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Background Colorectal cancer is the third most common and the third most lethal cancer in both men and women in developed countries. About 75% of cases are first diagnosed when the disease is classified as localized or regional, undergo potentially curative treatment and enter a post-treatment surveillance program. Although such programs drain significant resources from health systems, empirical evidence of their efficacy is scanty. Patients and methods Dukes B2-C colorectal cancer patients who had no evidence of disease at the end of their front-line treatment (surgery and adjuvant radiochemotherapy, if indicated) were eligible for the trial and randomized to two different surveillance programs. These programs differed greatly in the frequency of diagnostic imaging. They had similar schedules of physical examinations and carcinoembryonic antigen (CEA) assessments. Patients received baseline and yearly health-related quality-of-life (HR-QoL) questionnaires. Primary outcomes were overall survival (OS) and QoL. Results From 1998 to 2006, 1228 assessable patients were randomized, 933 with colon cancer and 295 with rectal cancer. More than 90% of patients had the expected number of diagnostic procedures. Median follow-up duration was 62 months [interquartile range (IQR) 51–86] in the minimal surveillance group and 62 months (IQR 50–85) in the intensive group. At primary analysis, 250 patients had recurred and 218 had died. Intensive surveillance anticipated recurrence, as shown by a significant difference in mean disease-free survival of 5.9 months. Comparison of OS curves of the whole intention-to-treat population showed no statistically significant differences. HR-QoL of life scores did not differ between regimens. Conclusion Our findings support the conclusions of other randomized clinical trials, which show that early diagnosis of cancer recurrence is not associated with OS benefit. ClinicalTrials.gov NCT02409472.

Background HER2 amplification (HER2+) is a therapeutic target for 2% (unselected)-6% (RAS wild-type) metastatic colorectal cancer (mCRC). In the HERACLES-A Trial of trastuzumab and lapatinib (Lancet Oncology, 2016), HER2+ KRAS wild-type mCRC patients achieved an overall objective response rate (CR+PR = ORR) of 30%. Based on this result and on preclinical trials, we activated HERACLES-B, evaluating a targeted chemotherapy precision approach by combining pertuzumab with T-DM1. Methods HERACLES-B is an open-label phase II trial in RAS/BRAF wild-type HER2+ mCRCs (as defined in Valtorta et al, 2015). ORR and Progression-Free Survival (PFS) are the primary and secondary end-points, respectively. With a Fleming/Hern design (H0=ORR 10%; α = 0.05; power=0.85), 7 OR/30 were required to demonstrate an ORR ≥30% (H1). Main inclusion criteria were: PS 0-1, progression after 5FU, oxaliplatin, irinotecan, and anti-EGFR containing regimens. Pertuzumab was dosed at 840 mg iv load, followed by 420 mg iv q3 weeks and T-DM1 at 3.6 mg/Kg q3 weeks. NGS-based molecular analyses of tumor tissue/plasma were performed. Results From 8/2016 to 3/2018, 30 patients were enrolled, treated and evaluable for efficacy. Patients received a median of 3 prior regimens. Data lock and centralized radiological revision were completed by 30/7/2019. ORR was 10% [95% CI: 0-28] and stable disease (SD) 70% [95% CI: 50-85]. Median PFS was 4.8 mos. [95% CI: 3.6-5.8]. Higher HER2 IHC score (3+ vs 2+) was associated with objective response/SD ≥4 mos. [p = 0.03]. Drug-related G3 adverse events were observed only in 2 patients (thrombocytopenia); G ≤ 2 events in 84% of cycles (N = 296), mainly nausea and fatigue. Conclusions Although HERACLES-B did not reach its primary endpoint, disease control was achieved in 80% of patients with a median PFS of 4.8 mos. that is superimposable to the 4.2 mos. achieved in the positive HERACLES-A trial. While the ORR could have been weakened by the lower trastuzumab dose delivered with T-DM1, the retained favorable PFS might be due the combined ‘broad-brush’ effect of emtansine and the synergy with pertuzumab. Preliminary molecular results will be presented. Clinical trial identification EudraCT: 2012-002128-33. Legal entity responsible for the study Fondazione del Piemonte per l’Oncologia IRCC di Candiolo. Funding Fondazione Piemontese per l’Oncologia - FPO funded by Associazione Italiana per la Ricerca sul Cancro (AIRC). Roche provided pertuzumab and T-DM1 for free. Disclosure A. Sartore-Bianchi: Advisory / Consultancy: Amgen; Advisory / Consultancy: Bayer; Advisory / Consultancy: Sanofi. A. Amatu: Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer; Advisory / Consultancy: Amgen. A. Ardizzoni: Advisory / Consultancy: Roche. L. Trusolino: Research grant / Funding (self): Symphogen; Research grant / Funding (self): Merus; Research grant / Funding (self): Servier; Research grant / Funding (self): Pfizer; Speaker Bureau / Expert testimony: Eli Lilly; Speaker Bureau / Expert testimony: AstraZeneca; Speaker Bureau / Expert testimony: Merck KGaA. S. Siena: Advisory / Consultancy: Amgen; Advisory / Consultancy: Bayer; Advisory / Consultancy: BMS; Advisory / Consultancy: CheckMab; Advisory / Consultancy: Celgene; Advisory / Consultancy: Clovis; Advisory / Consultancy: Daiichi Sankyo; Advisory / Consultancy: Incyte; Advisory / Consultancy: Merck; Advisory / Consultancy: Novartis; Advisory / Consultancy: Roche-Genentec; Advisory / Consultancy: Seattle Genetics. All other authors have declared no conflicts of interest.

