Donald M. O'Rourke

Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.

The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors.

SHP-2 is a ubiquitously expressed non-transmembrane tyrosine phosphatase with two SH2 domains. Multiple reverse-genetic studies have indicated that SHP-2 is a required component for organ and animal development. SHP-2 wild-type and homozygous mutant mouse fibroblast cells in which the N-terminal SH2 domain was target-deleted were used to examine the function of SHP-2 in regulating Phosphatidylinositol 3-Kinase (PI3K) activation by growth factors. In addition, SHP-2 and various mutants were introduced into human glioblastoma cells as well as SHP-2−/− mouse fibroblasts. We found that EGF stimulation and EGFR oncoprotein (ΔEGFR) expression independently induced the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Targeted deletion of the N-terminal SH2 domain of SHP-2 severely impaired PDGF- and IGF-induced Akt phosphorylation. Ectopic expression of SHP-2 in U87MG gliobastoma cells elevated EGF-induced Akt phosphorylation, and the effect was abolished by mutation of its N-terminal SH2 domain. Likewise, the reconstitution of SHP-2 expression in the SHP-2−/− cells enhanced Akt phosphorylation induced by EGF while rescuing that induced by PDGF and IGF. Further lipid kinase activity assays confirmed that SHP-2 modulation of Akt phosphorylation correlated with its regulation of PI3K activation. Based on these results, we conclude that SHP-2 is required for mediating PI3K/Akt activation, and the N-terminal SH2 domain is critically important for a ‘positive’ role of SHP-2 in regulating PI3K pathway activation.

Grading of CNS neoplasms using continuous arterial spin labeled perfusion MR imaging at 3 Tesla.

To differentiate glioma grade based on blood flow measured using continuous arterial spin labeled (CASL) perfusion MRI, implemented at 3 Tesla for improved signal‐to‐noise ratio (SNR) and spin labeling effect.

Differentiation between glioblastomas and solitary brain metastases using diffusion tensor imaging

The purpose of this study is to determine whether diffusion tensor imaging (DTI) metrics including tensor shape measures such as linear and planar anisotropy coefficients (CL and CP) can help differentiate glioblastomas from solitary brain metastases. Sixty-three patients with histopathologic diagnosis of glioblastomas (22 men, 16 women, mean age 58.4 years) and brain metastases (13 men, 12 women, mean age 56.3 years) were included in this study. Contrast-enhanced T1-weighted, fluid-attenuated inversion recovery (FLAIR) images, fractional anisotropy (FA), apparent diffusion coefficient (ADC), CL and CP maps were co-registered and each lesion was semi-automatically subdivided into four regions: central, enhancing, immediate peritumoral and distant peritumoral. DTI metrics as well as the normalized signal intensity from the contrast-enhanced T1-weighted images were measured from each region. Univariate and multivariate logistic regression analyses were employed to determine the best model for classification. The results demonstrated that FA, CL and CP from glioblastomas were significantly higher than those of brain metastases from all segmented regions (p<0.05), and the differences from the enhancing regions were most significant (p<0.001). FA and CL from the enhancing region had the highest prediction accuracy when used alone with an area under the curve of 0.90. The best logistic regression model included three parameters (ADC, FA and CP) from the enhancing part, resulting in 92% sensitivity, 100% specificity and area under the curve of 0.98. We conclude that DTI metrics, used individually or combined, have a potential as a non-invasive measure to differentiate glioblastomas from metastases.

Constitutive EGFR signaling confers a motile phenotype to neural stem cells

The epidermal growth factor receptor (EGFR) has been shown to play an important role in brain development, including stem and precursor cell survival, proliferation, differentiation, and migration. To further examine the temporal and spatial requirements of erbB signals in uncommitted neural stem cells (NSCs), we expressed the ligand-independent EGF receptor, EGFRvIII, in C17.2 NSCs. These NSCs are known to migrate and to evince a tropic response to neurodegenerative environments in vivo but for which an underlying mechanism remains unclear. We show that enhanced erbB signaling via constitutive kinase activity of EGFRvIII in NSCs sustains an immature phenotype and enhances NSC migration.

