Alexandros Ardavanis

Overexpression of matrix-metalloproteinase-9 in human breast cancer: a potential favourable indicator in node-negative patients.

Matrix metalloprotease-9 (MMP-9; 92 kDa type IV collaganase, gelatinase B) is regarded as, important for degradation of the basement membrane and extracellular matrix during cancer invasion and other tissue-remodelling events. In this study we evaluate the prognostic value of MMP-9, by immunoperoxidase staining in a series of 210 breast cancer tissues. The results were quantitated using the HSCORE system, which consider both staining intensity and the percentage of cells stained at given intensities. MMP-9 status was compared with the concentration of cytosolic Cathepsin-D and with other established prognostic factors, in terms of disease free survival and overall survival. The median follow-up period was 62 months. MMP-9 staining was observed primarily in cancer cells, and to a lesser degree in surrounding stromal cells. MMP-9 expression was not detected in normal breast tissue. Levels of MMP-9 expression below the cut-off point were more frequently observed in larger (P=0.014), invasive ductal histologic (P=0.037), progesterone receptor (PR)-negative and PR-strong positive tumours (P< 0.001), as well as samples belonging to patients with stage III-IV disease (P=0.009) and age 45–55 years (P=0.011). In univariate analysis, node-negative breast cancer patients with tumors positive for MMP-9 had a considerable reduction in risk for relapse (RR=0.45; P=0.039) or death (RR=0.32; P=0.009). Multivariate analysis indicated that MMP-9 status was an independent favourable predictor of OS (RR=0.47; P=0.034) in node-negative but not in node-positive patients. Our results suggest that MMP-9 may be an independent favourable prognostic factor in node-negative breast cancer patients. The overexpression of MMP-9 in breast cancer may be also used as a marker to subdivide node negative breast cancer patients in order to determine the optimal treatment modality.

Cardiotoxicity induced by tyrosine kinase inhibitors

Background. Cardiotoxicity is a serious side effect of drugs used to treat cancer patients. Older chemotherapy drugs such as the anthracyclins and new targeted therapies, mainly trastuzumab, have been implicated in causing clinically significant cardiac dysfunction, which may be irreversible for many patients. The advent of a new category of drugs, the tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia, gastrointestinal stromal tumors and renal cancer, while their indications include a variety of other types of tumors. Methods. Assessment of the incidence and severity of cardiac toxicity caused by the tyrosine kinase inhibitors and discussion on the molecular mechanisms and mode of diagnosis based on recent clinical trials. Review of related literature. Results. Cardiac toxicity can be caused by the tyrosine kinase inhibitors imatinib mesylate, dasatinib, nilotinib, sunitinib, sorafenib and lapatinib, while gefitinib and erlotinib have not been related to toxic effect on the heart. Although targeted therapies are considered less toxic and better tolerated by patients compared with classic chemotherapy drugs, certain complications can be very serious and as these agents have been in use for a limited period of time, the exact profile of side effects will be better defined in the years to come. Cardiac toxicity may range from asymptomatic subclinical abnormalities such as electrocardiographic changes and left ventricular ejection fraction decline to life threatening events like congestive heart failure and acute coronary syndromes. For patients with severe side effects, discontinuation of treatment is warranted. Conclusions. Careful cardiac monitoring and assessment by a cardiologist throughout the course of treatment with those TKIs that exert cardiac toxic effect is of primary importance.

CD4+CD25+ Regulatory T-Cell Frequency in HER-2/neu (HER)-Positive and HER-Negative Advanced-Stage Breast Cancer Patients

Purpose: CD4+CD25bright regulatory T cells (Tregs) are increased in patients with several malignancies and correlate with disease stage and prognosis. Breast cancer patients represent a heterogeneous population with unpredictable disease progression even at advanced stages. Circulating Tregs in correlation with HER-2/neu (HER) status and treatment with chemotherapy, either alone or in combination with trastuzumab therapy, were monitored in advanced-stage breast cancer patients. Experimental Design: Circulating Treg frequency and absolute counts of 46 HER+ and 28 HER−, stage III and IV, breast cancer patients before therapy and during trastuzumab therapy and/or chemotherapy have been compared with 24 healthy donors and correlated with plasma HER extracellular domain concentration and clinical outcome. Results: Treg frequency in HER+ patients was significantly increased compared with both HER− patients and healthy donors. Trastuzumab therapy, with or without combined chemotherapy, resulted in a progressive decrease of circulating Tregs. Percentage change in Tregs statistically correlated with percentage change in plasma HER extracellular domain. Furthermore, decrease in Tregs correlated with either objective clinical response or stable disease, whereas increased Treg frequency during trastuzumab therapy coincided with disease progression. No statistically significant change in Treg frequency following chemotherapy was observed in HER− patients. Conclusions: Treg cell frequency does not directly correlate with clinical stage in breast cancer, as stage III and IV HER+ and HER− patients exhibit significantly different Treg profiles. Trastuzumab therapy, either alone or combined with chemotherapy, results in decreased Treg frequency in HER+ advanced patients with an objective clinical response.

