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Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP).Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo.Results: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2+ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-β1.Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910–21. ©2014 AACR.

PDF file - 270KB, Supplementary Figure S1. Phenotype of in vitro generated mDCs. Supplementary Figure S2. Efficiency of transfection. Supplementary Figure S3. HuHuT-, HuRT- and RHuT-DCs from HS elicit anti-HER2 CD8 response. Supplementary Figure S4. RHuT-DCs from HER2-CP elicit anti-HER2 CD8 T cell response. Supplementary Figure S5. HuHuT DCs affects Treg cells activity. Supplementary Figure S6. HuHuT-DCs activate CD8 and CD4 T cells from CTRL-CP. Supplementary Table S1. HER2-overexpressing cancer patients. Supplementary Table S2. HER2-negative cancer patients.

Objectives: The aim of this study was to conduct a discrete choice experiment with patients affected by colorectal cancer to understand their preferences for different attributes of the chemotherapy supply. Our overall goal is to provide evidence on the relative importance of each attribute in order to tailor chemotherapy supply according to patients' priorities in the design or reorganization processes of cancer services. Methods: Focus groups were used to identify the attributes and levels for the discrete choice experiment. The attributes were: continuity of care, understanding, information, treatment choice, and time for therapy. Respondents were asked to choose between two mutually exclusive hypothetical alternatives of chemotherapy supply. Patients completed the discrete choice experiment along with the health-related quality of life and patients’ satisfaction questions. Conditional and mixed logistic models were used to analyses the data. Results: Patients with colorectal cancer treated with chemotherapy (n = 76) completed the survey. The most important aspects of chemotherapy supply were: “Providing detailed and complete information” and “High ability in understanding” patients. Preferences were also influenced by the availability of a trusted doctor. Except for one attribute (waiting time for therapy), all other characteristics significantly influenced respondents’ preferences. Conclusions: Results should support a policy of strengthening medical doctors’ capabilities to communicate with patients, providing them complete information and involving them in the clinical decisions. Specifically, the findings should be used to improve the current provision of cancer care by identifying areas of preferred intervention from the perspectives of patients in order to tailor the service supply accordingly.

Background Response shift (RS) has been defined as a change in the meaning of an individual’s self-evaluation that needs to be accounted for when assessing longitudinal changes in health-related quality of life (HRQoL). RS detection through structural equation modeling is accomplished by adopting Oort’s procedure based on a measurement model in which the observed variables are defined as reflective indicators of the HRQoL latent variable; that is, the latent variable causes the variation in the reflective indicators. This study aims to propose a procedure that assesses RS when formative indicators are used in measuring HRQoL; in this last case, the latent variable is considered to be a function of some formative indicators. A secondary aim is to compare the new procedure with Oort’s procedure to highlight similarities and differences. Methods The data were retrieved from a consecutive series of 258 patients newly diagnosed with colorectal cancer and undergoing chemotherapy and/or surgery. The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QOL-C30) was administered twice, once before and once six months after treatment. Structural equation modeling was used to evaluate RS and true change with the newly proposed method (in which fatigue and pain were defined as formative indicators) and with Oort’s procedure (in which fatigue and pain were defined as reflective indicators). Results According to the new procedure, there was no measurement bias, and on average, patients’ quality of life improved by 3.53 points (on a scale ranging from 0 to 100) at the 6-month follow-up. With Oort’s procedure, the loading of the pain indicator was not invariant across the two time points, suggesting the presence of reprioritization, whereas the estimation of true change was very similar to the previous one: 3.87. Conclusions RS and true change in HRQoL can be evaluated in the presence of formative indicators. Defining a measurement model by formative or reflective indicators can lead to different results.

Background Immune-inflammatory biomarkers (IIBs) showed a prognostic relevance in patients with metastatic CRC (mCRC). We aimed at evaluating the prognostic power of a new comprehensive biomarker, the Pan-Immune-Inflammation Value (PIV), in patients with mCRC receiving first-line therapy. Methods In the present pooled-analysis, we included patients enrolled in the Valentino and TRIBE trials. PIV was calculated as: (neutrophil count × platelet count × monocyte count)/lymphocyte count. A cut-off was determined using the maximally selected rank statistics method. Generalised boosted regression (GBR), the Kaplan–Meier method and Cox hazards regression models were used for survival analyses. Results A total of 438 patients were included. Overall, 208 patients (47%) had a low-baseline PIV and 230 (53%) had a high-baseline PIV. Patients with high PIV experienced a worse PFS (HR, 1.66; 95% CI, 1.36–2.03, P P Conclusion PIV is a strong predictor of survival outcomes with better performance than other well-known IIBs in patients with mCRC treated with first-line therapy. PIV should be prospectively validated to better stratify mCRC patients undergoing first-line therapy.

