Alternative NHEJ Pathway Components Are Therapeutic Targets in High-Risk Neuroblastoma.

UNLABELLED: In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. IMPLICATIONS: These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets.

Sentinel lymph node biopsy performed after neoadjuvant chemotherapy is accurate in patients with documented node-positive breast cancer at presentation.

BACKGROUND: The optimal strategy for incorporating lymphatic mapping and sentinel lymph node biopsy into the management of breast cancer patients receiving neoadjuvant chemotherapy remains controversial. Previous studies of sentinel node biopsy performed following neoadjuvant chemotherapy have largely reported on patients whose prechemotherapy, pathologic axillary nodal status was unknown. We report findings using a novel comprehensive approach to axillary management of node-positive-patients receiving neoadjuvant chemotherapy. METHODS: We evaluated 54 consecutive breast cancer patients with biopsy-proven axillary nodal metastases at the time of diagnosis that underwent lymphatic mapping with nodal biopsy as well as concomitant axillary lymph node dissection after receiving neoadjuvant chemotherapy. All cases were treated at a single comprehensive cancer center between 2001 and 2005. RESULTS: The sentinel node identification rate after delivery of neoadjuvant chemotherapy was 98%. Thirty-six patients (66%) had residual axillary metastases (including eight patients that had undergone resection of metastatic sentinel nodes at the time of diagnosis), and in 12 cases (31%) the residual metastatic disease was limited to the sentinel lymph node. The final, post-neoadjuvant chemotherapy sentinel node was falsely negative in three cases (8.6%). The negative final sentinel node accurately identified patients with no residual axillary disease in 17 cases (32%). CONCLUSIONS: Sentinel lymph node biopsy performed after the delivery of neoadjuvant chemotherapy in patients with documented nodal disease at presentation accurately identified cases that may have been downstaged to node-negative status and can spare this subset of patients (32%) from experiencing the morbidity of an axillary dissection.

Activation of the melanocortin-4 receptor mobilizes intracellular free calcium in immortalized hypothalamic neurons.

BACKGROUND: At least 4% of childhood obesity is due to mutations in the hypothalamic melanocortin-4 receptor. The melanocortin-4 receptor, a seven transmembrane G-protein-coupled receptor, is important in the regulation of feeding behavior and body weight. The specific pathways of intracellular signaling remain in investigative stages. To further understand its function, we hypothesized that the melanocortin-4 receptor activates the Galphaq/phospholipase C signaling pathway, resulting in alterations of cytoplasmic calcium in immortalized hypothalamic (GT1-1) neurons. MATERIALS AND METHODS: Changes in intracellular calcium were measured after loading GT1-1 cells with fura-2-AM. Cells were treated with NDP-alphaMSH, an alpha-melanocortin stimulating hormone analogue, and intracellular calcium changes were recorded. Cells treated with NDP-alpha-MSH were also treated with the melanocortin-4 receptor antagonist, SHU-9119. To assess the specific G-protein subunit involved, GT1-1 neurons were treated with the phospholipase C inhibitor U73122 and its inactive analogue, U73433. Experiments were also performed after inhibition of IP3 receptors with 2-aminoethoxydiphenylborate (2APB). Additional experiments were conducted in a calcium-depleted buffer environment. Data were analyzed by ANOVA with statistical significance of P < 0.05. RESULTS: Agonist treatment (0.01-1000 nm) of GT1-1 neurons resulted in dose-dependent increases in intracellular calcium. SHU-9119 (0.01-1000 nm) abolished the calcium response. Treatment with U73122 (10 microm) attenuated the calcium response, while U73433 (10 microm) had minimal effect. 2APB (200 microm) inhibited the calcium transient, and the use of calcium-free buffer did not affect the amplitude of the calcium spike. CONCLUSIONS: Our study demonstrates that, upon agonist binding, the melanocortin-4 receptor mediates increases in intracellular calcium through the Galphaq-protein/phospholipase C dependent signaling pathway. Understanding the physiological importance of calcium signaling by the melanocortin-4 receptor may be important for future development of therapeutic targets.

Lymphatic mapping and sentinel lymph node biopsy for patients with local recurrence after breast-conservation therapy.

BACKGROUND: Local recurrence (LR) after breast-conservation therapy for breast cancer occurs in 10% to 15% of cases. A subset of these represents biologically aggressive disease, yet prognostic features for identifying this high-risk category are lacking. We hypothesized that lymphatic mapping and sentinel lymph node biopsy would provide useful information regarding dominant lymphatic drainage patterns of patients with LR. METHODS: Breast cancer case records involving surgery for LR at the University of Michigan from 2002 to 2004 were reviewed. The lymphatic drainage patterns were compared with those of 117 patients who underwent mapping for primary breast cancer. RESULTS: Fourteen LR cases were identified (10 with initial axillary lymph node dissection, 2 with initial sentinel lymph nodes, and 2 with no axillary surgery at the time of primary cancer treatment); lymphatic mapping was performed in 10. The sentinel lymph node identification rate was 90%, the median number of lymph nodes retrieved was 3, and no metastases were detected. Significantly more cases of nonipsilateral axillary sentinel node drainage were observed in mapping procedures performed for LR compared with those for primary breast cancer (67% vs. 15%; P = .001). CONCLUSIONS: Lymphatic mapping is feasible in patients undergoing mastectomy for LR and is likely to identify aberrantly located sentinel lymph nodes that would otherwise be overlooked with a conventional completion mastectomy.