Centro di Riferimento Oncologico della Basilicata

BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

BACKGROUND: Current treatment recommendations for patients with polycythemia vera call for maintaining a hematocrit of less than 45%, but this therapeutic strategy has not been tested in a randomized clinical trial. METHODS: We randomly assigned 365 adults with JAK2-positive polycythemia vera who were being treated with phlebotomy, hydroxyurea, or both to receive either more intensive treatment (target hematocrit, <45%) (low-hematocrit group) or less intensive treatment (target hematocrit, 45 to 50%) (high-hematocrit group). The primary composite end point was the time until death from cardiovascular causes or major thrombotic events. The secondary end points were cardiovascular events, cardiovascular hospitalizations, incidence of cancer, progression to myelofibrosis, myelodysplasia or leukemic transformation, and hemorrhage. An intention-to-treat analysis was performed. RESULTS: After a median follow-up of 31 months, the primary end point was recorded in 5 of 182 patients in the low-hematocrit group (2.7%) and 18 of 183 patients in the high-hematocrit group (9.8%) (hazard ratio in the high-hematocrit group, 3.91; 95% confidence interval [CI], 1.45 to 10.53; P=0.007). The primary end point plus superficial-vein thrombosis occurred in 4.4% of patients in the low-hematocrit group, as compared with 10.9% in the high-hematocrit group (hazard ratio, 2.69; 95% CI, 1.19 to 6.12; P=0.02). Progression to myelofibrosis, myelodysplasia or leukemic transformation, and bleeding were observed in 6, 2, and 2 patients, respectively, in the low-hematocrit group, as compared with 2, 1, and 5 patients, respectively, in the high-hematocrit group. There was no significant between-group difference in the rate of adverse events. CONCLUSIONS: In patients with polycythemia vera, those with a hematocrit target of less than 45% had a significantly lower rate of cardiovascular death and major thrombosis than did those with a hematocrit target of 45 to 50%. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT01645124, and EudraCT number, 2007-006694-91.).

BACKGROUND: This open-label, randomized, phase 3 study compared melphalan at a dose of 200 mg per square meter of body-surface area plus autologous stem-cell transplantation with melphalan-prednisone-lenalidomide (MPR) and compared lenalidomide maintenance therapy with no maintenance therapy in patients with newly diagnosed multiple myeloma. METHODS: We randomly assigned 273 patients 65 years of age or younger to high-dose melphalan plus stem-cell transplantation or MPR consolidation therapy after induction, and 251 patients to lenalidomide maintenance therapy or no maintenance therapy. The primary end point was progression-free survival. RESULTS: The median follow-up period was 51.2 months. Both progression-free and overall survival were significantly longer with high-dose melphalan plus stem-cell transplantation than with MPR (median progression-free survival, 43.0 months vs. 22.4 months; hazard ratio for progression or death, 0.44; 95% confidence interval [CI], 0.32 to 0.61; P<0.001; and 4-year overall survival, 81.6% vs. 65.3%; hazard ratio for death, 0.55; 95% CI, 0.32 to 0.93; P=0.02). Median progression-free survival was significantly longer with lenalidomide maintenance than with no maintenance (41.9 months vs. 21.6 months; hazard ratio for progression or death, 0.47; 95% CI, 0.33 to 0.65; P<0.001), but 3-year overall survival was not significantly prolonged (88.0% vs. 79.2%; hazard ratio for death, 0.64; 95% CI, 0.36 to 1.15; P=0.14). Grade 3 or 4 neutropenia was significantly more frequent with high-dose melphalan than with MPR (94.3% vs. 51.5%), as were gastrointestinal adverse events (18.4% vs. 0%) and infections (16.3% vs. 0.8%); neutropenia and dermatologic toxic effects were more frequent with lenalidomide maintenance than with no maintenance (23.3% vs. 0% and 4.3% vs. 0%, respectively). CONCLUSIONS: Consolidation therapy with high-dose melphalan plus stem-cell transplantation, as compared with MPR, significantly prolonged progression-free and overall survival among patients with multiple myeloma who were 65 years of age or younger. Lenalidomide maintenance, as compared with no maintenance, significantly prolonged progression-free survival. (Funded by Celgene; ClinicalTrials.gov number, NCT00551928.).

