Phase II randomised trial of autologous tumour lysate dendritic cell plus best supportive care compared with best supportive care in pre-treated advanced colorectal cancer patients

European Journal of Cancer 64 (2016) 167e174

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.ejcancer.com

Original Research

Phase II randomised trial of autologous tumour lysate dendritic cell plus best supportive care compared with best supportive care in pre-treated advanced colorectal cancer patients Miguel Caballero-Ban˜os a,*, Daniel Benitez-Ribas b, Jaime Tabera c, Sara Varea d, Ramon Vilana e, Luis Bianchi e, Juan Ramo´n Ayuso f, Mario Page´s f, Gemma Carrera g, Miriam Cuatrecasas h, Marta Martin-Richard i, Joan Cid j, Miguel Lozano j, Antoni Castells b, Xabier Garcı´a-Albe´niz k, Joan Maurel l,1, Ramon Vilella a,1 a

Department of Immunology, Hospital Clinic, Barcelona, Spain Department of Gastroenterology, Hospital Clinic, iDIBAPS, CIBERhed, University of Barcelona, Barcelona, Spain c BST, Sant Boi De Llobregat, Spain d Clinical Trials Unit, Hospital Clinic, Barcelona, Spain e Department of Radiology, BCLC Group, Liver Unit, Hospital Clinic, University of Barcelona, Spain f Department of Radiology, Hospital Clinic, University of Barcelona, Spain g Department of Medical Oncology, Hospital Platon, Barcelona, Spain h Department of Pathology, Hospital Clinic Barcelona, Spain i Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain j Department of Haemotherapy, Hospital Clinic, Barcelona, Spain k Harvard T.H. Chan School of Public Health, Boston, MA, USA l Department of Medical Oncology, Hospital Clinic, Barcelona, Spain b

Received 15 April 2016; received in revised form 24 May 2016; accepted 7 June 2016 Available online 16 July 2016

KEYWORDS Cancer; Dendritic cells; Immunotherapy;

Abstract Background: Autologous tumour lysate dendritic cell vaccine (ADC) has T-cell stimulatory capacity and, therefore, potential antitumour activity. We designed a phase II randomised trial of ADC þ best supportive care (BSC) (experimental arm [EA]) compared with BSC (control arm [CA]), in pre-treated metastatic colorectal cancer (mCRC) patients.

* Corresponding author: Carrer Villarroel 170, 08036, Barcelona, Spain. Tel.: þ34 932275400x2367; fax: þ34 933129406. E-mail address: [email protected] (M. Caballero-Ban˜os). 1 Both authors contributed equally to the work. http://dx.doi.org/10.1016/j.ejca.2016.06.008 0959-8049/ª 2016 Elsevier Ltd. All rights reserved.

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Patients and methods: Patients with progressive mCRC, at least to two chemotherapy regimens and Eastern Cooperative Oncology Group performance status (ECOG PS) 0e2, were randomised to EA versus CA. Stratification criteria: ECOG PS (0e1 versus 2) and lactate dehydrogenase (ULN). EA was administered subcutaneously till progressive disease. Primary end-point was progression-free survival (PFS) at 4 months. Results: Fifty-two patients were included (28 EA/24 CA). An interim analysis recommended early termination for futility. No objective radiological response was observed in EA. Median PFS in EA was 2.7 months (95% confidence interval [CI], 2.3e3.2 months) versus 2.3 months (95% CI, 2.1e2.5 months) in CA (p Z 0.628). Median overall survival (OS) was 6.2 months (95% CI, 4.4 e7.9 months) in EA versus 4.7 months (95% CI, 2.3 e7 months) in CA (p Z 0.41). No ADC-related adverse events were reported. Immunization induces tumourspecific T-cell response in 21 of 25 (84%) patients. Responder patients have an OS of 7.3 months (95% CI, 5.2e9.4 months) versus 3.8 months (95% CI, 0.6e6.9 months) in non-responders; p Z 0.026). Conclusion: Our randomised clinical trial comparing ADC þ BSC versus BSC in mCRC demonstrates that ADC generates a tumour-specific immune response but not benefit on PFS and OS. Our results do not support the use of ADC alone, in a phase III trial. ª 2016 Elsevier Ltd. All rights reserved.

