Denis M. Collins

Trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in HER-2-non-amplified breast cancer cell lines

BACKGROUND Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD56+natural killer (NK) cells may contribute to the activity of trastuzumab in HER-2-amplified tumours. In this study, we investigated the possibility that trastuzumab might induce ADCC against HER-2-non-amplified breast cancer cells. METHODS Induction of NK cell-mediated ADCC was examined in trastuzumab-treated HER-2-non-amplified breast cancer cell lines. HER-2 protein levels were also determined in tumour and autologous normal tissue samples from patients with HER-2 negative breast cancer. RESULTS Trastuzumab induced a significant ADCC response in the HER-2-amplified HCC1954 and SKBR3 cell lines, and in all five of the non-amplified cell lines, which had low levels of detectable HER-2 by western blot (CAL-51, CAMA-1, MCF-7, T47D, and EFM19). Trastuzumab did not induce ADCC in the K562 control cell line or MDA-MB-468, which has very low levels of HER-2 detectable by enzyme-linked immunosorbent assay (ELISA) only. HER-2 protein was detected by ELISA in 14/15 patient tumour samples classified as HER-2-non-amplified. Significantly lower levels of HER-2 were detected in normal autologous tissue compared with tumour samples from the same patients. CONCLUSION Our results suggest that HER-2-non-amplified breast cancer cells, with low but detectable levels of HER-2 protein, can bind trastuzumab and initiate ADCC.

Tyrosine kinase inhibitors potentiate the cytotoxicity of MDR-substrate anticancer agents independent of growth factor receptor status in lung cancer cell lines

SummaryTo investigate the interactions of Epidermal Growth Factor Receptor (EGFR)-inhibiting tyrosine kinase inhibitors (TKIs) on P-gp-mediated drug resistance, we tested three TKIs, lapatinib, gefitinib and erlotinib in direct ATPase assays and in Non-Small Cell Lung Cancer (NCSLC) cell lines with defined low levels of growth factor receptor expression. The three TKIs potentiated the action of known P-gp substrate cytotoxic drugs at therapeutically-relevant concentrations. However, more detailed analysis revealed that the interaction of lapatinib with P-gp was distinct from that of gefitinib and erlotinib, and was characterised by direct inhibition of the stimulated P-gp ATPase activity. Lapatinib proved the most potent P-gp modulator of the TKIs examined. Drug transport studies in the P-gp-over-expressing A549-Taxol cell line showed that lapatinib and erlotinib are capable of increasing docetaxel accumulation at clinically achievable concentrations. Combination studies with P-gp substrate chemotherapeutic agents, demonstrated that all three TKIs have significant potential to augment cytotoxic activity against P-gp-positive malignancies, however, interestingly, these agents also potentiated the toxicity of epirubicin in non-P-gp resistant parental cells. Our observations suggest that the combination of lapatinib with a taxane or anthracycline warrants clinical investigation in NSCLC to examine if beneficial or detrimental interactions may result.

Membrane transport proteins in human melanoma: associations with tumour aggressiveness and metastasis.

Background:Malignant melanoma, generally described as incurable, is notoriously refractory to chemotherapy. The mechanisms contributing to this have not yet been defined and the contributions of drug efflux pumps, implicated in chemo-resistance of many other cancer types, have not been extensively investigated in melanoma.Methods:In this study, expression of multi-drug resistant (MDR1/P-gp and MRP-1) proteins was examined, by immunohistochemistry, in archival specimens from 134 melanoma patients. This included 92 primary tumours and 42 metastases.Results:On assessing all specimens, MRP-1 and MDR1/P-gp expression was found to be common, with the majority (81%) of melanomas expressing at least one of these efflux pumps. Although there is significant association between expression of these pumps (P=0.007), MRP-1 was found to be the predominant (67% of cases) form detected. χ2 analysis showed significant associations between expression of MRP-1 and/or MDR1/P-gp and the aggressive nature of this disease specifically increased Breslows depth, Clarks level and spread to lymph nodes. This association with aggressiveness and spread is further supported by the observation that a significantly higher percentage of metastases, than primary tumours, express MRP-1 (91% vs 57%; P<0.0001) and MDR1/P-gp (74% vs 50%; P=0.010).Conclusion:The predominant expression of these pumps and, in particular, MRP-1 suggests that they may be important contributors to the inherent aggressive and resistant nature of malignant melanoma.