Background The prognostic and predictive role of KRAS mutations in advanced nonsmall-cell lung cancer (NSCLC) is still unclear. TAILOR prospectively assessed the prognostic and predictive value of KRAS mutations in NSCLC patients treated with erlotinib or docetaxel in second line. Patients and methods NSCLC patients from 52 Italian hospitals were genotyped for KRAS and EGFR mutational status in two independent laboratories. Wild-type EGFR patients (N = 218) received first-line platinum-based chemotherapy and were randomly allocated at progression to erlotinib or docetaxel. Overall survival (OS) according to KRAS mutational status was the primary end point. Results KRAS mutations were present in 23% of TAILOR randomized cases. The presence of a KRAS mutation did not adversely affect progression-free (PFS) or overall (OS) survival [hazard ratio (HR) PFS = 1.01, 95% confidence interval (CI) 0.71–1.41, P = 0.977; OS = 1.24, 95% CI 0.87–1.77, P = 0.233], nor influenced treatment outcome (test for interaction: OS P = 0.965; PFS P = 0.417). Patients randomized to docetaxel treatment experienced longer survival independently from the KRAS mutational status of their tumors (HR: mutated KRAS 0.81, 95% CI 0.45–1.47; wild-type KRAS 0.79, 95% CI 0.57–1.10). Conclusion In TAILOR, KRAS was neither prognostic nor predictive of benefit for either docetaxel or erlotinib. Docetaxel remains superior independently from KRAS status for second-line treatment in EGFR wild-type advanced NSCLC patients. Clinical trial registration NCT00637910.