1. Introduction

2. Patient and methods

Patients with metastatic colorectal cancer (mCRC) have few therapeutic options once the tumour has progressed to two lines of therapy. Regorafenib is the only drug approved by the Food and Drug Administration in this setting [1], but due to the toxicity profile and limited efficacy, its benefit is controversial [2]. Cancer vaccines with dendritic cells (DCs) are an alternative therapeutic approach under study. These vaccines aim to stimulate tumour antigen-specific cytotoxic T lymphocytes that recognise and eliminate cancer cells in an antigen-specific way [3]. The active immunisation with DCs can elicit adaptive antitumour immunity even in metastatic patients, who are thought to be less immune responsive [4]. A systematic review of all published clinical trials showed a small clinical benefit of DC-based immunotherapy in terms of objective response and suggests a positive association between immune response induced by DCs and patient survival [5]. Three small prospective phase II trials that treated mCRC patients with peptide matured DCs [6] or autologous lysate matured DCs [7,8] showed promising results, with prolonged progression-free survival (PFS) and overall survival (OS). Due to the lack of control group in these studies, it is difficult to establish if the benefit is due to the therapy or the intrinsic behaviour of the disease. Here we present a randomised phase II trial comparing autologous tumour lysate dendritic cell vaccine (ADC) þ best supportive care (BSC) (experimental arm [EA]) versus BSC (control arm [CA]) in pre-treated mCRC patients upon stratifying by relevant prognostic factors, such as performance status (PS) and lactate dehydrogenase (LDH).

2.1. Study design The study was a randomised, open-label, phase II trial at the Clinic Hospital in Barcelona (Spain). The ethics committee at this institution and the Spanish regulatory board approved the clinical trial (Agencia Espan˜ola del Medicamento y Productos Sanitarios [AEMPS]; PEI 09e133) (NCT01413295). All patients provided written informed consent before randomisation. Eligible patients had metastatic CRC; measurable disease according RECIST1.1; progressive disease to at least two lines of therapy (including biological agents); adequate bone marrow, liver and renal function; age >18 years; Eastern Cooperative Oncology Group performance status  2; and adequate access to tumour tissue metastases for lysate production. Exclusion criteria included a history of other tumours, active infections and current immunosuppressive treatment or infection by HIV, HBV or HCV. Patients were stratified according LDH levels (<450 U/ml versus >450 U/ml) and PS (0e1 versus 2) to receive (1:1) the EA or CA. Patients assigned to the EA underwent aphaeresis to obtain peripheral blood leucocytes and an ultrasound/computed tomography (CT)guided needle biopsy of metastases to prepare the tumour lysate. These patients were planned to receive five doses of ADC (5  106 cells/dose) at days 0, 10, 20, 40, and 120 and every 6 months thereafter until progressive disease. PFS was defined as the time from randomisation to progression or death (whichever occurred first) or last follow-up for patients alive without progression. OS was defined as the time between randomisation and death or date of last follow-up

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for patients still alive. Radiological assessment of response was carried out every 8 weeks the first 6 months and thereafter every 3 months in both arms, until disease progression or withdrawal. Patients without a CT evaluation because of death before the established time for first evaluation, whatever the cause (rapidly progressive disease, treatment-related toxicity or severe adverse event not related to disease or treatment) were considered as having progressed at the time of death. Toxicity was recorded using the Common Toxicity Criteria (NCI-CTC). 2.2. Tumour sample and preparation of tumour lysate Biopsies to prepare tumour lysate were obtained from liver (n Z 14), lung (n Z 6), bone (n Z 4), peritoneum (n Z 3), and lymph node (n Z 1) from 28 patients; 0.5 mm3 of tumour was disrupted to obtain the lysate, with the GentleMACS disruptor device (Miltenyi Biotech, Bergisch Gladbach, Germany), followed by freezing/thawing procedures (five cycles), 25 KGy irradiation by cobalt-60, and subsequently cryopreserved at 20  C until needed for the preparation of DCs.

