Penny Johnson

Cancer cell adaptation to chemotherapy.

BackgroundTumor 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.MethodsCells 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.ResultsIn 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 IIα (TOPOIIα). Exposure to topotecan in vitro, resulted in >4-fold down-regulation of TOPOIIα in 6/7 colorectal tumors and 8/10 ovarian tumors.ConclusionThis 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.

Mechanisms of local immunosuppression in cutaneous melanoma.

Cutaneous melanoma is highly immunogenic, yet primary melanomas and metastases develop successfully in otherwise immunocompetent patients. To investigate the local immunosuppressive microenvironment, we examined the presence of suppressor T lymphocytes and tolerising dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFβ1 and TGFβ2) and the enzyme indoleamine 2,3-dioxygenase (IDO) using qRT–PCR and immunohistochemistry in primary skin melanomas, negative and positive sentinel lymph nodes (SLN), and lymph nodes with advanced metastases. Our results indicate that tolerogenic DCs and suppressor T lymphocytes are present in melanoma at all stages of disease progression. They express transforming growth factor β receptor 1 (TGFβR1), and are therefore susceptible to TGFβ1 and TGFβ2 specifically expressed by primary melanoma. We found that expression of IDO and interleukin 10 (IL-10) increased with melanoma progression, with the highest concentration in positive SLN. We suggest that negative SLN contain immunosuppressive cells and cytokines, due to preconditioning by tolerogenic DCs migrating from the primary melanoma site to the SLN. In primary melanoma, TGFβ2 is likely to render peripheral DCs tolerogenic, while in lymph nodes IDO and TGFβ1 may have a major effect. This mechanism of tumour-associated immunosuppression may inhibit the immune response to the tumour and may explain the discrepancy between the induction of systemic immunity by anti-melanoma vaccines and their poor performance in the clinic.

Targeting EGFR and PI3K pathways in ovarian cancer

Background:The epidermal growth factor receptor (EGFR) is expressed in ovarian cancer, but agents targeting this pathway have shown little effect as single agents. This may be due to the presence of alternative pathways, particularly activation of the PI3K/Akt/MTOR pathway.Methods:We have therefore examined the effect of inhibitors of this pathway (ZSTK474 and sirolimus) in combination with the EGFR inhibitors erlotinib and gefitinib in ovarian cancer primary cell cultures.Results:The single-agent EGFR inhibitors showed little activity, although some activity was seen with the single-agent PI3K inhibitor, ZSTK474. Combinations of ZSTK474 with EGFR inhibitors showed enhanced activity with some evidence of synergy, whereas sirolimus combinations were less active. The results were not explicable on the basis of PIK3CA mutation or amplification, or PTEN loss, although one tumour with a KRAS mutation showed resistance to EGFR inhibitors. However, there was correlation of the EGFR expression with sensitivity to EGFR and resistance to PI3K active agents, and inverse correlation in the sensitivity of individual tumours to agents active against these pathways, suggesting a mechanism of action for the combination.Conclusion:Phase I/II clinical trials with these agents should include further pharmacodynamic endpoints and molecular characterisation to identify patients most likely to benefit from this strategy.

Prognostic significance of cyclooxygenase-2 (COX-2) expression in patients with surgically resectable adenocarcinoma of the oesophagus.

BackgroundCOX-2 expression in tumour cells has been associated with poor prognosis in gastrointestinal and non-gastrointestinal cancers. The aim of our study was to test the hypothesis that higher levels of COX-2 expression are prognostically related to poor clinico-pathologic features in adenocarcinoma of the oesophagus.MethodsWe reviewed the records of 100 consecutive patients undergoing resection for adenocarcinoma of the oesophagus to collect data on T-stage, N-stage, tumour recurrence and survival. T & N-stage was further confirmed by histological examination. COX-2 protein expression was assessed by immunohistochemistry in all patients and COX-2 m-RNA expression was measured by quantitative RT-PCR in a small group of patients.ResultsHigher levels of COX-2 expression were associated with higher T stage (p = 0.008), higher N stage (p = 0.049), increased risk of tumour recurrence (p = 0.01) and poor survival (p = <0.001). A COX-2 score of >200 was associated with a median survival of 10 months compared to 26 months with a score of <200 (p = <0.001).ConclusionHigher levels of COX-2 expression are associated with poor clinico-pathologic features and poor survival in patients with oesophageal adenocarcinoma.

