Polyacetal-diethylstilboestrol: a polymeric drug designed for pH-triggered activation

Those polymer anticancer-drug conjugates currently undergoing clinical evaluation have a tripartite structure; a water-soluble polymer, an anticancer agent and a pendant linker. To simplify the construct it would be attractive to develop anticancer polymer therapeutics that contain the bioactive agent as an integral part of the polymer backbone. The aim of this study was to utilise the reaction between a divinyl ethers and diols, to synthesise polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumours would then trigger drug release eliminating the need for a biodegradable linker. A tert-polymerisation approach was used to incorporate non-steroidal oestrogen diethylstilboestrol (DES) into the mainchain of water-soluble polyacetals synthesised using as co-monomer PEG of Mw 2900 or 3400 g/mol. When PEG2900 was used the resultant polymer had a Mw of 18,900 g/mol, a Mw/Mn of 1.9 and a DES loading 4.3 wt.%. With PEG3400 the polymer Mw was 43,000 g/mol, Mw/Mn=1.8 and it had a DES loading 4.7 wt.%. 1H-NMR confirmed the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25-1.3(d) and 4.7-4.8(q) ppm and from DES at 1.55-1.6(d) and 5.4-5.5(q) ppm. These were consistent with the acetal signals observed for the non-water-soluble co-polymer DES: tri(ethylene glycol) divinyl ether (TEGDVE) (1 : 2, Mw=6859 g/mol, Mw/Mn=1.3). When evaluated in vitro, the DES-polyacetal displayed greater cytotoxicity than DES against human and murine tumour cell lines (IC50=48 and 420 microg/ml against MCF-7 human breast cancer cells and IC50=97 and 560 microg/ml against B16F10 murine melanoma cells, respectively). These polymers showed no significant haemolysis at concentrations up to 20 mg/ml confirming suitability for further in vivo evaluation. An enhanced rate of hydrolytic degradation of the polymer backbone was seen at pH 5.5, (65% trans-DES released in 96 h), compared to pH 7.4 (4% trans-DES released in 96 h). These bioresponsive DES-polyacetals tert-polymers are the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release of a drug incorporated into the polymer mainchain. Their in vitro characteristics suggest further in vivo evaluation is warranted.     

Her2/neu is not a commonly expressed therapeutic target in melanoma -- a large cohort tissue microarray study

Melanoma is among the most chemotherapy-resistant malignancies. Numerous new agents have been developed that target specific molecules on cancer cells, including the monoclonal antibody trastuzumab, which targets Her2/neu and has been very beneficial in the treatment of breast cancer. There are conflicting reports in the literature about Her2/neu expression in melanoma specimens, but all of the cohorts studied have been small. We therefore examined Her2/neu expression in a very large cohort of melanoma specimens in order to determine the value of exploring trastuzumab therapy for melanoma patients. Immunohistochemical staining was performed on two tissue microarrays, together containing 600 intact specimens. Expression was evaluated semi-quantitatively and correlated with tumour stage and other clinicopathological data. Of the 600 specimens in the cohort, 31 patients (5.2%) had positive Her2/neu expression. Among the primary cutaneous specimens (n=269), 7% had positive Her2/neu staining, while 3.6% of the recurrent or metastatic specimens (n=331) had positive Her2/neu staining (P=0.06). Among the primary lesions there was no significant correlation between Her2/neu expression, Clark level and ulceration; however, Her2/neu expression was associated with lesions with a Breslow depth of < 2 mm (P=0.05). Using this very large cohort of melanoma specimens, we found only a few cases with aberrant Her2/neu expression, many of them being primary cutaneous lesions rather than recurrent or metastatic lesions. Our findings suggest that drugs that specifically target Her2/neu are not likely to be useful for the treatment of metastatic melanoma or as adjuvant therapy for melanoma patients at high risk for recurrence.     

Cytotoxic T-cell immunity against telomerase reverse transcriptase in colorectal cancer patients

The catalytic subunit of telomerase (hTERT) has recently been proposed as a potential tumour-associated antigen capable of inducing T-cell mediated immunity in cancer patients. Before any attempts at vaccination with hTERT antigens can be made, one should establish if cancer patients possess cytotoxic T-lymphocytes (CTL) that can recognise hTERT epitopes. The T-cell response against two HLA-A2-specific epitopes of hTERT in 37 colorectal cancer patients and 12 normal controls was analysed using an interferon gamma (IFN-gamma) ELISPOT assay. For comparison the response to HLA-A2-restricted epitopes of CEA and influenza A matrix protein was also measured. CTL that recognised either of the two hTERT epitopes studied were found in 7 (19%) of colorectal cancer patients, with 2 (5%) possessing T-cells that recognised both these peptides. Four (11%) colorectal cancer patients had CTL that reacted to the CEA epitope. No relationship between cancer stage and the presence of specific CTL against hTERT or CEA was observed. None of the normal controls possessed T-cells capable of recognising either the hTERT or the CEA epitopes. However, a similar proportion of patients and normal controls had CTL reactive with the influenza A peptide. The results of this study demonstrate that CTL active against hTERT are present in approximately 20% of colorectal cancer patients irrespective of disease stage. Moreover, these cells are functional, able to secrete IFN-gamma when stimulated with the relevant peptide.     

