Mauro Magnani

KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer

Background:KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC.Methods:We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients.Results:Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients.Conclusion:Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.

PTEN Expression and KRAS Mutations on Primary Tumors and Metastases in the Prediction of Benefit From Cetuximab Plus Irinotecan for Patients With Metastatic Colorectal Cancer

PURPOSE PTEN, AKT, and KRAS are epidermal growth factor receptor (EGFR) downstream regulators. KRAS mutations confer resistance to cetuximab. This retrospective study investigated the role of PTEN loss, AKT phosphorylation, and KRAS mutations on the activity of cetuximab plus irinotecan in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS A cohort of patients with irinotecan-refractory mCRC who were treated with cetuximab plus irinotecan was tested for PTEN immunoreactivity (ie, immunohistochemistry; IHC), pAKT IHC, and KRAS mutations. Analyses were performed both on primary tumors and on related metastases, and the association among IHC, mutational results, and treatment outcomes was investigated. RESULTS One-hundred two patients were eligible. Ninety-six primary tumors, 59 metastases, and 53 paired samples were available. Forty-nine primary tumors (58% of assessable samples) had a preserved PTEN expression (PTEN-positive), whereas 35 (40% of assessable samples) were pAKT-positive. Levels of concordance between primary tumors and metastases were 60%, 68%, and 95% for PTEN, pAKT, and KRAS, respectively. PTEN status on primary tumors and pAKT status both on primary tumors and on metastases did not predict response or progression-free survival (PFS). On metastases, 12 (36%) of 33 patients with PTEN-positive tumors were responders compared with one (5%) of 22 who had PTEN-negative tumors (P = .007). The median PFS of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (hazard ratio [HR], 0.49; P = .005). Patients with PTEN-positive metastases and KRAS wild type had longer PFS compared with other patients (5.5 months v 3.8 months; HR, 0.42; P = .001). CONCLUSION PTEN loss in metastases may be predictive of resistance to cetuximab plus irinotecan. The combination of PTEN IHC and KRAS mutational analyses could help to identify a subgroup of patients with mCRC who have higher chances of benefiting from EGFR inhibition.

Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells☆

A simple and rapid method for the determination of ATP, ADP, AMP, NADP+, NAD+, NADPH, and NADH in human erythrocytes is described. A single-step extraction procedure employing alkaline medium and CF 50A Amicon ultrafiltration membranes allows a simultaneous and total recovery of the compounds of interest. Analysis is performed by reverse-phase high-performance liquid chromatography on a 5-micron Supelcosil LC-18 column and uv detection. Extraction and analysis require about 30 min. Levels of adenine and pyridine nucleotides in normal adults are also presented.

Pharmacogenetic Profiling in Patients With Advanced Colorectal Cancer Treated With First-Line FOLFOX-4 Chemotherapy

PURPOSE The objective is to investigate whether polymorphisms with putative influence on fluorouracil/oxaliplatin activity are associated with clinical outcomes of patients with advanced colorectal cancer treated with first-line oxaliplatin, folinic acid, and fluorouracil palliative chemotherapy. MATERIALS AND METHODS Consecutive patients were prospectively enrolled onto medical oncology units in Central Italy. Patients were required to have cytologically/histologically confirmed metastatic disease with at least one measurable lesion. Peripheral blood samples were used for genotyping 12 polymorphisms in thymidylate synthase, methylenetetrahydrofolate reductase, xeroderma pigmentosum group D (XPD), excision repair cross complementing group 1 (ERCC1), x-ray cross complementing group 1, x-ray cross complementing protein 3, glutathione S-transferases (GSTs) genes. The primary end point of the study was to investigate the association between genotypes and progression-free survival (PFS). RESULTS In 166 patients, ERCC1-118 T/T, XPD-751 A/C, and XPD-751 C/C genotypes were independently associated with adverse PFS. The presence of two risk genotypes (ERCC1-118 T/T combined with either XPD-751 A/C or XPD-751 C/C) occurred in 50 patients (31%). This profiling showed an independent role for unfavorable PFS with a hazard ratio of 2.84% and 95% CI of 1.47 to 5.45 (P = .002). Neurotoxicity was significantly associated with GSTP1-105 A/G. Carriers of the GSTP1-105 G/G genotype were more prone to suffer from grade 3 neurotoxicity than carriers of GSTP1-105 A/G and GSTP1-105 A/A genotypes. CONCLUSION A pharmacogenetic approach may be an innovative strategy for optimizing palliative chemotherapy in patients with advanced colorectal cancer. These findings deserve confirmation in additional prospective studies.

Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

ABSTRACT The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4+ T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8+ T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-γ, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8+CD127− T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4+ T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8+CD127− effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8+ T cells may be useful in the clinical management of HIV-infected individuals.

High concordance of KRAS status between primary colorectal tumors and related metastatic sites: implications for clinical practice.

PURPOSE Several studies have suggested that KRAS somatic mutations may predict resistance to cetuximab- and panitumumab-based treatments in metastatic colorectal cancer (CRC) patients. Nevertheless, most experiences were conducted on samples from primaries. The aim of this study was to evaluate the grade of concordance in terms of KRAS status between primaries and related metastases. PATIENTS AND METHODS We analyzed KRAS codon 12 and 13 mutations from formalin-fixed sections of 107 CRC primaries and related metastases. Eight pairs were excluded from the analysis because of the low amount of tumor tissue in the available samples. The main characteristics were: 50 men, 49 women; median age at diagnosis, 71 years (range, 41-84). The metastatic sites analyzed were the liver in 80 patients (80.8%), lung in seven patients (7.1%), and other sites in 12 patients (12.1%). RESULTS A KRAS mutation was found in 38 (38.4%) primary tumors and in 36 (36.4%) related metastases. The rate of concordance was 96.0% (95% confidence interval, 90.0%-98.9%). Discordance was observed in only four (4%) patients. CONCLUSIONS Our results indicate that the detection of KRAS mutations in either primary or metastatic tumors from patients with CRC is concordant and this assessment could be used to predict response to targeted therapies such as cetuximab and panitumumab.

