Kenneth D. Aldape

Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Epidermal Growth Factor Receptor Variant III Status Defines Clinically Distinct Subtypes of Glioblastoma

PURPOSE The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status. PATIENTS AND METHODS The expression of EGFRvIII was assessed by immunohistochemistry in 649 patients with newly diagnosed GBM. These data were then examined in conjunction with the expression of phospho-intermediates (in a subset of these patients) of downstream AKT and Ras pathways and YKL-40 as well as with known clinical risk factors, including the Radiation Therapy Oncology Groups recursive partitioning analysis (RTOG-RPA) class. RESULTS The RTOG-RPA class was highly predictive of survival in EGFRvIII-negative patients but much less predictive in EGFRvIII-positive patients. These findings were seen in both an initial test set (n = 268) and a larger validation set (n = 381). Similarly, activation of the AKT/MAPK pathways and YKL-40 positivity were predictive of poor outcome in EGFRvIII-negative patients but not in EGFRvIII-positive patients. Pair-wise combinations of markers identified EGFRvIII and YKL-40 as prognostically important. In particular, outcome in patients with EGFRvIII-negative/YKL-40-negative tumors was significantly better than the outcome in patients with the other three combinations of these two markers. CONCLUSION Established prognostic factors in GBM were not predictive of outcome in the EGFRvIII-positive subset, although this requires confirmation in independent data sets. GBMs negative for both EGFRvIII and YKL-40 show less aggressive behavior.

Direct Intracerebral Delivery of Cintredekin Besudotox (IL13-PE38QQR) in Recurrent Malignant Glioma: A Report by the Cintredekin Besudotox Intraparenchymal Study Group

PURPOSE Glioblastoma multiforme (GBM) is a devastating brain tumor with a median survival of 6 months after recurrence. Cintredekin besudotox (CB) is a recombinant protein consisting of interleukin-13 (IL-13) and a truncated form of Pseudomonas exotoxin (PE38QQR). Convection-enhanced delivery (CED) is a locoregional-administration method leading to high-tissue concentrations with large volume of distributions. We assessed the use of intracerebral CED to deliver CB in patients with recurrent malignant glioma (MG). PATIENTS AND METHODS Three phase I clinical studies evaluated intracerebral CED of CB along with tumor resection. The main objectives were to assess the tolerability of various concentrations and infusion durations; tissue distribution; and methods for optimizing delivery. All patients underwent tumor resection followed by a single intraparenchymal infusion (in addition to the intraparenchymal one following resection), with a portion of patients who had a preresection intratumoral infusion. RESULTS A total of 51 patients with MG were treated including 46 patients with GBM. The maximum tolerated intraparenchymal concentration was 0.5 microg/mL and tumor necrosis was observed at this concentration. Infusion durations of up to 6 days were well tolerated. Postoperative catheter placement appears to be important for optimal drug distribution. CB- and procedure-related adverse events were primarily limited to the CNS. Overall median survival for GBM patients is 42.7 weeks and 55.6 weeks for patients with optimally positioned catheters with patient follow-up extending beyond 5 years. CONCLUSION CB appears to have a favorable risk-benefit profile. CED is a complex delivery method requiring catheter placement via a second procedure to achieve accurate catheter positioning, better drug distribution, and better outcome.

EGFR overexpression and radiation response in glioblastoma multiforme

PURPOSE Recent studies have suggested relative radioresistance in glioblastoma multiforme (GM) tumors in older patients, consistent with their shorter survival. Two common molecular genetic abnormalities in GM are age related: epidermal growth factor receptor (EGFR) overexpression in older patients and p53 mutations in younger patients. We tested whether these abnormalities correlated with clinical heterogeneity in GM response to radiation treatment. METHODS AND MATERIALS Radiographically assessed radiation response (5-level scale) was correlated with EGFR immunoreactivity, p53 immunoreactivity, and p53 exon 5-8 mutation status in 170 GM patients treated using 2 prospective clinical protocols. Spearman rank correlation and proportional-odds ordinal regression were used for univariate and multivariate analysis. RESULTS Positive EGFR immunoreactivity predicted poor radiographically assessed radiation response (p = 0.046). Thirty-three percent of tumors with no EGFR immunoreactivity had good radiation responses (>50% reduction in tumor size by CT or MRI), compared to 18% of tumors with intermediate EGFR staining and 9% of tumors with strong staining. There was no significant relationship between p53 immunoreactivity or mutation status and radiation response. Significant relationships were noted between EGFR score and older age and between p53 score or mutation status and younger age. CONCLUSION The observed relative radioresistance of some GMs is associated with overexpression of EGFR.

Greater chemotherapy-induced lymphopenia enhances tumor-specific immune responses that eliminate EGFRvIII-expressing tumor cells in patients with glioblastoma

