6585 Background: Functional screening of T-ALL cell lines for sensitivity to NOTCH inhibitors has led to the discovery of frequent NOTCH1 gain-of-function mutations in the majority of human T-ALL. Gamma secretase catalyzes the cleavage of the Notch receptor within the transmembrane domain thereby releasing the Notch intracellular domain, which translocates to the nucleus to activate transcription of target genes. Gamma secretase inhibitors can induce G0/G1 arrest, decrease cell viability, and cause apoptosis of T-ALL cell lines carrying Notch activating mutations. MK-0752 is a potent gamma secretase inhibitor in clinical development (IC50 ∼50 nM). Methods: Patients with relapsed T-ALL and other leukemias were enrolled using an accelerated dose escalation scheme with 1 patient/dose level until ≥ grade 2 toxicity, followed by 3–6 patients/level. MK-0752 was administered by once-daily oral dosing in 28 day cycles. DLT was grade 3 or 4 non-hematologic toxicity during cycle 1. Plasma pharmacokinetic (PK) profiles and Abeta40 peptide levels were measured. Results: Six adult and two pediatric patients with leukemia (seven with T-ALL and one with AML) received MK-0752 dosed at 150, 225, and 300 mg/m2. Treatment duration ranged from 2 days to 56 days before patients discontinued for disease progression or drug-related toxicity. Dose-limiting toxicity (DLT) was Grade 3/4 diarrhea at 300 mg/m2. Four of the seven T-ALL patients had Notch activating mutations. One patient with T-ALL and a Notch activating mutation treated with 150 mg/m2 (300 mg) daily achieved a 45% reduction in a mediastinal mass at the end of 28 days as best response, but subsequently progressed by 56 days. Mean PK parameters at 150, 225, and 300 mg/m2 on Day 1 were AUC0–24hr = 723, 754, 1592 μM·hr, Cmax = 55, 40, 121 μM, C24hr = 18, 28, 59 μM, and tmax = 1, 8, 9 hr. Measurements of gamma secretase inhibition showed a 24–69% decrease in plasma Abeta40 peptide levels on Day 14 compared to predose. Conclusions: MK-0752 was well-tolerated in a limited number of patients below 300 mg/m2, and further enrollment is underway to establish the MTD. Plasma concentrations of MK-0752 at all doses were sufficient to inhibit gamma secretase. Leukemia samples will be assessed for Notch inhibition. [Table: see text]

Alterations in the gene copy numbers of the proto-oncogenes HER2/neu and c-myc in primary human breast cancer investigated in 73 patients. We detected amplification of HER2/neu in 17 patient samples and amplification of c-myc in 11, while amplification of both was seen in 6 samples. There was no correlation of age, hormone receptor positivity or tumour size with amplification of either proto-oncogene. Amplification of HER2/neu was significantly correlated with the stage of the disease. HER2/neu amplification was observed in 18.5% and 38% of node-negative and node-positive patients, respectively; the association between HER2/neu amplification and advanced stage of the disease was statistically significant (p = 0.05). Since this is a prospective study, the clinical significance of oncogene amplification is not known. The relatively high frequency of HER2/neu amplification points to a functional role in human breast cancer, particularly in the progression of the disease. The method used in our study allows oncogene amplification to be studied in conjunction with hormone receptor determination and thus may be of value in large clinical trials to determine the significance of oncogene abnormalities in breast cancer.

Gill disorders have emerged in recent years as a significant problem in the production of marine-stage Atlantic salmon Salmo salar L. The multi-aetiological condition 'proliferative gill inflammation' (PGI) has been reported to cause heavy losses in western Norway, yet reports of Scottish cases of the disease have remained anecdotal. In the present study, histopathological material from a marine production site in the Scottish Highlands experiencing mortalities due to a seasonal gill disease with proliferative-type pathology was examined using light microscopy, special staining techniques and transmission electron microscopy (TEM). The microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009 (syn. Paranucleospora theridion) was identified by staining using a Gram Twort method and TEM associated with distinctive proliferative and necrotic pathology confined to the interlamellar Malpighian cell areas of the primary filaments. Epitheliocystis was not a feature of the gill pathology observed. It is believed this is the first report of D. lepeophtherii being identified associated with pathology in a Scottish gill disease case, and supports anecdotal reports that a disease at least partly synonymous with PGI as described by Norwegian researchers is present in Scottish aquaculture.

