Alice Zoccoli

Early magnesium modifications as a surrogate marker of efficacy of cetuximab-based anticancer treatment in KRAS wild-type advanced colorectal cancer patients

BACKGROUND KRAS wild-type mutational status is necessary but not sufficient to get benefit from epidermal growth factor receptor inhibition. Predictive markers are currently being evaluated. In this study, we investigated early hypomagnesemia as a predictor of efficacy and outcome in terms of time to progression (TtP) and overall survival (OS) in a cohort of patients affected by advanced colorectal adenocarcinoma KRAS wild-type cetuximab-treated. PATIENTS AND METHODS One hundred and forty-three patients affected by stage IV colorectal adenocarcinoma KRAS wild type receiving cetuximab + irinotecan (CTX+IRI) as third-line anticancer treatment and resistant to oxaliplatin- and irinotecan-based chemotherapy were retrospectively included. Magnesium plasma levels were measured before the first day and 7, 14, 21 and 28 days after CTX+IRI infusion. RESULTS The median magnesium basal value showed a statistically significant decrease after the start of CTX+IRI treatment (at 28 days, P < 0.0001). Patients with an early decrease of magnesium levels >50% compared with the basal level had a higher tumor response rate (55.8% versus 16.7%, P < 0.0001), a longer TtP (6.3 versus 3.6, P < 0.0001) and a longer median OS (11.0 versus 8.1, P = 0.002). CONCLUSIONS We have shown that early hypomagnesemia could be a predictor of efficacy and outcome in those patients. Magnesium circulating level is an easy and inexpensive biomarker to routinely be detected in patients treated with cetuximab.

Emerging strategies to overcome the resistance to current mTOR inhibitors in renal cell carcinoma

The mammalian target of rapamycin (mTOR) has emerged as an attractive cancer therapeutic target. Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. Unfortunately, a number of potential mechanisms that may lead to resistance to mTOR inhibitors have been proposed. In this paper, we discuss the mechanisms underlying resistance to mTOR inhibitors, which include the downstream effectors of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway, the activation of hypoxia-inducible factor (HIF), the PIM kinase family, PTEN expression, elevated superoxide levels, stimulation of autophagy, immune cell response and ERK/MAPK, Notch and Aurora signaling pathways. Moreover, we present an updated analysis of clinical trials available on PubMed Central and www.clinicaltrials.gov, which were pertinent to the resistance to rapalogs. The new frontier of inhibiting the mTOR pathway is to identify agents targeting the feedback loops and cross talks with other pathways involved in the acquired resistance to mTOR inhibitors. The true goal will be to identify biomarkers predictive of sensitivity or resistance to efficiently develop novel agents with the aim to avoid toxicities and to better choose the active drug for the right patient.

Expression levels of MDR1, MRP1, MRP4, and MRP5 in peripheral blood mononuclear cells from HIV infected patients failing antiretroviral therapy

The aim of the study was to evaluate the mRNA expression of four relevant ABC‐transporter genes [MDR1 (P‐glycoprotein; Pgp), MRP1, MRP4, and MRP5] in HIV‐positive individuals failing treatment and analyze the association between the levels of their expression and viral load, CD4 cell count, and therapeutic history. Ninety‐eight HIV‐positive samples and 20 samples from healthy donors were analyzed, retrospectively. Peripheral blood mononuclear cells (PBMCs) from HIV1‐positive individuals were collected at the time of virological failure. Expression of mRNA of Pgp, MRP1, MRP4, and MRP5 in PBMCs was evaluated by real‐time PCR. A high inter‐individual variability was observed in both HIV‐positive individuals and healthy donors but the expression levels of all mRNA analyzed were significantly higher in the HIV‐infected group (P < 0.05). A weak but significant inverse correlation was observed between CD4 cell counts and expression levels of MRP4 and MRP5. Comparison of mRNA expression between individuals with different therapeutic histories showed that expression of MRP4 and MRP5 genes in patients who were both protease inhibitor (PI) and non‐nucleoside reverse transcriptase inhibitor (NNRTI)‐experienced was significantly higher than in patients who were PI experienced but NNRTI‐naïve. In conclusion, the mRNA expression of Pgp, MRP1, MRP4, and MRP5 varies among HIV‐infected patients and healthy donors but is significantly higher in HIV‐positive patients than in donors. The expression of MRP4 and MRP5 seems to correlate with CD4 cell counts. The same protein seems to be overexpressed in patients receiving NNRTIs. J. Med. Virol. 80:766–771, 2008.

