Ramon Colomer

Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation

Median survival of patients with brain metastases from nonsmall cell lung cancer (NSCLC) is poor and more effective treatments are urgently needed. We have evaluated the efficacy of erlotinib in this setting and its association with activating mutations in the epidermal growth factor receptor (EGFR) gene. We retrospectively identified patients with NSCLC and brain metastases treated with erlotinib. EGFR mutations in exons 19 and 21 were analysed by direct sequencing. Efficacy and tolerability were compared according to EGFR mutational status. 69 NSCLC patients with brain metastases were identified, 17 of whom harboured EGFR mutations. Objective response rate in patients with EGFR mutations was 82.4%; no responses were observed in unselected patients (p<0.001). Median (95% CI) time to progression within the brain for patients harbouring EGFR mutations was 11.7 (7.9–15.5) months, compared to 5.8 (5.2–6.4) months for control patients whose EGFR mutational status had not been assessed (p<0.05). Overall survival was 12.9 (6.2–19.7) months and 3.1 (2.5–3.9) months (p<0.001), respectively. The toxicity of erlotinib was as expected and no differences between cohorts were observed. Erlotinib is active in brain metastases from NSCLC; this clinical benefit is related to the presence of activating mutations in exons 19 or 21 of the EGFR gene.

Risk factors for treatment-related death in elderly patients with aggressive non-Hodgkin's lymphoma: results of a multivariate analysis.

PURPOSE It has been suggested that age is associated with chemotherapy-related death in patients with non-Hodgkins lymphoma (NHL) treated with doxorubicin-containing chemotherapy. The purpose of this study was to evaluate the relative influence of increasing age and other clinical parameters on the occurrence of treatment-related death in elderly patients with intermediate- or high-grade NHL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy. METHODS A retrospective study of patients 60 years of age or older with intermediate- or high-grade NHL treated with CHOP chemotherapy in a single cancer center. The following variables were recorded: age (60 to 69, 70 to 79, and 80 to 94 years), histology (Working Formulation [WF] D, E, F, G, and H), Ann Arbor stage, B symptoms, extranodal involvement, bulky disease (> 7 cm), performance status (Eastern Cooperative Oncology Group [ECOG] scale), International Prognostic Index (IPI score), serum lactate dehydrogenase (LDH) level and doxorubicin relative dose-intensity (RDI). The relationship between these features and treatment-related death was assessed in univariate and multivariate logistic regression analysis. RESULTS From 1982 to 1991, 267 consecutive patients were treated. Median age was 70 years (range, 60 to 94 years). There were 35 toxic deaths. Sixty-three percent of the deaths occurred after the first cycle. Infection accounted for 82% of the toxic deaths. In the univariate analysis, the features associated with an increased risk of toxic death were ECOG performance status 2 to 4 (relative risk [RR], 7.82), B symptoms (RR, 3.38), diffuse large-cell histology (RR, 3.06), bulky disease (RR, 2.58), serum levels of LDH (RR, 2.53), and IPI score (RR, 2.46). The age groups did not show significance. In the regression model, performance status 2 to 4 was the only independent predictor of treatment-related death (RR, 3.52; 95% confidence interval [CI], 2.98 to 4.06). CONCLUSION Our results show that in elderly patients with NHL treated with doxorubicin-based chemotherapy the risk for treatment-related death is associated with poor performance status rather than with increasing chronologic age.

Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells

Abstract.  Objectives: More than 50 years ago, we learned that breast cancer cells (and those of many other types of tumour) endogenously synthesize 95% of fatty acids (FAs) de novo, despite having adequate nutritional lipid supply. Today, we know that breast cancer cells benefit from this phenomenon in terms of enhanced cell proliferation, survival, chemoresistance and metastasis. However, the exact role of the major lipogenic enzyme fatty acid synthase (FASN) as cause, correlate or facilitator of breast cancer remains unidentified. Materials and methods: To evaluate a causal effect of FASN‐catalysed endogenous FA biosynthesis in the natural history of breast cancer disease, HBL100 cells (an SV40‐transformed in vitro model for near‐normal gene expression in the breast epithelium), and MCF10A cells (a non‐transformed, near diploid, spontaneously immortalized human mammary epithelial cell line) were acutely forced to overexpress the human FASN gene. Results: Following transient transfection with plasmid pCMV6‐XL4 carrying full‐length human FASN cDNA (gi: NM 004104), HBL100 cells enhanced their endogenous lipid synthesis while acquiring canonical oncogenic properties such as increased size and number of colonies in semisolid (i.e. soft‐agar) anchorage‐independent cultures. Anchorage‐dependent cell proliferation assays in low serum (0.1% foetal bovine serum), MTT‐based assessment of cell metabolic status and cell death ELISA‐based detection of apoptosis‐induced DNA‐histone fragmentation, together revealed that sole activation of endogenous FA biosynthesis was sufficient to significantly enhance breast epithelial cell proliferation and survival. When analysing molecular mechanisms by which acute activation of de novo FA biosynthesis triggered a transformed phenotype, HBL100 cells, transiently transfected with pCMV6‐XL4/FASN, were found to exhibit a dramatic increase in the number of phosphor‐tyrosine (Tyr)‐containing proteins, as detected by 4G10 antiphosphor‐Tyr monoclonal antibody. Phosphor‐Tyr‐specific antibodies recognizing the phosphorylation status of either the 1173 Tyr residue of epidermal growth factor receptor (HER1) or the 1248 Tyr residue of HER2, further revealed that FASN‐induced Tyr‐phosphorylation at ∼180 kDa region mainly represented that of these key members of the HER (erbB) network, which remained switched‐off in mock‐transfected HBL100 cells. ELISA and immunoblotting procedures demonstrated that FASN overactivation significantly increased (> 200%) expression levels of epidermal growth factor receptor and HER2 proteins in HBL100 cells. Proteome Profiler™ antibody arrays capable of simultaneously detecting relative levels of phosphorylation of 42 phospho‐receptor Tyr‐kinases (RTKs) confirmed that acute activation of endogenous FA biosynthesis specifically promoted hyper‐Tyr‐phosphorylation of HER1 and HER2 in MCF10A cells. This FASN‐triggered HER1/HER2‐breast cancer‐like phenotype was specifically inhibitable either by FASN inhibitor C75 or by Tyr‐kinase inhibitors (TKIs) gefitinib (Iressa™) and lapatinib (Tykerb™) but not by chemotherapeutic agents such as cisplatin. Transient overexpression of FASN dramatically increased HBL100 breast epithelial cells’ sensitivity to cytotoxic effects of C75, gefitinib and lapatinib (∼8, 10 and > 15 times, respectively), while significantly decreasing (∼3 times) cisplatin efficacy. Conclusions: Although we cannot definitely establish FASN as a novel oncogene in breast cancer, this study reveals for the first time that exacerbated endogenous FA biosynthesis in non‐cancerous epithelial cells is sufficient to induce a cancer‐like phenotype functionally dependent on the HER1/HER2 duo. These findings may perhaps radically amend our current perspective of endogenously synthesized fat, as on its own, it appears to actively increase signal‐to‐noise ratio in the HER1/HER2‐driven progression of human breast epithelial cells towards malignancy.

Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.

Circulating tumor marker levels in advanced breast carcinoma correlate with the extent of metastatic disease

The importance of the extent of metastatic disease in the circulating levels of CA 15‐3 and carcinoembryonic antigens (CEA) was studied in 173 patients with advanced breast carcinoma. Estimates of the extent of metastatic disease were obtained by an objective arbitrary scale. Patients were observed clinically after serum samples were obtained, and survival was recorded. Elevated values of CA 15‐3 (>40 U/ml) were seen in 130 patients and CEA values (>5 ng/ml) in 97 cases (75% versus 56%, P < .0001). Elevated CA 15‐3 values correlated with the estimated extent of metastatic disease (P < .0001), number of metastases (P = .0006), and survival from study entry (P = .01). Elevated CEA values correlated with extent of disease (P < .0001), but not with the number of metastases or with survival. No correlation was found between the elevated values of CA 15‐3 or CEA and age, menopausal status, and initial tumor size or nodal status. The combination of the elevated values of CA 15‐3 and CEA was more sensitive than CA 15‐3 alone (P = .04), but there were no significant improvements when subgroups were considered. Significant differences, that depended on which specific organ was affected dominantly by metastases, were seen in the mean levels of CA 15‐3 antigen. Similarly, patients with liver involvement had higher mean levels of CA 15‐3 than those without hepatic metastases. A stepwise regression analysis of the dominant site of metastases, liver involvement, and estimated extent of disease showed that only the latter parameter retained a significant correlation with CA 15‐3 antigen levels (P < .0001). Median survival of patients who showed abnormal CA 15‐3 levels was significantly shorter than that of patients with nonelevated CA 15‐3 (10.1 versus 18.0 months, P = .04). This difference was not appreciated with CEA levels (10.2 versus 12.2 months, P = .4). We conclude that tumor marker levels in patients with advanced breast carcinoma correlate with the extent of metastatic disease. In addition, the CA 15‐3 assay is more sensitive and correlates more accurately with the extent of disease than CEA. Finally, the observed CA 15‐3 differences by organ involvement are related to the extent of disease variations. The objective evaluation of the extent of metastatic disease provides a new approach in the study and comparison of breast cancer‐associated tumor markers.