patients allocated in EA were obtained by Histopaque gradient centrifugation (Lonza) and were slowly frozen to 80  C in foetal calf serum using a cryo-freezing container (Mr. Frosty; Nalgene, Rochester, NY, USA). To determine the effect of ADC in the immune response against tumour, the presence of tumour-specific T cells were evaluated, before (pre) and after (post) treatment (post 40, 25 patients; and post 120, 14 patients), using an autologous tumour mixed leucocyte reaction (ATMLR). After thawing the autologous DCs (pulsed with autologous tumour lysate) and PBMCs (days 0, 40 and 120) of each patient, co-cultures with 5  103 DCs and 1  105 PBMCs (triplicates of each condition) were done in 0.2 ml XeVIVO 15 (Lonza) in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark). Plates were incubated in a humid atmosphere of 5% CO2 at 37  C for 7 d. Eighteen hours before termination of culture, each well received 0.5 mCi of [methyl-3H] thymidine at 2 Ci/mmol (TRA310; Amersham Biosciences, Little Chalfont, UK). Uptake of thymidine into DNA was determined using a cell harvester (Perkin Elmer, Boston, Table 1 Baseline characteristics.

2.3. Preparation of autologous DCs Patients assigned to the EA underwent aphaeresis to obtain peripheral blood leucocytes (volume 80 ml). Autologous monocytes were selected by adherence to plastic and then differentiated to DCs by culturing plastic-adherent monocytes for 7 d in X-VIVO 15 (Lonza, Walkersville, MD, USA) supplemented with 2% autologous serum, 1000 U/ml granulocyteemacrophage colony-stimulating factor (Miltenyi Biotech) and 1000 U/ ml interleukin (IL)-4 (Miltenyi Biotec) and then for an additional 24 h in the presence of 20 ng/ml tumour necrosis factor-a (Miltenyi Biotech), 10 ng/ml IL1-b (Miltenyi Biotech), 20 ng/ml IL-6 (Miltenyi Biotech), 1 mg/ml prostaglandin E2 (Dinoprostona; Pfizer, New York, USA), 20 mg/ml poly (I:C) (Hiltonol; Oncovir Inc, Washington DC, USA) (a generous gift of Dr. Andres Salazar) and autologous tumour lysate, using good manufacturing practices standard procedures. Maturation was confirmed by assessing increases in the immunofluorescence of CD80 (BD Bioscience, San Diego, CA, USA), CD86 (BD Bioscience) and HLA-DR (BD Bioscience) and absence of CD3 (T lymphocytes) (BD Bioscience), CD14 (monocytes) (BD Bioscience) and CD19 (B lymphocytes) (BD Bioscience). Flow cytometry analysis was performed using FACS Canto (BD Bioscience). Release criteria included >80% CD80þ, CD86þ and negative microbial test. 2.4. Immunological monitoring Peripheral blood mononuclear cells (PBMC) and serum were collected at days 0, 40, and 120. PBMCs from

169

Median age (range) Sex Female ECOG performance status 0 1 2 Number of metastatic organs 1 2 >2 Liver metastasis KRAS mutation statusa No Yes Unknown Number of previous therapies 2 3 >3 Normal serum LDH concentration (1 ULN) Serum LDH concentration, mean (range) PALb, mean (range)

ADC þ BSC (n Z 28)

BSC (n Z 24)

63 (29e81)

63 (35e77)

11 (39%)

10 (42%)

4 (14%) 17 (61%) 7 (25%)

2 (8%) 17 (71%) 5 (21%)