Serum VEGF levels as predictive marker of bisphosphonate-related osteonecrosis of the jaw

Recent studies have been reported that angiogenesis suppression may play a role in developing bisphosphonate-related osteonecrosis of the jaw (B-ONJ). According to these evidence we evaluated the role of VEGF as predictive marker of B-ONJ onset. Of the 81 patients, 6 developed B-ONJ following bisphosphonate treatment. These patients showed a strongest decrease in VEGF circulating levels at day 7 and at day 21 after the first administration. These data demonstrated for the first time that the anti-angiogenic properties of bisphosphonates are directly linked to B-ONJ pathogenesis and serum VEGF levels could represent an effective early predictive marker.

Premetastatic niche: ready for new therapeutic interventions?

Introduction: Bone marrow-derived cells (BMDC) localize in premetastatic niche through chemokines and integrins signals and establish clusters that precede the arrival of even single metastatic tumor cell at distant site. CSCs demonstrate an increased metastatic propensity and would seem likely candidates for the acquisition of migratory capabilities and propagation of heterogeneous tumor cell populations to different target organs. Sonic Hedgehog (SHH), FOXM1 and Notch pathways and signaling molecules such as integrin and chemokine could dictate their fate. Areas covered: In this review, the molecular mechanisms of premetastatic niche onset are summarized. Expert opinion: Premetastatic niche is defined as a fertile microenvironment that forms in metastatic target organ and facilitates the invasion, survival and/or proliferation of metastatic tumor cells, providing a novel mechanism for the promotion of metastasis. Drugs targeting premetastatic niche could represent a new promising therapeutic approach in the treatment of bone metastases.

Longitudinal evaluation of vitamin D plasma levels during anthracycline- and docetaxel-based adjuvant chemotherapy in early-stage breast cancer patients

More than 30%–50% of women at breast cancer diagnosis are vitamin D deficient. The importance of preserving bone health in survivors of breast cancer has gained predominant priority because these women have higher than average rates of bone loss and fracture as they age and they have a 15% higher fracture risk than women without a history of breast cancer. Recently, prospective cohort studies reported an inverse association between serum 25-hydroxyvitamin D (25-OHD) levels and breast cancer prognosis [1]. We evaluate the fluctuations of vitamin D circulating levels during sequential anthracyclineand docetaxel-based adjuvant chemotherapy in early-stage breast cancer patients. The levels of 25-OHD at diagnosis (before chemotherapy) and during adjuvant chemotherapy (after four anthracyclinebased cycles and, at the end of adjuvant chemotherapy, after four docetaxel-based administrations) in seven preand 13 postmenopausal women (median age was 53 years) with early-stage breast cancer following radical surgery were assessed. Our results highlighted that all patients were deficient in vitamin D plasma levels at baseline. The median serum Annals of Oncology letters to the editor

Should Oncologists Be Aware in Their Clinical Practice of KRAS Molecular Analysis

