The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures.

The tricyclic antidepressants have previously been shown to exert activity against glioma cells in vitro. Initial studies in cell lines suggested that this might extend to melanoma cells. We have therefore conducted a study in primary cell cultures from metastatic cutaneous melanoma deposits using a well established ATP-based tumour chemosensitivity assay to confirm and extend these findings. Two cell lines and eight primary cell cultures from metastatic melanoma deposits were exposed to three tricyclic drugs, amitriptyline, nortriptyline and clomipramine, at concentrations ranging from 200 to 6.25 µmol/l in the ATP-based tumour chemosensitivity assay. All three drugs showed activity, although nortriptyline was more active than clomipramine or amitriptyline in both cell lines and primary cell cultures, with an IC50 of 9, 27 and 33 µmol/l, respectively. Tricyclic agents show activity against melanoma in vitro. This could be related to the lysosomal effects based on their cationic amphiphilic properties, or effects at the mitochondrial membrane.

The molecular basis of the chemosensitivity of metastatic cutaneous melanoma to chemotherapy.

BACKGROUND: Chemotherapy benefits relatively few patients with cutaneous melanoma. The assessment of tumour chemosensitivity by the ATP-based tumour chemosensitivity assay (ATP-TCA) has shown strong correlation with outcome in cutaneous melanoma, but requires fresh tissue and dedicated laboratory facilities. AIM: To examine whether the results of the ATP-TCA correlate with the expression of genes known to be involved in resistance to chemotherapy, based on the hypothesis that the molecular basis of chemosensitivity lies within known drug resistance mechanisms. METHOD: The chemosensitivity of 47 cutaneous melanomas was assessed using the ATP-TCA and correlated with quantitative expression of 93 resistance genes measured by quantitative reverse transcriptase PCR (qRT-PCR) in a Taqman Array after extraction of total RNA from formalin-fixed paraffin-embedded tissue. RESULTS: Drugs susceptible to particular resistance mechanisms showed good correlation with genes linked to these mechanisms using signatures of up to 17 genes. Comparison of these signatures for DTIC, treosulfan and cisplatin showed several genes in common. HSP70, at least one human epidermal growth factor receptor, genes involved in apoptosis (IAP2, PTEN) and DNA repair (ERCC1, XPA, XRCC1, XRCC6) were present for these agents, as well as genes involved in the regulation of proliferation (Ki67, p21, p27). The combinations tested included genes represented in the single agent signatures. CONCLUSIONS: These data suggest that melanoma chemosensitivity is influenced by known resistance mechanisms, including susceptibility to apoptosis. Use of a candidate gene approach may increase understanding of the mechanisms underlying chemosensitivity to drugs active against melanoma and provide signatures with predictive value.

The effectiveness of scoring and bipolar diathermy on ear cartilage behavior: ex vivo study.

A common unsatisfactory long-term outcome of otoplasty is undercorrection and residual deformity. Aggressive scoring with a scalpel blade can cause unattractive and painful ridging, especially in older patients with stiffer cartilage. We developed an ex vivo pig ear model to compare the effectiveness of bipolar diathermy with blade scoring for cartilage warping. Strips of cartilage harvested from Landrace pig ears were subjected to bipolar diathermy (group A), blade scoring (group B), and blade scoring combined with reverse-side bipolar diathermy (group C). The strip deflection and cartilage tension were measured for each group. No statistical difference (P

The effect of imatinib mesylate (Glivec) on human tumor-derived cells.

Imatinib mesylate is a specific inhibitor of the Bcr-Abl protein tyrosine kinase that competes with ATP for its specific binding site in the kinase domain. It has activity against platelet-derived growth factor receptor alpha and beta (PDGFR-alpha and -beta), and c-kit, the receptor for stem cell factor. We have used a standardized ATP-tumor chemosensitivity assay and immunohistochemistry to determine the cytotoxicity of imatinib mesylate in tumor-derived cells from cutaneous and uveal melanoma, and ovarian carcinoma. Imatinib mesylate was tested at concentrations ranging from 2.0 to 0.0625 micromol/l alone and in combination with a cytotoxic drug (cisplatin, doxorubicin, paclitaxel or treosulfan). Imatinib mesylate showed low inhibition (IndexSUM>300) across the range of concentrations tested in this study, with few tumors exhibiting increasing inhibition with increased drug concentration. The median IC90 values for cutaneous and uveal melanoma and ovarian carcinoma were 13.2 micromol/l (4.0-294.3 micromol/l), 12.0 micromol/l (2.0-285.4 micromol/l) and 7.71 micromol/l (6.51-11.02 micromol/l), respectively. Imatinib mesylate potentiated the effect of different cytotoxics in 9% (5/54) of cases and had a negative effect in 13% (7/54) of cases, with no effect in the remainder. No correlation of effect was noted with c-kit, platelet-derived growth factor receptor-alpha or platelet-derived growth factor receptor-beta expression, assessed by immunohistochemistry. The signaling pathways mediated by activation of c-kit or platelet-derived growth factor receptor may act as antiapoptotic survival signals in some cancers and inhibition of these pathways may potentiate the activity of some cytotoxic drugs by inhibiting the survival signal. Growth inhibition, however, may reduce the efficacy of cytotoxic drugs, which tend to target proliferating cells preferentially, and clinical effects are therefore difficult to predict.

