Identification of a new inhibitor of KRAS-PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer.

Oncogenic KRAS is considered a promising target for anti-cancer therapy. However, direct pharmacological strategies targeting KRAS-driven cancers remained unavailable. The prenyl-binding protein PDEδ, a transporter of KRAS, has been identified as a potential target for pharmacological inhibitor by selectively binding to its prenyl-binding pocket, impairing oncogenic KRAS signaling pathway. Here, we discovered a novel PDEδ inhibitor (E)-N'-((3-(tert-butyl)-2-hydroxy-6,7,8,9-tetrahydrodibenzo[b,dfuran-1-yl)methylene)-2,4-dihydroxybenzohydrazide(NHTD) by using a high-throughput docking-based virtual screening approach. In vitro and in vivo studies demonstrated that NHTD suppressed proliferation, induced apoptosis and inhibited oncogenic K-RAS signaling pathways by disrupting KRAS-PDEδ interaction in nonsmall cell lung cancer (NSCLC) harboring KRAS mutations. NHTD redistributed the localization of KRAS to endomembranes by targeting the prenyl-binding pocket of PDEδ and exhibited the suppression of abnormal KRAS function. Importantly, NHTD prevented tumor growth in xenograft and KRAS mutant mouse model, which presents an effective strategy targeting KRAS-driven cancer.

Preparing for events for physically challenged athletes.

The participation in sports for physically challenged athletes continues to expand in multiple domains from recreational, novice, and competitive to elite competitions such as the Paralympics. Physically challenged athletes have various disabilities such as visual impairments, spinal cord injuries, amputations, cerebral palsy, or other neuromuscular impairments and have different levels of functional ability within these broad categories. The spectrum of medical illnesses and musculoskeletal injuries seen with sports is similar to that of able-bodied athletes; however medical teams caring for athletes with disabilities need to be familiar with medical risks such as skin breakdown, thermoregulation problems, dehydration, autonomic dysreflexia, infections, orthotic and prosthetic issues, and psychiatric comorbidities that may be encountered. The medical team preparation for events involving physically challenged athletes should include obtaining appropriate medical supplies, ensuring disability-compatible access to medical areas, and preparing for emergency extraction from adaptive equipment.

Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells.

Diabetes mellitus is characterized by either the inability to produce insulin (type 1 diabetes) or as insensitivity to insulin secreted by the body (type 2 diabetes). In either case, the body is unable to move blood glucose efficiently across cell membranes to be used. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Here, we describe a potential cure for diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced pluripotent stem (iPS) cells, we were able to derive beta-like cells similar to the endogenous insulin-secreting cells in mice. These beta-like cells secreted insulin in response to glucose and corrected a hyperglycemic phenotype in two mouse models of type 1 and 2 diabetes via an iPS cell transplant. Long-term correction of hyperglycemia was achieved, as determined by blood glucose and hemoglobin A1c levels. These data provide an initial proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2.

Activating killer-cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands are significantly increased in autism.

Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis. Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p = 0.00003 [Odds ratio = 2.87]). This information ties together two major immune gene complexes, the human leukocyte complex and the leukocyte receptor complex, and may partially explain immune abnormalities observed in many subjects with autism.

Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells.

Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis.

Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells.

Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls.

Generation of murine hepatic lineage cells from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated from somatic cells of individuals by retrodifferentiation using defined transcription factors. Similar to embryonic stem (ES) cells, iPS cells can be differentiated into a variety of specific cell types. However, to date, no detailed hepatic differentiation of mouse iPS cells has been reported. In this study, we successfully developed a stepwise protocol to induce hepatic differentiation of iPS cells reprogrammed from mouse tail tip fibroblasts. At day 25 of differentiation, the iPS cell-derived hepatocytes morphologically resemble mouse primary hepatocytes with a distinct polygonal shape. Immunostaining and reverse transcription-polymerase chain reaction analysis revealed expression of specific hepatic markers including alpha-fetoprotein, albumin and alpha-1-anti-trypsin. In addition, these iPS cell-derived hepatocytes successfully demonstrated mature liver cell functions in vitro. Furthermore, in vivo assays revealed that the mouse iPS cell-derived hepatocytes successfully engrafted into the recipient livers with typical hepatic morphology. Thus, iPS cell-derived hepatocytes may hold great promise as a unique system for basic liver research and liver regeneration in the near future.

SALL4 is a key regulator of survival and apoptosis in human leukemic cells.