Genetic Activation of the MET Pathway and Prognosis of Patients With High-Risk, Radically Resected Gastric Cancer

PURPOSE To investigate whether prognosis of patients with high-risk gastric cancer may depend on MET copy number gain (CNG) or an activating truncation within a deoxyadenosine tract element (DATE) in the promoter region of the MET ligand HGF. PATIENTS AND METHODS A single-institution cohort of 230 patients with stage II/III gastric cancer was studied. Formalin-fixed paraffin-embedded tumor specimens were used for DNA extraction. Quantitative polymerase chain reaction (qPCR) for MET CNG and sequencing for HGF DATE truncation (< 25 deoxyadenosines instead of 30) were used. Results were analyzed for association with disease-free survival (DFS) and overall survival (OS). To assess the reliability of the qPCR measurement, a random sample of cases was reanalyzed using an alternative assay (fluorescent in situ hybridization [FISH]) with calculation of the intracorrelation coefficient (ICC). RESULTS In 216 assessable patients, MET CNG five or more copies and homozygous HGF-truncated DATE occurred in 21 patients (10%) and 30 patients (13%), respectively. Patients with MET CNG five or more copies (MET-positive) showed significantly worse prognosis with multivariate hazard ratio (HR) of 3.02 (95% CI, 1.71 to 5.33; P < .001) for DFS and multivariate HR of 2.91 (95% CI, 1.65 to 5.11; P < .001) for OS. The agreement between qPCR and FISH was high, with ICC = 0.9% (95% CI, 0.81% to 0.95%; the closer the ICC is to 1, the greater is the agreement). HGF-truncated DATE did not show relevant prognostic effect. CONCLUSION In this study, qPCR revealed approximately 10% of white patients with gastric cancer harboring MET CNG of five or more copies. This marker was significantly associated with unfavorable prognosis. This information is relevant to the current clinical development of anti-MET compounds.

A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk

Intoxications and infections caused by food-borne pathogens represent an increasing public health problem, and diagnostic tests in multiplex format are needed for the rapid identification of food contaminations caused by more than one microbial species. We have developed a multiple PCR-based platform for the simultaneous detection of the widespread milk-associated pathogens Salmonella spp., Listeria monocytogenes and Escherichia coli O157. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens, a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (mRT-PCR), or on melting curve analysis (mHRM). The second, producing a distinct peak for each amplification product, allows the qualitative assessment of pathogen presence. Moreover, the internal amplification control (IAC) included in the reaction, ensuring the reliability of results, complies with quality management programmes. Inclusivity and exclusivity were 100% each, with a detection limit of 1 CFU for each pathogen in a total of five 25 ml-aliquots of raw milk, and a duration of two working days. The assay represents an alternative approach for the qualitative detection of the cited bacterial species, suitable for a relatively inexpensive screening of several milk samples, reducing the turnaround time and the workload.

Pharmacogenetic Profiling for Cetuximab Plus Irinotecan Therapy in Patients With Refractory Advanced Colorectal Cancer

PURPOSE Regulation of epidermal growth factor receptor (EGFR) signaling pathways may play a relevant role in determining the activity of cetuximab therapy in patients with metastatic colorectal cancer (MCRC). We investigated possible associations between genetic variants and clinical outcomes of MCRC patients treated with cetuximab-irinotecan salvage therapy. PATIENTS AND METHODS Patients who underwent cetuximab-irinotecan salvage therapy after disease progression during or after first-line bolus/infusional fluorouracil, leucovorin, and oxaliplatin chemotherapy and a second-line irinotecan-based regimen were considered eligible for analysis of polymorphisms with putative influence on cetuximab-related pathways. Epidermal growth factor (EGF) 61A>G, EGF receptor (EGFR) 216G>T, EGFR 497G>A, EGFR intron-1 (CA)(n) dinucleotide short (S)/long (L) variant, cyclin-D1 870A>G, immunoglobulin-G fragment-C receptors RIIIa 158G>T, and RIIa 131G>A were studied for a possible association with overall survival (OS) as the primary end point. Additional analyses were addressed at possible associations among polymorphisms and EGFR expression, toxicity, and response. RESULTS In 110 assessable patients, significant association with favorable OS was observed for EGFR intron-1 S/S and EGF 61 G/G genotypes. In the multivariate model, EGFR intron-1 S/S and EGF 61 G/G genotypes showed a hazard ratio of 0.41 (95% CI, 0.21 to 0.78; P = .006) and 0.44 (95% CI, 0.23 to 0.84; P = .01), respectively. EGFR intron-1 S/S carriers showed more frequent G2-G3 skin toxicity (chi(2) test = 12.7; P = .001) and treatment response (chi(2) test = 9.45; P = .008) than EGFR intron-1 L/L carriers. CONCLUSION Although additional studies are required for confirmation, our findings could optimize the use of cetuximab in MCRC patients.