Gastrointestinal perforation in metastatic carcinoma: a complication of bevacizumab therapy.

Bevacizumab (Avastin , Genentech, Inc.) is a humanised monoclonal antibody directed against Vascular Endothelial Growth Factor (VEGF). Developed by Napoleone Ferrara and colleagues, it was the first commercially available antiangiogenesis therapy to demonstrate efficacy and improved survival in patients with metastatic colorectal carcinoma. Bevacizumab is relatively new to clinical usage. It is FDA approved since 2004, and is generally well tolerated. As the glycoprotein VEGF is produced by normal tissues, antiVEGF therapy has been associated with notable adverse effects including hypertension, haemorrhage, arterial thrombosis and poor wound healing. Anecdotally, increased frequency of gastrointestinal perforation has been reported. Recent randomised trials evaluating Bevacizumab in combination with 5-FU based chemotherapy have demonstrated GI perforation rates of up to 3.6%. Much more data is needed to elucidate this issue owing to confounding factors. For example, the reported incidence of perforated colorectal cancer varies between 2.6 and 9%, therefore, in patients treated with bevacizumab, are these perforations simply a reflection of the natural history of the cancer due to improved survival following therapy? This is unlikely, as GI perforation not only occurs at the tumour site but also at remote anatomical locations. In addition, data regarding whether the primary tumour is intact is not reported in over 50% of the available trials.

Cytomegalovirus pneumonia in a patient with breast cancer on chemotherapy: Case report and review of the literature

Cytomegalovirus (CMV) pneumonia in the setting of non-transplantation patients is a rarity. We present a case of CMV pneumonitis in a woman with stage IV breast cancer, with brain metastases, receiving both chemotherapy and systemic corticosteroids. A review of the literature reveals this as a unique case. Potential viral etiologies should therefore be considered in cancer patients with pneumonia receiving non-transplantation chemotherapy-regimens, particularly if steroids are a component of their therapy.

Modulation of P-gp expression by lapatinib

SummaryChemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

Direct estrogen receptor (ER) / HER family crosstalk mediating sensitivity to lumretuzumab and pertuzumab in ER+ breast cancer

Bidirectional cross talk between members of the human epidermal growth factor family of receptors (HER) and the estrogen receptor (ER) is believed to underlie resistance mechanisms that develop in response to treatment with anti-HER agents and endocrine therapy. We investigated the interaction between HER2, HER3 and the ER in vitro using human embryonic kidney cells transfected with human HER2, HER3, and ERα. We also investigated the additive efficacy of combination regimens consisting of anti-HER3 (lumretuzumab), anti-HER2 (pertuzumab), and endocrine (fulvestrant) therapy in vivo. Our data show that both HER2 and HER3 can directly complex with the ER and can mediate phosphorylation of the ER. Phosphorylation of the ER was only observed in cells that expressed both HER2 and ERα or in heregulin-stimulated cells that expressed both HER3 and ERα. Using a mouse xenograft model of ER+/HER2-low (HER2 immunohistochemistry 1+ or 2+ without gene amplification) human breast cancer we show that the combination of lumretuzumab and pertuzumab is highly efficacious and induces long-lasting tumor regression in vivo and adding endocrine therapy (fulvestrant) to this combination further improved efficacy. In addition, a prolonged clinical response was observed with the combination of lumretuzumab and pertuzumab in a patient with ER+/HER2-low breast cancer who had failed endocrine therapy. These preclinical data confirm that direct cross talk exists between HER2/HER3 and ER which may explain the resistance mechanisms to endocrine therapy and monoclonal antibodies that target HER2 and HER3. Our data also indicate that the triplet of anti-HER2, anti-HER3, and endocrine therapy might be an efficacious combination for treating patients with ER+/HER2-low breast cancer, which is an area of significant unmet medical need.

Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.

Innovative Technologies Changing Cancer Treatment

Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional therapeutic options in cancers with poor survival outcomes. New therapeutic strategies involving areas like energy metabolism and extracellular vesicles along with advances in immunotherapy and nanotechnology are driving the next generation of cancer treatments. The development of fields such as theranostics in nanomedicine is also opening new doors for targeted drug delivery and nano-imaging. Here we discuss the use of innovative technologies presented at the Irish Association for Cancer Research (IACR) Annual Meeting, highlighting examples of where new approaches may lead to promising new treatment options for a range of cancer types.

Abstract P5-11-03: Tumor CXCL16/CXCR6 expression and soluble CXCL16 in HER2+ breast cancer (BC)

Background: CXCL16 is a pro-inflammatory chemokine associated with chemotaxis of CXCR6-expressing lymphocytes (T cells, NKT cells, NK cells) and the promotion of breast cancer cell proliferation, migration, and invasion in vitro . CXCL16 exists in a transmembrane or a cleaved, soluble form. There is limited clinically relevant data available on the CXCL16/CXCR6 signaling axis in HER2+ BC. This preliminary study examines tumor CXCL16 and CXCR6 mRNA expression and patient outcome in publicly available datasets and examines soluble CXCL16 in the plasma of 32 HER2+ BC patients enrolled in ICORG 10-05 (neo-adjuvant chemotherapy (docetaxel/carboplatin) +/- trastuzumab, lapatinib or trastuzumab/lapatinib). Methods: CXCL16 and CXCR6 mRNA expression was interrogated in publicly available datasets using BreastMark, a web-based tool for preliminary assessment of putative biomarkers in breast cancer.A median cut-off was used for all analyses. Pre-treatment and post-treatment (2 weeks pre-surgery) blood samples were collected from patients enrolled in ICORG 10-05. Plasma CXCL16 levels were determined by Luminex xMAP assay. Pre- and post-treatment levels of CXCL16 were compared directly or based on response (pathological complete response, CR, n=14 or non-pathological complete response, nCR, n=18). Stromal lymphocyte (SL) and tumor-infiltrating lymphocyte (TIL) levels were determined from Haematoxylin and Eosin-, AE1/AE3- and CD45FFPE- stained formalin-fixed paraffin embedded tissue. Pre-treatment lymphocyte levels were correlated with pre- and post-treatment levels of CXCL16 (Pearson9s product-moment correlation). Results: In BC as a whole, analysis of publicly available datasets reveals tumor CXCL16 expression is not associated with outcome (n=1238, HR=0.9953, p=0.9516) but high tumor CXCR6 expression is (n=2652, HR=1.127, p=0.0476). Within a HER2+ cohort, inverse correlative analysis suggests high CXCR6/low CXCL16 tumor expression is significantly associated with a worse outcome (n=61, HR=2.731, p=0.01). For ICORG 10-05 patients , circulating CXCL16 plasma levels were significantly higher post-treatment (p Conclusions: Our preliminary results suggest tumor levels of CXCL16/CXCR6 are associated with patient outcome and circulating levels of soluble CXCL16 are altered by treatment and correlate with tumor immune infiltrate in HER2+ BC patients. Further examination of tumor CXCL16/CXCR6 expression and circulating CXCL16 as potential biomarkers of response is warranted in a larger cohort of HER2+ BC patients. Citation Format: Collins DM, Madden SF, Eustace AJ, Toomey S, Kay EW, Fay J, O9Donovan N, Gallagher WM, Hennessy B, Crown J. Tumor CXCL16/CXCR6 expression and soluble CXCL16 in HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-11-03.