Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer.

Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Met(mt)) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Met(mt) mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Met(mt), significantly increased tumor penetrance over Met(mt) or Trp53 loss alone. Unlike Met(mt) tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Met(mt) tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.

Genotype-phenotype association studies of chromosome 8p inverted duplication deletion syndrome.

Individuals diagnosed with chromosome 8p inverted duplication deletion (invdupdel(8p)) manifest a wide range of clinical features and cognitive impairment. The purpose of this study is to employ array CGH technology to define more precisely the cytogenetic breakpoints and regions of copy number variation found in several individuals with invdupdel(8p), and compare these results with their neuropsychological characteristics. We examined the cognitive-behavioral features of two male and two female children, ages 3-15 years, with invdupdel(8p). We noted cognitive deficits that ranged from mild to severe, and adaptive behavior composites that ranged from significantly to substantially lower than adequate levels. CARS scores, a measure of autistic behavior, identified three children with autism or autistic-like features. Three of the four children exhibited attention deficits and hyperactivity consistent with a DSM-IV-TR diagnosis of ADHD. One child showed extreme emotional lability. Interestingly, intellectual disability was not correlated with deletion size, nor was the deletion location associated with the autistic phenotype. On the other hand, the duplication length in 8p21.1/8p22 was associated with cognitive deficit. In addition, a small locus of over-expression in 8p21.3 was common for all three participants diagnosed as autistic. A limitation of the study is its small sample size. Further analyses of the deleted and over-expressed regions are needed to ascertain the genes involved in cognitive function and, possibly, autism.

Altered gene expression and function of peripheral blood natural killer cells in children with autism.

Immune related abnormalities have repeatedly been reported in autism spectrum disorders (ASD), including evidence of immune dysregulation and autoimmune phenomena. NK cells may play an important role in neurodevelopmental disorders such as ASD. Here we performed a gene expression screen and cellular functional analysis on peripheral blood obtained from 52 children with ASD and 27 typically developing control children enrolled in the case-control CHARGE study. RNA expression of NK cell receptors and effector molecules were significantly upregulated in ASD. Flow cytometric analysis of NK cells demonstrated increased production of perforin, granzyme B, and interferon gamma (IFNgamma) under resting conditions in children with ASD (p<0.01). Following NK cell stimulation in the presence of K562 target cells, the cytotoxicity of NK cells was significantly reduced in ASD compared with controls (p<0.02). Furthermore, under similar stimulation conditions the presence of perforin, granzyme B, and IFNgamma in NK cells from ASD children was significantly lower compared with controls (p<0.001). These findings suggest possible dysfunction of NK cells in children with ASD. Abnormalities in NK cells may represent a susceptibility factor in ASD and may predispose to the development of autoimmunity and/or adverse neuroimmune interactions during critical periods of development.

Prospective Evaluation of the Concordance of Commercial Circulating Tumor DNA Alterations with Tumor-Based Sequencing across Multiple Soft Tissue Sarcoma Subtypes.

Soft tissue sarcomas (STS) are diverse tumors with heterogenous alterations. Platforms to detect circulating tumor DNA (ctDNA) have rapidly increased in popularity as they may avoid invasive biopsy morbidity. However, ctDNA profiling concordance with standard solid tumor comprehensive genomic profiling (CGP) is poorly characterized. Here, we report the outcomes of a single-institution experience comparing mutational results from commercial ctDNA and solid tumor CGP in advanced STS subjects. We identified STS subjects who had undergone solid tumor based CGP in four distinct cohorts: Dedifferentiated liposarcoma (DDLPS), leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), and gastrointestinal stromal tumor (GIST). Subjects with radiographically measurable tumor were profiled using a commercial ctDNA CGP panel. Overlapping genes/exons on both biopsy panels were analyzed. Twenty-four subjects completed both ctDNA and solid tumor CGP. ctDNA was detected in 18/24 subjects. Subject level concordance rates in all overlapping genes were: LMS = 4/6; UPS = 2/6; DDLPS = 1/6; GIST = 0/6. Copy number alterations were notably poorly concordant. For subjects with short variant alterations and detectable tumor fractions, concordance with solid tumor CGP was 76% (13/17). LMS subjects had the highest median tumor fraction and concordance. No correlation was seen between tumor fraction or radiographic tumor volume largely driven by low estimated tumor fraction. A limitation of the study is that only targeted sequencing was performed. However, given the poor concordance in commonly altered genes, ctDNA panels in sarcoma cannot be broadly applied. Further, more extensive studies will need to be performed.