We conducted a pooled analysis of 869 individual newly diagnosed elderly patient data from 3 prospective trials. At diagnosis, a geriatric assessment had been performed. An additive scoring system (range 0-5), based on age, comorbidities, and cognitive and physical conditions, was developed to identify 3 groups: fit (score = 0, 39%), intermediate fitness (score = 1, 31%), and frail (score ≥2, 30%). The 3-year overall survival was 84% in fit, 76% in intermediate-fitness (hazard ratio [HR], 1.61; P = .042), and 57% in frail (HR, 3.57; P < .001) patients. The cumulative incidence of grade ≥3 nonhematologic adverse events at 12 months was 22.2% in fit, 26.4% in intermediate-fitness (HR, 1.23; P = .217), and 34.0% in frail (HR, 1.74; P < .001) patients. The cumulative incidence of treatment discontinuation at 12 months was 16.5% in fit, 20.8% in intermediate-fitness (HR, 1.41; P = .052), and 31.2% in frail (HR, 2.21; P < .001) patients. Our frailty score predicts mortality and the risk of toxicity in elderly myeloma patients. The International Myeloma Working group proposes this score for the measurement of frailty in designing future clinical trials. These trials are registered at www.clinicaltrials.gov as #NCT01093136 (EMN01), #NCT01190787 (26866138MMY2069), and #NCT01346787 (IST-CAR-506).

Antibiotics are essential drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. Antibiotic resistance is a serious challenge and has led to the need for new alternative molecules less prone to bacterial resistance. Antimicrobial peptides (AMPs) have aroused great interest as potential next-generation antibiotics, since they are bioactive small proteins, naturally produced by all living organisms, and representing the first line of defense against fungi, viruses and bacteria. AMPs are commonly classified according to their sources, which are represented by microorganisms, plants and animals, as well as to their secondary structure, their biosynthesis and their mechanism of action. They find application in different fields such as agriculture, food industry and medicine, on which we focused our attention in this review. Particularly, we examined AMP potential applicability in wound healing, skin infections and metabolic syndrome, considering their ability to act as potential Angiotensin-Converting Enzyme I and pancreatic lipase inhibitory peptides as well as antioxidant peptides. Moreover, we argued about the pharmacokinetic and pharmacodynamic approaches to develop new antibiotics, the drug development strategies and the formulation approaches which need to be taken into account in developing clinically suitable AMP applications.

Smoking increases mortality from all causes and has a crucial role in atherosclerotic cardiovascular disease (ASCVD). Active smoking and secondhand smoke exposure determine more than 30% of coronary heart disease (CHD) mortality. The exact mechanisms of cardiovascular damages are not well known, but the detrimental effect of smoking on endothelial function has long been recognized. Smoking elicits oxidative processes, negatively affects platelet function, fibrinolysis, inflammation and vasomotor function; all these proatherogenic effects double the 10-year risk of fatal events in smokers compared to non smokers. An intriguing issue about smoking is the vulnerability of female gender. The mortality from cardiovascular diseases (CVDs) is higher in female than male smokers and female smokers show a 25% higher risk of developing CHD than men with the same exposure to tobacco smoke. This female vulnerability seems to be related to genes involved in thrombin signaling. The effects of smoking cessation have also been extensively studied. Cessation at an early age (40 years) has an impressive 90% reduction in the excess risk of death. In this review we report recent data about the causal link between smoking and CVDs and about the benefits of smoking cessation.

