Andrew K.W. Wood

Identification of ALK as a major familial neuroblastoma predisposition gene

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23–24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

Inhibition of ALK Signaling for Cancer Therapy

Paradigm shifting advances in cancer can occur after discovering the key oncogenic drivers of the malignant process, understanding their detailed molecular mechanisms, and exploiting this transdisciplinary knowledge therapeutically. A variety of human malignancies have anaplastic lymphoma kinase (ALK) translocations, amplifications, or oncogenic mutations, including anaplastic large cell lymphoma, inflammatory myofibroblastic tumors, non–small cell lung cancer, and neuroblastoma. This finding has focused intense interest in inhibiting ALK signaling as an effective molecular therapy against diseases with ALK-driven pathways. Recent progress in the elucidation of the major canonical signaling pathways postulated to be activated by NPM-ALK signaling has provided insight into which pathways may present a rational therapeutic approach. The identification of the downstream effector pathways controlled by ALK should pave the way for the rational design of ALK-inhibition therapies for the treatment of a subset of human cancers that harbor ALK aberrations. (Clin Cancer Res 2009;15(18):5609–14)

A Review of Low-Intensity Ultrasound for Cancer Therapy

The literature describing the use of low-intensity ultrasound in four major areas of cancer therapy-sonodynamic therapy, ultrasound-mediated chemotherapy, ultrasound-mediated gene delivery and anti-vascular ultrasound therapy-was reviewed. Each technique consistently resulted in the death of cancer cells, and the bio-effects of ultrasound were attributed primarily to thermal actions and inertial cavitation. In each therapeutic modality, theranostic contrast agents composed of microbubbles played a role in both therapy and vascular imaging. The development of these agents is important as it establishes a therapeutic-diagnostic platform that can monitor the success of anti-cancer therapy. Little attention, however, has been given either to the direct assessment of the mechanisms underlying the observed bio-effects or to the viability of these therapies in naturally occurring cancers in larger mammals; if such investigations provided encouraging data, there could be prompt application of a therapy technique in the treatment of cancer patients.

The Disruption of Murine Tumor Neovasculature by Low-intensity Ultrasound—Comparison Between 1- and 3-MHz Sonication Frequencies

RATIONALE AND OBJECTIVES The goal was to determine whether the tumor vascular disrupting actions of low-intensity ultrasound were frequency dependent. MATERIALS AND METHODS The effect of the frequency (1 MHz at 2.2 W/cm2 or 3 MHz at 2.4 W/cm2) of low-intensity ultrasound as a neovascular disrupting modality was investigated in 15 murine melanomas (K1735(22)) insonated for 3 minutes after the intravenous injection of a microbubble contrast agent (Definity). In contrast-enhanced power Doppler observations of each tumor (before and after treatment), measurements were made of the size of the area of the tumor that was perfused with blood containing the ultrasound contrast agent (percentage area of flow [PAF]), and the volume of contrast agent flowing through the unit volume of the tumor (color-weighted fractional area [CWFA]). During insonation of the tumor, the temperature was measured with a fine wire thermocouple in an additional eight mice. RESULTS The antivascular action of low-intensity ultrasound was significantly enhanced (PAF by 64%; CWFA by 106%) when the tumor was treated with 3-MHz ultrasound rather than 1 MHz (analysis of variance: PAF, P=.02; CWFA, P=.04). The average rate of tumor temperature increase was 2.6+/-1.3 degrees C/min for 1 MHz and 5.0+/-1.7 degrees C/min for 3 MHz; these increases were significantly different (P=.04). CONCLUSIONS Insonation of the tumor at a higher frequency amplified the heating of the neoplasm and led to greater disruption of the tumor vasculature; 3-MHz ultrasound was more efficacious than 1 MHz for antivascular cancer therapy.

Serial Transcriptome Analysis and Cross-Species Integration Identifies Centromere-Associated Protein E as a Novel Neuroblastoma Target

Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in nine hyperplastic ganglia harvested at three time points and four tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN-amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized three targets [centromere-associated protein E (Cenpe), Gpr49, and inosine monophosphate dehydrogenase type II] with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC(50), 41 nmol/L; range, 27-266 nmol/L) and delayed tumor growth in three xenograft models (P values ranged from P < 0.0001 to P = 0.018). We provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets and identify CENPE as a novel therapeutic candidate in neuroblastoma.

Prolonged General Anesthesia in MR Studies of Rats

RATIONALE AND OBJECTIVES Magnetic resonance (MR) imaging of laboratory animals may require general anesthesia to minimize body movements over many hours. The anesthetization technique should allow physiologic parameters to remain as close to normal as possible, permit fast recovery, allow safe, repeated use, and avoid attachment of ferrous metal components to the animal. The purpose of this study was to evaluate an anesthetization technique that was developed to meet each of these qualifications. MATERIALS AND METHODS In 15 rats (280-483-g body weight), general anesthesia was induced (with intramuscular ketamine hydrochloride, xylazine hydrochloride, acepromazine maleate, and atropine), a tail vein catheter was inserted, and preimaging surgical procedures were performed. A face mask was applied, the animal was positioned in a dorsal recumbent position on an acrylic board, and an isothermal heating pad was placed on the ventral aspect of the abdominal wall. The rat, on the board, was then inserted into a trough that contained a custom-built, linearly polarized birdcage head coil and placed in the bore of a 4.7-T horizontal-bore magnet. The face mask was connected to a non-rebreathing gaseous anesthetic system, and anesthesia was maintained with 1.5-2.0 L/min oxygen and 0.25%-1.50% isoflurane. Oxygen saturation, heart rate, and rectal temperature were continuously monitored. RESULTS The duration of intramuscular anesthesia was 110 minutes +/- 12, and the duration of gaseous anesthesia was 106 minutes +/- 43. The monitoring equipment permitted display of vital signs. CONCLUSION The method appeared safe, was easy to perform, maintained a stable physiologic state for the parameters monitored, and could be used for repeated anesthesia in the same animal.

