Bonnie LaFleur

Expression profiling of medulloblastoma : PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease

Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor α (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor α (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.

Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α

Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Reduction of frequent otitis media and pressure-equalizing tube insertions in children after introduction of pneumococcal conjugate vaccine

OBJECTIVE. Streptococcus pneumoniae is an important cause of otitis media in children. In this study we estimated the effect of routine childhood immunization with heptavalent pneumococcal conjugate vaccine on frequent otitis media (3 episodes in 6 months or 4 episodes in 1 year) and pressure-equalizing tube insertions. PATIENTS AND METHODS. The study population included all children who were enrolled at birth in TennCare or selected upstate New York commercial insurance plans as of July 1998 and continuously followed until 5 years old, loss of health plan enrollment, study outcome, or end of the study. We compared the risk of developing frequent otitis media or having pressure-equalizing tube insertion for 4 birth cohorts (1998–1999, 1999–2000, 2000–2001, and 2001–2002) by using Cox regression analysis. We used data from the National Immunization Survey to estimate the heptavalent pneumococcal conjugate vaccine uptake for children in these 4 birth cohorts in Tennessee and New York. RESULTS. The proportion of children in Tennessee and New York who received at least 3 doses of heptavalent pneumococcal conjugate vaccine by 2 years of age increased from ≤1% for the 1998–1999 birth cohort to ∼75% for the 2000–2001 birth cohort. By age 2 years, 29% of Tennessee and New York children born in 2000–2001 had developed frequent otitis media, and 6% of each of these birth cohorts had pressure-equalizing tubes inserted. Comparing the 2000–2001 birth cohort to the 1998–1999 birth cohort, frequent otitis media declined by 17% and 28%, and pressure-equalizing tube insertions declined by 16% and 23% for Tennessee and New York children, respectively. For the 2000–2001 to the 2001–2002 birth cohort, frequent otitis media and pressure-equalizing tubes remained stable in New York but increased in Tennessee. CONCLUSIONS. After heptavalent pneumococcal conjugate vaccine introduction, children were less likely to develop frequent otitis media or have pressure-equalizing tube insertions.

An Expression Signature Classifies Chemotherapy-Resistant Pediatric Osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFbeta1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor-driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.

Augmenting Chemosensitivity of Malignant Melanoma Tumors via Proteasome Inhibition: Implication for Bortezomib (VELCADE, PS-341) as a Therapeutic Agent for Malignant Melanoma

Melanoma poses a great challenge to patients, oncologists, and biologists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor κB is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor κB activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor κB by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1–10 nm and in combination with the chemotherapeutic agent temozolomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strikingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma.

Use of a Live Attenuated Varicella Vaccine to Boost Varicella-Specific Immune Responses in Seropositive People 55 Years of Age and Older: Duration of Booster Effect

Varicella-zoster virus (VZV)-specific T cell immunity was measured in 130 persons > or = 55 years of age 6 years after they received a live attenuated VZV vaccine. Circulating T cells, which proliferated in vitro in response to VZV antigen, were enumerated (VZV responder cell frequency assay). Six years after the booster vaccination, the VZV-responding cell frequency (1/61,000 circulating cells) was still significantly (P < .05) improved over the baseline measurements (1/70,000) and appears to have diminished the expected decline in frequency as these vaccinees aged (to 1/86,000). Ten herpes-zoster--like clinical events were recorded. Although the frequency of these events, approximately 1/100 patient-years, is within the expected range of such events for this age cohort, the number of lesions was small, there was very little pain, and there was no postherpetic neuralgia. These results support the development of a vaccine to prevent or attenuate herpes zoster.

Randomized Phase II Trial of the Clinical and Biological Effects of Two Dose Levels of Gefitinib in Patients With Recurrent Colorectal Adenocarcinoma

PURPOSE The clinical objective of this trial was to evaluate gefitinib in patients with metastatic colorectal cancer that had progressed despite prior treatment. Serial tumor biopsies were performed when possible and analyzed for activation of the epidermal growth factor receptor (EGFR) signaling pathway. Serial serum samples were measured for amphiregulin and transforming growth factor-alpha (TGFalpha). PATIENTS AND METHODS One hundred fifteen patients were randomly assigned to receive gefitinib 250 or 500 mg orally once a day. One hundred ten patients were assessable for clinical efficacy. Biologic evaluation was performed on paired tumor samples from 28 patients and correlated with clinical outcome. RESULTS Median progression-free survival was 1.9 months (95% CI, 1.8 to 2.1 months) and 4-month progression-free survival rate was 13% +/- 5%. One patient achieved a radiographic partial response (RR = 1%; 95% CI, 0.01% to 5%). Median survival was 6.3 months (95% CI, 5.1 to 8.2 months). The most common adverse events were skin rash, diarrhea, and fatigue. In the biopsy cohort, expression of total or activated EGFR, activated Akt, activated MAP-kinase, or Ki67 did not decrease following 1 week of gefitinib. However, a trend toward decreased post-treatment levels of activated Akt and Ki67 was observed in patients with a PFS higher than the median, although these did not reach the .05 level of significance. CONCLUSION Gefitinib is inactive as a single agent in patients with previously treated colorectal cancer. In tumor samples, gefitinib did not inhibit activation of its proximal target, EGFR. Trends were observed for inhibition of downstream regulators of cellular survival and proliferation in patients achieving longer progression-free survival.

