Pierluigi Pilati

Quantitative real-time PCR: a powerful ally in cancer research

In this era of the Human Genome Project, quantitation of gene expression in tumor or host cells is of paramount importance for investigating the gene patterns responsible for cancer development, progression and response or resistance to treatment. Quantitative real-time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that supports its use as a routine bioinstrumentation for gene level measurement. Several applications have already been implemented in the field of cancer research, and others are being validated, showing that this molecular biology tool can provide both researchers and clinicians with precious information concerning the behavior of tumors. Knowledge of the biochemical principles underlying this biotechnology can be of great value to interpret correctly qrt-PCR data.

Gastrointestinal stromal tumors: From a surgical to a molecular approach

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. These tumors span a wide clinical spectrum from benign to malignant and have long been recognized for their nearly absolute resistance to chemotherapy and radiation treatment. We reviewed the worldwide experience on GIST diagnosis, prognosis and treatment and describe our own series. PubMed was searched for references using the terms gastrointestinal stromal tumor, GIST and gastrointestinal sarcoma. Recent reports were given emphasis because GIST is a novel clinical entity and older published work on gastrointestinal sarcomas might be contaminated with other histologic tumor types. At present, surgery is the standard treatment for primary resectable GIST. To increase the activity of conventional chemotherapeutic agents, locoregional therapies are being implemented in the clinical setting. A major breakthrough is the development of a new class of anticancer agents targeting tumor‐specific molecular abnormalities. Preliminary results on administration of imatinib mesylate, a signal transduction inhibitor, are particularly encouraging, showing potent activity of this drug against metastatic GIST. Molecular targeting of the critical pathogenetic mechanism underlying GIST might not only revolutionize the strategy to treat locally advanced and metastatic GIST but also improve disease control after macroscopically radical surgery.

Meta-Analysis of Hepatic Arterial Infusion for Unresectable Liver Metastases From Colorectal Cancer: The End of an Era?

PURPOSE The treatment of unresectable liver-confined metastatic disease from colorectal cancer (CRC) is a challenging issue. Although locoregional treatments such as hepatic arterial infusion (HAI) claim the advantage of delivering higher doses of anticancer agents directly into the affected organ, the benefit in terms of overall survival (OS) is unclear. We quantitatively summarized the results of randomized controlled trials (RCT) comparing HAI with systemic chemotherapy (SCT). METHODS To date, 10 RCTs have been published, for a total of 1,277 patients enrolled. For tumor response rates, relative risks (RR) and their 95% CIs were obtained from raw data; for OS, hazard ratios (HRs) and their 95% CIs were extrapolated from the Kaplan-Meier survival curves. RESULTS HAI regimens were based on floxuridine (FUDR) in nine of 10 RCTs, whereas in one RCT, fluorouracil (FU) + leucovorin was used. SCT consisted of FUDR, FU, FU + leucovorin, or a miscellany of FU and best supportive care in three, one, four, and two studies, respectively. Pooling the data, tumor response rate was 42.9% and 18.4% for HAI and SCT, respectively (RR = 2.26; 95% CI, 1.80 to 2.84; P < .0001). Mean weighted median OS times were 15.9 and 12.4 months for HAI and SCT, respectively; the meta-risk of death was not statistically different between the two study groups (HR = 0.90; 95% CI, 0.76 to 1.07; P = .24). CONCLUSION Currently available evidence does not support the clinical or investigational use of fluoropyrimidine-based HAI alone for the treatment of patients with unresectable CRC liver metastases, at least as a first-line therapy.

Cytoreductive Surgery Combined With Hyperthermic Intraperitoneal Intraoperative Chemotherapy for Peritoneal Carcinomatosis Arising From Colon Adenocarcinoma

AbstractBackground: Hyperthermic intraoperative intraperitoneal chemotherapy (HIIC) has been recently proposed to treat peritoneal carcinomatosis arising from colon adenocarcinoma, which is usually regarded as a lethal clinical entity. The purpose of this study was to evaluate the clinical outcome of this combined treatment. Methods: A retrospective study of 46 patients treated for peritoneal carcinomatosis from colon adenocarcinoma was performed. Thirty-four patients were treated with complete cytoreductive surgery immediately followed by intraoperative HIIC with mitomycin C and cisplatin. The clinical outcome of these 34 patients was analyzed; the median follow-up period was 14.5 months. Results: No postoperative deaths were reported. The postoperative morbidity rate was 35%. No severe locoregional or systemic toxicity was observed. The 2-year overall survival was 31%, and the median survival time and the median time to local disease progression were 18 and 13 months, respectively. Survival and local disease control in patients with well- and moderately differentiated colon adenocarcinoma were significantly better than in those with poorly differentiated tumors. Conclusions: Considering the dismal prognosis of this condition, HIIC seems to achieve encouraging results in a selected group of patients affected with resectable peritoneal carcinomatosis arising from colon adenocarcinoma. These findings support the conduction of formal phase III randomized trials.

Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis.

BackgroundQualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed.ResultsMost changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples.ConclusionsUsing a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.