The European Myeloma Network provides recommendations for the management of the most common complications of multiple myeloma. Whole body low-dose computed tomography is more sensitive than conventional radiography in depicting osteolytic disease and thus we recommend it as the novel standard for the detection of lytic lesions in myeloma (grade 1A). Myeloma patients with adequate renal function and bone disease at diagnosis should be treated with zoledronic acid or pamidronate (grade 1A). Symptomatic patients without lytic lesions on conventional radiography can be treated with zoledronic acid (grade 1B), but its advantage is not clear for patients with no bone involvement on computed tomography or magnetic resonance imaging. In asymptomatic myeloma, bisphosphonates are not recommended (grade 1A). Zoledronic acid should be given continuously, but it is not clear if patients who achieve at least a very good partial response benefit from its continuous use (grade 1B). Treatment with erythropoietic-stimulating agents may be initiated in patients with persistent symptomatic anemia (hemoglobin <10g/dL) in whom other causes of anemia have been excluded (grade 1B). Erythropoietic agents should be stopped after 6-8 weeks if no adequate hemoglobin response is achieved. For renal impairment, bortezomib-based regimens are the current standard of care (grade 1A). For the management of treatment-induced peripheral neuropathy, drug modification is needed (grade 1C). Vaccination against influenza is recommended; vaccination against streptococcus pneumonia and hemophilus influenza is appropriate, but efficacy is not guaranteed due to suboptimal immune response (grade 1C). Prophylactic aciclovir (or valacyclovir) is recommended for patients receiving proteasome inhibitors, autologous or allogeneic transplantation (grade 1A).

In a recent phase 3 trial, bortezomib-melphalan-prednisone-thalidomide followed by maintenance treatment with bortezomib-thalidomide demonstrated superior efficacy compared with bortezomib-melphalan-prednisone. To decrease neurologic toxicities, the protocol was amended and patients in both arms received once-weekly instead of the initial twice-weekly bortezomib infusions: 372 patients received once-weekly and 139 twice-weekly bortezomib. In this post-hoc analysis we assessed the impact of the schedule change on clinical outcomes and safety. Long-term outcomes appeared similar: 3-year progression-free survival rate was 50% in the once-weekly and 47% in the twice-weekly group (P > .999), and 3-year overall survival rate was 88% and 89%, respectively (P = .54). The complete response rate was 30% in the once-weekly and 35% in the twice-weekly group (P = .27). Nonhematologic grade 3/4 adverse events were reported in 35% of once-weekly patients and 51% of twice-weekly patients (P = .003). The incidence of grade 3/4 peripheral neuropathy was 8% in the once-weekly and 28% in the twice-weekly group (P < .001); 5% of patients in the once-weekly and 15% in the twice-weekly group discontinued therapy because of peripheral neuropathy (P < .001). This improvement in safety did not appear to affect efficacy. This study is registered at http://www.clinicaltrials.gov as NCT01063179.

The initial analysis of the oral combination melphalan, prednisone, and thalidomide (MPT) in newly diagnosed patients with myeloma showed significantly higher response rate and longer progression-free survival (PFS) than did the standard melphalan and prednisone (MP) combination and suggested a survival advantage. In this updated analysis, efficacy and safety end points were updated. Patients were randomly assigned to receive oral MPT or MP alone. Updated analysis was by intention to treat and included PFS, overall survival (OS), and survival after progression. After a median follow-up of 38.1 months, the median PFS was 21.8 months for MPT and 14.5 months for MP (P = .004). The median OS was 45.0 months for MPT and 47.6 months for MP (P = .79). In different patient subgroups, MPT improved PFS irrespective of age, serum concentrations of beta(2)-microglobulin, or high International Staging System. Thalidomide or bortezomib administration as salvage regimens significantly improved survival after progression in the MP group (P = .002) but not in the MPT group (P = .34). These data confirm activity of MPT for PFS but failed to show any survival advantage. New agents in the management of relapsed disease could explain this finding. The study is registered at www.clinicaltrials.gov as #NCT00232934.