Cancer cell adaptation to chemotherapy.

BACKGROUND: Tumor resistance to chemotherapy may be present at the beginning of treatment, develop during treatment, or become apparent on re-treatment of the patient. The mechanisms involved are usually inferred from experiments with cell lines, as studies in tumor-derived cells are difficult. Studies of human tumors show that cells adapt to chemotherapy, but it has been largely assumed that clonal selection leads to the resistance of recurrent tumors. METHODS: Cells derived from 47 tumors of breast, ovarian, esophageal, and colorectal origin and 16 paired esophageal biopsies were exposed to anticancer agents (cisplatin; 5-fluorouracil; epirubicin; doxorubicin; paclitaxel; irinotecan and topotecan) in short-term cell culture (6 days). Real-time quantitative PCR was used to measure up- or down-regulation of 16 different resistance/target genes, and when tissue was available, immunohistochemistry was used to assess the protein levels. RESULTS: In 8/16 paired esophageal biopsies, there was an increase in the expression of multi-drug resistance gene 1 (MDR1) following epirubicin + cisplatin + 5-fluorouracil (ECF) chemotherapy and this was accompanied by increased expression of the MDR-1 encoded protein, P-gp. Following exposure to doxorubicin in vitro, 13/14 breast carcinomas and 9/12 ovarian carcinomas showed >2-fold down-regulation of topoisomerase IIalpha (TOPOIIalpha). Exposure to topotecan in vitro, resulted in >4-fold down-regulation of TOPOIIalpha in 6/7 colorectal tumors and 8/10 ovarian tumors. CONCLUSION: This study suggests that up-regulation of resistance genes or down-regulation in target genes may occur rapidly in human solid tumors, within days of the start of treatment, and that similar changes are present in pre- and post-chemotherapy biopsy material. The molecular processes used by each tumor appear to be linked to the drug used, but there is also heterogeneity between individual tumors, even those with the same histological type, in the pattern and magnitude of response to the same drugs. Adaptation to chemotherapy may explain why prediction of resistance mechanisms is difficult on the basis of tumor type alone or individual markers, and suggests that more complex predictive methods are required to improve the response rates to chemotherapy.

Presence and maturity of dendritic cells in melanoma lymph node metastases.

Immune avoidance mechanisms play a key role in the successful dissemination of melanoma. One mechanism whereby this could be achieved is by interfering with dendritic cell (DC) presentation of tumour-associated antigens to naïve T cells. In particular, immature DCs characterized by the absence of accessory molecules are known to be immunosuppressive and to be involved in the induction of tolerance. The present study has investigated the presence and activation status of DCs within melanoma metastases in the regional lymph nodes. Using image analysis techniques, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA-DR and the morphological features of DCs were examined in paraffin sections from 26 lymph nodes containing melanoma metastases. DCs expressing FXIIIa were found in 70% of the lymph nodes. The number of DCs identified was generally small but there were more concentrated areas of DCs designated as hotspots. In these areas of high FXIIIa staining, the percentage area occupied by DCs varied between 0.1% and 10%. The majority of FXIIIa-positive cells did not express the DC maturation markers CD83 or CD40 and morphologically were rounded with few dendrites, indicating that they were immature. The cells did, however, express high levels of HLA-DR, suggesting that they have the ability to present antigen but lack the accessory molecules required to initiate an immune response. Immature DCs, characterized by phenotypic and morphological features, are therefore present within the tumour deposits in lymph nodes infiltrated by melanoma and may specifically modulate the anti-melanoma immune response.

Macroscopic quality control improves the reliability of blue dye-only sentinel lymph node biopsy in breast cancer.

BACKGROUND: One of the problems of sentinel lymph node (SLN) biopsy is the risk of false negatives. At the Institut Curie, to reduce the false-negative rate, we have developed a histological quality control of the SLN performed by blue dye alone, which consists of verification of the SLN blue stain by the pathologist. METHODS: A total of 324 patients underwent an SLN biopsy procedure with patent blue dye only followed by an immediate axillary dissection. Initially, SLNs were checked to ensure that they were blue by macroscopic examination. Finally, a search for immunohistochemistry micrometastasis was performed. RESULTS: In 277 (85.5%) of 324 patients, an SLN was identified by the surgeon. After standard examination, the false-negative rate was 11.1% (10 of 90). After macroscopic checking of the 197 negative SLNs, 167 of the 197 were confirmed blue, and there were 5 false negatives, which brought the false-negative rate down to 5.6% (5 of 90). Sixty SLNs out of the 167 confirmed blue SLNs were then proved to be immunohistochemically micrometastatic, and there were 3 false negatives, giving a final false-negative rate of 2.2% (2 of 90; P =.002). CONCLUSIONS: In this series, the procedure of pathologic analysis of the SLN has resulted in a significant reduction of the false-negative rate.