Gemcitabine and docetaxel as first-line treatment for advanced urothelial carcinoma: a phase II study

The purpose of the study was to investigate the toxicity and efficacy of the combination of gemcitabine and docetaxel in untreated advanced urothelial carcinoma. Patients with previously untreated, locally advanced/recurrent or metastatic urothelial carcinoma stage-IV disease were eligible. Patients with Performance status: PS ECOG >3 or age >75 years or creatinine clearance <50 ml min−1 were excluded. Study treatment consisted of docetaxel 75 mg m−2 (day 8) and gemcitabine 1000 mg m−2 (days 1+8), every 21 days for a total of six to nine cycles. A total of 31 patients with urothelial bladder cancer, 25 men and six women, aged 42–74 (median 64) years were enrolled. The majority of patients had a good PS (51.6%; PS 0). In all, 15 (48.3%) patients had locally advanced or recurrent disease only and 16 (54.8%) presented with distant metastatic spread, with multiple site involvement in 22.5%. Toxicity was primarily haematologic, and the most frequent grade 3–4 toxicities were anaemia 11 (6.7%) thrombocytopenia eight (4.9%), and neutropenia 45 (27.6%), with 10 (6.1%) episodes of febrile neutropenia. No toxic deaths occurred. A number of patients had some cardiovascular morbidity (38.7%). Nonhaematological toxicities except alopecia (29 patients) were mild. Overall response rate was 51.6%, including four complete responses (12.9%) and 12 partial responses (38.7%), while a further five patients had disease stabilisation (s.d. 16.1%). The median time to progression was 8 months (95% CI 5.1–9.2 months) and the median overall survival was 15 months (95% CI 11.2–18.5 months), with 1-year survival rate of 60%. In conclusion, this schedule of gemcitabine and docetaxel is very active and well tolerated as a first-line treatment for advanced/relapsing or metastatic urothelial carcinoma. Although its relative efficacy and tolerance as compared to classic MVAC should be assessed in a phase III setting, the favourable toxicity profile of this regimen may offer an interesting alternative, particularly in patients with compromised renal function or cardiovascular disease.

Clinical evaluation of PRMT1 gene expression in breast cancer

Methylation of arginine residues has been implicated in many cellular activities like mRNA splicing, transcription regulation, signal transduction and protein–protein interactions. Protein arginine methyltransferases are the enzymes responsible for this modification in living cells. The most commonly used methyltransferase in man is protein arginine methyltransferase 1 (PRMT1). Since methylation processes appear to interfere in the emergence of several diseases, including cancer, we investigated the localisation of the protein in cancer tissue and, for the first time, the relation that possibly exists between the expression of PRMT1 gene and breast cancer progression. We used tumour specimens from 62 breast cancer patients and semi-quantitative RT-PCR to determine the expression of PRMT1 gene and was found to be associated with patient’s age (p = 0.002), menopausal status (p = 0.006), tumour grade (p = 0.03), and progesterone receptor status (p = 0.001). Survival curves revealed that PRMT1-v1 status-low expression relates to longer disease-free survival (DFS; p = 0.036). To the contrary, PRMT1-v2 status is not associated neither with the clinical or pathological parameters nor with DFS (p = 0.31). PRMT1-v3 was not statistically significantly expressed in breast cancer tissue. Selected cancer and normal breast samples were stained for PRMT1. In both normal and cancerous breast tissues, staining was in the cytoplasm and only in rare cases the cell nucleus appeared stained. Present results show a potential use for this gene as a marker of unfavourable prognosis for breast cancer patients.

Human kallikrein-related peptidase 12 (KLK12) splice variants expression in breast cancer and their clinical impact

Kallikrein-related peptidases (KLKs) are a group of 15 serine proteases, hormonally regulated, and localized on chromosome 19q13.4. Alternative splicing is a process that plays significant role in the development, physiology, and different diseases, like cancer. Kallikrein family numbers more than 82 alternative transcripts. Understanding the role that those gene transcripts play in various cancer types, could lead to the discovery of diagnostic markers or drug targets. The present study was designed to analyze the expression profile of the splice variants of kallikrein-related peptidase 12 (KLK12) in breast cancer patients and to evaluate their clinical significance. KLK12 splice variants (KLK12sv3 and KLK12sv1/KLK12sv2) were examined in 69 tissue samples of breast cancer using quantitative real-time PCR as well as semi-quantitative PCR. Relative quantitative expression of KLK12 was statistically associated to clinicopathological parameters. From the splice variants examined, statistical associations with clinicopathological parameters were obtained only from KLK12sv3 variant. KLK12sv3 is more frequently expressed in tumors of lower grade (p = 0.040), early patient TNM stage (p = 0.024), and smaller tumor size (p = 0.023). Positive KLK12sv3 expression is associated with longer patient disease-free survival (DFS) (p = 0.042) and higher progesterone receptor concentration (p = 0.008). KLK12sv1/KLK12sv2 expression is statistically associated with KLK12sv3 expression (p = 0.001). KLK12sv3 can be regarded as a marker of good prognosis in breast cancer.