Differentiation between Glioblastomas, Solitary Brain Metastases, and Primary Cerebral Lymphomas Using Diffusion Tensor and Dynamic Susceptibility Contrast-Enhanced MR Imaging

More on the eternal question: what can we use to differentiate preoperatively glioblastomas, metastases, and lymphomas? Here, the authors investigated whether diffusion tensor imaging and gadolinium perfusion studies could be used for this purpose. They evaluated 26 GBMs, 25 brain metastases, and 16 primary cerebral lymphomas with these techniques. Basically, GBMs showed lower fractional anisotropy and higher perfusion patterns. The best predictive data obtained were the apparent diffusion coefficients from enhancing tumor regions and the perfusion (cerebral blood volume) from the peritumoral regions. Although this is probably something that we all use on a daily basis, it is nice to see it reported in such an organized and careful fashion. BACKGROUND AND PURPOSE: Glioblastomas, brain metastases, and PCLs may have similar enhancement patterns on MR imaging, making the differential diagnosis difficult or even impossible. The purpose of this study was to determine whether a combination of DTI and DSC can assist in the differentiation of glioblastomas, solitary brain metastases, and PCLs. MATERIALS AND METHODS: Twenty-six glioblastomas, 25 brain metastases, and 16 PCLs were retrospectively identified. DTI metrics, including FA, ADC, CL, CP, CS, and rCBV were measured from the enhancing, immediate peritumoral and distant peritumoral regions. A 2-level decision tree was designed, and a multivariate logistic regression analysis was used at each level to determine the best model for classification. RESULTS: From the enhancing region, significantly elevated FA, CL, and CP and decreased CS values were observed in glioblastomas compared with brain metastases and PCLs (P < .001), whereas ADC, rCBV, and rCBVmax values of glioblastomas were significantly higher than those of PCLs (P < .01). The best model to distinguish glioblastomas from nonglioblastomas consisted of ADC, CS (or FA) from the enhancing region, and rCBV from the immediate peritumoral region, resulting in AUC = 0.938. The best predictor to differentiate PCLs from brain metastases comprised ADC from the enhancing region and CP from the immediate peritumoral region with AUC = 0.909. CONCLUSIONS: The combination of DTI metrics and rCBV measurement can help in the differentiation of glioblastomas from brain metastases and PCLs.

Arterial spin-labeling and MR spectroscopy in the differentiation of gliomas.

BACKGROUND AND PURPOSE: Noninvasive grading of gliomas remains a challenge despite its important role in the prognosis and management of patients with intracranial neoplasms. In this study, we evaluated the ability of cerebral blood flow (CBF)-guided voxel-by-voxel analysis of multivoxel proton MR spectroscopic imaging (1H-MRSI) to differentiate low-grade from high-grade gliomas. MATERIALS AND METHODS: A total of 35 patients with primary gliomas (22 high grade and 13 low grade) underwent continuous arterial spin-labeling perfusion-weighted imaging (PWI) and 1H-MRSI. Different regions of the gliomas were categorized as “hypoperfused,” “isoperfused,” and “hyperperfused” on the basis of the average CBF obtained from contralateral healthy white matter. 1H-MRSI indices were computed from these regions and compared between low- and high-grade gliomas. Using a similar approach, we applied a subgroup analysis to differentiate low- from high-grade oligodendrogliomas because they show different physiologic and genetic characteristics. RESULTS: Choglioma (G)/white matter (WM), GlxG/WM, and Lip+LacG/CrWM were significantly higher in the “hyperperfused” regions of high-grade gliomas compared with low-grade gliomas. ChoG/WM and Lip+LacG/CrWM were also significantly higher in the “hyperperfused” regions of high-grade oligodendrogliomas. However, metabolite ratios from the “hypoperfused” or “isoperfused” regions did not exhibit any significant differences between high-grade and low-grade gliomas. CONCLUSION: The results suggest that 1H-MRSI indices from the “hyperperfused” regions of gliomas, on the basis of PWI, may be helpful in distinguishing high-grade from low-grade gliomas including oligodendrogliomas.

Distinct Domains in the SHP-2 Phosphatase Differentially Regulate Epidermal Growth Factor Receptor/NF-κB Activation through Gab1 in Glioblastoma Cells

ABSTRACT The transcription factor nuclear factor κB (NF-κB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-κB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-κB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-κB activation. We also show that EGFR-induced NF-κB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-κB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-κB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-κB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-κB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-κB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-κB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-κB activation.