Population-Based Impact of Pneumococcal Conjugate Vaccine in Young Children

Objective. To determine the population impact of pneumococcal conjugate vaccine (PCV) on pneumococcal-related diseases, including pneumonia and otitis media. Methods. Using administrative data from Tennessee Medicaid and 3 commercial insurance plans in upstate New York, we measured annual rates of medical visits for pneumococcal-related diseases (pneumococcal and nonspecific pneumonia and invasive disease; otitis media) and pneumococcal-unrelated diseases (other acute respiratory illnesses). Disease rates before (1995–2000 in Tennessee; 1998–2000 in New York) and after (2000–2002) PCV licensure were calculated for children aged <2 years (eligible for PCV) and those 3 to 5 years (not routinely given PCV). Because annual variations should affect both age groups similarly and vaccine-related outcomes should preferentially decline in younger children, ratios (<2:3–5 years) of disease rates before and after PCV licensure were compared. Expected disease rates were calculated for children aged <2 years in each postvaccine year. The difference between observed and expected disease rates was the estimated vaccine effect. Results. In 2001–2002, there were 67 380 and 9485 child-years of observation for Tennessee and New York children aged <2 years, respectively. We observed fewer visits for pneumonia and invasive disease per 1000 children than expected in both regions: 20 fewer emergency department or outpatient visits in Tennessee and 33 fewer outpatient visits in New York. Otitis media visits declined by 118 and 430 per 1000 children in Tennessee and New York, respectively. Conclusions. Adding PCV to the childhood immunization schedule was associated with a 10-fold greater reduction in pneumonia and a 100-fold greater reduction in otitis media than the previously reported reduction in culture-confirmed invasive pneumococcal diseases of 1.3 episodes per 1000 children aged <2 years. Although additional studies are needed to confirm the impact of routine immunization with PCV on pneumococcal-related disease, these results suggest that its impact is substantially greater than the effects on invasive disease alone.

Gene expression correlates of neurofibrillary tangles in Alzheimer's disease.

Neurofibrillary tangles (NFT) constitute one of the cardinal histopathological features of Alzheimers disease (AD). To explore in vivo molecular processes involved in the development of NFTs, we compared gene expression profiles of NFT-bearing entorhinal cortex neurons from 19 AD patients, adjacent non-NFT-bearing entorhinal cortex neurons from the same patients, and non-NFT-bearing entorhinal cortex neurons from 14 non-demented, histopathologically normal controls (ND). Of the differentially expressed genes, 225 showed progressively increased expression (AD NFT neurons > AD non-NFT neurons > ND non-NFT neurons) or progressively decreased expression (AD NFT neurons < AD non-NFT neurons < ND non-NFT neurons), raising the possibility that they may be related to the early stages of NFT formation. Immunohistochemical studies confirmed that many of the implicated proteins are dysregulated and preferentially localized to NFTs, including apolipoprotein J, interleukin-1 receptor-associated kinase 1, tissue inhibitor of metalloproteinase 3, and casein kinase 2, beta. Functional validation studies are underway to determine which candidate genes may be causally related to NFT neuropathology, thus providing therapeutic targets for the treatment of AD.

Restaurant Inspection Scores and Foodborne Disease

Restaurants in the United States are regularly inspected by health departments, but few data exist regarding the effect of restaurant inspections on food safety. We examined statewide inspection records from January 1993 through April 2000. Data were available from 167,574 restaurant inspections. From 1993 to 2000, mean scores rose steadily from 80.2 to 83.8. Mean inspection scores of individual inspectors were 69–92. None of the 12 most commonly cited violations were critical food safety hazards. Establishments scoring <60 had a mean improvement of 16 points on subsequent inspections. Mean scores of restaurants experiencing foodborne disease outbreaks did not differ from restaurants with no reported outbreaks. A variety of factors influence the uniformity of restaurant inspections. The restaurant inspection system should be examined to identify ways to ensure food safety.