Background The DiMaio (D), EPSILoN (E) and plasma microRNA signature classifier (MSC), are 3 diverse clinico-biochemical and molecular scores able to independently predict prognosis in advanced non-small cell lung cancer (aNSCLC) patients (pts) treated with immunotherapy (IO). By assessing the ability of each test a combined score (SC) called DEMo was developed. The study aims to prospectively evaluate the prognostic value of DEMo in aNSCLC pts treated with IO. Methods We included in the analyses 166 aNSCLC pts treated in 1L (n = 47) and further-lines (n = 119) with IO at Istituto Nazionale Tumori of Milan. For all pts we obtained complete necessary data for both clinical SC: D (sex, histology, ECOG-PS stage, uses of platinum-based therapy at 1L and response to 1L) and E (ECOG-PS, Smoke, Liver, LDH, NL-ratio). MSC was prospectively evaluated in plasma samples collected at baseline IO and the risk level was assessed. Endpoints were median overall survival (mOS), progression-free survival (mPFS) and overall response rate (ORR). Kaplan-Meier and Cox model were used to generate survival curves and multivariate analyses, respectively. Results In multivariate analysis adjusted for age, sex, smoke and ECOG-PS each score remain independently significant for both PFS (D: HR = 1.99, CI95% 1.21–3.03, p = 0.007; E: HR = 1.87 CI95% 1.12 – 3.10, p = 0.016; MSC: HR = 1.56, CI95% 1.03–2.37, p = 0.0370) and OS (D: HR = 3.12, CI95% 1.80–5.41, p = 0.0001; E: HR = 2.21, CI95% 1.28–3.79, p = 0.0041; MSC: HR = 2.03, CI95% 1.30–3.17, p = 0.0019). DEMo separated patients in 4-risk groups (gr) based on the presence of 3–2–1–0 poor prognostic SC. Strata had 0%–7%–20%–46% 18 months (mo) PFS (p Conclusions We created a composite clinic-molecular combined biomarker classifier able to better predict prognosis compared to each single SC and to select patients who less likely benefit from IO. DEMo could be a useful tool to guide choices in aNSCLC. Clinical trial identification The authors. Legal entity responsible for the study Fondazione IRCCS Istituto Nazionale Tumori of Milan, Italy. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background Hypoxia induced by antiangiogenic agents could cause a functional impairment of homologous recombination, thus sensitizing wild-type (wt) BRCA tumor cells to PARP inhibition. In a phase II study the combination of cediranib-olaparib increased progression free survival (PFS) in women with recurrent platinum sensitive OC with respect to olaparib. Methods 123 patients were allocated in a 1:1:1 ratio to receive: 80 mg/m2 weekly paclitaxel up to 24 weeks (control), olaparib 600 mg tablet (300 mg twice daily) together with 20 mg cediranib daily (continuous schedule) or 20 mg cediranib given 5 days/week (intermittent schedule) until progression. PFS comparison between experimental schedules and the control arm (alpha one-sided 5%; power 80% to detect a HR of 0.5) was the primary objective. Results Median platinum-free interval was 1.8 mos, 59% of patients were pretreated with >3 chemotherapy lines. Median PFS for paclitaxel, the continuous, and the intermittent schedules were 3.1, 5.7, and 3.8 mos. Estimated HR for PFS in continuous arm vs control was 0.76 (90% CI: 0.49-1.17), p = 0.28 by log-rank test. HR for PFS in intermittent arm vs. control was 1.08 (90% CI: 0.71-1.64), p = 0.76 by log-rank test. In the subgroup gBRCA wt (n = 109) the median PFS for paclitaxel, the continuous, and the intermittent schedules were 2.1, 5.8 and 3.8 mos and HR for PFS in continuous arm vs control was 0.63 (95% CI: 0.36 to 1.10; p = 0.10). The toxicity profile of the study arms was as expected and similar between experimental arms. 11%, 18%, and 7% in control, continuous and intermittent arm discontinued treatment for adverse events. Five serious adverse drug reactions occurred and two of these were fatal: one in the control and one in the continuous arm. Conclusions The combination of cediranib and olaparib is effective in heavily pretreated PROC patients with the advantage of an oral administration and good tolerability. The continuous schedule of cediranib-olaparib showed a promising trend towards improved PFS in comparison with weekly paclitaxel particularly in the BRCA wt population. Clinical trial identification IRFMN-OVA-7289, EudraCT: 2016-003964-38, NCT03314740. Legal entity responsible for the study Istituto di Ricerche Farmacologiche Mario Negri IRCCS. Funding AstraZeneca. Disclosure N. Colombo: Honoraria (self), Advisory / Consultancy: Roche/Genentech; Honoraria (self), Advisory / Consultancy: PharmaMar; Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Tesaro; Advisory / Consultancy: Clovis Oncology; Advisory / Consultancy: Pfizer; Advisory / Consultancy: MSD Oncology; Advisory / Consultancy: BioCad; Advisory / Consultancy: Takeda. G. Tognon: Advisory / Consultancy: Amgen; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Speaker Bureau / Expert testimony, Research grant / Funding (institution): AstraZeneca; Speaker Bureau / Expert testimony: Tesaro. M. Ratti: Travel / Accommodation / Expenses: Tesaro Bio. All other authors have declared no conflicts of interest.

