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Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab

Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate... Background: Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome. Methods: Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit. Results: Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) + + + was observed, which in the case of BDCA1/CD1c cDC1 and MDC8 /6-sulfoLacNAc inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related + − adverse events. High pre-treatment levels of CD14 HLA-DR monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction + + with our previously identified lymphoid biomarker of high pretreatment CD4 CTLA4 T cell frequencies. Conclusions: Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment. Keywords: Ipilimumab, Prostate GVAX, Biomarker, Patient selection, Survival prediction * Correspondence: td.degruijl@vumc.nl Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article © 2014 Santegoets et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 2 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Background To study whether PBDC and MDSC subsets are af- Prostate cancer is the third leading cause of cancer- fected by prostate GVAX/ipilimumab therapy, extensive related death in men worldwide [1]. Curative treatment myeloid subset monitoring was performed. Myeloid sub- options are only available for localized disease. In pa- sets were analyzed prospectively and followed during tients that develop metastatic castration-resistant prostate treatment, after which cut-off points for response to cancer (mCRPC) the median survival is 16-21 months treatment and/or survival were determined in retrospect. [2-4]. Recent advances have led to novel immunotherapy Our data demonstrate that PBDC are activated by pros- options with proven clinical efficacy in patients with tate GVAX/ipilimumab therapy and that a specific mye- metastatic CRPC (mCRPC), such as PROSTVAC and loid lineage marker profile (i.e. high post-treatment cDC Sipuleucel-T [1,3,4]. In addition, the CTLA-4 blocking activation and low pre-treatment frequencies of mono- antibody ipilimumab has shown clinical activity in a cytic MDSC) proved predictive for clinical benefit after variety of cancer types, including prostate cancer [5-7]. Prostate GVAX and/or ipilimumab immunotherapy. CTLA-4 blockade enhanced antitumor efficacy when combined with other immunomodulating agents, in- Results cluding Granulocyte Macrophage-Colony Stimulating Clinical results Factor (GM-CSF) and GM-CSF-secreting cancer vac- mCRPC patients (n = 28) received 13 bi-weekly injec- cines, e.g. GVAX immunotherapy [8-10]. In line with tions of the prostate GVAX vaccine and 6 four-weekly this, we recently reported the combined immunotherapy infusions of ipilimumab. As described previously [11], of a GM-CSF-engineered allogeneic tumor cell-based vac- five patients experienced serum-PSA-based PR with PSA cine (Prostate GVAX) and ipilimumab in patients with declines of more than 50% and 12 demonstrated disease mCRPC to be safe and clinically active [11]; clinical results stabilization (SD); PR/SD was significantly correlated included partial responses (PR) and a relatively long sur- with prolonged overall survival (med. survival of 41 ver- vival as compared to survival rates observed in control sus 21 months; p = 0.0034). Nine patients, all of which arms of recent Phase III trials [2-4]. received 3 or 5 mg/kg ipilimumab and five of which ex- Recent clinical findings have indicated that the induc- perienced a PR, developed immune-related adverse tion of an effective antitumor immune response relies events (IRAE) [11]. Interestingly, although IRAE were on the proper differentiation, maturation and functional- more frequent in patients that benefited from treatment ity of myeloid antigen-presenting cells (APC), and that (i.e. PR and SD; p = 0.0015), the development of IRAE the accumulation of myeloid-derived suppressor cells was not associated with survival ([11]; Additional file 1: (MDSC) and functionally impaired (immature) dendritic Figure S1). cells (DC) in tumor, blood or lymph nodes of cancer pa- tients is a poor prognostic