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioblastoma multiforme (GBM) and other neoplasms, but absent from normal tissues. Immunotherapeutic targeting of EGFRvIII could eliminate neoplastic cells more precisely but may be inhibited by concurrent myelosuppressive chemotherapy like temozolomide (TMZ), which produces a survival benefit in GBM. A phase II, multicenter trial was undertaken to assess the immunogenicity of an experimental EGFRvIII-targeted peptide vaccine in patients with GBM undergoing treatment with serial cycles of standard-dose (STD) (200 mg/m(2) per 5 days) or dose-intensified (DI) TMZ (100 mg/m(2) per 21 days). All patients receiving STD TMZ exhibited at least a transient grade 2 lymphopenia, whereas those receiving DI TMZ exhibited a sustained grade 3 lymphopenia (<500 cells/μL). CD3(+) T-cell (P = .005) and B-cell (P = .004) counts were reduced significantly only in the DI cohort. Patients in the DI cohort had an increase in the proportion of immunosuppressive regulatory T cells (T(Reg); P = .008). EGFRvIII-specific immune responses developed in all patients treated with either regimen, but the DI TMZ regimen produced humoral (P = .037) and delayed-type hypersensitivity responses (P = .036) of greater magnitude. EGFRvIII-expressing tumor cells were also eradicated in nearly all patients (91.6%; CI(95): 64.0%-99.8%; P < .0001). The median progression-free survival (15.2 months; CI(95): 11.0-18.5 months; hazard ratio [HR] = 0.35; P = .024) and overall survival (23.6 months; CI(95): 18.5-33.1 months; HR = 0.23; P = .019) exceeded those of historical controls matched for entry criteria and adjusted for known prognostic factors. EGFRvIII-targeted vaccination induces patient immune responses despite therapeutic TMZ-induced lymphopenia and eliminates EGFRvIII-expressing tumor cells without autoimmunity.

Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative polymerase chain reaction assay.

The combined loss of chromosomes 1p and 19q has recently emerged as a genetic predictor of chemosensitivity in anaplastic oligodendrogliomas. Here, we describe a strategy that uses a novel method of real-time quantitative polymerase chain reaction, quantitative microsatellite analysis (QuMA), for the molecular analysis of 1p and 19q loss in oligodendrogliomas and oligoastrocytomas in archival routinely processed paraffin material. QuMA is performed on the ABI 7700 and based on amplifications of microsatellite loci that contain (CA)n repeats where the repeat itself is the target for hybridization by the fluorescently labeled probe. This single probe can therefore be used to determine copy number of microsatellite loci spread throughout the human genome. In genomic DNA prepared from paraffin-embedded brain tumor specimens, QuMA detected combined loss of 1p and 19q in 64% (21 of 32) of oligodendrogliomas and 67% (6 of 9) of oligoastrocytomas. We validate the use of QuMA as a reliable method to detect copy number by showing concordance between QuMA and fluorescence in situ hybridization at 37 of 45 chromosomal arms tested. These results indicate that QuMA is an accurate, high-throughput assay for the detection of copy number at multiple loci; as many as 31 loci of an individual tumor can be analyzed on a 96-well plate in a single 2-hour run. In addition, it has advantages over standard allelic imbalance/loss of heterozygosity assays in that all loci are potentially informative, paired normal tissue is not required, and gain can be distinguished from loss. QuMA may therefore be a powerful molecular tool to expedite the genotypic analysis of human gliomas in a clinical setting for diagnostic/prognostic purposes.

Chromosomal Abnormalities Subdivide Ependymal Tumors into Clinically Relevant Groups

Ependymoma occurs most frequently within the central nervous system of children and young adults. We determined relative chromosomal copy-number aberrations in 44 ependymomas using comparative genomic hybridization. The study included 24 intracranial and 20 spinal cord tumors from pediatric and adult patients. Frequent chromosomal aberrations in intracranial tumors were gain of 1q and losses on 6q, 9, and 13. Gain of 1q and loss on 9 were preferentially associated with histological grade 3 tumors. On the other hand, gain on chromosome 7 was recognized almost exclusively in spinal cord tumors, and was associated with various other chromosomal aberrations including frequent loss of 22q. We conclude that cytogenetic analysis of ependymomas may help to classify these tumors and provide leads concerning their initiation and progression. The relationship of these aberrations to patient outcome needs to be addressed.

Using the molecular classification of glioblastoma to inform personalized treatment

Glioblastoma is the most common and most aggressive diffuse glioma, associated with short survival and uniformly fatal outcome, irrespective of treatment. It is characterized by morphological, genetic and gene‐expression heterogeneity. The current standard of treatment is maximal surgical resection, followed by radiation, with concurrent and adjuvant chemotherapy. Due to the heterogeneity, most tumours develop resistance to treatment and shortly recur. Following recurrence, glioblastoma is quickly fatal in the majority of cases. Recent genetic molecular advances have contributed to a better understanding of glioblastoma pathophysiology and disease stratification. In this paper we review basic glioblastoma pathophysiology, with emphasis on clinically relevant genetic molecular alterations and potential targets for further drug development. Copyright

Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors

We amplified various amounts of DNA derived from frozen SF210 and U251NCI human glioblastoma cells, carried out comparative genomic hybridization (CGH) using degenerate oligonucleotide primed-PCR (DOP-PCR) products as test probes, and compared results to analyses performed with probes prepared by standard nick translation. Next we extracted DNA from hematoxylin-eosin (HE)- and methyl green (MG)-stained, microdissected sections of formalin-fixed and paraffin-embedded U251NCI cells, amplified and labeled it by DOP-PCR, and subjected it to CGH. Finally, we used the same methods in multiple samples from a single human mixed glioma tissue. DOP-PCR products from 50 pg to 250 ng of DNA were equally effective in generating the same CGH profiles as the standard method. DOP-PCR products from microdissected pieces of MG-stained cells were effective probes for CGH, but HE-stained samples were not desirable. As the proportion of HE-stained sample increased, CGH profiles deteriorated. DOP-PCR products from microdissected pieces of MG-stained paraffin sections of glioma tissue produced CGH profiles compatible with their histological features. CGH performed with DOP-PCR products from microdissected paraffin blocks allows for the accurate investigation of the cytogenetic characteristics from invasive tumors and of cytogenetic heterogeneity within neoplastic tissue.

Beyond BRAFV600: Clinical Mutation Panel Testing by Next-Generation Sequencing in Advanced Melanoma

The management of melanoma has evolved due to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent event in tumors with BRAFV600 and NRAS mutations. Melanomas with BRAFNon-V600 mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.