The majority of women with ovarian cancer develop recurrent disease. For patients with a platinum-free interval of >6 months, platinum-based chemotherapy is a treatment of choice. The benefit of platinum-based combination chemotherapy in randomized trials varies, and a meta-analysis was carried out to gain more secure information on the size of the benefit of this treatment. Materials and methods: We initiated a systematic review and meta-analysis following a pre-specified protocol to determine whether combination chemotherapy is superior to single-agent platinum chemotherapy in women with relapsed platinum-sensitive ovarian cancer. Results: A total of five potentially eligible randomized trials were identified that had used combination-platinum chemotherapy versus single-agent platinum chemotherapy in women with relapsed platinum-sensitive ovarian cancer. For one trial (190 patients), adequate contact with the investigators could not be established. Therefore, four trials that randomly assigned 1300 patients were included, with a median follow-up of 36.1 months. Overall survival (OS) analyses were based on 865 deaths and demonstrated evidence for the benefit of combination-platinum chemotherapy (HR = 0.80; 95% CI, 0.64-1.00; P = 0.05). Progression-free survival (PFS) analyses were based on 1167 events and demonstrated strong evidence for the benefit of combination-platinum chemotherapy (HR = 0.68; 95% CI, 0.57-0.81; P < 0.001). There was no evidence of a difference in the relative effect of combination-platinum chemotherapy on either OS or PFS in patient subgroups defined by previous paclitaxel (Taxol) treatment (OS, P = 0.49; PFS, P = 0.66), duration of treatment-free interval (OS, P = 0.86; PFS, P = 0.48) or the number of previous lines of chemotherapy (OS, P = 0.21; PFS, P = 0.27). Conclusions: In this individual patient data (IPD) meta-analysis, we have demonstrated that combination-platinum chemotherapy improves OS and PFS across all subgroups. This provides the strongest evidence to date of the benefit of combination-platinum over single-agent platinum. © The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.

Background: We designed a prospective study to test epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC) in resected stage I–IIIA non-small-cell lung cancer (NSCLC) and to correlate overexpression with survival. Patients and methods: EGFR expression was evaluated in 130 consecutive NSCLC patients after radical surgery (60 squamous cell carcinomas, 48 adenocarcinomas, 22 large cell carcinomas: stage I, 41 (31%); stage II, 37 (29%) and stage IIIA, 52 (40%). Results: Overall, 101 of 130 (78%) specimens expressed EGFR, and with a cut-off value of 10% positive cells 48 cases (37%) were classified as positive. At univariate analysis, EGFR was significantly more expressed in stage III (50%) than stage I (20%) and stage II (25%) (P

Background: The results of the Italian part of an international survey on therapeutic preferences and opinions about prognosis of patients affected by non-small-cell lung cancer (NSCLC) are shown. Patients and methods: The investigation was conducted by the means of a postal questionnaire aiming to gather information on preferences about treatment and beliefs about survival of three hypothetical patients affected by NSCLC in different stages (T2N1M0, T2N3M0, M1); three sources of Italian physicians potentially treating patients affected by NSCLC were the target population: participants in the Adjuvant Lung Project Italy (Alpi) trial, a 20% random sample of the Italian Medical Oncology Association (AIOM) and representatives of almost all the pneumology wards in Italy. Results: Overall, there were 287 evaluable responses, 89% of respondents were males, mean age was 46 years, years from graduation 21 and charge of patients per clinician 82. The most important result is the wide variation of answers both about therapy and prognosis. Expectations about size of prognosis improvement with a new chemotherapy seem to be excessive. Conclusions: The results are discussed in relation to the twin surveys of Canada and England and Wales and to the meta-analyses on the efficacy of chemotherapy as an adjunct to primary treatment and on postoperative radiotherapy in non-small-cell lung cancer.