3 (11%) 14 (50%) 11 (39%) 17 (61%)

7 (29%) 12 (50%) 5 (21%) 17 (71%)

13 (46%) 14 (50%) 1 (4%)

11 (45%) 13 (55%) 0 (0%)

14 (58%) 10 (36%) 4 (14%) 12 (43%)

10 (42%) 8 (33%) 6 (25%) 12 (50%)

690.8 (273e3779) 925.2 (312 e6578) 348.4 (131e930) 653.2 (138 e3092)

Previous biologic therapies Bevacizumab 17 (61%) Panitumumab, cetuximab or both 15 (53%) Time from diagnosis of metastases to study entry 24 months 11 (39%) >24 months 17 (61%)

13 (54%) 10 (48%) 10 (42%) 14 (58%)

ADC: autologous tumour lysate dendritic cell vaccine; BSC: best supportive care; ECOG: Eastern Cooperative Oncology Group; LDH: lactate dehydrogenase; ULN: Upper Limit of Normal. a Exon 2 analysis only. b Six patients not analyzed.

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MA, USA) involving filtration of lysed cells onto 96-well filter plates (PHDTM cell harvester; Cambridge Tec, Cambridge, MA, USA) and addition of scintillation to each well followed by counting in a TopCount. We calculated the geometric mean of three replicates. ATMLR was performed followed by immune staining and fluorescence-activated cell sorting analyses to test the polarisation of the immune response against the tumour generated by the vaccination. After thawing the autologous DCs (pulsed with autologous tumour lysate) and PBMCs (days 0, 40 and 120) of each patient, cocultures were done with 5  104 DCs and 1  106 PBMCs (triplicates of each condition) in 2 ml X-VIVO 15 (Lonza) in 24-well culture plates (Nunc). Plates were incubated in a humid atmosphere of 5% CO2 at 37  C for 7 d. Eighteen hours before termination of culture, each well received phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 mg/ml). After that, staining (human CD4-PERCP-CY5.5, human IL17-PE, human IFN-g-FITC, human IL4-APC, CD45-PCy7, and CD3APC-Alexa [BD Bioscience]) and analysis (FACS Canto) were made following manufacturer instructions.

increase of 4-month PFS from 10% in the CA to 40% in the EA (alpha Z 0.05 and beta Z 0.2). PFS and OS were measured from the date of randomization and were estimated to the KaplaneMeier method and compared by a stratified log-rank test. We fitted Cox regression modelling for OS and PFS. Analysis was performed with the R statistical software. 3. Results 3.1. Patient characteristics Between August 5th, 2011, and October 31st, 2013 (date when the futility analysis recommended to stop recruitment), 61 patients were screened and 52 patients were randomised to (EA) (n Z 28) or (CA) (n Z 24). All randomised patients were included with intention-totreat (ITT) analysis for the primary end-point. Baseline characteristics of the patients were well balanced in both arms (Table 1). In the EA 50% of patients (14 of 28) completed the treatment (five doses), receiving at least four doses 90% of patients included. The reasons for treatment discontinuation are reported in Fig. 1.

2.5. Statistical analysis

3.2. Toxicity

The primary end-point of the study was to determine PFS. Secondary end-points were PFS and OS, toxic effects, and the proportion of patients with an objective response. A post hoc analysis of PFS and OS was done according to the immune response in the EA. We planned to recruit 38 patents per arm to detect an

The intradermal administration of DCs was well tolerated and no significantly severe adverse events were related to the treatment with EA compared with CA (Table 2).

61 paƟents assessed for eligibility

9 paƟents excluded 8 did not meet the inclusion criteria 6 posiƟve virus (VIH, HBV) 2 no biopsiable disease 1 declined to parƟcipate

52 paƟents randomized ITT populaƟon

28 paƟents allocated to ADC+BSC

24 paƟents allocated to BSC 1 paƟent did not iniƟate treatment

27 paƟents received treatment

8 paƟents received potenƟal acƟve treatment+BSC

16 paƟents received BSC only

Fig. 1. Consort diagram. ADC: autologous tumour lysate dendritic cell vaccine; BSC: best supportive care.