The 1100delAT BRCA1 and the 8765delAG BRCA2 Mutations: Occurrence in High-Risk Non-Ashkenazi Jews and Haplotype Comparison of Jewish and Non-Jewish Carriers

Few mutations have been described in BRCA1 and BRCA2 in high-risk non-Ashkenazi Jews. In a Libyan family the 1100delAT BRCA1 mutation was detected and the 8765delAG BRCA2 mutation was previously described in two Jewish-Yemenite-families. In this study, the rate of these mutations in high-risk Jews of North African and Yemenite origin was assessed, and the BRCA1 -linked haplotype of Jewish and non-Jewish 1100delAT mutation carriers were compared. Genotyping included 64 high-risk Yemenite women (tested only for the BRCA 2 mutation) and 147 high-risk North African women, tested for both mutations. PCR amplification was followed by either restriction enzyme digestion or DGGE or dHPLC analyses and direct sequencing. For haplotyping, 5 BRCA1 -linked markers were used. Neither the 1100delAT BRCA1 nor the 8765delAG BRCA2 mutations were detected in any non-Ashkenazi individual. The haplotype of the non-Jewish 1100delAG mutation carrier differed from that of the Jewish-Libyan mutation carriers. We conclude that both1100delAT BRCA1 and 8765delAG BRCA2 mutations occur rarely in high-risk non-Ashkenazi Jews, and while the latter seems to be a founder mutation in some populations, the former occurs on a different background in ethnically diverse families.     

Cell-specific effects of RB or RB/p107 loss on retinal development implicate an intrinsically death-resistant cell-of-origin in retinoblastoma

Retinogenesis involves expansion of pluripotent progenitors, specification of postmitotic precursors, and terminal differentiation. Rb or Rb/p107 loss causes retinoblastoma in humans or mice, respectively. One model suggests that Rb- or Rb/p107-deficient retinal precursors have infinite proliferative capacity but are death-prone and must acquire an antiapoptotic mutation. Indeed, we show that Rb/p107 loss does not affect progenitor proliferation or precursor specification, but perturbs cell cycle exit in all seven retinal precursors. However, three precursors survive Rb/p107-loss and stop proliferating following terminal differentiation. Tumors arise from precursors that escape this delayed growth arrest. Thus, retinoblastoma arises from a precursor that has extended, not infinite, proliferative capacity, and is intrinsically death-resistant, not death-prone. We suggest that additional lesions common in retinoblastoma overcome growth arrest, not apoptosis.     

Pubertal maturation in girls and the relationship to anthropometric changes: pathways through puberty

OBJECTIVE: Patterns of pubertal maturation may have an impact on several risk factors associated with adult morbidity and mortality, such as obesity. We examined the relationship of the initial manifestation of puberty in girls with anthropometric measures, as well as age at menarche. METHODS: White females (n = 1166, ages 9 and 10 at intake) were followed with annual visits for 10 years. Physical examinations included height, weight, skinfold thicknesses, and pubertal maturation assessment. RESULTS: During the course of the study, 443 of 859 eligible females (51.6%) were observed to have asynchronous maturation in the development of puberty, that is, initial areolar/breast (thelarche pathway) or pubic hair (adrenarche pathway) development, without development of the other characteristic. Using a longitudinal regression model, significant interactions were noted between initial pubertal manifestation and years since onset of puberty on the following outcomes: sum of skinfolds thickness, percent body fat, waist-to-hip ratio, and body mass index (BMI). However, age of onset of pubertal maturation was the same in the 2 groups (10.7 years). Females in the thelarche pathway had earlier menarche (12.6 vs 13.1 years) as well as greater skinfolds, body fat, and BMI at the time of menarche. Females in the thelarche pathway also had greater body fat and BMI 1 year before puberty and throughout puberty compared with those in the adrenarche pathway. CONCLUSIONS: Females who enter puberty through the thelarche pathway, as compared with the adrenarche pathway, had greater sum of skinfold thicknesses, BMI, and percent body fat 1 year before the onset, as well as throughout, puberty. Because larger body composition and earlier age of menarche of females in the thelarche pathway parallel the epidemiologic profiles of women who are obese or at risk for obesity, these females may be at greater risk for adult obesity.     

Excretion of serotype G1 rotavirus strains by asymptomatic staff: a possible source of nosocomial infection

This study supports the hypothesis that feces from asymptomatic adults may provide a vehicle for the transmission of rotavirus, in addition to aerosols, hands, and fomites. The observed preferential carriage of serotype G1 strains in the adult gastrointestinal tract may explain G1 predominance and persistence in epidemiologic studies worldwide.