Citations

BACKGROUND The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography–tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D2], 2.2 nmol/L [0.9 μg/L, 25(OH)D3], and 10 nmol/L [4 μg/L, 25(OH)D2], 5 nmol/L [2 μg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%–15.2%. Spiked recoveries were 94%–119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n = 1733) and 25(OH)D3 (n = 7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

Genome-wide association studies have identified an association between two intronic single nucleotide polymorphisms (SNPs), rs12722489 and rs2104286, in the interleukin-2 receptor alpha-chain gene ( IL2RA) and susceptibility to multiple sclerosis (MS). We studied these SNPs in association with susceptibility to and severity of MS in a population-based cohort of 220 patients from Olmsted County, Minnesota, compared with 442 matched controls. We sequenced the exons, splice sites and 5’ and 3’ untranslated regions in 27 randomly selected MS patients (powered for allele frequency ≥0.04) to search for mutations. No novel missense mutation was identified. Two patients (7.5%) had an exon 2 SNP ( rs4308625) and two patients had an exon 4 SNP ( rs2228149), both synonymous.

Abstract Purpose: To describe the demographic and clinical features and outcomes for children and adolescents with primary CNS lymphoma (PCNSL). Experimental Design: A retrospective series of children and adolescents with PCNSL was assembled from 10 cancer centers in 3 countries. Results: Twenty-nine patients with a median age of 14 years were identified. Sixteen (55%) had Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 1 or greater. Frontline therapy consisted of chemotherapy only in 20 patients (69%), while 9 (31%) had chemotherapy plus cranial radiotherapy. Most patients received methotrexate (MTX)-based regimens. Overall response rate was 86% (complete remission 69%, partial remission 17%). The 2-year progression-free survival (PFS) and overall survival (OS) rates were 61% and 86%, respectively; the 3-year OS was 82%. Univariate analyses were conducted for age (≤14 vs. >14 years), PS (0 or 1 vs. >1), deep brain lesions, MTX dose, primary treatment with chemotherapy alone, intrathecal chemotherapy, and high-dose therapy. Primary treatment with chemotherapy alone was associated with better overall response rates with an odds ratio (OR) of 0.125 (P = 0.02). There was a marginally significant relationship between higher doses of MTX and response (OR = 1.5, P = 0.06). ECOG-PS of 0 to 1 was the only factor associated with better outcome with hazard ratios of 0.136 (P = 0.017) and 0.073 (P = 0.033) for PFS and OS, respectively. Conclusion: This is the largest series collected of pediatric PCNSL. The outcome of children and adolescents seems to be better than in adults. PS of 0 to 1 is associated with better survival. Clin Cancer Res; 17(2); 346–52. ©2011 AACR.

Background— Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell–T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. Methods and Results— Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. Conclusions— Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.

By the time Harvey Cushing entered medical school, nerve reconstruction techniques had been developed, but peripheral nerve surgery was still in its infancy. As an assistant surgical resident influenced by Dr. William Halsted, Cushing wrote a series of reports on the use of cocaine for nerve blocks. Following his residency training and a hiatus to further his clinical interests and intellectual curiosity, he traveled to Europe and met with a variety of surgeons, physiologists, and scientists, who likely laid the groundwork for Cushing's increased interest in peripheral nerve surgery. Returning to The Johns Hopkins Hospital in 1901, he began documenting these surgeries. Patient records preserved at Yale's Cushing Brain Tumor Registry describe Cushing's repair of ulnar and radial nerves, as well as his exploration of the brachial plexus for nerve repair or reconstruction. The authors reviewed Harvey Cushing's cases and provide 3 case illustrations not previously reported by Cushing involving neurolysis, nerve repair, and neurotization. Additionally, Cushing's experience with facial nerve neurotization is reviewed. The history, physical examination, and operative notes shed light on Cushing's diagnosis, strategy, technique, and hence, his surgery on peripheral nerve injury. These contributions complement others he made to surgery of the peripheral nervous system dealing with nerve pain, entrapment, and tumor.