Sentinel Lymph Node Molecular Ultrastaging in Patients With Melanoma: A Systematic Review and Meta-Analysis of Prognosis

PURPOSE Molecular biology-based ultrastaging of cancer is already part of the standard management of patients with hematologic malignancies, whereas the evidence for solid tumors is much more debated. Polymerase chain reaction (PCR) -based detection of melanoma cells in sentinel lymph nodes (SLN) of patients with melanoma represents an appealing prognostic tool. However, no consensus exists on the clinical implementation of this prognostic indicator for the management of these patients. METHODS Twenty-two studies enrolling 4,019 patients who underwent SLN biopsy for clinical stage I to II cutaneous melanoma were reviewed. Correlation of PCR status with TNM stage, disease recurrence rates, and survival was assessed by means of association statistics and formal meta-analysis, respectively. RESULTS PCR status correlated with both TNM stage (stage I to II v III; PCR positivity, 95.1% v 46.6%; P < .0001) and disease recurrence (PCR positive v negative; relapse rate, 16.8% v 8.7%; P < .0001). PCR positivity was also associated with worse overall (hazard ratio [HR], 5.08; 95% CI, 1.83 to 14.08; P = .002) and disease-free (HR, 3.41; 95% CI, 1.86 to 6.24; P < .0001) survival. Statistical heterogeneity was significant, underscoring the variability among overall effect estimates across studies; metaregression and subgroup analysis did not identify clear-cut sources of heterogeneity, although some study design variables were suggested as potential causes. CONCLUSION PCR status of SLN appears to have a clinically valuable prognostic power in patients with melanoma. Although the heterogeneity of the studies so far published warrants caution to avoid overestimating the favorable results of pooled data, our findings strongly support additional investigation in this field.

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.

Patterns of Recurrence after Resection of Colorectal Liver Metastases: Prediction by Models of Outcome Analysis

Abstract. Various series have reported similar survival and recurrence rates after resection of colorectal liver metastases (CRLM). If outcomes were predictable, indications for surgery could be improved. This hypothesis was tested in 135 consecutive patients with CRLM who underwent “curative” resection from 1977 to 1997. Among the 132 patients available for follow-up, three groups were identified on the basis of outcome: (1) survival of more than 5 years disease-free (n= 32; 24%); (2) diffuse recurrences within the first 6 months (n= 24; 18%); and (3) discrete recurrences for which reresection was performed (n= 16; 12%). As our results are similar to those reported in the literature, we assumed that about 50% of patients with resectable lesions have recognizable patterns of recurrence. At multivariate analysis, factors significant for disease-free survival (DFS) were the percentage of liver invasion, metastases to lymph nodes at the primary site, number of metastases, preoperative glutamic pyruvic transaminase (GPT) level, and type of liver resection. On the basis of the relative risk (RR) expressed by significant prognostic factors, a score model was developed, and three prognostic groups were defined: Group A, with the best prognostic score, included 23 of 32 (72%) patients who survived more than 5 years, and that with the worst prognostic score (group C) included 22 of 24 (92%) patients with early diffuse recurrences. Extreme (especially unfavorable) outcomes can therefore be predicted. By using improved models of outcome analysis, many patients could be spared surgery as first-line treatment, and stratification criteria could be worked out for future trials.

Hyperthermic Isolated Limb Perfusion With Low-Dose Tumor Necrosis Factor-α and Melphalan for Bulky In-Transit Melanoma Metastases

Background: Melphalan (L-PAM) hyperthermic isolated limb perfusion (HILP) is currently considered the standard treatment for patients with in-transit metastases from cutaneous melanoma. We here report on the results of L-PAM and low-dose tumor necrosis factor (TNF)α HILP in patients with bulky disease.Methods: Twenty patients underwent TNFα (1 mg) and L-PAM (10 mg/L) HILP. Perfusion was performed for 90 minutes, and systemic leakage was strictly monitored. Locoregional toxicity was evaluated according to Wieberdink’s criteria, whereas tumor response was evaluated with physical examination and ultrasound scan with or without fine-needle aspiration of any suspected recurrence.Results: In all cases, systemic leakage was <5%. No postoperative deaths occurred, and locoregional toxicity was mild (grade 1 or 2) in 95% of patients. A complete tumor response was obtained in 14 patients (70%), and partial responses were obtained in 5 patients (25%). After a median follow-up of 18 months, six patients are alive and disease free, seven are alive with local or distant recurrence or both, and seven have died of disease.Conclusions: Low-dose TNFα HILP can achieve tumor responses comparable with those reported with higher doses of cytokine. Moreover, this drug regimen is associated with acceptable local toxicity, carries a smaller risk of systemic toxicity, and incurs lower costs.

Molecular detection of circulating tumor cells is an independent prognostic factor in patients with high‐risk cutaneous melanoma

Detection of circulating tumor cells (CTCs) might improve current staging procedures by identifying a subgroup of patients with minimal residual disease and thus a higher risk of disease recurrence. Forty patients with ≥2‐mm‐thick cutaneous melanoma with or without lymph node metastasis were enrolled. After standard radical surgery and adjuvant therapy in case of lymph node metastasis, patients were followed up with routine physical and radiologic assessments as well as serial PCR‐based analysis of CTCs using 2 melanoma markers (tyrosinase and Melan‐A/Mart‐1). After a median follow‐up of 30 months, 18 patients had disease recurrence and 28 were PCR‐positive before the disease became clinically evident. The sensitivity of the molecular test was 83%. Median time to PCR positivity and median PCR‐to‐relapse time were 12 and 8 months, respectively. At multivariate analysis, PCR positivity was an independent predictor of disease recurrence (hazard ratio = 2.06, 95% CI 1.07–3.35; p = 0.03). Among high‐risk melanoma patients, serial PCR‐based analysis of CTCs can identify a subgroup at higher risk of disease recurrence, with clinically significant advance. Therefore, CTC detection might be employed for the selection of patients for adjuvant treatment and during follow‐up for early indication of therapeutic failure.