Selective high-affinity ligand antibody mimics for cancer diagnosis and therapy: initial application to lymphoma/leukemia.

PURPOSE: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. EXPERIMENTAL DESIGN: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. RESULTS: These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1. CONCLUSIONS: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Enhancement of the therapeutic index: from nonmyeloablative and myeloablative toward pretargeted radioimmunotherapy for metastatic prostate cancer.

PURPOSE: New strategies that target selected molecular characteristics and result in an effective therapeutic index are needed for metastatic, hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: A series of preclinical and clinical studies were designed to increase the therapeutic index of targeted radiation therapy for prostate cancer. (111)In/90Y-monoclonal antibody (mAb), m170, which targets aberrant sugars on abnormal MUC1, was evaluated in androgen-independent prostate cancer patients to determine the maximum tolerated dose and efficacy of nonmyeloablative radioimmunotherapy and myeloablative combined modality radioimmunotherapy with paclitaxel. To enhance the tumor to liver therapeutic index, a cathepsin degradable mAb linkage ((111)In/90Y-peptide-m170) was used in the myeloablative combined modality radioimmunotherapy protocol. For tumor to marrow therapeutic index improvement in future studies, anti-MUC1 scFvs modules were developed for pretargeted radioimmunotherapy. Anti-MUC1 and anti-DOTA scFvs were conjugated to polyethylene glycol scaffolds tested on DU145 prostate cancer cells and prostate tissue arrays, along with mAbs against MUC1 epitopes. RESULTS: The nonmyeloablative maximum tolerated dose of 90Y-m170 was 0.74 GBq/m2 for patients with not more than 10% axial skeleton involvement. Metastatic prostate cancer was targeted in all 17 patients; mean radiation dose was 10.5 Gy/GBq and pain response occurred in 7 of 13 patients reporting pain. Myeloablative combined modality radioimmunotherapy with 0.4 GBq/m2 of 90Y-peptide-m170 and paclitaxel showed therapeutic effects in 4 of 6 patients and 30% less radiation to the liver per unit of activity. Neutropenia was dose limiting without marrow support and patient eligibility was a major limitation to dose escalation. Hypoglycosylated MUC1 epitopes were shown to be abundant in prostate cancer and to increase with disease grade. Anti-MUC1 scFvs binding to prostate cancer tissue and live cells were developed into di-scFv binding modules. CONCLUSIONS: The therapeutic index enhancement for prostate radioimmunotherapy was achieved in clinical studies by the addition of cathepsin cleavable linkers to 90Y-conjugated mAbs and the use of paclitaxel. However, the need for marrow support in myeloablative combined modality radioimmunotherapy restricted eligible patients. Therefore, modular pretargeted radioimmunotherapy, aiming at improving the tumor to marrow therapeutic index, is being developed.

Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format.

We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in. x 3 in. (2.54 cm x 7.62 cm) microscope slide.

Exposure of myeloid dendritic cells to exogenous or endogenous IL-10 during maturation determines their longevity.

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-x(L) expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-x(L) in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.

Global alteration of gene expression in human keratinocytes by inorganic arsenic.

