Lapatinib with whole brain radiotherapy in patients with brain metastases from breast and non-small cell lung cancer: a phase II study of the Hellenic Cooperative Oncology Group (HeCOG).

Small molecules, mainly tyrosine kinase inhibitors, are currently used in various malignancies. Lapatinib, a dual inhibitor of EGFR/HER2 tyrosine kinases, has demonstrated effectiveness in brain metastases from HER2-overexpressing breast cancer. It also appears to sensitize EGFR-expressing cell lines to radiation. To evaluate the efficacy of lapatinib in combination with whole brain radiotherapy (WBRT) in patients with brain metastases from non-small cell lung cancer (NSCLC) and breast cancer, as assessed by volumetric analysis by MRI. 81 patients were treated with WBRT (30 Gy in ten fractions) in combination with lapatinib 1250 mg once daily, followed by lapatinib 1500 mg once daily for a total 6 weeks. 21 patients had primary breast cancer and 60 patients NSCLC. Pre- and post-treatment MRI scans in a compact disk for central volumetric assessment were available for 43 patients. 27 patients (62.8%) achieved partial response, 15 patients (34.9%) had stable disease and only one patient (2.3%) had disease progression. Response was not associated to EGFR protein expression. All 81 patients were assessed for safety. The large majority of the adverse events were mild. Eight deaths occurred, four of which were considered related to the study drugs but there were also other contributing factors. Nine cases of serious infections were observed in eight patients, but they were also receiving dexamethasone. Lapatinib in combination with WBRT in patients with brain metastases from breast cancer and NSCLC is a feasible approach that can be further studied in larger clinical trials.

Gemcitabine Combined with the mTOR Inhibitor Temsirolimus in Patients with Locally Advanced or Metastatic Pancreatic Cancer. A Hellenic Cooperative Oncology Group Phase I/II Study.

BACKGROUND: The prognosis of patients with advanced pancreatic cancer is dismal, and there is a need for novel and effective treatments. OBJECTIVES: Tο determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of a novel gemcitabine (G) and temsirolimus (T) combination (phase I) and estimate the 6-month progression-free survival (PFS) in patients treated with the T + G combination (phase II). PATIENTS AND METHODS: Eligible patients with histologically confirmed inoperable or metastatic pancreatic carcinoma (MPC) were entered into the trial. G was given bi-weekly and T weekly in a 4-week cycle. The first dose level was set at G 800 mg/m2 and T 10 mg. G was escalated in increments of 200 mg/m2 and T in increments of 5 mg until DLT was reached, and the recommended dose was used for the phase II part. RESULTS: Thirty patients were enrolled in the phase I component at the pre-planned six dose levels; one bilirubin DLT of grade III occurred at the first dose level. The MTD was established as the approved doses of both drugs. Fifty-five patients were entered into the phase II component. Median relative dose intensities administered in the first cycle were 0.75 for T and 0.99 for G. Grade 3-4 hematological toxicities were recorded in 87.3% of patients. The most common non-hematological adverse events were metabolic disorders (81.8%) followed by gastrointestinal disorders (63.6%). Median PFS was 2.69 months (95% CI 1.74-4.95) and median OS was 4.95 months (95% CI 3.54-6.85), while the 6-month PFS rate was 30.9%. CONCLUSIONS: Combination of G and T is feasible in patients with locally advanced or MPC with manageable side effects, but lacks clinical efficacy. The study was registered in the Australian New Zealand Clinical Trials Registry (ACTRN12611000643976).

AMALTHEA: Prospective, Single-Arm Study of the Hellenic Cooperative Oncology Group (HeCOG) Evaluating Efficacy and Safety of First-Line FOLFIRI + Aflibercept for 6 Months Followed by Aflibercept Maintenance in Patients With Metastatic Colorectal Cancer.