Introduction: Telemedicine options for symptom assessment include both telephonic means (call centers) and Internet sites. Although symptom assessment call centers have been available for decades, symptom assessment over the Internet is relatively new. It is not well known what types of symptoms Internet users are seeking assessment for and whether extant telephone triage algorithms would be applicable to Internet users. Methods: Symptom assessments on a heavily used Internet site ( MayoClinic.com ) were compared with symptom calls to a U.S. call center (Ask Mayo Clinic). Results: Internet users sought symptom assessments about adult symptoms 13 times more often than children's symptoms. In contrast, over the telephone, adult symptoms were addressed 2.1 times more frequently than children's symptoms. Despite the differences in frequencies of adult and child symptom assessments, users of the Internet and telephone callers asked about specific symptoms with similar relative frequencies. Analysis of 20 adult symptom types shared by the Web and call center revealed that by excluding only 2 assessments (nasal symptoms and leg pain) the remaining 18 showed a significant correlation in counts of use ( r 2 = 0.68, p < 0.001 for linear trend). Conclusions: Internet users have symptoms assessed in similar proportions to callers, with a few exceptions. Compared with callers, Web users are much more likely looking for information about adult symptoms. Callers are proportionally asking more about acute symptoms, whereas Internet users appear more interested in symptom assessment of chronic conditions.

Abstract Abstract #404 Background: The oral HDACi vorinostat (V) sensitizes breast cancer cells to tubulin polymerizing agents and anti-VEGF directed therapies in vitro. We sought to determine the safety and efficacy of V plus paclitaxel (P) and bevacizumab (B) as first-line therapy for MBC.&#x2028; Methods: Eligible patients (pts) with measurable disease, ECOG PS 0 or 1, and no prior chemotherapy for MBC, received P (90mg/m2) on days 1, 8, 15 and B (10mg/kg) on days 1,15 every 28 days, plus V 200 mg (N=3) or 300 mg (N=28) BID orally for 3 days given the day before, day of, and day after each P dose. Response was assessed after every 3 cycles (RECIST). The trial was designed to distinguish between a response rate of &lt; 40% vs. &gt; 60% (alpha=0.10, beta=0.10) using Simon's optimal 2-stage design. At least 12 responses were required in the first 28 evaluable pts in the 1st stage and 21 of 41 in the 2nd stage.&#x2028; Results: There were no dose-limiting toxicities among the first 6 pts treated at the 1st (200 mg BID; N=3) and 2nd (300 mg BID; N=3) dose levels. Among the 26 evaluable pts treated at the recommended phase II dose of 300mg bid in the 1st stage, 14 patients had a partial response (PR-54%; 90% confidence interval [C.I.] 36%-71%), 6 had stable disease (SD) at 3 months and remain on treatment, 2 had SD as the best response with progressive disease (PD) at 11.8 and 15 months, and 4 had PD as their best response. Additionally, 2 of 3 pts treated at the first V dose level had a PR, indicating PR in 16 of 29 overall (55%, 90% C.I 38%-71%). Only 11 of 29 evaluable pts (38%) have progressed after a median followup of 10.5 months (R-2.3-14.9). Two pts had tumor biopsies and peripheral blood mononuclear cells (PBMC) collected before and 4 hours after the 3rd V dose (300 mg BID) which showed increased acetylation of K69 lysine residue of the chaperone protein Hsp 90, upregulation of Hsp70, and downregulation of AKT by Western Blot, providing evidence of Hsp90 inhibition in tumor and PBMC.&#x2028; &#x2028; Gr 3-4 toxicities included neutropenia (26%), neuropathy (23%), fatigue (16%), thrombosis (10%), vomiting (6%), hypertension (3%), and diarrhea (3%).&#x2028; Conclusions: Our preliminary findings indicate that the HDACi V inhibits Hsp90 and downregulates AKT in breast cancer in vivo, and enhances the effectiveness of P plus B in MBC. Accrual is nearly complete and the final efficacy analysis will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 404.