Oscar Alabiso

Increased Detection Sensitivity for KRAS Mutations Enhances the Prediction of Anti-EGFR Monoclonal Antibody Resistance in Metastatic Colorectal Cancer

Purpose:KRAS mutations represent the main cause of resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) in metastatic colorectal cancer (mCRC). We evaluated whether highly sensitive methods for KRAS investigation improve the accuracy of predictions of anti-EGFR MoAbs efficacy. Experimental Design: We retrospectively evaluated objective tumor responses in mCRC patients treated with cetuximab or panitumumab. KRAS codons 12 and 13 were examined by direct sequencing, MALDI-TOF MS, mutant-enriched PCR, and engineered mutant-enriched PCR, which have a sensitivity of 20%, 10%, 0.1%, and 0.1%, respectively. In addition, we analyzed KRAS codon 61, BRAF, and PIK3CA by direct sequencing and PTEN expression by immunohistochemistry. Results: In total, 111 patients were considered. Direct sequencing revealed mutations in codons 12 and 13 of KRAS in 43/111 patients (39%) and BRAF mutations in 9/111 (8%), with almost all of these occurring in nonresponder patients. Using highly sensitive methods, we identified up to 13 additional KRAS mutations compared with direct sequencing, all occurring in nonresponders. By analyzing PIK3CA and PTEN, we found that of these 13 patients, 7 did not show any additional alteration in the PI3K pathway. Conclusions: The application of highly sensitive methods for the detection of KRAS mutations significantly improves the identification of mCRC patients resistant to anti-EGFR MoAbs. Clin Cancer Res; 17(14); 4901–14. ©2011 AACR.

Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma

Anthocyanins extracted from the berries of Phillyrea latifolia L., Pistacia lentiscus L., and Rubia peregrina L., three evergreen shrubs widely distributed in the Mediterranean area, were examined for their antioxidant and anticancer activity. The P. lentiscus anthocyanins showed the highest H2O2 and 1,1-diphenyl-2-picryl-hydrazil radical scavenging effects, indicating that these compounds can be considered as an alternative source of natural antioxidants for food and pharmaceutical products. Here, we also report a novel function of anthocyanins: the induction of autophagy, a process of subcellular turnover involved in carcinogenesis. Autophagy was characterized by the up-regulation of eIF2α, an autophagy inducer, and down-regulation of mTOR and Bcl-2, two autophagy inhibitors. This led to the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Anthocyanin-induced autophagy switched to apoptosis, as shown by the activation of Bax, cytochrome c and caspase 3, terminal deoxynucleotide transferase–mediated dUTP nick-end labeling–positive fragmented nuclei, and cells with sub-G1 DNA content, which were prevented by z-VAD. Inhibition of autophagy by either 3-methyladenine or Atg5 small interfering RNA enhanced anthocyanin-triggered apoptosis. This provided evidence that autophagy functions as a survival mechanism in liver cancer cells against anthocyanin-induced apoptosis and a rationale for the use of autophagy inhibitors in combination with dietary chemopreventive agents. [Mol Cancer Ther 2008;7(8):2476–85]

TAILOR: A phase III trial comparing erlotinib with docetaxel as the second-line treatment of NSCLC patients with wild-type (wt) EGFR.