Increasing studies suggest that SALL4 may play vital roles in leukemogenesis and stem cell phenotypes. We have mapped the global gene targets of SALL4 using chromatin immunoprecipitation followed by microarray hybridization and identified more than 2000 high-confidence, SALL4-binding genes in the human acute promyelocytic leukemic cell line, NB4. Analysis of SALL4-binding sites reveals that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (P < .05). These genes include 38 important apoptosis-inducing genes (TNF, TP53, PTEN, CARD9, CARD11, CYCS, LTA) and apoptosis-inhibiting genes (Bmi-1, BCL2, XIAP, DAD1, TEGT). Real-time polymerase chain reaction has shown that expression levels of these genes changed significantly after SALL4 knockdown, which ubiquitously led to cell apoptosis. Flow cytometry revealed that reduction of SALL4 expression in NB4 and other leukemia cell lines dramatically increased caspase-3, annexin V, and DNA fragmentation activity. Bromodeoxyuridine-incorporation assays showed decreased numbers of S-phase cells and increased numbers of G1- and G2-phase cells indicating reduced DNA synthesis, consistent with results from cell proliferation assays. In addition, NB4 cells that express low levels of SALL4 have significantly decreased tumorigenecity in immunodeficient mice. Our studies provide a foundation in the development of leukemia stem cell-specific therapy by targeting SALL4.

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex.

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.

A randomized controlled study of human serum albumin and serum substitute supplement as protein supplements for IVF culture and the effect on live birth rates.

BACKGROUND: It has been speculated that the addition of proteins more complex than human serum albumin (HSA) to culture media may improve IVF outcomes. Whether the expense, labor and risk of adding additional human-derived protein to IVF media are warranted is a question unanswered. METHODS: In a randomized controlled trial with couples undergoing routine IVF or ICSI, 528 patients were assigned to one of two treatment groups. Embryos were cultured in either media supplemented with HSA as a solitary protein supplement or in media supplemented with HSA+serum substitute supplement (SSS) from the 2PN stage until the time of embryo transfer. Clinical end-points monitored included implantation (total 1151 embryos) and live birth rates (total 528 patients). RESULTS: The transfer of embryos cultured in HSA+SSS resulted in higher embryo implantation (289/571, 50.6% versus 254/580, 43.8%; difference 6.8% with 95% CI 1.0-12.7, P = 0.042) and live birth rates (167/266, 62.8% versus 142/262, 54.2%; difference 8.6% with 95% CI 0.1-17.3, P = 0.043) when compared with those of women whose embryos were cultured with HSA as the sole protein supplement. CONCLUSIONS: SSS added to commercial HSA-supplemented embryo culture media resulted in an overall increase in implantation and live birth rates. It remains uncertain whether the use of human-derived blood products in culture media and the requirement for ultra-rigorous quality control measures make these findings applicable to the average IVF laboratory. Protein enrichment of media may significantly improve the blastocyst implantation rate, creating opportunities to transfer single blastocysts without compromising the live birth rate. The study was registered at clinicaltrials.gov. NCT00708383.

Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts.

We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and alpha/beta adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.

Diagnostic markers for early detection of ovarian cancer.

PURPOSE: Early detection would significantly decrease the mortality rate of ovarian cancer. In this study, we characterize and validate the combination of six serum biomarkers that discriminate between disease-free and ovarian cancer patients with high efficiency. EXPERIMENTAL DESIGN: We analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All six markers were evaluated in a training set (181 samples from the control group and 113 samples from OC patients) and a test set (181 sample control group and 43 ovarian cancer). RESULTS: Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves were good enough to differentiate healthy versus cancer cells. However, the combination of the six markers provided a better differentiation than CA-125. Four models with <2% classification error in training sets all had significant improvement (sensitivity 84%-98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221 out of 224 specimens in the test set, with a classification accuracy of 98.7%. CONCLUSIONS: We describe the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. This novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.

A Unique Partnership Between the University of Hawai'i Cancer Center and the University of Guam: Fifteen Years of Addressing Cancer Health Disparities in Pacific Islanders in Hawai'i and Guam.

This manuscript describes the efforts in research, education, and outreach of a unique partnership between the University of Hawai'i Cancer Center and the University of Guam in addressing cancer health disparities faced by Pacific Islanders in Hawai'i, Guam, and other parts of Micronesia. Significant accomplishments of this 15-year collaboration in research, training Micronesian students, and impact on the local communities are highlighted.

Development of a rapid diagnostic assay for methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococcus.