Background: Several studies have investigated the possible role of the adjuvant chemotherapy after curative resection for gastric cancer failing to show a clear indication; previous meta-analyses suggested small survival benefit of adjuvant chemotherapy, but the statistical methods used were open to criticisms. Materials and methods: Randomised trials were identified by means of Medline and CancerLit and by selecting references from relevant articles. Systematic review of all randomised clinical trials of adjuvant chemotherapy for gastric cancer compared with surgery alone, published before January 2000, were considered. Pooling of data was performed using the fixed effect model. Death for any cause was the study endpoint. The hazard ratio and its 95% confidence intervals (95% CI), derived according to the method of Parmar, were the statistics chosen for summarising the relative benefit of chemotherapy versus control. Results: Overall 20 articles (21 comparisons) were considered for analysis. Three studies used single agent chemotherapy, seven combination of 5-fluorouracil (5-FU) with anthracyclin, ten combination of 5-FU without anthracyclines. Information on 3658 patients, 2180 deaths, was collected. Chemotherapy reduced the risk of death by 18% (hazard ratio 0.82, 95% CI: 0.75–0.89, P Conclusions: Chemotherapy produces a small survival benefit in patients with curatively resected gastric cancer. However, taking into account the limitations of literature based meta-analyses, adjuvant chemotherapy is still to be considered as an investigational approach.

Summary Background Several prognostic factors for stage I ovarian carcinoma have been analyzed. Some of them are biological and clinical in nature, but others such as the thoroughness of the staging procedure, the extent of the surgery and the philosophy of treatment, are defined by the human element. Patients and methods We reviewed the records of 351 patients with Stage I ovarian cancer who had been treated from 1981 to 1991. For all patients the following information was available: age, size of the tumor, FIGO sub-stage, tumor grade, histologic type, rupture of the tumor, cytology, extent of the staging and of the surgery (hysterectomy and bilateral salpingo-oophorectomy vs. fertility-conserving surgery) and use of adjuvant treatments. The thoroughness of the staging was defined as: optimal staging: total abdominal hysterectomy and bilateral salpingooophorectomy or fertility-conserving surgery, peritoneal cytology or washing, omentectomy, multiple peritoneal biopsies, sampling of the retroperitoneal nodes or formal lymphadenectomy, peritoneal staging: all the criteria described above were met with the exception of retroperitoneal sampling, incomplete staging: lack of any of the previously-cited criteria. Results An optimal staging was performed in 100 patients, a peritoneal staging in 107 and an incomplete staging in 144. Radical surgery was performed in 295 women and fertility-conserving surgery in 56. With a median follow-up of 108 months (range 14–184) 64 patients had recurrence of the tumor. Fifty-three died of the disease, two are currently alive with disease and nine were salvaged by surgery and/or chemotherapy. In a multivariate analysis only the tumor grade and the type of staging were significant independent prognostic factors for both disease-free and overall survival. Conclusions As described by other authors, we confirm that tumor grade is the single most important biological prognostic factor in early ovarian carcinoma. The thoroughness of the staging impacts significantly on survival, particularly in poorly differentiated carcinomas. Fertility-sparing surgery is not associated with a worse outcome than standard radical surgery.

Summary Background From 1983 to 1990, 271 consecutive patients with stage I ovarian cancer entered two randomised trials, aimed at assessing the role of adjuvant chemotherapy after radical surgery in early stages of ovarian cancer.Trial I compared cisplatin (50 mg/m2 with repeated courses every 28 days for 6 cycles) to no further therapy in F.I.G.O.stage la & b Grade U-IH patients; trial II compared cisplatin (same dose and schedule) to 32P in Ian & bii and Ic patients Methods Both studies were multicentric and centrally randomized. Treatment was allocated by phone and stratified by center. All patients satisfying major eligibility criteria (histological diagnosis of ovarian carcinoma, correct F.I.G.O. stage and grade, no previous neoplasms) were analysed according to treatment allocated by randomisation. Results With a median observation time of 16 months, cisplatin significantly reduced the relapse rate by 65% (HR = 0.35; 95% CI = 0.14–0.89, p = 0.028; Cox Model) in trial I and 61% (HR = 0.39; 95% CI = 0.19–0.77, p = 0.007; Cox Model) in trial II. Survival was not significantly different (trial I – Kaplan-Meier overall 5-year survival: cisplatin = 88%, control = 82%, HR = 1.15; 95% CI = 0.44 – 2.98; p = 0.773; Cox Model); trial II – overall 5-year survival: cisplatin = 81%, 32P = 79%, HR = 0.72; 95% CI = 0.37–1.43; p = 0.354; Cox model). In both studies the risk of dying after relapse increased for patients originally randomized to the cisplatin arms: in trial I, 6 of 7 patients in the cisplatin relapsed arm and died of tumor compared with 8 of 14 patients in the control arm. In trial II, 11 of 12 patients on cisplatin, and 18 of 26 on 32P succumbed to tumor recurrence. Conclusion Adjuvant cisplatin treatment in early ovarian cancer significantly prevents relapse in comparison to 32P in Stage IC patients or to no immediate treatment in earlier Stage women. The impact of cisplatin adjuvant treatment on survival remains, however, unclear.

