Angelo Paradiso

Signatures of mutational processes in human cancer

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, ‘kataegis’, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Multicenter Validation of a Gene Expression–Based Prognostic Signature in Lymph Node–Negative Primary Breast Cancer

PURPOSE We previously identified in a single-center study a 76-gene prognostic signature for lymph node-negative (LNN) breast cancer patients. The aim of this study was to validate this gene signature in an independent more diverse population of LNN patients from multiple institutions. PATIENTS AND METHODS Using custom-designed DNA chips we analyzed the expression of the 76 genes in RNA of frozen tumor samples from 180 LNN patients who did not receive adjuvant systemic treatment. RESULTS In this independent validation, the 76-gene signature was highly informative in identifying patients with distant metastasis within 5 years (hazard ratio, [HR], 7.41; 95% CI, 2.63 to 20.9), even when corrected for traditional prognostic factors in multivariate analysis (HR, 11.36; 95% CI, 2.67 to 48.4). The actuarial 5- and 10-year distant metastasis-free survival were 96% (95% CI, 89% to 99%) and 94% (95% CI, 83% to 98%), respectively, for the good profile group and 74% (95% CI, 64% to 81%) and 65% (53% to 74%), respectively for the poor profile group. The sensitivity for 5-yr distant metastasis-free survival was 90%, and the specificity was 50%. The positive and negative predictive values were 38% (95% CI, 29% to 47%) and 94% (95% CI, 86% to 97%), respectively. The 76-gene signature was confirmed as a strong prognostic factor in subgroups of estrogen receptor-positive patients, pre- and postmenopausal patients, and patients with tumor sizes 20 mm or smaller. The subgroup of patients with estrogen receptor-negative tumors was considered too small to perform a separate analysis. CONCLUSION Our data provide a strong methodologic and clinical multicenter validation of the predefined prognostic 76-gene signature in LNN breast cancer patients.

Cytoskeleton and paclitaxel sensitivity in breast cancer : the role of β-tubulins

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the β subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I β‐tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel‐binding site of β‐tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of β‐tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC50. The Class I β‐tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF‐7 and SK‐BR‐3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III β‐tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III β‐tubulin could at least partially increase paclitaxel‐chemosensitivity. The hypothesis of a relationship between β‐tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel‐based chemotherapy. Thirty‐five percent (95% CI: 45–31) of patients with high Class III β‐tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III β‐tubulin tumor expression could be considered a predictive biomarker of paclitaxel‐clinical resistance for breast cancer patients.

Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

Purpose: In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions. Experimental Design: Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRβ1, RARβ2, HIC1, APC, CCND2, and CDH1 genes. Results: The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRβ1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARβ2 locus (P < 0.03). Conclusions: Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Cyclohexylpiperazine derivative PB28, a σ2 agonist and σ1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer

σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ2 agonist/σ1 antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ2 receptor expression, by high and low σ1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ2 receptor affinity and low σ1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC50 in nanomolar range), a consistent time exposure-independent increase of G0-G1-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ2 agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]

Multicenter Study Using Paraffin-Embedded Tumor Tissue Testing PITX2 DNA Methylation As a Marker for Outcome Prediction in Tamoxifen-Treated, Node-Negative Breast Cancer Patients

PURPOSE We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor-positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. PATIENTS AND METHODS Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor-positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). RESULTS Reproducibility of the PCR assay in replicate measurements (r(s) > or = 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (r(s) = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). CONCLUSION PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.