Alteration of gene expression by inorganic arsenic has been studied in cultured human keratinocytes derived from normal epidermis, a premalignant lesion and a malignant tumor. The purpose was to find whether these cells displayed common alterations in gene expression that might elucidate the mechanism of arsenic action. Global analysis of approximately 12 000 genes by microarray showed that approximately 30% were expressed. Of these, transcription of a substantial fraction (up to 12%) was altered, nearly twice as many being suppressed as stimulated by 2-fold or more at 2 micro M sodium arsenite or 6 micro M arsenate, which did not affect cell growth. At 0.67 micro M arsenite (50 p.p.b.), effects on transcription were less pronounced but clearly evident. Genes whose transcription was altered in common among all the treated keratinocytes included those induced by reactive oxygen, of which heme oxygenase-1 displayed the highest fold induction. Genes indicative of reactive oxygen generation were detected at the earliest time examined, raising the possibility this feature drives subsequent cellular responses. Unlike some agents that produced transient induction of heme oxygenase-1, arsenicals produced sustained induction. Comparison with other agents producing reactive oxygen in the cells, as reflected in heme oxygenase-1 induction, suggested cellular differentiation was suppressed by sustained but not transient generation of reactive oxygen. Sustained global changes in gene expression were seen in target cells treated chronically with inorganic arsenic at concentrations consumed by millions of humans in contaminated drinking water.

Transformation and normalization of oligonucleotide microarray data.

MOTIVATION: Most methods of analyzing microarray data or doing power calculations have an underlying assumption of constant variance across all levels of gene expression. The most common transformation, the logarithm, results in data that have constant variance at high levels but not at low levels. Rocke and Durbin showed that data from spotted arrays fit a two-component model and Durbin, Hardin, Hawkins, and Rocke, Huber et al. and Munson provided a transformation that stabilizes the variance as well as symmetrizes and normalizes the error structure. We wish to evaluate the applicability of this transformation to the error structure of GeneChip microarrays. RESULTS: We demonstrate in an example study a simple way to use the two-component model of Rocke and Durbin and the data transformation of Durbin, Hardin, Hawkins and Rocke, Huber et al. and Munson on Affymetrix GeneChip data. In addition we provide a method for normalization of Affymetrix GeneChips simultaneous with the determination of the transformation, producing a data set without chip or slide effects but with constant variance and with symmetric errors. This transformation/normalization process can be thought of as a machine calibration in that it requires a few biologically constant replicates of one sample to determine the constant needed to specify the transformation and normalize. It is hypothesized that this constant needs to be found only once for a given technology in a lab, perhaps with periodic updates. It does not require extensive replication in each study. Furthermore, the variance of the transformed pilot data can be used to do power calculations using standard power analysis programs. AVAILABILITY: SPLUS code for the transformation/normalization for four replicates is available from the first author upon request. A program written in C is available from the last author.

Investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans.

A bioassay for zinc status in humans has been sought due to the importance of zinc, an essential trace metal, for many divergent functions in the human body; however, a sensitive bioassay for zinc status in humans is lacking. To address this issue, we established gene expression profiles of human lymphoblastoid cells treated with 0 or 30 micro mol/L ZnSO(4) using microarray technology. A limited number of genes were responsive to 30 micro mol/L zinc based on the analysis of Affymetrix human genome U133A GeneChips. We also examined the gene expression patterns of zinc transporters in human lymphoblastoid cells using quantitative RT-PCR analysis. ZNT1 was upregulated in lymphoblastoid cells, whereas ZIP1 was downregulated in response to the increased zinc concentrations in the culture media. To evaluate the potential applications of using both zinc transporter genes as biomarkers of zinc status, we measured the expression levels of ZIP1 and ZNT1 in the peripheral leukocytes collected from 2 different age groups of Korean women. After administration of a zinc supplement (22 mg zinc gluconate/d for 27 d), ZIP1 expression decreased by 17% (P < 0.01) and 21% (P < 0.05) in the peripheral leukocytes collected from 15 young (20-25 y) and 10 elderly (64-75 y) subjects, respectively. ZNT1 expression was not affected by taking the zinc supplement. These data suggest a potential application of ZIP1 as a biomarker of zinc status in humans.