LBA7501 Background: While the benefit of EGFR tyrosine kinase inhibitors in the treatment of patients with NSCLC harboring EGFR mutations has been widely established, their value in treating patients with wt EGFR is still debated. To assess the role of erlotinib in these patients, we performed an independent multicenter phase III trial (Tarceva Italian Lung Optimization Trial [TAILOR] NCT00637910), comparing erlotinib to docetaxel in second line treatment, having overall (OS) and progression free survival (PFS) as principal and secondary endpoints, respectively. METHODS EGFR and KRAS mutational status were assessed by direct sequencing in all eligible patients; only patients with wt EGFR NSCLC (exons 19 and 21) at progression, and previously treated with a first line platinum-based regimen, were randomized to receive either erlotinib 150 mg daily or docetaxel 75 mg/m2 (3-weekly) or 35 mg/m2 (weekly) until disease progression or unacceptable toxicity occurred. To detect an hazard ratio of 0.67 (2-sided 5% significance level for the log-rank test and a power of 80%), 199 events were required for both OS and PFS evaluation. RESULTS On the planned analysis date (March 30, 2012), 221 patients had been randomized and 218 were evaluable (docetaxel 110, erlotinib 108; three major violations excluded). At a median follow-up of 20 months, 199 relapses and 157 deaths were recorded. The Kaplan-Meier PFS curves showed a highly significant increase favoring docetaxel (HR 0.70 with 95% CI 0.53-0.94; p = 0.016) over erlotinib regimen. The HR translated into an estimated absolute difference in 6-months PFS of 12% (16% vs 28%). Data concerning toxicity were consistent with the literature. CONCLUSIONS In terms of PFS, our results indicate a clear superiority of docetaxel over erlotinib as second line treatment for patients without EGFR mutations in exons 19 or 21. Analysis of OS will be conducted as far as the planned number of 199 deaths is reached.

Continuous 5-fluorouracil infusion plus long acting octreotide in advanced well-differentiated neuroendocrine carcinomas. A phase II trial of the Piemonte Oncology Network

BackgroundWell-differentiated neuroendocrine carcinomas are highly vascularized and may be sensitive to drugs administered on a metronomic schedule that has shown antiangiogenic properties. A phase II study was designed to test the activity of protracted 5-fluorouracil (5FU) infusion plus long-acting release (LAR) octreotide in patients with neuroendocrine carcinoma.MethodsTwenty-nine patients with metastatic or locally advanced well-differentiated neuroendocrine carcinoma were treated with protracted 5FU intravenous infusion (200 mg/m2 daily) plus LAR octreotide (20 mg monthly). Patients were followed for toxicity, objective response, symptomatic and biochemical response, time to progression and survival.ResultsAssessment by Response Evaluation Criteria in Solid Tumors (RECIST) criteria showed partial response in 7 (24.1%), stable disease in 20 (69.0%), and disease progression in 2 patients. Response did not significantly differ when patients were stratified by primary tumor site and proliferative activity. A biochemical (chromogranin A) response was observed in 12/25 assessable patients (48.0%); symptom relief was obtained in 9/15 symptomatic patients (60.0%). There was non significant decrease in circulating vascular epithelial growth factor (VEGF) over time. Median time to progression was 22.6 months (range, 2.7-68.5); median overall survival was not reached yet. Toxicity was mild and manageable.ConclusionContinuous/metronomic 5FU infusion plus LAR octreotide is well tolerated and shows activity in patients with well-differentiated neuroendocrine carcinoma. The potential synergism between metronomic chemotherapy and antiangiogenic drugs provides a rationale for exploring this association in the future.Trial registrationNCT00953394

A Phase II Study of Three-Weekly Docetaxel and Weekly Trastuzumab in HER2-Overexpressing Advanced Breast Cancer

Background: To test safety and activity of 3-weekly doses of docetaxel and a weekly dose of trastuzumab in women with HER2-overexpressing advanced breast cancer. Patients and Methods: Forty-two women, median age 53 years (range 36–73 years), with HER2-overexpressing advanced breast cancer were enrolled in a study of docetaxel, 75 mg/m2 q3w for 6 cycles, and trastuzumab, 4 mg/kg loading dose, 2 mg/kg weekly thereafter. Thirty-four patients (81%) had visceral metastatic involvement. Thirty-five patients had received prior chemotherapy as part of their treatment: adjuvant/neoadjuvant (26), metastatic (2) and both (7). Thirty-one patients had been previously exposed to an anthracycline and 11 to paclitaxel. Four patients had previously received high-dose chemotherapy followed by autologous stem cell transplant. Results: 226 cycles (median 6, range 1–6) were administered. The median delivered dose intensity for docetaxel was 24 mg/m2/week (range 16–25 mg/m2/week). The intent to treat overall response rate was 67% (95% confidence interval, 52–79%). Median progression-free survival, time to treatment failure, and duration of response were 9, 8 and 12 months, respectively. Symptomatic cardiotoxicity (grade 3) occurred in 1 patient. The most common grade 3/4 toxicity was neutropenia (76% of the patients), although febrile neutropenia did not occur. Conclusions: Three-weekly doses of docetaxel and a weekly dose of trastuzumab is an active and safe combination in patients with HER2-overexpressing advanced breast cancer.