Posttreatment recurrence of malignant brain neoplasm: Accuracy of relative cerebral blood volume fraction in discriminating low from high malignant histologic volume fraction

PURPOSE To determine the accuracy of relative cerebral blood volume (rCBV) fraction for distinguishing high-grade recurrent neoplasm from treatment-related necrosis (TRN) in enhancing masses identified on surveillance magnetic resonance (MR) images following treatment for primary or secondary brain neoplasm. MATERIALS AND METHODS This institutional review board approved and HIPAA-compliant retrospective study included 30 patients undergoing resection of recurrent enhancing mass appearing after treatment with surgery and radiation, with or without chemotherapy. The enhancing mass volume was manually segmented on three-dimensional T1-weighted images. The rCBV maps were created by using T2-weighted dynamic susceptibility contrast perfusion MR imaging and registered to T1-weighted images, and the fraction of enhancing mass with rCBV above a range of thresholds was calculated. A receiver operating characteristic (ROC) curve was created by calculating sensitivity-specificity pairs at each threshold for rCBV fraction (< or = 20% or > 20%) by using percentage of malignant features at histologic evaluation as the reference criterion. Relationships between rCBV and probability of recurrence were estimated by using logistic regression analysis. RESULTS ROC analysis showed excellent discriminating accuracy of rCBV fraction (area under the ROC curve, 0.97 +/- 0.03 [standard error]) and high efficiency (93%) with an rCBV threshold of 1.8 times that of normal-appearing white matter. Logistic regression analysis showed that a unit increase of rCBV is associated with a 254-fold increase (95% confidence interval: 43, 1504, P < .001) of the odds that enhanced tissue is recurrence, adjusting for age, treatment, volume of enhancing tissue, and time to suspected recurrence. CONCLUSION The fraction of malignant histologic features in enhancing masses recurring after treatment for brain neoplasms can be predicted by using the rCBV fraction, with improved differentiation between recurrent neoplasm and TRN.

Experimental traumatic brain injury modulates the survival, migration, and terminal phenotype of transplanted epidermal growth factor receptor-activated neural stem cells.

OBJECTIVE:We have previously shown that constitutively active epidermal growth factor receptor signaling enhances the survival and motility of engrafted neural stem cells (NSCs) when transplanted into normal adult brain. In the present study, using the C17.2 NSC line stably transfected with the constitutively active epidermal growth factor receptor vIII, we sought to evaluate the phenotype of NSCs after engraftment into the milieu of traumatic head injury. METHODS:We performed intracerebral NSC transplantation with C17.2 NSCs overexpressing the active epidermal growth factor receptor vIII receptor into the ipsilateral (n = 17) or contralateral (n = 19) corpus callosum at 48 hours after severe experimental traumatic brain injury (TBI) or after sham injury (n = 12) in rats. RESULTS:All sham-injured animals (100%) showed NSC graft survival, compared with 65% of brain-injured animals receiving ipsilateral NSC transplants, and only 10% of brain-injured animals had surviving transplants after engraftment into the contralateral uninjured corpus callosum. A marked elevation of nerve growth factor (pg/mg protein) was observed at 72 hours after injury in the injured hemisphere (x = 80 ± 10 pg/mg) compared with the contralateral uninjured hemisphere (35 ± 0 pg/mg) (P < 0.05), and this elevation of nerve growth factor may have contributed to enhanced survival of engrafted NSCs. In uninjured control animals, NSC transplants proliferated actively, as evidenced by incorporation of bromodeoxyuridine. After TBI, however, transplanted NSCs failed to proliferate, regardless of the site of implantation. Morphologically, NSCs transplanted into the injured brain showed extensive process formation suggestive of a more differentiated phenotype, in contrast to NSCs engrafted into uninjured brain that appear undifferentiated, with round soma and no processes. NSCs transplanted into the corpus callosum of brain-injured animals also expressed NG2, a pro-oligodendrocyte marker that was not seen in cells transplanted into uninjured brain. Although migration of NSCs was much more pronounced in the uninjured brain, 2 weeks after TBI, NSCs transplanted into the ipsilateral corpus callosum were found to have migrated to the injury cavity. Moreover, NSCs transplanted into the corpus callosum contralateral to the site of injury were observed crossing the corpus callosum by 2 weeks after transplantation. CONCLUSION:Our results suggest that the environment associated with acute experimental TBI can significantly modulate the phenotype and migratory patterns of the engrafted NSC. These findings have particularly important implications for transplantation of NSCs into the traumatically injured nervous system.