factor for survival [12-16]. PBDC and monocyte frequency and activation in relation Therefore, cancer immunotherapeutic approaches aiming to survival and IRAE at the normalization of myeloid differentiation are of inter- To assess the effects of prostate GVAX/ipilimumab treat- est for clinical application in support of immunotherapy. ment on circulating myeloid DC subsets, frequency and We have reported the effects of prostate GVAX/ activation status of circulating conventional DC (cDC) ipilimumab immunotherapy on circulating T cell sub- subsets cDC1, cDC2, cDC3 and plasmacytoid DC (pDC) sets [17]. However, the net effect of combined prostate were determined before, during and after treatment. cDC1 hi − − + GVAX/ipilimumab immunotherapy on DC and MDSC were identified as CD11c CD19 CD14 BDCA-1/CD1c ; + − + hi subsets in peripheral blood is currently unknown. It cDC2 as CD11c CD14 BDCA-3 ; cDC3 as CD11c C- lo + − − hi has been described that under proper maturational D14 MDC8 and pDC as CD11c CD14 CD123 BDCA- conditions, peripheral blood DC (PBDC) have the po- 2 (see also Additional file 2: Figure S2 for gating tential to develop into functional DC with the capacity strategies). Similar to previous observations in cancer to induce antitumor T-cell responses [18,19]. In par- patients by us and by others [12,15,27,28], frequencies ticular GM-CSF has been implicated in the recruit- and activation status of circulating DC and monocytes ment and activation of DC in vivo [20,21]. Importantly were generally lower in CRPC patients as compared however, GM-CSF has also been linked to the systemic in- with healthy individuals (see Additional file 3: Figure S3A duction/expansion of MDSC in mice and man [22,23]. and 3B). On-treatment activation (shown in Figure 1A by Furthermore, CTLA-4 antibody blockade has been shown CD40 expression levels) was observed for all DC subsets to reduce MDSC suppressive potency in vitro,and in vivo (as previously reported by us for cDC1 [11]). Interestingly, in a murine ovarian carcinoma model, and this effect was this activation was paralleled by decreases in cDC1, cDC2, achieved both indirectly through inhibiting T cell-MDSC and pDC frequencies (Figure 1B). These decreases were interaction [24,25] and directly through binding to CTLA- observed as early as four weeks after start of treatment 4 expressed on MDSC [26]. and were maintained during treatment (Figure 1B). Of Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 3 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 25 25 1.25 AB ** ** 1.00 20 20 ** ** cDC3 0.75 * * 0.50 15 15 * * cDC1 0.5 0.4 ** 10 10 ** ** ** ** 0.3 ** pDC 5 5 cDC1 cDC1 0.2 ** * ** * ** * BDCA1/CD1c BDCA1/CD1c 0.1 0.10 cDC2 50 50 0.05 ** * * * * 0.00 40 40 w0 w4 w8 w12 w16 w20 w24 fu 30 30 0.025 *** 20 20 0.020 cDC3 10 10 cDC2 cDC2 0.015 BDCA3/CD141 BDCA3/CD141 0 0 cDC1 0.010 20 20 * * 0.005 pDC 15 15 0.0015 * * 0.0010 cDC2 10 10 0.0005 0.0000 w0 w4 w8 w16 fu 5 5 cDC3 cDC3 dim dim CD14 CD14 /MDC8 /MDC8 cDC1 and cDC3 activation cDC1 and cDC3 activation 0 0 100 100 Activation Activation of both of both 20 20 Activation Activation of of one one or or none none * * 75 75 15 15 N=15; med N=15; med. . surv surv.. 52 52 mths mths ** ** 10 10 50 50 5 5 P<0.0001 P<0.0001 pDC pDC 25 25 BDCA2/CD123 BDCA2/CD123 N=13; med N=13; med. . surv surv.. w0 w4 w8 w12 w16 w20 w24 fu 16mths 16mths 0 0 0 0 20 20 40 40 60 60 80 80 100 100 months months Figure 1 Prostate GVAX/ipilimumab therapy-induced activation of peripheral blood (PB) DC subsets is associated with prolonged survival. PBDC activation and frequency was determined before (week 0/visit 1 (w0v1)), during (w4v3, w8v5, w12v7, w16v9, w20v11, w24v13 and after (follow-up (fu)) prostate GVAX/ipilimumab therapy by flow cytometry. A) Activation state over treatment –by Median Fluorescence Index hi − lo + + − + + lo + − − hi + (MFI)ofCD40ofCD11c CD19 CD14 BDCA1 cDC1, CD11c CD14 BDCA-3 cDC2, CD11c CD14 MDC8 cDC3 and CD11c CD14 CD123 BDCA-2 pDC. Grey bars denote the mean ± SEM range at baseline. B) DC subset frequencies (as percentages of PBMC) and C) absolute numbers per ml blood, over treatment, cDC1: solid black squares, cDC2: open black squares, cDC3: solid grey squares, pDC: open grey squares. Means ± SEM of 28 patients are shown. D) Kaplan Meier curve for on-treatment increases in cDC1 and cDC3 activation. Number of patients and corresponding median survival for each group is given. Differences between pre- and on- or post-treatment were analyzed with the repeated measures ANOVA with a post-hoc Dunnett’s multiple comparisons test. Differences were considered significant when p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01) within the respective squares. Statistical significance of the survival distribution was analyzed by log-rank testing. note, decreases in absolute cDC1, CDC2, and pDC num- reached significance (Figure 1C). These differences be- bers per volume blood were much less pronounced and tween DC frequencies and absolute numbers may in large on-treatment increased absolute cDC3 numbers even part be explained by a sustained increase in absolute CD40 MedFI absolute count (in 1x10e6 per ml of blood) % cells (as % PBMC) Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 4 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 lymphocyte numbers over the course of treatment (see activation were significantly higher in patients that eventu- Additional file 4: Figure S4). Increased PBDC activation ally developed IRAE, suggesting that these treatment- was generally maintained during treatment (see Figure 1A) induced increases might serve as an indicator for risk of and increases of >70% of CD40 med. FI on the cDC1 IRAE. and cDC3 subsets (see for representative histograms Additional file 2: Figure S2) were associated with signifi- MDSC frequencies in relation to survival cantly prolonged overall survival (OS; median survival To assess the effects of prostate GVAX/ipilimumab 38.5 vs. 15.5 months, p = 0.0004 and median survival 40 treatment on circulating MDSC, the frequency of mono- ־ + ־/lo vs. 19 months, p = 0.0031, respectively (Table 1). Survival cytoid MDSC (mMDSC; Lin CD14 HLA-DR ,see benefit was even more pronounced for patients who dis- Figure 4A) was determined. Significantly higher levels played treatment-induced activation of both cDC1 and of circulating mMDSC were detected in CRPC patients cDC3 subsets (median survival 52 vs. 16 months, p < compared with age- and sex-matched healthy individ- 0.0001; Figure 1C). No relationship with survival was uals. Significant post-treatment increases in mMDSC found for either pDC or cDC2. frequencies were observed (Figure 4B), yet these in- When patients were divided by treatment response, creases were only modest and did not correlate with re- decreases in the frequency of monocytes were found to sponse to treatment (not shown) or survival (Table 1). be selectively associated with PR (Figure 2A), which, like In contrast, patients who displayed high pre-treatment observed for PBDC subsets, was paralleled by increased levels of mMDSC had a significantly shorter OS than pa- activation (Figure 2B). Of note, although these were tients who did not (Figure 4C, median survival 20 vs. 52 clear trends, they did not reach statistical significance. months, HR = 4.26, 95% CI = 1.37 - 13.25, p = 0.0046). Similarly to the cDC1/BDCA1 and cDC3/SLAN-DC subsets, treatment-induced increases of CD40 med. FI A myeloid marker profile of high DC activation and low on CD14 monocytes was associated with prolonged OS frequencies of MDSC is predictive for survival (median survival 57 vs. 21 months, p = 0.0749; Figure 2C To assess whether clinical prognosis impacted the puta- and Table 1). tive predictive value for treatment outcome of any of the To determine whether changes in DC and monocyte identified myeloid parameters, the median Halabi Pre- activation status could possibly serve as early marker for dicted Survival (HPS) was determined for the patient the ipilimumab-associated development of IRAE, we groups above and below the designated cut-offs [29]. No compared the rise in DC or monocyte activation status at significant differences were observed, indicating that bet- week four of treatment (i.e. after two GVAX administra- ter prognosis before treatment was not the determining tions and only one ipilimumab infusion) between patients factor for any of these parameters (Table 