TO THE EDITOR: KRAS mutations have been implicated in the pathogenesis of numerous tumors and detected in approximately 40% to 45% of colorectal adenocarcinomas. The mutational status of KRAS has emerged as a predictive factor for patients with metastatic colorectal cancer (mCRC) undergoing treatment with epidermal growth factor receptor (EGFR) –targeted therapy. In the clinical setting, a consistent correlation between presence of a KRAS mutation in codon 12 or 13 and lack of response to anti-EGFR monoclonal antibody therapy in mCRC patients has been described. 1-3 Based on these findings, anti-EGFR therapies were approved by the US Food and Drug Administration, as well as the European Medicinal Agency, for use exclusively in patients with mCRC harboring KRAS wild-type status and in combination with irinotecan after progression or as first line with standard chemotherapy. Thus, KRAS mutational status actually represents a criterion to select patients who would benefit from anti-EGFR therapy. Point mutations in KRAS codons 12 and 13 represent two hotspots that include more than 95% of mutations in this gene. 4 Different methods forKRAS mutation analysis have been used in experimental settings or as part of clinical trials. Instead, in relation to KRAS mutational status for routine use, comparison of diverse techniques and their diagnostic applicability is being recently analyzed and needs further critical evaluation. Current international recommendations for KRAS mutational status detection advice well-established molecular assays use. 5 All of these tests have to be sensitive, specific, and reliable DNA-based assays. Among them, direct sequencing analysis and real-time polymerase chain reaction (PCR) are commonly used 5 whereas pyrosequencing has recently emerged as a new powerful sequencing methodology for single nucleotide polymorphism/mutation analysis. Direct sequencing method is able to detect all the mutations in KRAS gene but it has lower sensitivity compared with other methods. Real-time PCR uses oligonucleotide primers that bind specifically to the most common mutations in codons 12 and 13 and it is sufficiently sensitive to identify KRAS mutants represented at a relatively low frequency. The detection limit of these two methods is around 20% of the mutation rate. 6 The pyrosequencing-based assay detects KRAS mutations in codons 12, 13 and it is more sensitive than traditional sequencing methods or real-time PCR being able to detect mutation rate represented in fewer than 20% of the analyzed sample. Differences in detection limits and quantitation among KRAS testing platforms represent a relevant issue for pathologists to face with, since it is currently unknown what level of test sensitivity is unambiguously required to provide useful and predictive information in clinical practice. In our institution, pyrosequencing platform is routinely used to detectKRAS mutational status in patients with mCRC. We tested 100 consecutive patients and we observed a higher percentage, attested on more than 50% of KRAS mutated (codon 12/13) patients than that usually reported (40% to 45%). To explore this difference of approximately 20%, we retrospectively obtained archival tissue samples from patients previously resulted wild-type for KRAS codon 12/13 by real time-PCR. All these samples were reanalyzed by performing real time-PCR and pyrosequencing methods at the same time, in the same laboratory and in a blinded way. We aimed to identify the diagnostic and clinical implications in using the two methods in KRAS mutational status detection as mandatory criterion to select patients to receive anti-EGFR therapy. Formalin-fixed paraffin-embedded tumor samples from 29 patients with primary colorectal adenocarcinomas KRAS wild type for codon 12/13 were identified. Other main selection criteria were: tumor tissue availability, clinical response (partial or complete response; taking into account the best clinical response according to Response Evaluation Criteria in Solid Tumors [RECIST]) after cetuximab irinotecan after progression on prior irinotecan-based therapy. Of 29 patients, three (10.3%) were identified as KRAS mutant in codon 12 G for 12D mutation by pyrosequencing whereas all of them were reconfirmedKRAS wild-type by real-time PCR. Consistent with the findings in the literature, mutations in codon 12 were far more common and all of them were G12D. All three patients who were mutant by pyrosequencing have been previously treated with anti-EGFR therapy according to wild-type status reported by real time-PCR. All of these patients with mutations showed partial response according to RECIST as best response during cetuximab-based chemotherapy. Moreover, the percentage of mutation rate at pyrosequencing resulted of 18.6%, 20.%, and 18.2%, respectively, which are under the general detection limit of real time-PCR. Our concerns are the clinical implications of using the most recent technologies in diagnostic routine for KRAS status detection. Until pyrosequencing introduction in diagnostic setting, the detection limit of the other molecular tests was not inferior to 20%. Our experience suggests that in those patients with low KRAS mutational rate by pyrosequencing ( 20%), critical clinical interpretation of the result is needed. The identification of a very low frequency of KRAS mutations in patients could not influence negatively tumor anti-EGFR response, as demonstrated by our three mutated responder patients. These observations would advise oncologists in considering critically pyrosequencing as routine diagnostic test to mandatory discriminate patients eligible to receive anti-EGFR therapy in clinical practice. Prospective studies using pyrosequencing are needed to evaluate the role of different percentages of mutated alleles in terms of response to anti-EGFR treatment in larger number of patients.