The objective of this study was to evaluate the clinical features, prognostic factors, and efficacy of treatments in patients with blastic plasmacytoid dendritic cell neoplasm with a leukemic presentation at onset of the disease. In order to do this, a retrospective multicenter study was performed from 2005-2011 in 28 Italian hematology divisions in which 43 cases were collected. Forty-one patients received an induction therapy, consisting of an acute myeloid leukemia-type regimen in 26 patients (60%) and acute lymphoid leukemia/lymphoma-type regimen in 15 patients (35%). Six patients (14%) underwent allogeneic hematopoietic stem cell transplantation. Seventeen patients (41%) achieved a complete remission: seven after acute myeloid leukemia-type treatment and 10 after an acute lymphoid leukemia/lymphoma-type regimen, with a significant advantage for acute lymphoid leukemia/lymphoma-type chemotherapy (P=0.02). Relapse occurred in six of the 17 patients (35%) who achieved complete remission, more frequently after acute lymphoid leukemia/lymphoma-type chemotherapy. The median overall survival was 8.7 months (range, 0.2-32.9). The patients treated with an acute myeloid leukemia-type regimen had an overall survival of 7.1 months (range, 0.2-19.5), whereas that of the patients receiving acute lymphoid leukemia/lymphoma-type chemotherapy was 12.3 months (range, 1-32.9) (P=0.02). The median overall survival of the allogeneic hematopoietic stem cell transplant recipients was 22.7 months (range, 12-32.9), and these patients had a significant survival advantage compared to the non-transplanted patients (median 7.1 months, 0.2-21.3; P=0.03). In conclusion, blastic plasmacytoid dendritic cell neoplasm with bone-marrow involvement is an aggressive subtype of high-risk acute leukemia. The rarity of this disease does not enable prospective clinical trials to identify the better therapeutic strategy, which, at present, is based on clinicians' experience.

In a randomized, phase 3 study, superior complete/near-complete response (CR/nCR) rates and extended progression-free survival were demonstrated with bortezomib-thalidomide-dexamethasone (VTD) versus thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autologous stem cell transplantation for newly diagnosed myeloma patients (intention-to-treat analysis; VTD, n = 236; TD, n = 238). This per-protocol analysis (VTD, n = 160; TD, n = 161) specifically assessed the efficacy and safety of consolidation with VTD or TD. Before starting consolidation, CR/nCR rates were not significantly different in the VTD (63.1%) and TD arms (54.7%). After consolidation, CR (60.6% vs 46.6%) and CR/nCR (73.1% vs 60.9%) rates were significantly higher for VTD-treated versus TD-treated patients. VTD consolidation significantly increased CR and CR/nCR rates, but TD did not (McNemar test). With a median follow-up of 30.4 months from start of consolidation, 3-year progression-free survival was significantly longer for the VTD group (60% vs 48% for TD). Grade 2 or 3 peripheral neuropathy (8.1% vs 2.4%) was more frequent with VTD (grade 3, 0.6%) versus TD consolidation. The superior efficacy of VTD versus TD as induction was retained despite readministration as consolidation therapy after double autologous transplantation. VTD consolidation therapy significantly contributed to improved clinical outcomes observed for patients randomly assigned to the VTD arm of the study. The study is registered at www.clinicaltrials.gov as #NCT01134484.

The role of thalidomide for previously untreated elderly patients with multiple myeloma remains unclear. Six randomized controlled trials, launched in or after 2000, compared melphalan and prednisone alone (MP) and with thalidomide (MPT). The effect on overall survival (OS) varied across trials. We carried out a meta-analysis of the 1685 individual patients in these trials. The primary endpoint was OS, and progression-free survival (PFS) and 1-year response rates were secondary endpoints. There was a highly significant benefit to OS from adding thalidomide to MP (hazard ratio = 0.83; 95% confidence interval 0.73-0.94, P = .004), representing increased median OS time of 6.6 months, from 32.7 months (MP) to 39.3 months (MPT). The thalidomide regimen was also associated with superior PFS (hazard ratio = 0.68, 95% confidence interval 0.61-0.76, P < .0001) and better 1-year response rates (partial response or better was 59% on MPT and 37% on MP). Although the trials differed in terms of patient baseline characteristics and thalidomide regimens, there was no evidence that treatment affected OS differently according to levels of the prognostic factors. We conclude that thalidomide added to MP improves OS and PFS in previously untreated elderly patients with multiple myeloma, extending the median survival time by on average 20%.

