Background. Amplification of erb‐B2 and myc shows prognostic value in patients with operable breast cancer. Amplification is usually detected in tumor samples remaining after pathologic work‐up, preventing the examination of small tumors. Methods. Tumor imprints that contained low numbers of contaminating normal cells were obtained from small tumors. The prognostic value of erb‐B2 and myc amplification in imprint breast preparations was examined, using the polymerase chain reaction (PCR) to determine gene copy number. Tumor material was obtained from 162 patients with breast cancer operated 1975‐1976. Results. Amplification of erb‐B2 existed in 21% and myc in 16% of the patients. Both erb‐B2 and myc amplification showed prognostic significance in univariate analysis for survival and relapse free survival. In multivariate analysis, erb‐B2 was a significant factor. In small tumors less than 13 mm in greatest dimension, erb‐B2 showed prognostic significance for survival but not for relapse free survival. Conclusions. The use of imprints/PCR allows the detection of gene amplification in breast cancer. The procedure is suitable for the analysis of small tumors and can be used to examine very small tumors, such as those detected mammographically. Cancer 1995;75:2681–7.

In Bloom's syndrome (BS) the regulation of uracil-DNA glycosylase, an enzyme involved in the repair of DNA containing 5-FU, is altered. 5-FU induces higher levels of DNA fragmentation in BS cells than in non-BS cells. The increase in DNA fragmentation is connected to the cytotoxic mechanism where 5-FU is incorporated into DNA. When 5-FU induces DNA fragmentation by a mechanism not involving the incorporation of drug into DNA, the levels of DNA fragmentation in BS and non-BS cells remain similar.

DNA replication intermediates in human melanoma cells have been investigated by using the drug aphidicolin, which inhibits DNA polymerase a. In untreated cells, Okazaki fragments and 10-kilobase (kb) DNA intermediates are formed. In aphidicolin-treated cells, the replication fork is stopped and there is no formation of DNA replication intermediates. However, 10-kb DNA intermediates formed before the drug blockade are ligated to high molecular weight DNA whereas already formed Okazaki fragments accumulate in the cell. Moreover, in cells released from aphidicolin inhibition there is preferential labeling of 10-kb DNA compared to Okazaki fragments. The 10-kb DNA and the Okazaki fragments, therefore, respond -differently to aphidicolin.Our knowledge of the different steps in the synthesis of DNA in mammalian cells is very poor. Because of the size and complexity of the mammalian genome, most work has focused on small viral DNAs which replicate in mammalian cells (1,2 For experiments, the cells were seeded in small culture dishes (35 x 10 mm) containing 3 ml of medium 24 hr before the addition of 100 ACi of [3H]thymidine (22 Ci/mmol; 1 Ci = 3.7 X 1010 Bq; Amersham) and the incubation was performed for the desired length of time. Aphidicolin was dissolved in ethanol prior to addition to the cell cultures. Ethanol without drug was always added to control cultures incubated in parallel to the drug-treated cell cultures. The aphidicolin was a gift from Imperial Chemical Industries.Cell Lysis. The incubation medium was drained from the culture dish and the cells were rinsed twice in cold phosphatebuffered saline. Cell lysis was performed in the dark at 0C by the addition of 2.25 ml of 0.03 M NaOH. After 30 min the solution was neutralized by the addition of 0.9 ml of 0.067 M HCl/ 0.02 M NaH2PO4. Finally, the solution was made 0.5% in NaDodSO4 and analyzed after 60 min at room temperature (9). Gel Electrophoresis. The 0.75% agarose flat bed gels were made as described (12). The labeled DNA was separated in the agarose gels by using an LKB Multiphor electrophoretic system. DNAs of known molecular weights, used as markers, were obtained from New England Nuclear. The gels were cut into 1-mm-thick slices that were assayed for radioactivity with a toluene-based scintillation fluid containing 3% Soluene 100 in a Packard scintillation counter.Digestion with S1 Nuclease. S1 nuclease (Sigma) was added, to a concentration of 200 units/ml, to 0.03 M sodium acetate, pH 4.6/0.05 M zinc acetate/0.075 M sodium chloride conAbbreviation: kb, kilobase(s). 3996The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

BackgroundUveal melanoma is the most common primary intraocular malignancy in adults. Despite successful control of the primary tumor, metastatic disease will ultimately develop in approximately 50% of patients, with the liver being the most common site for metastases. The median survival for patients with liver metastases is between 6 and 12 months, and no treatment has in randomized trials ever been shown to prolong survival. A previous phase II trial using isolated hepatic perfusion (IHP) has suggested a 14-month increase in overall survival compared with a historic control group consisting of the longest surviving patients in Sweden during the same time period (26 versus 12 months).Methods/DesignThis is the protocol for a multicenter phase III trial randomizing patients with isolated liver metastases of uveal melanoma to IHP or best alternative care (BAC). Inclusion criteria include liver metastases (verified by biopsy) and no evidence of extra-hepatic tumor manifestations by positron emission tomography–computed tomography (PET-CT). The primary endpoint is overall survival at 24 months, with secondary endpoints including response rate, progression-free survival, and quality of life. The planned sample size is 78 patients throughout five years.DiscussionPatients with isolated liver metastases of uveal melanoma origin have a short expected survival and no standard treatment option exists. This is the first randomized clinical trial to evaluate IHP as a treatment option with overall survival being the primary endpoint.Trial registrationClinicalTrials.gov registration number: NCT01785316 (registered 1 February 2013). EudraCT registration number: 2013-000564-29.