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.

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. Copyright

Presence and phenotype of dendritic cells in uveal melanoma

Background: Uveal melanoma arises in an immune-privileged site and can itself add to the immunosuppressive environment. Previous studies on cutaneous melanoma have shown the presence of tolerogenic dendritic cells (DCs), which could play an important role in the progression of the tumour. Aim: To examine the presence and functional status of DCs in a small series of uveal melanomas. Methods: 10 cases of uveal melanoma were examined for the expression of FXIIIa, CD68, human leucocyte antigen (HLA)-DR, CD40, CD83, transforming growth factor βR1 and indolamine 2,3 dioxygenase by immunohistochemical analysis on sections embedded in paraffin wax. Results: CD68-positive macrophages were present in all of the tumours and were evenly distributed throughout. DCs expressing FXIIIa-positive were seen in 7 cases, and were often found concentrated in foci within the tumour mass. These cells were dendritic and expressed high levels of HLA-DR. The DCs did not express the maturation markers CD83 or CD40. In one case, concentration of DCs around the area of tumour necrosis was observed, and some of these cells expressed CD83. Conclusion: Numerous tolerising antigen-presenting cells may play a role in melanoma-related immunosuppression in the eye, although activation of DCs may be associated with tumour necrosis.

Ex vivo reversal of chemoresistance by tariquidar (XR9576).

The expression of P-glycoprotein (P-gp) has been demonstrated to confer resistance to several anticancer drugs, including anthracyclines, taxanes and vinca alkaloids. Tariquidar is a novel inhibitor of P-gp that has been shown to reverse resistance to cytotoxic drugs in tumor cell lines and mouse xenografts. We have used an ATP-based chemosensitivity assay (ATP-TCA) to compare the activity of cytotoxic drugs in combination with tariquidar against a variety of solid tumors (n=37). The expression of P-gp was determined in a subset of solid tumor samples by immunohistochemistry (n=16). Resistance was seen in 20 of 37 (54%) tumors tested with doxorubicin, in 27 of 34 (79%) samples tested with paclitaxel and 17 of 31 (55%) with vinorelbine. Tariquidar alone showed no activity over a wide range of concentrations up to 2 μM (n=14). The median IC90s for doxorubicin, paclitaxel and vinorelbine, alone were 2.57, 27.4 and 15.5 μM. These decreased to 1.67 (p<0.0005), 20.6 (p<0.05) and 9.5 μM (p<0.001), respectively, in combination with tariquidar. Tariquidar also significantly decreased resistance in 14 of 20 (70%), six of 27 (22%) and six of 17 (35%) samples tested with doxorubicin, paclitaxel and vinorelbine, respectively. Immunohistochemical staining for P-gp was positive in nine of 16 (56%) samples and in all of these cases addition of tariquidar improved the activity of the cytotoxic. The results show that tariquidar is able to decrease resistance in a number of solid tumors resistant to cytotoxic drugs known to be P-gp substrates. These data support the introduction of tariquidar in combination with chemotherapy to clinical trials of patients expressing P-gp.

Activity of EGFR, mTOR and PI3K inhibitors in an isogenic breast cell line model

BackgroundThe epidermal growth factor receptor family is expressed in breast cancer, and agents targeting this pathway have single agent effects (e.g. traztuzumab). Development of resistance may be due to the presence of alternative pathways, particularly activation of the PI3K/Akt/MTOR pathway. We have therefore examined the effect of inhibitors of this pathway (ZSTK474 and sirolimus) in combination with the epidermal growth factor (EGFR) inhibitors erlotinib and gefitinib in breast MCF10a isogenic cell lines with EGFR, BRAF, AKT, and PI3K mutations.ResultsPI3K mutation conferred increased activity of EGFR inhibitors against MCF10a cells in comparison with the parental cell line and other mutations studied. Combination of EGFR inhibitors with either the PI3K inhibitor ZSTK474 or the MTOR inhibitor sirolimus showed increased activity.ConclusionsThese results are encouraging for the use of combinations targeting the PI3K and EGFR pathway simultaneously.