We describe here a rapid assay for the detection of the tuf gene for the identification of Staphylococcus genus, the femB gene for the identification of Staphylococcus aureus species, and the mecA gene for the identification of methicillin resistance directly from BACTEC blood culture bottles showing Gram-positive cocci in clusters. The test, configured on a thin-film biosensor platform, allows for detection of genomic DNA from blood culture samples without the need for nucleic acid amplification. In an initial study to validate the technology, 107 consecutive positive blood cultures were tested on the thin-film biosensor, and the assay exhibited 100% concordance in comparison with standard microbiological methods for identifying methicillin-susceptible and methicillin-resistant S. aureus and for identifying methicillin-susceptible and methicillin-resistant coagulase-negative Staphylococcus. Results were obtained within 90 min directly from signal positive bottles with no instrumentation required.

Inhibition of KRAS-dependent lung cancer cell growth by deltarasin: blockage of autophagy increases its cytotoxicity.

Deltarasin is a recently identified small molecule that can inhibit KRAS-PDEδ interactions by binding to a hydrophobic pocket on PDEδ, resulting in the impairment of cell growth, KRAS activity, and RAS/RAF signaling in human pancreatic ductal adenocarcinoma cell lines. Since KRAS mutations are the most common oncogene mutations in lung adenocarcinomas, implicated in over 30% of all lung cancer cases, we examined the ability of deltarasin to inhibit KRAS-dependent lung cancer cell growth. Here, for the first time, we document that deltarasin produces both apoptosis and autophagy in KRAS-dependent lung cancer cells in vitro and inhibits lung tumor growth in vivo. Deltarasin induces apoptosis by inhibiting the interaction of with PDEδ and its downstream signaling pathways, while it induces autophagy through the AMPK-mTOR signaling pathway. Importantly, the autophagy inhibitor, 3-methyl adenine (3-MA) markedly enhances deltarasin-induced apoptosis via elevation of reactive oxygen species (ROS). In contrast, inhibition of ROS by N-acetylcysteine (NAC) significantly attenuated deltarasin-induced cell death. Collectively, these observations suggest that the anti-cancer cell activity of deltarasin can be enhanced by simultaneously blocking "tumor protective" autophagy, but inhibited if combined with an anti-oxidant.

Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies.

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic -794 CATT(5-8) repeat and -173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO-peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with 'naked' oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO-peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.

Common Genetic Variants Found in HLA and KIR Immune Genes in Autism Spectrum Disorder.

The "common variant-common disease" hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased vs. matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the "common variant-common disease" hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics. Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14 bp-indel) frequencies are significantly increased by more than 5% over control populations (Table 2). The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2). Three activating KIR genes: 3DS1, 2DS1, and 2DS2 have increased frequencies of 15, 22, and 14% in autism populations, respectively. There is a 6% increase in total activating KIR genes in autism over control subjects. And, more importantly there is a 12% increase in activating KIR genes and their cognate HLA alleles over control populations (Torres et al., 2012a). These data suggest the interaction of HLA ligand/KIR receptor pairs encoded on two different chromosomes is more significant as a ligand/receptor complex than separately in autism.

Detection of chromosomal instability in paired breast surgery and ductal lavage specimens by interphase fluorescence in situ hybridization.

PURPOSE: Ductal lavage is a new modality for collecting exfoliated breast cells with the goal of detecting early neoplasia. The purpose of our study was to evaluate the correlation between cancer-associated abnormalities in breast lesions and exfoliated breast cells collected by ductal lavage. EXPERIMENTAL DESIGN: We performed histopathologic, cytologic, and molecular cytogenetic analyses on 39 paired cases of surgically excised breast lesions and ductal lavage specimens collected immediately before surgery. RESULTS: Abnormal cytology was detected in 7 of 15 (47%) of the evaluable lavages collected from malignant cases, versus 4 of 19 (21%) of the evaluable lavages harvested from benign cases for a sensitivity and specificity of 47 and 79%, respectively. Interphase fluorescence in situ hybridization analysis of all evaluable lavages revealed numeric changes on chromosomes 1, 8, 11, and/or 17 in 10 of 14 (71%) specimens from malignant cases versus 2 of 18 (11%) from benign cases for a sensitivity and specificity of 71 and 89%, respectively. CONCLUSIONS: Our study demonstrates that cytologic and genetic abnormalities associated with breast cancer progression can be detected in ductal lavage cells collected from women with in situ and invasive breast cancer and suggests that fluorescence in situ hybridization may have superior sensitivity and specificity compared with conventional cytology.