CCR5 Expression Influences the Progression of Human Breast Cancer in a p53-dependent Manner

Chemokines are implicated in tumor pathogenesis, although it is unclear whether they affect human cancer progression positively or negatively. We found that activation of the chemokine receptor CCR5 regulates p53 transcriptional activity in breast cancer cells through pertussis toxin–, JAK2-, and p38 mitogen–activated protein kinase–dependent mechanisms. CCR5 blockade significantly enhanced proliferation of xenografts from tumor cells bearing wild-type p53, but did not affect proliferation of tumor xenografts bearing a p53 mutation. In parallel, data obtained in a primary breast cancer clinical series showed that disease-free survival was shorter in individuals bearing the CCR5Δ32 allele than in CCR5 wild-type patients, but only for those whose tumors expressed wild-type p53. These findings suggest that CCR5 activity influences human breast cancer progression in a p53-dependent manner.

Overexpression of c‐erbB‐2 in epithelial ovarian cancer. Prognostic value and relationship with response to chemotherapy

Background. Overexpression of the c‐erbB‐2 protein has been reported in tumors from approximately 25% of patients with epithelial ovarian cancer. However, its clinical significance has not been well established.

A Single-Nucleotide Polymorphism in the Aromatase Gene Is Associated with the Efficacy of the Aromatase Inhibitor Letrozole in Advanced Breast Carcinoma

Purpose: To evaluate the efficacy of treatment with the aromatase inhibitor letrozole in breast cancer patients segregated with respect to DNA polymorphisms of the aromatase gene CYP19. Patients and Methods: Postmenopausal patients (n = 67) with hormone receptor–positive metastatic breast cancer were treated with the aromatase inhibitor letrozole. PCR allelic discrimination was used to examine three single-nucleotide polymorphisms (SNP) in DNA obtained from breast carcinoma tissue. Two SNPs analyzed (rs10046 and rs4646) were located in the 3′ untranslated region and one (rs727479) was in the intron of the aromatase CYP19 gene. The primary end point of treatment efficacy was time to progression (TTP). Results: Median age was 62 years and median number of metastatic sites was 2. Observed allelic SNP frequencies were rs10046, 71%; rs4646, 46%; and rs727479, 63%. Of the 67 patients, 65 were evaluable for efficacy. Median TTP was 12.1 months. We observed no relationship between TTP and the rs10046 or rs727479 variants. In contrast, we found that TTP was significantly improved in patients with the rs4646 variant, compared with the wild-type gene (17.2 versus 6.4 months; P = 0.02). Conclusion: In patients with hormone receptor–positive metastatic breast cancer treated with the aromatase inhibitor letrozole, the presence of a SNP in the 3′ untranslated region of the CYP19 aromatase gene is associated with improved treatment efficacy. Testing for the CYP19 rs4646 SNP as a predictive tool for breast cancer patients on antiaromatase therapy deserves prospective evaluation.

Patient selection in high-dose chemotherapy trials: relevance in high-risk breast cancer.