Complete response (CR) was an uncommon event in elderly myeloma patients until novel agents were combined with standard oral melphalan-prednisone. This analysis assesses the impact of treatment response on progression-free survival (PFS) and overall survival (OS). We retrospectively analyzed 1175 newly diagnosed myeloma patients, enrolled in 3 multicenter trials, treated with melphalan-prednisone alone (n = 332), melphalan-prednisone-thalidomide (n = 332), melphalan-prednisone-bortezomib (n = 257), or melphalan-prednisone-bortezomib-thalidomide (n = 254). After a median follow-up of 29 months, the 3-year PFS and OS were 67% and 27% (hazard ratio = 0.16; P < .001), and 91% and 70% (hazard ratio = 0.15; P < .001) in patients who obtained CR and in those who achieved very good partial response, respectively. Similar results were observed in patients older than 75 years. Multivariate analysis confirmed that the achievement of CR was an independent predictor of longer PFS and OS, regardless of age, International Staging System stage, and treatment. These findings highlight a significant association between the achievement of CR and long-term outcome, and support the use of novel agents to achieve maximal response in elderly patients, including those more than 75 years. This trial was registered at www.clinicaltrials.gov as #NCT00232934, #ISRCTN 90692740, and #NCT01063179.

PURPOSE We investigated the effect on minimal residual disease, by qualitative and real-time quantitative polymerase chain reaction (RQ-PCR), of a consolidation regimen that included bortezomib, thalidomide, and dexamethasone (VTD) in patients with multiple myeloma (MM) responding to autologous stem-cell transplantation (auto-SCT). PATIENTS AND METHODS Patients achieving at least very good partial response who had an available molecular marker based on the immunoglobulin heavy-chain rearrangement received four courses of treatment every month: four infusions per month of bortezomib at 1.6 mg/m(2), thalidomide at 200 mg/d, and dexamethasone at 20 mg/d on days 1 to 4, 8 to 11, and 15 to 18. Patients were studied with tumor-clone-specific primers by qualitative nested PCR and RQ-PCR. Results Of 39 patients enrolled, 31 received the four VTD courses. Immunofixation complete responses increased from 15% after auto-SCT to 49% after VTD. Molecular remissions (MRs) were 3% after auto-SCT and 18% after VTD. Median time to maximum response was 3.5 months. So far, no patient in MR has relapsed (median follow-up, 42 months). VTD consolidation induced an additional depletion of 4.14 natural logarithms of tumor burden by RQ-PCR. Patients with a tumor load less than the median value after VTD had outcomes better than those who had tumor loads above the median value after VTD (at median follow-up: progression-free survival, 100% v 57%; P < .001). CONCLUSION To the best of our knowledge, this study is the first to document the occurrence of persistent MRs in a proportion of MM patients treated without allogeneic transplantation. Moreover, the major reduction in tumor load recorded by RQ-PCR after VTD suggests that unprecedented levels of tumor cell reduction can be achieved in MM thanks to the new nonchemotherapeutic drugs.

The association between serum uric acid and hypertension was evaluated in a sample of male workers in southern Italy enrolled in the Olivetti Heart Study, an ongoing longitudinal epidemiological investigation on risk factors for coronary heart disease carried out at the Olivetti factory in the suburban area of Naples. Participants were screened at baseline (1975) and at five year (1980) and 12 year (1987) follow-up examinations. The present report focuses on 619 male workers for whom information on coronary heart disease risk factors was available both at baseline and 12 year follow-up examination. At baseline, after excluding hypertensive participants (systolic blood pressure (SBP) > or = 140 mmHg and/or diastolic blood pressure (DBP), > or = 90 mmHg and/or on antihypertensive therapy; n = 72), serum uric acid was positively and significantly related to age, SBP, DBP, body mass index (BMI), serum total cholesterol (CHOL) and serum triglycerides (TG) in 547 normotensive participants. At 12 year follow-up examination, hypertension was defined by SBP > or = 140 mmHg and/or DBP > or = 90 mmHg and/or being on antihypertensive therapy. Multiple logistic regression analysis showed an independent positive association between serum uric acid levels and development of hypertension (RR = 1.23, 95% CI = 1.07-1.39; p = 0.011) after adjustment for age, BMI, CHOL and TG. Furthermore, according to more severe degrees of hypertension (SBP > or = 160 mmHg and/or DBP > or = 95 mmHg and/or being on antihypertensive therapy), the relative risk to develop hypertension was still significant (RR = 1.19; CI = 1.01-1.38; p = 0.051).(ABSTRACT TRUNCATED AT 250 WORDS)