BACKGROUND: The efficacy and safety of the FOLFIRI (leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin) regimen combined with aflibercept has not been studied in the first-line management of patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: In the context of a prospective single-arm trial (NCT02129257), patients with mCRC received standard doses of a maximum of 12 cycles of FOLFIRI combined with aflibercept (4 mg/kg body weight delivered intravenously) every 2 weeks, followed by aflibercept maintenance. Endpoints were 12-month progression-free survival rate, efficacy, and toxicity. RESULTS: Seventy-three fit patients were enrolled onto the study between 2014 and 2016. Median relative dose intensities administered were 0.80 for irinotecan and 1.0 for aflibercept. The most common grade 3/4 adverse events were neutropenia (13 patients, 18%), febrile neutropenia (3 patients, 4%), diarrhea (11 patients, 15%), hypertension (19 patients, 26%), proteinuria (8 patients, 11%), infections (8 patients, 11%), and mucositis (6 patients, 8%), with no toxic deaths. The objective response rate was 46.6%, significantly associated with the presence of right-sided primary, synchronous metastases, and a relapse-free interval of < 12 months (odds ratio = 3.00, 2.92, and 3.75 respectively, P ≤ .05). Intermediate infiltration by stromal core lymphocytes correlated with progression-free survival (hazard ratio = 0.40, [95% confidence interval (CI), 0.19-0.83], P = .014). At a median follow-up of 24.5 months, 12-month progression-free survival rate was 21.9% (median overall survival 20.9 months [95% CI, 16.6-29], median progression-free survival 8.4 months [95% CI, 7.4-9.3]). CONCLUSION: The FOLFIRI + aflibercept regimen is active and tolerable; however, it failed to improve historical benchmarks of efficacy in chemonaive patients with mCRC. Preliminary data hint that this regimen has cytoreductive activity in disease with adverse biology.

TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 alterations are not associated with microsatellite instability or p53 mutations in invasive urothelial carcinoma of the urinary bladder.

AIM: The aim of this study was to determine the potential synchronous contribution of alterations in TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 genes to the development and clinical outcome of bladder cancer, in relation to p53 mutations, microsatellite status and hMLH1/hMSH2 expression. METHODS: Molecular biology techniques as well as immunohistochemistry were applied in 69 samples from patients with urothelial carcinoma. RESULTS: Microsatellite alterations were observed in TGF-betaRII(A)(10 )(16%) and BAX(G)(8 )(3%), irrespective of the presence of p53 mutations, but not in IGFIIR(G)(8), caspase-5(A)(10, ) hMSH3(A)(8) and hMSH6(C)(8). A statistically significant correlation could be found only between hMLH1 expression and the presence of microsatellite instability (Fisher's exact test, p = 0,013). Survival analysis indicated that apart from grade and T-category, hMLH1 expression was the only parameter significantly affecting overall survival (p = 0.021 in univariate and p = 0.015 in multivariate analysis) and recurrence-free survival (p = 0.0463 in univariate and p = 0.022 in multivariate analysis). CONCLUSIONS: We conclude that alterations of the examined target genes of MSI are rare in urinary bladder carcinoma and they are not associated with microsatellite instability or the presence of p53 mutations.

Novel histone deacetylase inhibitors in the treatment of thyroid cancer.

Histone deacetylases (HDAC) and histone acetyltransferases exert opposing enzymatic activities that modulate the degree of acetylation of histones and other intracellular molecular targets, thereby regulating gene expression, cellular differentiation, and survival. HDAC inhibition results in accumulation of acetylated histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bis-hydroxamide, on thyroid carcinoma cell lines, including lines originating from anaplastic and medullary carcinomas. In these models, both SAHA and m-carboxycinnamic acid bis-hydroxamide induced growth arrest and caspase-mediated apoptosis and increased p21 protein levels, retinoblastoma hypophosphorylation, BH3-interacting domain death agonist cleavage, Bax up-regulation, down-regulation of Bcl-2, A1, and Bcl-x(L) expression, and cleavage of poly(ADP-ribose) polymerase and caspase-8, -9, -3, -7, and -2. Transfection of Bcl-2 cDNA partially suppressed SAHA-induced cell death. SAHA down-regulated the expression of the apoptosis inhibitors FLIP and cIAP-2 and sensitized tumor cells to cytotoxic chemotherapy and death receptor activation. Our studies provide insight into the tumor type-specific mechanisms of antitumor effects of HDAC inhibitors and a framework for future clinical applications of HDAC inhibitors in patients with thyroid cancer, including histologic subtypes (e.g., anaplastic and medullary thyroid carcinomas) for which limited, if any, therapeutic options are available.

Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing.