Safety and activity of docetaxel and trastuzumab in HER2 overexpressing metastatic breast cancer: a pilot phase II study.

We conducted a pilot phase II trial of trastuzumab administered concurrently with docetaxel in women with HER2-overexpressing advanced breast cancer. Twenty-five women with HER2-positive (3+ by immunohistochemistry = 16, 2+ = 9) metastatic breast cancer received docetaxel (75 mg/m2 every 3 weeks for 6 cycles) and trastuzumab (4 mg/kg loading dose, 2 mg/kg weekly thereafter). Twenty-three patients (92%) had visceral metastatic involvement. Twenty-three patients had received prior chemotherapy as part of adjuvant (18), metastatic (2), and both (3) treatment. The number of cycles administered was 121 (median 6, range 1–6). Symptomatic cardiotoxicity (GIII) occurred in one patient. The most common grade GIII/IV toxicity was neutropenia (80% of the cycles), although febrile neutropenia did not occur. No other GIII/IV toxicities were observed. Response rate was 70% (1 complete response and 15 partial responses) in 23 evaluable patients. The combination of docetaxel and trastuzumab is well tolerated and has clinically meaningful antitumor activity.

Involvement of autophagy in ovarian cancer: a working hypothesis

Autophagy is a lysosomal-driven catabolic process that contributes to preserve cell and tissue homeostases through the regular elimination of damaged, aged and redundant self-constituents. In normal cells, autophagy protects from DNA mutation and carcinogenesis by preventive elimination of pro-oxidative mitochondria and protein aggregates. Mutations in oncogenes and oncosuppressor genes dysregulate autophagy. Up-regulated autophagy may confer chemo- and radio-resistance to cancer cells, and also a pro-survival advantage in cancer cells experiencing oxygen and nutrient shortage. This fact is the rationale for using autophagy inhibitors along with anti-neoplastic therapies. Yet, aberrant hyper-induction of autophagy can lead to cell death, and this phenomenon could also be exploited for cancer therapy. The actual level of autophagy in the cancer cell is greatly affected by vascularization, inflammation, and stromal cell infiltration. In addition, small non-coding microRNAs have recently emerged as important epigenetic modulators of autophagy. The present review focuses on the potential involvement of macroautophagy, and on its genetic and epigenetic regulation, in ovarian cancer pathogenesis and progression.

The Role of Lung Metastasis Resection in Improving Outcome of Colorectal Cancer Patients: Results From a Large Retrospective Study

BACKGROUND The role of surgery for lung metastases (LM) secondary to colorectal cancer (CRC) remains controversial. The bulk of evidence is derived from single surgical series, hampering any definitive conclusions. The aim of this study was to compare the outcomes of CRC patients with LM submitted to surgery with those who were not. PATIENTS AND METHODS Data from 409 patients with LM as the first evidence of advanced disease were extracted from a database of 1,411 patients. Patients were divided into three groups: G1, comprised of 155 patients with pulmonary and extrapulmonary metastases; G2, comprised of 104 patients with LM only and no surgery; G3, comprised of 50 patients with LM only and submitted to surgery. RESULTS No difference in response rates emerged between G1 and G2. Median progression-free survival (PFS) times were: 10.3 months, 10.5 months, and 26.2 months for G1, G2, and G3, respectively. No difference in PFS times was observed between G1 and G2, whereas there was a statistically significant difference between G2 and G3. Median overall survival times were 24.2 months, 31.5 months, and 72.4 months, respectively. Survival times were longer in resected patients: 17 survived >5 years and three survived >10 years. In patients with LM only and no surgery, four survived for 5 years and none survived >10 years. CONCLUSIONS Even though patients with resectable LM are more likely to be those with a better outcome, our study provides evidence suggesting an active role of surgery in improving survival outcomes in this patient subset.

Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer

Autophagy is dysregulated in cancer and might be involved in ovarian carcinogenesis. BECLIN-1, a protein that interacts with either BCL-2 or PI3k class III, plays a critical role in the regulation of both autophagy and cell death. Induction of autophagy is associated with the presence of vacuoles characteristically labelled with the protein LC3. We have studied the biological and clinical significance of BECLIN 1 and LC3 in ovary tumours of different histological types. The positive expression of BECLIN 1 was well correlated with the presence of LC3-positive autophagic vacuoles and was inversely correlated with the expression of BCL-2. The latter inhibits the autophagy function of BECLIN 1. We found that type I tumours, which are less aggressive than type II, were more frequently expressing high level of BECLIN 1. Of note, tumours of histologic grade III expressed low level of BECLIN 1. Consistently, high level of expression of BECLIN 1 and LC3 in tumours is well correlated with the overall survival of the patients. The present data are compatible with the hypotheses that a low level of autophagy favours cancer progression and that ovary cancer with upregulated autophagy has a less aggressive behaviour and is more responsive to chemotherapy.

Aberrant somatic hypermutation in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMLBCL) is recognized as a subtype of diffuse large B-cell lymphoma (DLBCL) arising in the mediastinum. However, the clinical, morphological and molecular peculiarities of PMLBCL have been taken to suggest that the disease may represent a distinct clinico-pathologic entity. Clinically, PMLBCL patients display younger median age (35 vs 60 years) and a higher female:male ratio when compared to DLBCL. Also, PMLBCL rarely has extrathoracic disease at diagnosis, and at relapse preferentially involves extranodal sites. Pathologically, PMLBCL is typically associated with various degrees of sclerosis and consists of a diffuse proliferation of large cells expressing B-lineage markers but lacking surface immunoglobulins. At the molecular level, PMLBCL exhibits several genetic abnormalities, including gains of 9p and X that are otherwise absent in DLBCL, and lack genetic lesions, such as BCL-2 and BCL-6 rearrangements that are typically associated with DLBCL. An aberrant activity of the somatic hypermutation (SHM) mechanism, targeting the 50 sequences of PIM-1, PAX-5, RhoH/ TTF and c-MYC proto-oncogenes, has been advocated as a molecular feature distinctive of DLBCL. Aberrant SHM affects 450% of DLBCL of immunocompetent hosts, while it is rare or absent among other B-cell malignancies. The involvement of aberrant SHM in PMLBCL has been suggested by a recent report based on a limited number of cases. These observations prompted our comprehensive analysis aimed at exploring the involvement of aberrant SHM of PIM-1, PAX-5, RhoH/TTF and c-MYC in a sizeable panel of PMLBCL and at comparing the mutational spectrum with that of systemic DLBCL. This study was based on 19 primary samples of PMLBCL. The clinical characteristics of this series were consistent with PMLBCL diagnosis (median age of 37 years; male:female ratio of 0.8; Ann Arbor stage I–II). Diagnosis was pathologically confirmed by three expert hematopathologists (MP, AC and SAP) according to the World Health Organization classification of hematopoietic tumours. Malignant cells expressed CD20 in all cases, CD30 in 16 cases and CD23 in 13 cases, but lacked CD15. For comparison, 19 systemic DLBCL, matched for stage, sex and age, were also analysed. Mutations of PIM-1, PAX-5, RhoH/TTF and c-MYC genes were detected by DNA direct sequencing. The presence of intraclonal heterogeneity, indicative of ongoing SHM, was assessed by sequencing cloned PCR products generated using the proof-reading Pfu Turbo polymerase. The prevalence of aberrant SHM in PMLBCL and DLBCL was compared by the w test. Differences in the