To investigate effects of ablation margins on local tumour progression-free survival (LTPFS) according to RAS status in patients with colorectal liver metastases (CLM).This two-institution retrospective study from 2005-2016 included 136 patients (91 male, median age 60 years) with 218 ablated CLM. LTPFS was performed using the Kaplan-Meier method and evaluated with the log-rank test. Uni/multivariate analyses were performed using Cox-regression models.Three-year LTPFS rates for CLM with minimal ablation margin ≤10 mm were significantly worse than those with10 mm in both mutant-RAS (29% vs. 48%, p=0.038) and wild-type RAS (70% vs. 94%, p=0.039) subgroups. Three-year LTPFS rates of mutant-RAS were significantly worse than wild-type RAS in both CLM subgroups with minimal ablation margin ≤10 mm (29% vs. 70%, p0.001) and10 mm (48% vs. 94%, p=0.006). Predictors of worse LTPFS were ablation margins ≤10 mm (HR: 2.17, 95% CI 1.2-4.1, p=0.007), CLM size ≥2 cm (1.80, 1.1-2.8, p=0.017) and mutant-RAS (2.85, 1.7-4.6, p0.001).Minimal ablation margin and RAS status interact as independent predictors of LTPFS following CLM ablation. While minimal ablation margins10 mm should be always the procedural goal, this becomes especially critical for mutant-RAS CLM.• RAS and ablation margins are predictors of local tumour progression-free survival. • Ablation margin10 mm, always desirable, is crucial for mutant RAS metastases. • Interventional radiologists should be aware of RAS status to optimize LTPFS.

ObjectiveMutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC).DesignGenome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy.ResultsMethylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival.ConclusionThis five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

3511 Background: Preop CTRT is considered the standard of care in the management of LARC. RT can induce antigen release from a low neoantigen-burden tumor (such as a mismatch repair proficient colorectal cancer) and activate dendritic cells leading to a CD8+ T lymphocyte-mediated anticancer immune response. In LARC patients, neoadjuvant CTRT increases PD-L1 expression in tumor cells, strongly suggesting a neoadjuvant combinatory strategy with RT and PD-1/PD-L1 pathway blockade. Based on such considerations, we have designed the AVANA study to investigate the role of Ave in combination with preop CTRT in LARC. Methods: This is an Italian multi-center, phase II study. Pts with resectable LARC, defined by the presence of at least one of the following features: cN+, cT4, high risk cT3, received standard preop CTRT (capecitabine 825 mg/sqm/bid 5 days/week+ 50.4 Gy in 28 fractions over 5.5 weeks) plus 6 cycles of Ave 10 mg/Kg every 2 weeks. Surgery with total mesorectal excision was performed at 8-10 weeks after the end of CTRT. The primary end-point was the pCR rate, defined as complete histological regression with no available tumor cells ypT0N0. Secondary end-points were R0 resection rate, tumor downstaging, local recurrence, sphincter preservation rate, progression-free survival, overall survival, safety profile, and the evaluation of exploratory predictive and/or prognostic biomarkers. Assuming as null hypothesis p0 a pCR rate of 15%, a significance level of 5% (one-side), and a power of 80%, a sample size of 101 pts was needed to detect an absolute increment of 10% in pCR rate (from 15% to 25%). The experimental regimen is considered for further studies if, in at least 22 pts, we observe a pCR. Results: From April 2019 to November 2020, a total of 101 resectable LARC pts were enrolled in 10 Italian Centers. The median age was 63 years (23-82), 62 (61.4%) pts were male, 93 (92%) had ECOG PS 0. At baseline, 94 (93%) and 16 (16%) pts had cN+ and cT4 LARC, respectively. All pts completed the induction phase. Out of 96 pts evaluable for pathological response, 22 (23%) pts achieved a pCR and 59 (61.5%) pts a major pathological response (a central review is ongoing). At this time, microsatellite status is available only in 39 pts, of which only one was instable. The rate of grade 3-4 non-immune and immune-related adverse events was 8% and 4%, respectively. Avelumab was early interrupted in 9 pts out 101, mainly due to toxicity. Conclusions: The combination of preop CTRT plus Ave showed a promising activity and a feasible safety profile. According to our statistical considerations, the experimental regimen will be considered for further studies. Updated results will be presented during the Congress. Sponsored by GONO and partially supported by Merck. EUDRACT 2017-003582-10. Clinical trial information: NCT03854799.