Antivascular Ultrasound Therapy Extends Survival of Mice With Implanted Melanomas

The goal of this murine investigation was to evaluate the effect of an antivascular ultrasound treatment on the growth of an implanted melanoma and the consequent survival rate. After the intravenous injection of 0.2 mL ultrasound contrast agent (Definity), therapy (n = 15) was performed on 1-mL tumors for 3 min with low-intensity continuous ultrasound (3 MHz; 2.4 +/- 0.1 W cm(-2) [I(SATA)]); control mice (n = 17) received a sham treatment. Mice were euthanized once the tumor had reached 3 mL, and then survival percentage vs. time curves were plotted. The median survival time (time for tumor to reach 3 mL) for the treated group was 23 d and for the control group was 18 d; the difference was statistically significant (p <or= 0.0001). Antivascular ultrasound therapy reduced the growth rate of an implanted melanoma and increased survival time. The ultrasound therapy provides a further example of tumor vascular disruption, and its future clinical potential should be investigated.

Gadodiamide T1 relaxivity in brain tissue in vivo is lower than in saline.

In vivo measurements of gadodiamide (Gd‐DTPA‐BMA) T1 relaxivity were performed at 4.7 T in injured and normal rat brains. Cerebral lesions were induced in nine rats by a localized freezing method. T1 maps of the lesions were generated before and after injection of Gd‐DTPA‐BMA (0.1–0.6 mmol/kg). Samples of normal and necrotic brain were collected postmortem; the wet and dry weights were determined, and Gd content was measured by inductively coupled plasma mass spectroscopy. The in vivo relaxivity was determined by a linear fit of a plot of the change in relaxation rate following injection of the contrast agent as a function of Gd content. This analysis yielded a relaxivity in the injured brain of 2.8 sec−1 mmol−1 kg tissue water at 36°C. The water weight fraction was 0.90 ± SD 0.02 wt/wt in injured brain and 0.79 ± 0.02 in normal brain. Relaxivity measurements were also performed on solutions of Gd‐DTPA‐BMA (0.0–0.6 mmol) and albumin (0–30% wt/wt) in normal saline at room and physiologic temperatures. The relaxivity in the albumin/saline increased with increasing solids content with values of 4.0–4.9 sec−1 mmol−1kg at 21°C and 3.4–4.5 sec−1 mmol−1 kg at 37°C. The relaxivity of the tissues differed significantly from that of the saline solutions of comparable solids content, suggesting that the solids content of a tissue is not the only factor that determines in vivo relaxivity. Magn Reson Med 53:35–40, 2005.

A novel method for analysis of TOMROP data

To develop an efficient method for extracting maps of the corrected T1 from images generated using the T One by Multiple Read Out Pulses (TOMROP) sequence.

Delta-projection imaging on contrast-enhanced ultrasound to quantify tumor microvasculature and perfusion.

RATIONALE AND OBJECTIVES The aim of this study was to assess the Delta-projection image processing technique for visualizing tumor microvessels and for quantifying the area of tissue perfused by them on contrast-enhanced ultrasound images. MATERIALS AND METHODS The Delta-projection algorithm was implemented to quantify perfusion by tracking the running maximum of the difference (Delta) between the contrast-enhanced ultrasound image sequence and a baseline image. Twenty-five mice with subcutaneous K1735 melanomas were first imaged with contrast-enhanced grayscale and then with minimum-exposure contrast-enhanced power Doppler (minexCPD) ultrasound. Delta-projection images were reconstructed from the grayscale images and then used to evaluate the evolution of tumor vascularity during the course of contrast enhancement. The extent of vascularity (ratio of the perfused area to the tumor area) for each tumor was determined quantitatively from Delta-projection images and compared to the extent of vascularity determined from contrast-enhanced power Doppler images. Delta-projection and minexCPD measurements were compared using linear regression analysis. RESULTS Delta-projection was successfully performed in all 25 cases. The technique allowed the dynamic visualization of individual blood vessels as they filled in real time. Individual tumor blood vessels were distinctly visible during early image enhancement. Later, as an increasing number of blood vessels were filled with the contrast agent, clusters of vessels appeared as regions of perfusion, and the identification of individual vessels became difficult. Comparisons were made between the perfused area of tumors in Delta-projections and in minexCPD images. The Delta-projection perfusion measurements were correlated linearly with minexCPD. CONCLUSION Delta-projection visualized tumor vessels and enabled the quantitative assessment of the tumor area perfused by the contrast agent.