighted patterns these indicators predicted disposition decisions of 70 clinicians in five psychiatric emergency rooms over 251 cases. A concurrent measure of perceived dangerousness, Clinician's Global Ratings of patients on these criteria, yielded similar results. We conclude that clinicians in California psychiatric emergency rooms apply a shared concept of dangerousness that can be described in behavioral terms.

Civil commitment in the psychiatric emergency room. II. Mental disorder indicators and three dangerousness criteria.

Proponents of return to a "need for treatment" standard for civil commitment contend that the current dangerousness standard forces psychiatrists to neglect severely ill patients in favor of those who are less ill but dangerous to others. Among 198 psychiatric emergency patients in five facilities, those rated as most dangerous on Three Ratings of Involuntary Admissibility, a reliable index of indicators employed by clinicians in evaluating danger to self, danger to others, and grave disability, were also most severely ill on diagnostic and symptomatic assessments of mental disorder. Clinicians' Global Ratings of patient dangerousness on the three criteria were similarly related to severity of diagnosis and symptoms. Perceived dangerousness was associated with major mental disorder and severity of most symptom types, especially impulsivity. Danger to self was the criterion related to the fewest indicators of mental disorder.