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171

Table 2 Toxicity profile. BSC (n Z 24)b

ADC þ BSC (n Z 28)a Any event Anaemia Leucopaenia Thrombocytopaenia Diarrhoea Hypertension Nausea Asthenia Hand-foot Rash Oral mucositis Flu-like symptoms Chills Myalgia Local reaction

Any grade

Grade 3

Grade 4

Any grade

Grade 3

Grade 4

16 5 2 0 0 0 1 12 0 0 0 2 0 0 0

1 0 0 0 0 0 0 1 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

14 3 1 2 1 0 3 5 1 1 0 0 0 0 0

1 0 0 0 0 0 0 0 1 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

(59%) (18%) (7%) (0%) (0%) (0%) (4%) (44%) (0%) (0%) (0%) (7%) (0%) (0%) (0%)

(4%) (0%) (0%) (0%) (0%) (0%) (0%) (4%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

(0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

(66%) (14%) (5%) (9%) (5%) (0%) (14%) (23%) (5%) (5%) (0%) (0%) (0%) (0%) (0%)

(5%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (5%) (0%) (0%) (0%) (0%) (0%) (0%)

(0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

ADC: autologous tumour lysate dendritic cell vaccine; BSC: best supportive care. a One patient was not assessed because of death before treatment. b Three patients were not assessed because they were treated in other centers in phase I trials.

A Pr ogr ession-fr ee sur vival (% )

At the data cut-off, median PFS and 4-month PFS in EA was 2.7 months (95% confidence interval [CI], 2.3e3.2 months) and 15% versus 2.3 months (95% CI, 2.1e2.5 months) and 21% in CA (p Z 0.628). Overall, 50 patients died, 27 (96%) in EA and 23 (96%) in CA. The median OS was 6.2 months (95% CI, 4.4e7.9 months) and 4.7 months (95% CI, 2.3e7 months) in EA and CA, respectively. Two-year OS was 7% in EA and 4% in CA (p Z NS) (Fig. 2). Among variables entered in the multivariate model for survival, only LDH level (<1 versus >1 Upper Limit of Normal [ULN], p Z 0.0004; hazard ratio 3.34; 95% CI 1.71e0.6.52) remains significant. No objective radiological response was observed in EA (0 in 25). One patient treated in the CA who received panitumumab obtained a partial response (1 in 19, 5%). Three disease stabilizations were observed in EA (9%) and six (32%) in the CA (p Z 0.12). In CA, eight patients were treated up-front with conventional therapies (five patients) or phase I studies (three patients). Finally, two patients treated with BSC at progressive disease were treated with other therapies (capecitabine plus bevacizumab [1p] and panitumumab plus irinotecan [1p]). Therefore, a total of ten patients (42%) were treated with different schedules in CA. Five patients received additional therapies (FOLFIRI [1p], capecitabine plus bevacizumab [3p] and irinotecan plus capecitabine [1p]) in EA (18%).

25, 84.0%) was 3.5 months (95% CI, 2.4e4.6 months). Median PFS in patients (4 in 25, 16.0%) who did not show increment of ATMLR was 3 months (95% CI, 2.6e3.3 months). Three patients were not evaluable for ATMLR (because death before day 40; Fig. 4). Patients with an increase of ATMLR showed an improvement in OS of 7.3 months (95% CI, 5.2e9.4 months) versus 3.8 months (95% CI, 0.6e6.9 months) (p Z 0.026; Fig. 5).