Clinical biochemistry has long been utilized in human and veterinary medicine as a vital diagnostic tool, but despite occasional studies showing its usefulness in monitoring health status in Atlantic salmon (Salmo salar L.), it has not yet been widely utilized within the aquaculture industry. This is due, in part, to a lack of an agreed protocol for collection and processing of blood prior to analysis. Moreover, while the analytical phase of clinical biochemistry is well controlled, there is a growing understanding that technical pre‐analytical variables can influence analyte concentrations or activities. In addition, post‐analytical interpretation of treatment effects is variable in the literature, thus making the true effect of sample treatment hard to evaluate. Therefore, a number of pre‐analytical treatments have been investigated to examine their effect on analyte concentrations and activities. In addition, reference ranges for salmon plasma biochemical analytes have been established to inform veterinary practitioners and the aquaculture industry of the importance of clinical biochemistry in health and disease monitoring. Furthermore, a standardized protocol for blood collection has been proposed.

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10574 Introduction: Activation of Notch signaling occurs in ∼40% of human BCs and high Notch expression is associated with poor outcome. Inhibition of Notch inhibits BC cell proliferation in vitro. Notch signaling requires gamma secretase (GS), which cleaves Notch, releasing the Notch intracellular domain (NICD) to activate transcription of target genes. MK-0752 is a potent GS inhibitor. Methods: In Part 1 of the study, pts with advanced solid tumors were enrolled using an accelerated dose escalation with 1 pt/dose level until the occurrence of ≥Grade 2 toxicity, then 3–6 pts/dose level. MK-0752 was administered by once-daily oral dosing in 28-day cycles. Once a maximum tolerated dose (MTD) was established, an additional 22 pts with advanced BC were to be enrolled in Part 2. Six-point PK plasma profiles were collected over 24 hours on Days 1 and 28 and assayed by LC/MS/MS. PD measurement of plasma Abeta40 peptide (another gamma secretase substrate) was performed pre/post dose on Days 1 and 28. Tumor biopsies on Days 1 and 28 were obtained from a subset of pts to assess changes in Notch activity by immunohistochemical analysis of NICD. Results: In Part 1, two pts were enrolled at 450mg daily, and five pts at 600mg daily. Dose-limiting toxicities (DLTs) were Grade 3 diarrhea, constipation, nausea, and abdominal cramping at 600 mg. In Part 2, an additional 14 pts with BC were enrolled at 450mg daily. In this cohort, Grade 2/3 fatigue requiring dose reduction occurred in 6 pts. Grade 3 diarrhea (1pt), nausea (1 pt) and elevated liver transaminases (2 pt) were also observed. Mean PK parameters (at 450mg, 600 mg) on Day 1 were AUC0–24hr = 1036, 1065μM·hr, Cmax = 72, 61μM, C24hr = 25, 32μM, and tmax = 3, 7 hr. PD measurements of GS inhibition showed a 12–78% (mean 46%) decrease in plasma Abeta40 at 4 hours on Day 1 compared to predose. Conclusion: Continuous dosing of MK-0752 at 450mg daily in pts with BC was associated with significant toxicity, predominantly fatigue, and cannot be considered a MTD. An intermittent dosing schedule is being explored. MK-0752 at all doses inhibited GS, as demonstrated by decreases in plasma Abeta40. Analysis of efficacy and intratumoral Notch inhibition will be reported at the meeting. [Table: see text]

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The cloning of established human leukemia and lymphoma cell lines at limiting dilutions in microwells and soft agar is described. Growth of most cell lines was improved by the addition of human AB serum and irradiated human bone marrow stromal cells. In general, the cloning efficiency in microwells was greater than in soft agar. The effect of bone marrow stromal cells appeared to be caused by a diffusable factor(s), but close cellular interaction could not be excluded since cloning in microwells produced consistent and optimal cell growth compared to growth in soft agar. It was concluded that cloning of leukemia and lymphoma cell lines in microwells was the preferred method and that similar techniques could improve the cloning of fresh leukemia and lymphoma cells.