PURPOSE To evaluate the impact of the selection criteria that are used in current high-dose consolidation chemotherapy (HDCT) trials on the outcome of high-risk breast cancer patients treated with conventional adjuvant chemotherapy. PATIENTS AND METHODS From 1975 to 1995, 265 breast cancer patients at our institution showed involvement of ten or more positive axillary lymph nodes. Of these, 171 received standard adjuvant combination chemotherapy, but not HDCT consolidation, and were the subjects of our study. One hundred twenty-eight patients met the standard selection criteria for HDCT with hematological support (< 60 years, no significant concomitant disease, and no progression during adjuvant treatment), whereas 43 did not. Clinical outcome was analyzed by using disease-free survival (DFS) and overall survival (OS) as endpoints. To provide an assessment of the short-term efficacy of HDCT, we also evaluated the outcome in a cohort of 39 patients from the last 4 years who met the criteria for, and were actually treated with, HDCT after adjuvant chemotherapy. RESULTS With a median follow-up of 4.4 years (range, 0.7 to 17.2 years), 112 of the 171 patients have had a relapse, and 87 have died. The estimated 5-year DFS was 32.3%, and OS was 49.4%. DFS was significantly higher for patients who met the HDCT criteria (36.6% at 5 years) than for those who did not (15.8% at 5 years; P < .05). OS was also significantly more favorable in patients meeting HDCT criteria (55.4% at 5 years) than in patients not meeting HDCT criteria (22.7% at 5 years; P < .01). We performed a multivariate analysis to adjust for other potential prognostic factors and found that meeting the HDCT criteria and having undergone locoregional radiotherapy were the only significant independent predictors for DFS and OS. Finally, we compared the outcome of the 128 patients who met the HDCT criteria and were treated with conventional adjuvant chemotherapy only with that of the 39 patients who met the criteria and who actually underwent HDCT, and we did not observe significant differences in the DFS or OS between these groups. CONCLUSIONS Meeting HDCT inclusion criteria is an independent indicator of favorable prognosis in high-risk breast cancer patients. The selection of patients by these criteria may explain, at least in part, the promising short-term results of nonrandomized adjuvant HDCT trials in high-risk breast cancer.

Pharmacological and small interference RNA-mediated inhibition of breast cancer- associated fatty acid synthase (oncogenic antigen-519) synergistically enhances taxol (Paclitaxel)-induced cytotoxicity

The relationship between breast cancer‐associated fatty acid synthase (FAS; oncogenic antigen‐519) and chemotherapy‐induced cell damage has not been studied. We examined the ability of C75, a synthetic slow‐binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule‐interfering agent Taxol™ (paclitaxel) in SK‐Br3, MDA‐MB‐231, MCF‐7 and multidrug‐resistant MDR‐1 (P‐Glycoprotein)‐overexpressing MCF‐7/AdrR breast cancer cells. When the combination of C75 with Taxol™ in either concurrent (C75 + Taxol™ 24 hr) or sequential (C75 24 hr → Taxol™ 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median‐effect plot analyses, co‐exposure of C75 and Taxol™ mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol™ mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule‐dependent in MCF‐7 cells, we next evaluated the effects of C75 on Taxol™‐induced apoptosis as well as Taxol™‐activated cell death and cell survival‐signaling pathways in this breast cancer cell model. An ELISA for histone‐associated DNA fragments demonstrated that C75 and Taxol™ co‐exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre‐treatment did not enhance the apoptosis‐inducing activity of Taxol™. Co‐exposure to C75 and Taxol™ induced a remarkable nuclear accumulation of activated p38 mitogen‐activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor‐suppressor protein that was phosphorylated at Ser46, a p38 MAPK‐regulated pro‐apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol™ induced a significant stimulation of the proliferation and cell survival mitogen‐activated protein kinase extracellular signal‐regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol™ inactivated the anti‐apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down‐regulation of AKT phosphorylation at its activating site Ser473 without affecting AKT protein levels. To rule out a role for non‐FAS C75‐mediated effects, we finally used the potent and highly sequence‐specific mechanism of RNA interference (RNAi) to block FAS‐dependent signaling. Importantly, SK‐Br3 and multi‐drug resistant MCF‐7/AdrR cells transiently transfected with sequence‐specific double‐stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol™‐induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti‐mitotic drug Taxol™ against breast cancer cells and that this chemosensitizing effect is schedule‐dependent. We suggest that the alternate activation of both the pro‐apoptotic p38 MAPK‐p53 signaling and the cytoprotective MEK1/2 → ERK1/2 cascade, as well as the inactivation of the anti‐apoptotic AKT activity may explain, at least in part, the sequence‐dependent enhancement of Taxol™‐induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes‐based chemotherapy in human breast cancer.