The Na + -H + exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells

IntroductionAn increasing body of evidence shows that the tumour microenvironment is essential in driving neoplastic progression. The low serum component of this microenvironment stimulates motility/invasion in human breast cancer cells via activation of the Na+–H+ exchanger (NHE) isoform 1, but the signal transduction systems that underlie this process are still poorly understood. We undertook the present study to elucidate the role and pattern of regulation by the Rho GTPases of this serum deprivation-dependent activation of both NHE1 and subsequent invasive characteristics, such as pseudopodia and invadiopodia protrusion, directed cell motility and penetration of normal tissues.MethodsThe present study was performed in a well characterized human mammary epithelial cell line representing late stage metastatic progression, MDA-MB-435. The activity of RhoA and Rac1 was modified using their dominant negative and constitutively active mutants and the activity of NHE1, cell motility/invasion, F-actin content and cell shape were measured.ResultsWe show for the first time that serum deprivation induces NHE1-dependent morphological and cytoskeletal changes in metastatic cells via a reciprocal interaction of RhoA and Rac1, resulting in increased chemotaxis and invasion. Deprivation changed cell shape by reducing the amount of F-actin and inducing the formation of leading edge pseudopodia. Serum deprivation inhibited RhoA activity and stimulated Rac1 activity. Rac1 and RhoA were antagonistic regulators of both basal and stimulated tumour cell NHE1 activity. The regulation of NHE1 activity by RhoA and Rac1 in both conditions was mediated by an alteration in intracellular proton affinity of the exchanger. Interestingly, the role of each of these G-proteins was reversed during serum deprivation; basal NHE1 activity was regulated positively by RhoA and negatively by Rac1, whereas RhoA negatively and Rac1 positively directed the stimulation of NHE1 during serum deprivation. Importantly, the same pattern of RhoA and Rac1 regulation found for NHE1 activity was observed in both basal and serum deprivation dependent increases in motility, invasion and actin cytoskeletal organization.ConclusionOur findings suggest that the reported antagonistic roles of RhoA and Rac1 in cell motility/invasion and cytoskeletal organization may be due, in part, to their concerted action on NHE1 activity as a convergence point.

Cell kinetics and hormonal receptor status in inflammatory breast carcinoma. Comparison with locally advanced disease.

The biological and prognostic role of hormone receptor status and proliferative activity have been studied in two series of patients affected by inflammatory breast carcinoma (IBC, 28 patients) and locally advanced breast cancer (LABC, 50 patients). Estrogen receptor (ER) and progesterone receptor (PgR) were measured by dextran‐coated charcoal (DCC) method whereas proliferative activity was measured by 3H‐thymidine autoradiographic labeling index (LI). The percentages of ER+ and PgR+ cases resulted lower in IBC than in LABC (ER+, 44% versus 64%; PgR+, 30% versus 51%, respectively), pertaining to both premenopausal and postmenopausal women. Inflammatory breast carcinoma showed a higher median LI value than LABC (3.5% versus 1.6%; P = 0.006). Regarding clinical aspects, time to progression (TTP) in IBC patients was not affected by hormone receptor status (19 evaluable patients) or by LI (17 evaluable patients); PgR+ status and low LI resulted important for individualizing women with a longer median overall survival (OS). Inflammatory breast carcinoma has been verified to be a heterogeneous biological entity for which hormone receptors and cell kinetics could be useful in identifying patients with different prognoses and therefore candidates for a personalized therapy.

Changes in CpG Islands Promoter Methylation Patterns during Ductal Breast Carcinoma Progression

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

p53 and PCNA expression in advanced colorectal cancer: Response to chemotherapy and long‐term prognosis

In a series of 71 patients with advanced colorectal cancer treated with biochemically modulated 5‐fluorouracil (5‐FU) and methotrexate (MTX), we investigated the relationship between the proliferating‐cell nuclear antigen (PCNA) (PC10) and p53 (Pab 1801) primary‐tumor immunohistochemical expression with respect to clinical response and long‐term prognosis. Nuclear p53 expression was demonstrated in 44% of samples (any number of positive tumor cells) while all tumors showed a certain degree of PCNA immunostaining. PCNA immunostaining was correlated with histopathologic grade and p53 expression, while p53 was not correlated with any of the parameters considered. The probability of clinical response to biochemically modulated 5‐FU was independent of p53 and PCNA expression. p53 expression (all cut‐off values) was not associated with short‐ or long‐term clinical prognosis, whereas patients with higher PCNA primary‐tumor expression showed longer survival from treatment and survival from diagnosis, according to univariate and multivariate analysis, particularly in the sub‐set of colon‐cancer patients. We conclude that the clinical response of advanced‐colorectal‐cancer patients to biochemically modulated 5‐FU and MTX cannot be predicted by PCNA and p53 primary‐tumor expression, but high PCNA expression appears to be independently related to long‐term prognosis.