Neoadjuvant therapy for locally advanced breast cancer: Focus on chemotherapy and biological targeted treatments' armamentarium.

Despite progress achieved in diagnosis and therapy in recent years, locally advanced breast cancer (LABC) remains a major clinical issue. Biological characteristics and clinical behavior varies widely, ranging from indolent to locally aggressive or generalized disease. In depth knowledge of biology of cancer progression and cancer could lead to the identification of tumor characteristics associated with outcome. Neoadjuvant chemotherapy (NCT) integrated into a multimodality program is nowadays the established treatment in LABC. Although our efforts in this research task are ongoing, of special clinical interest is the integration of anti-HER2 and other biological therapies, as anti-angiogenesis targeted treatments, that may further improve the long term control of LABC. Clinical management of LABC could be modified based on molecular biology and an approach tailored to each patient will optimize therapy.

Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide AE37 vaccine in breast cancer patients to prevent recurrence

BACKGROUND AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccines efficacy. METHODS Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed. RESULTS The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12). CONCLUSION The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted.

Quantitative analysis of BCL2 mRNA expression in nasopharyngeal carcinoma: an unfavorable and independent prognostic factor

Programmed cell death plays a vital role in a wide variety of physiological processes. Defects in apoptotic cell death contribute to neoplastic diseases by preventing or delaying normal cell death. BCL2 (Bcl-2) is an anti-apoptotic gene with marked up-regulation in various malignancies, such as breast cancer, in which expression of the BCL2 protein has been proposed as a prognostic tumor biomarker. The purpose of the current study was to investigate mRNA expression of the BCL2 gene in nasopharyngeal carcinoma (NPC) biopsies and assess its prognostic value. For this purpose, total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time PCR methodology for BCL2 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as an endogenous control gene. Relative quantification analysis was performed using the comparative CT (2−∆∆CT) method. High BCL2 mRNA levels were detected in advanced-stage nasopharyngeal tumors (p = 0.030). Furthermore, BCL2 mRNA expression was strongly associated with lymph node involvement (p = 0.009) and presence of distal metastases (p = 0.013). Survival analysis demonstrated that patients with BCL2-positive nasopharyngeal tumors have significantly shorter disease-free and overall survival (p = 0.011 and p = 0.028, respectively). The major contribution of the current study is the quantification and evaluation, for the first time, of the prognostic significance of the BCL2 mRNA expression in nasopharyngeal carcinoma (NPC) patients. Our results suggest that mRNA expression levels of BCL2 may represent a novel unfavorable and independent tumor biomarker for nasopharyngeal carcinoma.

Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide GP2 vaccine in breast cancer patients to prevent recurrence

GP2 is a HER2-derived, HLA-A2+ restricted peptide. Phase I studies showed GP2 administered with GM-CSF to be safe and immunogenic. Here we report the primary analysis of a prospective, randomized, multicenter phase II adjuvant trial conducted to determine the vaccines efficacy. The trial enrolled HLA-A2+, clinically disease-free, node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2 (immunohistochemistry[IHC] 1+-3+). Patients were randomized to GP2+GM-CSF versus GM-CSF alone. Disease-free survival (DFS) was analyzed in intention-to-treat (ITT) and per-treatment cohorts; pre-specified subgroup analyses were performed for patients with IHC 3+ or FISH+ disease. The trial enrolled 180 patients; 89 received GP2+GM-CSF and 91 received GM-CSF alone. The groups were well-matched for clinicopathologic characteristics. Toxicities have been minimal. The Kaplan-Meier estimated 5-year DFS rate in the ITT analyses was 88% (95% CI:78-94%) in vaccinated vs. 81% (95% CI:69-89%) (P = 0.43) in control patients after a 34 month median follow-up. In the per-treatment analysis, the estimated 5-year DFS rates were 94% (95% CI:83-98%) and 85% (73-92%) (P = 0.17). In IHC 3+/FISH+ patients, the estimated 5-year DFS rate was 94% (82-98%) in vaccinated patients (n = 51) vs. 89% (71-96%) in control patients (n = 50), (P = 0.86) in the ITT analyses and 100% vs. 89% (71-96%) in vaccinated vs. control patients in the per-treatment analyses (P = 0.08). While the overall ITT analysis did not demonstrate benefit to vaccination, this trial confirmed that the GP2 vaccine is safe and suggests that vaccination may have clinical activity, particularly in patients with HER2 overexpression who received the full vaccine series (ie per-treatment group).