Association between NOD2/CARD15 polymorphisms and coronary artery disease: a case–control study

Inflammation and immune response play an important role in the pathogenesis of atherosclerosis. In this prospective study we tested the hypothesis of whether polymorphic variations in the NOD2/CARD15 gene may influence the risk of developing clinically evident coronary artery disease (CAD). ARG702TRP, GLY908ARG, and Leu1007fsinsC NOD2/CARD15 polymorphisms were analyzed in 109 consecutive patients with angiographically documented CAD and in 109 age- and sex-matched healthy controls. The ARG702TRP, GLY908ARG, and Leu1007fsinsC polymorphisms were analyzed by polymerase chain reaction followed by restriction digestion. The prevalence of the Leu1007fsinsC polymorphism was significantly increased in CAD patients compared with controls (11.9% vs 1.8%; odds ratios (OR) 7.2, 95% confidence interval (95% CI) 1.5-32.9; p = 0.01), especially in those presenting with an acute coronary syndrome (OR 5.7; 95% CI 1.1-39.7; p = 0.034 vs stable angina). In CAD patients the frequency of GLY908ARG polymorphism was significantly lower (1.8% vs 6.4% in controls; OR 0.05, 95% CI 0.01-0.69; p = 0.031, at multivariable analysis) and the prevalence of the ARG702TRP polymorphism was higher compared with controls (10.1% vs 3.7%; OR 2.9, 95% CI 0.91-9.6; p = 0.07). We report in a Caucasian population that NOD2/CARD15 polymorphisms influence the development of clinically evident and angiographically documented coronary artery disease. In particular, the Leu1007fsinsC polymorphism was associated with an increased risk of clinically evident and angiographically documented coronary atherosclerosis and clinical destabilization of coronary plaques, whereas the GLY908ARG polymorphism demonstrated a protective effect on coronary atherogenesis. These correlations were independent of cardiovascular risk factors at multivariable analysis. These findings may contribute to the identification of a novel genetic approach for the stratification of cardiovascular risk profile.

The Synergistic Effect of Everolimus and Chloroquine on Endothelial Cell Number Reduction Is Paralleled by Increased Apoptosis and Reduced Autophagy Occurrence

Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.

Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

Introduction: Activating mutations of K-Ras gene have a well-established role as predictors of resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) patients. Their prognostic value is controversial, and no data regarding the prognostic value of mutation rate, defined as the percentage of mutated alleles/tumor sample, are available. We aimed to evaluate the prognostic value of K-Rasmutation rate in a homogenous cohort of mCRC patients receiving first-line doublet plus bevacizumab. Patients and Methods: This retrospective study enrolled 397 K-Ras mutant mCRC patients from 6 Italian centers, and 263 patients were fully evaluable for our analysis. K-Ras mutation rate was assessed by pyrosequencing. Patients with less than 60% of cancer cells in tumor tissue were excluded. No patients received anti-EGFR containing anticancer therapy, at any time. Median mutation rate was 40% and was adopted as cut-off. The primary and secondary endpoints were PFS and OS respectively. Results: At univariate analysis, K-Ras mutation rate higher than 40% was significantly associated with lower PFS (7.3 vs 9.1 months; P < 0.0001) and OS (21 vs 31 months; P = 0.004). A multivariate model adjusted for age at diagnosis, site of origin of tumor tissue (primary vs metastases), referral center, number of metastatic sites, and first-line chemotherapy backbone, showed that K-Ras mutation rate remained a significant predictor of PFS and OS in the whole population. Discussion: Our data demonstrate an association between K-Ras mutation rate and prognosis in mCRC patients treated with bevacizumab-containing first-line therapy. These data deserve to be verified in an independent validation set.