3534 Background: Based on retrospective experiences, gender seems to affect the safety profile of chemotherapy (CT), with a higher incidence of CT-related adverse events (AEs) among females than males. Here we focus on the impact of gender on the toxicity of FOLFOXIRI/bevacizumab (bev) as compared with doublets (FOLFOX or FOLFIRI)/bev in two randomized phase III studies by GONO: TRIBE and TRIBE2. Methods: The risk of experiencing CT-related AEs in males and females was estimated in univariable analysis in the overall safety population and according to treatment arms (doublets/bev and FOLFOXIRI/bev). In order to assess the independent weight of gender on the risk of developing AEs, multivariable logistic regression models were built. Results: Among 1187 patients enrolled in TRIBE and TRIBE2 studies, 1176 (684 males, 58%, and 492 females, 42%) were included in the safety population. Overall, women had a significantly higher risk of CT-related AEs, in particular gastrointestinal and hematologic AEs, asthenia and alopecia, independently of the treatment arm. The risk of CT-related AEs was increased with FOLFOXIRI/bev vs doublets/bev independently of gender (p for interaction: 0.329). Notably, among women treated with FOLFOXIRI/bev 50% and 68% experienced any grade of vomiting and nausea, respectively. Conclusions: Female mCRC patients have a higher risk to develop CT-related AEs. In women treated with FOLFOXIRI/bev the high incidence of nausea and vomiting may suggest the need for an intensification of the antiemetic prophylaxis. [Table: see text]

To test the hypothesis that irradiated volume of specific subregions of pelvic active bone marrow as detected by (18)FDG-PET may be a predictor of decreased blood cells nadirs in anal cancer patients undergoing concurrent chemoradiation, we analyzed 44 patients submitted to IMRT and concurrent chemotherapy. Several bony structures were defined: pelvic and lumbar-sacral (LSBM), lower pelvis (LPBM) and iliac (IBM) bone marrow. Active BM was characterized employing (18)FDG-PET and characterized in all subregions as the volume having standard uptake values (SUVs) higher than SUVmean. All other regions were defined as inactive BM. On dose-volume histograms, dosimetric parameters were taken. Endpoints included white blood cell count (WBC), absolute neutrophil count (ANC), hemoglobin (Hb) and platelet (Plt) nadirs. Generalized linear modeling was used to find correlations between dosimetric variables and blood cells nadirs. WBC nadir was significantly correlated with LSBM mean dose (β = -1.852; 95 % CI -3.205/-0.500; p = 0.009), V10 (β = -2.153; 95 % CI -4.263/-0.721; p = 0.002), V20 (β = -2.081; 95 % CI -4.880/-0.112; p = 0.003), V30 (β = -1.971; 95 % CI -4.748/-0.090; p = 0.023) and IBM V10 (β = -0.073; 95 % CI -0.106/-0.023; p = 0.016). ANC nadir found to be significantly associated with LSBM V10 (β = -1.878; 95 % CI -4.799/-0.643; p = 0.025), V20 (β = -1.765; 95 % CI -4.050/-0.613; p = 0.030) and IBM V10 (β = -0.039; 95 % CI -0.066/-0.010; p = 0.027). Borderline significance was found for correlation between Plt nadir and LSBM V30 (β = -0.056; 95 % CI -2.748/-0.187; p = 0.060), V40 (β = -0.059; 95 % CI -3.112/-0.150; p = 0.060) and IBM V30 (β = -0.028; 95 % CI -0.074/-0.023; p = 0.056). No inactive BM subsites were found to be correlated with any blood cell nadir. (18)FDG-PET is able to define active bone marrow within pelvic osseous structures. LSBM is the strongest predictor of decreased blood cells nadirs in anal cancer patients undergoing concurrent chemoradiation.

3505Background: Since doublets plus anti-EGFRs were continued until progressive disease (PD) in mCRC first-line registration trials, there is scarce evidence if de-escalating treatment intensity after an anti-EGFR-based induction might be not inferior in terms of disease control, while reducing toxicity burden and improving quality of life. In MACRO-2, maintenance with cetuximab alone achieved a 9-month progression-free survival (PFS) rate non inferior than continued mFOLFOX + cetuximab in KRAS ex. 2 wt patients. Methods: RAS wt unresectable mCRC patients, stratified for prior adjuvant therapy and metastatic sites (1 vs ≥2), were randomized and received FOLFOX + Pan induction (8 cycles) followed by maintenance with Pan (arm B) or 5FU/LV + Pan (arm A) until PD/unacceptable toxicity/death. The primary endpoint was non-inferiority of PFS of arm B vs A. An overall sample size of 224 subjects (112 per arm) achieved 90% power to detect a 50% 10-month PFS rate in arm A and a maximum 15% difference in arm B, with...

Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP). Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo. Results: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2+ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-β1. Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910–21. ©2014 AACR.