PURPOSE: Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L are members of the TNFalpha family that can trigger apoptosis in susceptible cells via respective death receptors (DRs). FasL cross-links its receptor Fas, resulting in recruitment and proteolytic activation of caspase-8, which initiates the downstream apoptotic cascade. TRAIL signals through its receptors DR4 and DR5, which can activate caspase-8 as well. This study was undertaken to investigate the functional status of the FasL and TRAIL apoptotic pathways in retinoblastoma (Rb) cells. METHODS: The human Rb cell lines Y79 and WERI-Rb1 were evaluated for their response to the Fas cross-linking antibody CH11 and recombinant TRAIL, as well as for cell surface presence and mutational status of Fas, DR4, and DR5 by flow cytometry and genomic DNA sequencing, respectively. The expression of caspase-8 and its inhibitor FLIP, as well as their recruitment to the DR signaling complex were studied by immunoblot analysis. RESULTS: Rb cells express Fas, DR4, and DR5 on their surfaces, yet were resistant to DR-mediated apoptosis. This was not due to DR mutations or secretion of the soluble decoy Fas, antiapoptotic NF-kappaB activity, or FLIP overexpression, but to the absence of caspase-8 expression. The demethylating agent 5-aza-2'-deoxycytidine restored caspase-8 expression and sensitivity to DR-mediated apoptosis. CONCLUSIONS: Rb cells are resistant to DR-mediated apoptosis because of a deficiency in caspase-8 expression secondary to epigenetic gene silencing by overmethylation. The data help delineate the apoptotic pathways in Rb cells and suggest that the combination of demethylating agents with DR-activating modalities, such as TRAIL receptor monoclonal antibodies, may benefit patients with retinoblastoma.

Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators.

Evaluation of cell cycle regulators has gained special interest in the effort to increase the amount of prognostic information in malignant tumors. Minichromosome maintenance proteins (MCMs) drive the formation of prereplicative complexes, which is the first key event during G1 phase. Therefore, altered MCM expression may be a hallmark of cell cycle deregulation, which is supposed to be the most essential mechanism in the development and progression of bladder cancer. Our aim was to investigate the value of MCMs as proliferation markers and prognostic indicators in detrusor muscle-invasive urothelial bladder carcinomas. We analyzed immunohistochemically the expression of MCM-2 and MCM-5 in 65 patients with detrusor muscle-invasive urothelial bladder carcinomas in relation with clinicopathologic parameters, patients' overall and disease-free survival, and the expression of the conventional proliferation index Ki-67 and other cell cycle modulators (p53, pRb, p21(WAF1), and p27(Kip1)). The levels of MCM-2 and MCM-5 were significantly higher in high-grade (P < .0001), advanced-stage (P = .001), and nonpapillary tumors (P < .0001). The expression of MCM-2 and MCM-5 significantly associated with the conventional proliferation index Ki-67 (P = .0001 for each protein). The expression of MCM-2 or MCM-5 positively correlated with p53 labeling index (P = .014 and P = .009, respectively). Also, median p21(WAF1) labeling index was higher in MCM-5 high expressors (P = .028). Finally, both MCM-2 and MCM-5 associated significantly with adverse patients' outcome in both univariate (P = .0072 and P = .0074, respectively) and multivariate (P = .0001) analysis. In conclusion, MCM-2 and MCM-5 appear to be reliable proliferation indexes and useful prognostic markers in patients with muscle-invasive urothelial bladder carcinomas.

c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations.

OBJECTIVES: To investigate the incidence of Fas (exon 9) mutations and the expression of Fas, Fas-Fas ligand (FasL) system, and cellular FLICE-like inhibitory protein (c-FLIP) in relation to standard clinicopathologic parameters and patient outcome in bladder carcinoma. Disruption of apoptotic cell death has been implicated in tumor aggressiveness in bladder urothelial carcinomas. The FasL system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an important endogenous inhibitor of Fas and other death receptor-mediated apoptosis. METHODS: The expression of Fas, FasL, and c-FLIP was quantified immunohistochemically in paraffin-embedded tissues from 53 patients for whom clinical information was available. DNA extracted from the same samples was screened for mutations in Fas exon 9 by single-strand conformation polymorphism and sequencing. The effect of Fas, FasL, and c-FLIP on clinical outcome was assessed by univariate and multivariate analyses. RESULTS: Positive immunostaining was detected for Fas, FasL, and c-FLIP in 72%, 66%, and 81% of cases, respectively. Concurrent expression of Fas and FasL was seen in 27 samples (51%), of which 22 (81.5%) also displayed c-FLIP positivity. FasL and c-FLIP expression increased with advancing stage but was absent from normal urothelium. None of the 53 urothelial carcinoma samples analyzed showed evidence of mutations by polymerase chain reaction single-strand conformation polymorphism and direct sequencing. Survival analysis demonstrated that although both FasL and c-FLIP expression adversely affected survival, only c-FLIP remained statistically significant on multivariate analysis. CONCLUSIONS: The frequent expression and coexpression of Fas, FasL, and c-FLIP in urothelial carcinomas implicates c-FLIP as an inhibitor of the Fas-FasL-induced death pathway in these tumors. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.