Daniele Spada

Targeted inhibition of the epidermal growth factor receptor‐tyrosine kinase by ZD1839 (‘Iressa’) induces cell‐cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 (‘Iressa’), an orally active EGFR—tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen‐responsive (LNCaP) and androgen‐independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell‐cycle and on protein expression levels, respectively. ZD1839 caused a dose‐ and time‐dependent growth inhibition in all three cell lines. A dose‐dependent supra‐additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G0/G1 phase of the cell‐cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27Kip1, both in the cytoplasmic and nuclear fractions. The increase in p27Kip1 was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR‐associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer. J. Cell. Physiol. 201: 97–105, 2004.

Increased expression of dystroglycan inhibits the growth and tumorigenicity of human mammary epithelial cells.

Dystroglycan (DG) is an adhesion molecule formed by two subunits, _ (extracellular) and _ (transmembrane) DG, which are codified by a single gene and form a continuous link from the extracellular matrix to the intracellular cytoskeleton. Reduction or loss of expression of DG has been observed in human cancer cell lines and primary tumors and has been suggested to promote tumor development and invasiveness. In this study, the human breast epithelial non-tumorigenic MCF10F and the breast cancer MCF7 cell lines were engineered to stably express an exogenous DG cDNA and the effects on the phenotype of both cell lines were evaluated. The MCF10F transfected cells displayed an increased expression of both DG subunits which was associated with inhibition of the anchorage-dependent growth, accumulation of cells in the G0/G1 phase of the cell cycle and increased adhesion to a substratum. The MCF7 transfected cells were unable to restore _-DG despite an increased expression of the _-DG subunit. Anchorage-dependent and independent growth and the in vivo tumorigenicity were reduced in these derivatives that also displayed a reduced adhesion to a substratum and were shown to release _-DG in the culture medium. These findings confirm and extend previous evidence that transformation of mammary epithelial cells is associated with loss of their ability to retain _-DG on the cell membrane. Moreover, they indicate that DG is involved in cell functions other than cell adhesion to the extracellular matrix, and that its loss of function might predispose to tumor progression by compromising regulatory controls over cell growth and proliferation.

Contribution of KRAS mutations and c.2369C > T (p.T790M) EGFR to acquired resistance to EGFR-TKIs in EGFR mutant NSCLC: a study on circulating tumor DNA.

Introduction KRAS oncogene mutations (MUTKRAS) drive resistance to EGFR inhibition by providing alternative signaling as demonstrated in colo-rectal cancer. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations (MUTEGFR). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS, or restoration of EGFR activity due to additional MUTEGFR, i.e., the c.2369C > T (p.T790MEGFR). Aim The aim of this study was to investigate the appearance of MUTKRAS during EGFR-TKI treatment and their contribution to drug resistance. Methods This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 MUTKRAS and p.T790MEGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI. Results p.T790MEGFR was detected in 11 (33.3%) patients, MUTKRAS at codon 12 in 3 (9.1%) while both p.T790MEGFR and MUTKRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type (WTKRAS) and negative for p.T790MEGFR. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790MEGFR and 37.5% for MUTKRAS. The analysis of time to progression (TTP) and overall survival (OS) in WTKRAS vs. MUTKRAS were not statistically different, even if there was a better survival with WTKRAS vs. MUTKRAS, i.e., TTP 14.4 vs. 11.4 months (p = 0.97) and OS 40.2 vs. 35.0 months (p = 0.56), respectively. Conclusions MUTKRAS could be an additional mechanism of escape from EGFR-TKI inhibition and cftDNA is a feasible approach to monitor the molecular development of drug resistance.

Bevacizumab-based neoadjuvant chemotherapy for colorectal cancer liver metastases: Pitfalls and helpful tricks in a review for clinicians

Bevacizumab added to chemotherapy has shown encouraging efficacy in the neoadjuvant therapy of colorectal cancer liver metastases. In absence of biological predictor factors of efficacy to bevacizumab-based treatment, the assessment of response may be a crucial point to select patients who may benefit the most from surgery. At the same time the pathological response after liver resection could represent a guide for the next therapeutic plan. In the pre-surgical phase, conventional computed tomography and response evaluation with RECIST criteria may underestimate the response to anti-angiogenic drugs. Modified computed tomography criteria of response, morphologic changes as well as novel imaging techniques and metabolic assessment by fluorodeoxyglucose positron emission tomography seem to be promising methods for the assessment of response and for leading the clinical choices. Pathological response at the time of surgery is an important prognostic factor and a surrogate of survival for resected patients. Different classification criteria to assess pathological response have been developed, residual viable tumor, tumor regression grade (TRG), modified TRG and tumor thickness at the tumor-normal interface, but to date a superiority of one approach over the others has not been clearly established. In this review, we evaluate the available data with the aim to help the clinicians in the pre- and post-surgical care of patient with colorectal cancer liver metastases treated with bevacizumab-based neoadjuvant strategy.

1616PLIQUID BIOPSY TO MONITOR THE EVOLUTION OF NSCLC EGFR+DURING TREATMENT WITH GEFINTINIB

ABSTRACT Aim: Resistance to EGFR tyrosine kinase inhibitors (TKI) is a clinically relevant problem that needs to be addressed by the use of appropriate technological platforms to discover the early appearance of mutations during treatment and before clinical disease progression. However, there is a lack of standardised approaches to monitor the molecular evolution of the disease under the selective pressure exerted by targeted treatments. One potential, not yet fully validated approach, is the analysis of gene mutations conferring drug resistance on cell-free circulating tumor DNA (cctDNA) released into the peripheral blood from primary tumor and metastatic sites. Periodic monitoring of the genetic evolution of the tumor, unfeasible with repeated biopsies, will greatly contribute to a better understanding and clinical management of drug resistance in cancer patients. Methods: In order to validate this approach in real-life conditions, we examined a case of acquired resistance to EGFR TKI to ascertain whether the technical platform chosen to perform this analysis is appropriate in terms of sensitivity and specificity and if it fulfills our need for molecular monitoring of the disease to improve our ability to personalise treatment. Therefore, in order to understand the cause of resistance to EGFR TKIs, a peripheral blood sample (6 ml) was drawn in a patient at first documented complete response to chemotherapy and at the three following disease progression after gefitinib. cctDNA was extracted from plasma with QIAamp Circulating Nucleic Acid Kit (Qiagen®) and molecular analysis of KRAS G12D, G12V, G12A, G12C, G12R, G12S, BRAF V600E, EGFR T790M was performed with a Digital Droplet PCR (Bio-Rad). Results: At first sampling the patient was wild type for all the tested mutations. At second sampling the patient developed the KRAS G12R and the BRAF V600E mutations. At third sampling also the the EGFR T790M was detected and the analysis confirmed the previous KRAS and BRAF mutations. Finally, the fourth sampling confirmed all three mutations. Conclusions: The liquid biopsy approach is feasible and uncovers the complexity of secondary mutations occurring in NSCLC patients treated with targeted treatments and undescores the role of the KRAS pathway in the development of resistance. Disclosure: All authors have declared no conflicts of interest.