Summary Background We wished to assess the clinical value in terms of treatment choice and establishment of the prognosis of the ACTG classification modified (TNM-TIS) according to the recent guidelines of CDC for the classification of the HIV infection in patients with HIV-related epidemic Kaposi's sarcoma (EKS). Patients We retrospectively studied 296 HIV-positive individuals with EKS. Patients were initially classified according to the NYU system and then reclassified according to the TNM-TIS proposal which considers three major parameters: T, anatomical extent of the lesion; I, immune system status; S, HIV-related systemic illness. Methods Survival analyses according to patient characteristics and the different TNM-TIS classification stages were performed; curves were compared using the Kaplan-Meyer method, and predictive factors for survival using the Cox model. Results Of the parameters considered in the TNM-TIS staging system, the T variable was not predictive of survival. Conversely, I and S variables revealed predictive value in the survival analyses, when considered separately and together. Conclusions The extent of cutaneous or mucosal lesions of Kaposi's sarcoma did not correlate with prognosis. However, both CD4+ cell count and history of systemic illness were predictive of survival. Indicators of HIV infection must be included in the clinical evaluation of EKS patients and taken into account when choosing optimal treatment.

Purpose Over the last decade a dramatic improvement in the treatment and prognosis of human epidermal growth factor receptor-2 (HER2) positive metastatic breast cancer (MBC) has been achieved. This study aimed to describe pattern, timing of metastases, and time to progression (TTP) of MBC patients (pts) treated with multiple lines of therapy with trastuzumab and/or lapatinib.

Background: Phase III trial aiming to compare first line pharmacogenomic-driven chemotherapy based on Excision-Repair-Cross-Complementing-1 (ERCC1, E), Ribonucleotide Reductase subunit M1 (RRM1, R) and Thymidylate Synthase (TS, T) gene expression versus standard treatment in elderly patients (pts) with advanced NSCLC. Trial design: pts aged ≥70 years, ECOG Performance Status 0 or 1, EGFR negative mutational status, chemonaive stage IV NSCLC will be evaluated. Pts will be randomized (2:1) to experimental arm (A) or standard arm (B). In arm A, treatment will be based on histology, E, R and T mRNA expression. The cut-off for high or low expression have been previously defined. Treatments for pts with squamous NSCLC: carboplatin for E low/R high, gemcitabine for E high/R low, carboplatin and gemcitabine for E low/R low, docetaxel or vinorelbine for E high/R high. Treatments for non-squamous NSCLC pts: carboplatin for E low/T high, pemetrexed for E high/T low, carboplatin and pemetrexed for E low/T low, gemcitabine for E high/T high/R low, docetaxel or vinorelbine for E high/T high/R high. In arm B treatment will be at the investigator discretion. The primary endpoint is overall survival. The secondary endpoints are progression-free survival, response rate (RECIST 1.1) and tolerability (CTCAE version 4.0). Feasibility of treatment selection based on pharmacogenomic parameters will be also assessed. Treatment will continue to a maximum of 6 cycles if tolerated or until disease progression. Assuming an exponential survival distribution for both arms and a median survival time of 8 months in the control arm, we anticipate to detect an improvement of 3 months in the median survival time in the experimental arm. To have 90% power to detect a three-month improvement in median survival at a significance level of 5% (2-sided) and assuming a 10% failure rate in gene analyses or loss to follow-up rate, a sample size of 567 patients is planned. Conclusion: To our knowledge, this is the first prospective pharmacogenomic-driven trial designed in elderly advanced NSCLC population. Disclosure: D. Cortinovis: Consultant for AZ, BI, Roche and Lilly; M. Papotti: Honoraria have been received from Eli Lilly; G. Scagliotti: Honoraria have been received from Eli Lilly, Roche, Pfizer and Astrazeneca. All other authors have declared no conflicts of interest.