Aims and background Multimodal therapy is a keystone of care in advanced esophageal cancer. Although neoadjuvant chemoradiotherapy is known to provide a survival advantage in selected cases, reliable prognostic and response predictive factors remain elusive. We report the outcome in a series of esophageal cancer patients treated at our center and the results of a retrospective analysis of epidermal growth factor receptor (EGFR) expression and EGFR/HER2 gene copy numbers taken as possible prognostic and predictive factors. Methods and study design Between 2001 and 2009, a total of 40 consecutive patients (34 men and 6 women; median age, 59 years) were treated for esophageal cancer. Treatment: cisplatin, 80 mg/m2 day 1, and 5-fluorouracil, 800 mg/m2/24 h on days 1–5, every 21 days, concomitant with 3D-conformal radiotherapy (54–59.4 in 30–33 fractions) for three up to four cycles. Surgery was performed in eligible patients 6–8 weeks after chemoradiation. EGFR expression and EGFR/HER2 amplification and gene copy number were studied by immunohistochemical analysis and fluorescence in situ hybridization, respectively. Results Acceptable toxicity following chemoradiation was recorded, with G3–G4 hematological toxicity in 20% of patients and G3–G4 dysphagia in less than 10%; 14 (35%) patients achieved complete response and 19 (48%) partial response; 18 underwent surgery after chemoradiation, of which 8 (20%) achieved pathologic complete response. The median survival was 29 months (95% CI, 25.7–32.1): 42 months for the resected and 20 for the unresected patients. EGFR and HER2 analysis in 28 patients showed that 89% had immunohistochemical EGFR expression, with 5 cases of EGFR and 10 of HER2 gene gain without a significant difference in response rate and survival in these patient subgroups. Conclusions Our results suggest a better outcome in patients who underwent surgery after chemoradiation. A larger sample size is necessary to clarify the role of EGFR and HER2 gene gain in predict response and survival.

The nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy. Using RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically. Target gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (p

Aims Several randomised trials have tested adjuvant regimens using concomitant high-dose cisplatin and radiotherapy to improve outcome in high-risk locally advanced squamous cell head and neck cancer (HNSCC), showing a substantial increase in locoregional control and disease-free survival, despite a higher and eventually detrimental toxicity profile. The aim of the present phase II single-stage prospective study was to investigate whether a weekly cisplatin-based chemoradiotherapy regimen might be able to improve patients' compliance compared with standard-dose cisplatin with similar outcome results. Materials and methods Between January 2004 and November 2008, 54 patients with high-risk locally advanced HNSCC were enrolled on to this phase II trial. Patient characteristics were: median age 59.7 years, Eastern Cooperative Oncology Group performance status 1 in 72% of patients and stage IV disease in 82%, extracapsular nodal spread in 67% and positive/close surgical margins in 37%. Patients received cisplatin (30mg/m 2 ) once a week for 7–8 weeks concurrent with external beam radiotherapy delivered with a median dose of 66.6Gy (1.8Gy each day; five fractions/week) on the primary site and 50Gy (2Gy each day) for the lower neck. Results Major acute toxicity of the combined treatment, defined as grade 3–4 mucositis, was observed in 35.2% of patients. No fatal complications occurred, with 81.5% of patients completing the planned regimen. Late reactions were mild (total 16% with a grade 3 dysphagia rate of 12%). The locoregional control rate was 82%; 5 year overall and disease-free survival were 63 and 62%, respectively. Conclusions Concomitant adjuvant chemoradiotherapy with weekly cisplatin seems to be a feasible and well-tolerated therapeutic approach in ‘unfit' patients. Clinical results seem to be at least comparable with those previously reported. However, to draw any definitive conclusion, large confirmatory phase III randomised trials are demanded.

Although Ki67 index suffers from poor reproducibility, it is one of the most important prognostic markers used by oncologists to select the treatment of estrogen receptor (ER) positive breast cancer patients. In this study, we aim to establish the optimal Ki67 cut-offs for stratifying patient prognosis and to create a comprehensive prognostic index for clinical applications. A mono-institutional cohort of 1.577 human epidermal growth factor receptor 2 negative/ER+ breast cancer patients having complete clinical, histological, and follow-up data was collected. The 14 and 20 % Ki67 cut-offs were correlated to disease-free interval (DFI) and disease-specific survival (DSS). To create a comprehensive prognostic index, we used independent variables selected by uni/multivariate analyses. In terms of DFI and DSS, patients bearing tumors with Ki67 20 % showed the poorest prognosis. Moreover, to tumor size, the number of metastatic lymph nodes and Ki67 > 20 % was given a score value, varying depending on definite cut-offs and used to create a prognostic index, which was applied to the population. Patients with a prognostic index ≥3 were characterized by significant risk of relapse [DFI: Hazard Ratio (HR) = 4.74, p