An extensive body of literature describes anticancer property of dichloroacetate (DCA), but its effective clinical administration in cancer therapy is still limited to clinical trials. The occurrence of side effects such as neurotoxicity as well as the suspicion of DCA carcinogenicity still restricts the clinical use of DCA. However, in the last years, the number of reports supporting DCA employment against cancer increased also because of the great interest in targeting metabolism of tumour cells. Dissecting DCA mechanism of action helped to understand the bases of its selective efficacy against cancer cells. A successful coadministration of DCA with conventional chemotherapy, radiotherapy, other drugs, or natural compounds has been tested in several cancer models. New drug delivery systems and multiaction compounds containing DCA and other drugs seem to ameliorate bioavailability and appear more efficient thanks to a synergistic action of multiple agents. The spread of reports supporting the efficiency of DCA in cancer therapy has prompted additional studies that let to find other potential molecular targets of DCA. Interestingly, DCA could significantly affect cancer stem cell fraction and contribute to cancer eradication. Collectively, these findings provide a strong rationale towards novel clinical translational studies of DCA in cancer therapy.

Misuse and overuse of antibiotics have contributed in the last decades to a phenomenon known as antibiotic resistance which is currently considered one of the principal threats to global public health by the World Health Organization. The aim to find alternative drugs has been demonstrated as a real challenge. Thanks to their biodiversity, insects represent the largest class of organisms in the animal kingdom. The humoral immune response includes the production of antimicrobial peptides (AMPs) that are released into the insect hemolymph after microbial infection. In this review, we have focused on insect immune responses, particularly on AMP characteristics, their mechanism of action and applications, especially in the biomedical field. Furthermore, we discuss the Toll, Imd, and JAK-STAT pathways that activate genes encoding for the expression of AMPs. Moreover, we focused on strategies to improve insect peptides stability against proteolytic susceptibility such as D-amino acid substitutions, N-terminus modification, cyclization and dimerization.

Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1(super)) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.

To determine the frequency and distribution of mitochondrial DNA mutations in breast cancer, 18 primary breast tumors were analyzed by direct sequencing. Twelve somatic mutations not present in matched lymphocytes and normal breast tissues were detected in 11 of the tumors screened (61%). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303-315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or noncoding regions (D-loop) of the mitochondrial genome. In three cases (25%), the mutations detected in coding regions led to amino acid substitutions in the protein sequence. We then screened an additional 46 primary breast tumors with a rapid PCR-based assay to identify poly-C alterations in D310, and we found seven more cancers with alterations. Using D310 mutations as clonal marker, we detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes. The high frequency of D310 alterations in primary breast cancer combined with the high sensitivity of the PCR-based assays provides a new molecular tool for cancer detection.

One of the aims of the EPIC study is to produce accurate descriptions of the dietary habits of the participants recruited in the 27 EPIC centers of 10 European countries. To do this, different dietary assessment instruments were developed and applied to capture the wide range of diets characterizing the different European populations. Three different food frequency questionnaires were developed for Italy: one for the centers of Varese, Turin and Florence, one for Ragusa and one for Naples. These inquired about eating habits over the previous year and were completed by 46,839 Italian EPIC participants. Specially developed software analyzed the responses and linked them to food composition tables in order to provide a nutritional breakdown of individual and collective diets. A further aim of EPIC was to develop a method of rendering data from different dietary questionnaires comparable. To do this, dietary data were collected from a sample of about 8% of the Italian EPIC cohort, using a standardized computer-driven 24-hour dietary recall interview, and then compared with the dietary data collected by the questionnaires. This paper provides an extensive description of the technical features and performance of the food frequency questionnaires and the 24-hour recall interview, including a comparison of estimates of the intake of different food groups provided by the two instruments. From this comparison, the repeatability and reliability of consumption estimates was assessed, resulting in indications for improving data comparability. The paper also presents food frequency questionnaire estimates of the daily intake of foods and nutrients by center, sex and age group, as well as information on dietary habits such as place and time of intake, and food preparation and preservation methods as provided by the 24-hour recall interview. The picture that emerged is that Italian eating habits are undergoing marked changes, with a tendency to less healthy eating. Documentation of these changes in relation to age, sex and region provides an essential starting point for investigating the influence of diet on the development of cancer and other chronic diseases.