mutation frequency between PMLBCL and DLBCL were analysed by the t-test. Mutation frequencies were normalized based on the nucleotide composition of the sequences analysed. The normalized mutation frequency of nucleotides occurring in the context of an RGYW/WRCY motif was compared to the expected mutation frequency by the goodness-of fit w test. Mutation analysis of PAX-5, Rho/TTF, PIM-1 and c-MYC is summarized in Table 1. Overall, the prevalence of aberrant SHM did not significantly differ between PMLBCL and DLBCL. Mutations targeting at least one of the four proto-oncogenes were found in 14/19 (73.6%) PMLBCL and 13/19 (68.4%) DLBCL, while mutations targeting more than one gene were found in 7/19 (36.8%) PMLBCL and 9/19 (47.3%) DLBCL. Each of the four genes analysed was altered in a significant fraction of PMLBCL and DLBCL, since PAX-5 was mutated in 9/19 (47.3%) PMLBCL and 7/19 (36.8%) DLBCL, RhoH/TTF in 6/19 (31.5%) PMLBCL and 8/19 (42.1%) DLBCL, PIM-1 in 3/19 (15.7%) PMLBCL and 7/19 (36.8%) DLBCL, and c-MYC in 6/19 (31.5%) PMLBCL and 5/19 (26.3%) DLBCL. The detailed characterization of PAX-5, RhoH/TTF, PIM-1 and c-MYC mutations is reported in Tables 1 and 2. Overall, the molecular profile of mutations was similar between PMLBCL and DLBCL. A total of 74 mutational events were detected in PMLBCL. The overwhelming majority of the mutations included single base-pair substitutions (n1⁄4 66), whereas deletions of a short DNA stretch were observed in eight instances. Of the 66 single base-pair substitutions observed, 41 were transitions and 25 were transversions, with a transition/transversion ratio of 1.64 (expected 0.5; P1⁄4 0.001). When considering all the nucleotide substitutions observed, analysis of the nucleotide exchange pattern showed that GþC base pairs were targeted 3.33-fold more frequently than Aþ T (expected 1.28; P1⁄4 0.009). In all, 19 of 66 single base-pair substitutions detected in PMLBCL felt within RGYW/WRCY motifs. Considering all the genes together, the frequency of mutations targeting RGYW/ WRCY motifs was significantly higher than the frequency of mutations occurring outside RGYW/WRCY motifs (2.3 vs 1.3%; P1⁄4 0.03). As mutations introduced by physiological SHM preferentially affect specific dinucleotides, we also analysed the distribution of mutations within dinucleotide motifs. Considering together all the four proto-oncogenes, the mutation frequency was higher than expected in the following motifs: AA (4.3 vs 1.3%; Po0.0001), AG (3.0 vs 1.3%; P1⁄4 0.028), GC (5.1 vs 1.1%; Po0.0001), GG (4.2 vs 1.1%; Po0.0001) and TA (4.6 vs 1.3%; P1⁄4 0.003). Among DLBCL, a total of 87 mutational events were detected. Mutations were preferentially represented by single base-pair substitutions (n1⁄4 81), whereas only four deletions and two insertions of a short DNA stretch were observed. Of the 81 single base-pair substitutions, 42 were transitions and 39 were transversions, with a transition/transversion ratio of 1.07 (expected 0.5; P1⁄4 0.02). Analysis of the nucleotide exchange pattern showed that GþC base pairs were targeted 1.89-fold more frequently compared to Aþ T (expected 1.28; P1⁄4 ns). In all, 41 out of 81 single base-pair substitutions felt within RGYW/WRCY motifs. The frequency of mutations targeting RGYW/WRCY motifs was significantly higher than the frequency of mutations occurring outside RGYW/WRCY motifs (5.0 vs 1.7%; Po0.0001). Similarly to PMLBCL, also in DLBCL the mutation frequency was higher than expected in specific dinucleotide motifs: AA (6.4 vs 1.5%; Po0.0001), AC (5.4 vs 1.4%; Po0.0001), AG (4.5 vs 1.6%; Po0.0001), AT (4.2 vs 1.7%; P1⁄4 0.033), CT (4.4 vs 1.6%; Received 6 April 2005; accepted 6 September 2005; published online 6 October 2005 Correspondence: Professor G Gaidano, Division of Hematology, Department of Medical Sciences & IRCAD, Amedeo Avogadro University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy; Fax þ 39 0321 620 421; E-mail: gaidano@med.unipmn.it Correspondence