100

BSC ADC + BSC

75 50 25 0

0

100

200

300

Days after randomization

B 100

Overall Survival (%)

3.3. Efficacy

BSC ADC + BSC

75 50 25 0

0

200

400

600

800

1000

Days after randomization

3.4. Immunologic response Patients treated with EA increased tumour-specific Tcell response detected by ATMLR (Fig. 3). Median PFS in patients who showed an increase of ATMLR (21 in

Fig. 2. Survival following treatment with ADC þ BSC or BSC. (A) KaplaneMeier curves for progression-free survival and (B) KaplaneMeier curves for overall survival in the intention-to-treat population. ADC: autologous tumour lysate dendritic cell vaccine; BSC: best supportive care.

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p=0,0067

15000

p<0,0001

CPM

10000

5000

12 0 ST PO

PO

ST

40

PR E

0

Fig. 3. Autologous tumour mixed leucocyte reaction at day 0 (PRE), 40 (POST 40) and 120 (POST 120). The mean of counts per minute (cpm) before treatment was 2479 cpm (limits 388e24,959 cpm). In those patients treated with ADC þ BSC, the frequency of tumour-specific T cells increased: day 40 (POST 40) mean 8138 cpm (limits 467e40,245 cpm, p < 0.0001); day 120 (POST 120) mean 8676 cpm (limits 554e59,476 cpm, p Z 0.0067).

Furthermore, ADC treatment polarised the immune response to a type 1 cellular response (p < 0.0001; Fig. 6). 4. Discussion

Patient

Our trial is the first randomised clinical trial comparing ADC þ BSC versus BSC in heavily pre-treated mCRC

patients. Our study was designed to capture large differences between both groups. This methodology would allow, if results were positive, to be more confident to progress in a large phase III trial. Unfortunately, we were unable to demonstrate substantial benefit in PFS or OS compared with BSC. To determine the effect of ADC in the immune response against tumour, we checked the presence of the tumour-specific T cells by cell proliferation, before and after treatment, using the ATMLR. In our opinion, this is an appropriate methodology to demonstrate the specificity of the immune response induced by the vaccination with autologous DCs. It reflects in vitro the efficacy in vivo because the same DCs were used to measure the proliferation induced by these cells, on autologous PBMCs. The higher proliferation seen in post-vaccination samples suggests an increment of specific T lymphocytes due to vaccination. Furthermore, the treatment induces a polarisation of the immune response towards a Th1-cellular response. In fact, this type of immune response is considered to be the most effective in the elimination of tumour cells. Despite the in vitro data, we observed a discrepancy with clinical efficacy. In mismatch repair, proficient tumours comprise 95% of mCRC patients [9], and at least, two mechanisms can hamper efficacy of ADC therapy: (1) immune tolerance among tumour-specific T cells by programmed death 1 (PD-1) pathway [10]; (2) release of chemokines and cytokines by proliferating fibroblast and activated macrophages that deplete T cells in the tumour [11,12]. The major challenge is to understand

2,007 3,004 2,018 4,002 2,003 2,012 2,022 4,006 2,014 2,016 4,005 2,021 2,011 3,006 1,011 2,019 3,001 4,003 1,017 1,007 1,005 1,013 1,003 2,008 1,016 2,001 1,010 1,001

Time to progression ATMLR increase Non-ATMLR increase Non-Evaluated

0

200

400

600

800

1000

Days after randomization Fig. 4. Time to progression and overall survival in ADC-treated patients according to ATMLR response. ADC: autologous tumour lysate dendritic cell vaccine; ATMLR: autologous tumour mixed leucocyte reaction; ATMLR increase: patients who showed an increase of the proliferative response against the tumour; non-ATMLR increase: patients who did not show an increase of the proliferative response against the tumour; non-evaluated: patients who were not tested due to exitus before the obtention of the post-treatment samples (POST 40/POST 120).

M. Caballero-Ban˜os et al. / European Journal of Cancer 64 (2016) 167e174 OS Overall Survival (%)

100

ATMLR increase Non-ATMLR increase

80

p<0,02

60 40 20 0

0

200

400

600

800

1000

Days after randomization

Fig. 5. OS in patients treated with ADC according to ATMLR response. KaplaneMeier curves for OS. ADC: autologous tumour lysate dendritic cell vaccine; ATMLR increase: patients who showed an increase of the proliferative response against the tumour; non-ATMLR increase: patients who did not showed an increase of the proliferative response against the tumour; OS: overall survival.

