Heterogeneous expression or amplification is a challenge to HER2 diagnostics. A guideline defines heterogeneity as the presence of between 5% and 50% cells with HER2/CEP17 ratios of more than 2.20. We audited the frequency of such cells and their clinical impact in the results from 2 pathology laboratories combined with data from the TEAM [Tamoxifen vs Exemestane Adjuvant Multicentre] pathology study. HER2 reports were scanned and the percentages of amplified cells reported. Of 6,461 eligible cases, 754 (11.7%) exhibited 50% or more cells with ratios of more than 2.20, which is "amplified" by College of American Pathologists guidelines. Of the cases, 2,166 (33.5%) exhibited more than 5% but less than 50% of cells with HER2/CEP17 ratios of more than 2.20, or "heterogeneous amplification." No prognostic impact was observed when fewer than 30% of cells exhibited ratios of more than 2.20. All amplified cases with 30% to 50% of cells with ratios more than 2.20 were identified as such by United Kingdom guidelines. The percentage of tumor cells with HER2/CEP17 ratios more than 2.20 does not identify cases with heterogeneous amplification or poor outcome. A modified approach for identification of true heterogeneous amplification is suggested.

A high-performance liquid chromatography (HPLC) method is described for the determination of reactive nitrogen mustard anticancer drugs in plasma after derivatization with diethyl-dithiocarbamic acid (DDTC). Three compounds were studied: two reactive species (mechlorethamine (HN2) and galactose 6-mustard (G-6-M] and a less reactive species (melphalan (L-PAM] included for validation experiments. Mass and NMR spectrometry confirmed that one molecule of DDTC reacts with each arm of the mustard, displacing a chlorine atom to form a stable disubstituted adduct. With the reactive mustards a 30-min incubation at 37 degrees C is recommended for greater than 90% derivatization efficiency. Gradient elution was employed to analyze all three compounds using the same conditions with a microBondapak C18 10-microns particle size column (30 cm by 3.8 mm i.d.). The retention time (tR) of HN2-DDTC2 was 13.1 min +/- 1.5% within day CV; tR of L-PAM was 7.6 min and L-PAM-DDTC2 was 14.6 min +/- 0.8% CV. G-6-M-DDTC2 yielded a double peak, tR = 10.7 min and 10.9 min +/- 2.9% CV. The limit of detection on column was 0.5 ng for HN2, 1 ng for L-PAM, and 5 ng for G-6-M. A solid-phase sample preparation technique using "Bond Elut" phenyl is described that extracts from plasma G-6-M-DDTC2 with greater than 74% efficiency and HN2-DDTC2 with greater than 90% efficiency. When the drugs were derivatized in plasma, recovery remained high for G-6-M (greater than 84%) but dropped to 50% for HN2.(ABSTRACT TRUNCATED AT 250 WORDS)

HPLC has been applied to determine the stability of mechlorethamine hydrochloride (nitrogen mustard) formulated as an ointment in white soft paraffin (10 mg drug, 50 g paraffin and 1 mL acetone). A new solubilization technique is described for extraction of the drug from the ointment for HPLC analysis which has an extraction efficiency of 76.1% with a coefficient of variation of 10.4%. Stored at 4 degrees C the drug content of the ointment remained stable for at least 84 days and stored at 37 degrees C the drug content remained stable for at least 40 days. In comparison aqueous solutions of mechlorethamine at the same concentration, fully degraded after 4 days. These data will aid hospital pharmacists decide on a standardized protocol for the controlled usage of the ointment which is both safe and cost-effective.

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Levels of antagonist G required to produce a small but significant effect on the growth of SCLC cell lines in vitro are in the region of 4-7 microM. Taking into account metabolites, a peak concentration of 4.1 microgram/g (4.3 microM) was achieved in the H69 xenograft. These studies reveal a favourable preclinical pharmacology profile for antagonist G and offer hope that anticancer activity may be achievable in man.

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Summary 3,5-Dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (penclomedine, NSC 338720, CRC 88-04) is an a-picoline derivative with anti-tumour activity in preclinical models. Penclomedine administration by 1-h intravenous infusion on 5 consecutive days was repeated 3 weekly in the absence of dose-limiting toxicity (DLT) or disease progression. Five dose levels were investigated (22.5-340 mg m-2 day-'). Eight men and eight women were entered, median age 59 years (range 39-73 years), with good performance status (ECOG 0/1) in 11 patients. A total of 13 out of 16 patients had received previous chemotherapy. Common toxicity criteria grade (CTCg) II vomiting was recorded at all dose levels. Neurotoxicity (cerebellar ataxia and dizziness) was the DLT, CTCg IlIl toxicity occurring in three out of three patients treated at 340 mg m-2 day-'. CTCg IlIl dizziness was noted in one out of three patients at 250 mg m-2 day-1. Neurotoxicity developed during the 1-h infusion and persisted for a variable period (maximum 5 h) after infusion. Prophylactic antiemetic drugs appeared to reduce associated vomiting but did not prevent ataxia. No antiproliferative toxicities were noted and no anti-tumour responses were documented. Penclomedine pharmacokinetic studies confirmed preclinical evidence of extensive apparent distribution (931 m-2) and rapid clearance (41 1 h-1m-2). Purkinje cell loss has been identified in preclinical models after intraperitoneal administration (O'Reilly et al, 1 996a) and further clinical development of penclomedine will focus on oral administration.Keywords: phase l; penclomedine; neurotoxicity Penclomedine (3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl}-pyridine) was originally synthesized by Helen K Tobol, Dow Chemical, USA, as part of a programme to develop effective herbicides. It was identified as a potential anti-tumour agent by the NCI in vivo P388 leukaemia prescreen. After evidence of good activity against murine and human breast tumours, mouse CD8F, mammary carcinoma and human MX-1 mammary tumour xenograft (Plowman et al, 1989), penclomedine was selected for further studies and subsequent clinical development. The mechanism of action of penclomedine is unclear, although studies have been performed that suggest that it undergoes metabolism to yield reactive species that bind to DNA (Plowman et al, 1989; Reid et al, 1992;Benvenuto et al, 1995.) Penclomedine is poorly soluble in water but soluble in nonpolar solvents and lipids (Prankerd et al, 1989). Therefore, an experimental formulation of penclomedine as a 10% oil in water emulsion was developed for intravenous (i.v.) administration. Using this formulation, penclomedine was active against the advanced stage MX-1 mammary carcinoma: oral treatment with doses of 135 mg kg-' day-' for 5 days resulted in ten out of ten tumour-free survivors. The potency increased after intraperitoneal administration and the drug was most potent when administered i.v. (Harrison et al, 1991). No clear schedule dependency was been observed with the oral administratio...