% cells Th1 (Ifn-g +)

20 p<0,0001 15 10 5

12 0

induction of tumour-specific T lymphocytes is not sufficient and would require combination with other treatments. We are aware that our study has some weaknesses. First, the limited sample size and the early accrual termination due to futility and, second, the selected primary end-point (PFS instead of OS) hampered our results [13,14]. Despite this, the random nature with an adequate CA (BSC) and the optimal stratification criteria (LDH was the unique variable with value in the multivariate analysis for survival) supports the strength of our conclusions. Our results do not support the use of ADC alone, in a phase III trial. Statement of translational relevance Small non-randomised prospective phase II trials in metastatic colorectal cancer (mCRC) patients treated with peptide matured DCs or autologous lysate matured dendritic cells (DCs), showed promising results, with prolonged progression free survival (PFS) and overall survival (OS). Due to the lack of control group in these studies, it is difficult to establish if the benefit is due to the therapy or the intrinsic behaviour of the disease. We present the first randomised clinical trial in mCRC patients, that progressed after all available therapies, that compared two strategies: Autologous dendritic cells (ADC) plus best supportive care (BSC) (Experimental Arm; EA) vs BSC alone (Control Arm; CA). Despite that in the EA the frequency of tumour specific T cells increased, we were unable to demonstrate a substantial benefit in PFS or OS compared with CA.

PO

ST

40 ST PO

PR E

0

173

Funding Fig. 6. Th1 polarisation of the immune response at days 0, 40 and 120. Treatment showed the capacity to polarise the immune response to a type 1 e cellular response (Th1þ cells). The mean percentages of Th1þ cells (IFN-gþ) before the treatment (PRE) was 6.7%, and after the treatment (post 40), the value was increased to 12.4% (p < 0.0001). No differences were shown at day 120 (POST 120).

This study was funded by the projects TRA-082 (EnsClinicos 09) and EC11-156 (EnsClinicos 11) of the Spanish Ministry of Health. Conflict of interest statement

that the biology of the tumour cells and the immune competence of the host, will suffer changes over time therapies, such as chemotherapy and especially biological agents. How tumour cells and microenvironment in heavily pre-treated mCRC patients can influence the response to new immune therapies (anti-PD-1/PD-L1, ADC or anti-stromal therapies) would be critical in new clinical trial design on mCRC patients. In future studies, we recommend to obtain biopsies from primary tumour by colonoscopy to provide biological information for patient selection or stratification. It is important to highlight that ADC was well tolerated and although the trial did not reach significant differences in PFS and OS, we observed that administration of tumour-loaded mature DCs induce tumourspecific T lymphocytes. These results suggest that the

None declared.

Acknowledgements We thank Lucia Millan and Jordi Mila for technical assistance. We thank Dr. Andres Salazar for the generous gift of Hiltonol. We thank to Dr. Estela Pineda, Dr. Laura Visa, Dr. Cristina Nadal and Dr. Nuria Pardo (all from HCB) for including patients in the trial. We thank Dr. Joaquim Perez de Oleguer (Hospital Plato´n), Dr. Alejandro Martinez-Fernandez (Hospital del Mar), Dr. Montserrat Gay (Hospital de Vic), Dr. Esther Casado (Hospital de Manresa), Dr. David Paez and Dr. Maria Toben˜a (Hospital Sant Pau Barcelona), Dr. Bernardo Queralt (Hospital ICO

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Girona), Dr. Susana Martinez Peralta (Hospital de Mataro´), Dr. Jordi Alfaro (Hospital Consorci Sanitari de Terrassa), and Dr. Joaquim Bosch (Hospital de Palamo´s) for sending patients into the trial.

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