1). with or without IRAE. As shown in Figure 3, treatment- We previously identified a lymphoid biomarker profile induced increases in cDC1, cDC3, and monocyte with predictive value for OS benefit, which was dominated Table 1 Characteristics and survival distribution of treatment-induced changes in PBDC activation and in mMDSC frequencies † § ׀׀ Immune parameter Mean treatment- Cut-off Median survival between p-value Hazard ratio Mean Halabi p-value induced increase groups‡ (# of patients (95% CI HR) Predicted * ¶ (range) in each group) Survival CD40 on cDC1 313% (-67 to +1194) 70% 38.5 vs. 15.5(22 vs. 6) 0.0004 0.052 (0.010-0.267) 18.7 vs. 16.6 0.614 CD40 on cDC2 170% (-52 to +612) 150% 36.5 vs. 25.5 (10 vs. 18) 0.397 0.688 (0.299-1.582) 16.8 vs. 19.1 0.338 CD40 on cDC3 160% (-16 to +765) 70% 40.0 vs. 19.0 (19 vs. 9) 0.0031 0.179 (0.057-0.560) 18.6 vs. 17.7 0.684 CD40 on pDC 290% (-19 to +1814) 450% 26.0 vs. 31.5 (4 vs. 26) 0.739 1.226 (0.373-4.032) 14.2 vs. 19.0 0.168 CD40 on monocytes 243% (-28 to +1001) 160% 57.0 vs. 21.0 (9 vs. 19) 0.0749 0.469 (0.204-1.079) 20.1 vs. 17.4 0.192 + − Lin-CD14 HLA-DR mMDSC 87% (-71 to +543) 60% 35.0 vs. 36.0 (8 vs. 11) 0.270 0.559 (0.198-1.572) 16.7 vs. 21.4 0.139 Mean and range of treatment-induced increases are given in percentages relative to pre-treatment values. Cut-off points for survival prediction were determined using the Cox regression model and the relative increments are given as percentage of pre-treatment values. Median Survival for both groups was calculated using the Kaplan-Meier method and given in months. Statistical significance of the survival distribution was analyzed by log-rank testing and considered significant when p < 0.05 (in bold). Mean Halabi Predicted Survival (HPS) ± standard error (in months) was determined for patients with biomarker increments above or under designated cut-offs. NB: Halabi scores were determined based on Halabi et al. J. Clin. Oncol. 2003 [29], but Halabi scores based on Halabi et al. J. Clin. Oncol. 2014 [53] similarly showed prognosis before treatment (based on HPS) not to be the determining factor for any of these myeloid markers in terms of prediction of median survival upon treatment (not shown). ׀׀ Differences in HPS between groups were determined by Mann-Whitney U test and were considered significant when p < 0.05 (in bold). Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 5 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 PR SD PD w0 w4 w8 w12 w16 w20 w24 fu1 w0 w4 w8 w12 w16 w20 w24 fu1 v1 v3 v5 v7 v9 v11 v13 v1 v3 v5 v7 v9 v11 v13 160% increase < 160% increase N=9; med.surv. 57 mths N=17; med.surv. 21 p = 0.0749 mths Cut-off 160% 0 20 40 60 80 100 months Figure 2 Increased monocyte activation following Prostate GVAX/ipilimumab therapy is associated with prolonged survival. Frequencies and activation status of circulating CD14 monocytes were determined before (w0), during (w4, w8, w12, w16, w20, w24) and after (fu) Prostate GVAX/ ipilimumab treatment by flowcytometric analysis. A) Mean percentage (of PBMC) ± SEM and B) mean activation ± SEM of CD14+ monocytes is shown before, during and after Prostate GVAX/ipilimumab treatment for 28 patients, divided by clinical PSA response: partial PSA response (PR; black squares), disease stabilization or (SD; white squares) or disease progression (PD; grey squares). Activation is given as med. Fluorescence Index (med. FI) and calculated by dividing the med. fluorescence (med. fl) of CD40 antibody by the med. fl of the isotype control antibody. C) Kaplan Meier curve for treatment-induced increases in activation of monocytes. Number of patients and corresponding median survival for each group are given. Differences between pre- and on- or post-treatment were analyzed with the repeated measures ANOVA with a post-hoc Dunnett’s multiple comparisons test. Differences were considered significant when p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01). + + by high CD4 CTLA4 T cell frequencies prior to therapy Discussion [17]. When combining all predictive lymphoid [17] and PBDC and MDSC profiling of patients with CRPC re- myeloid markers in an unsupervised cluster analysis, we ceiving combined Prostate GVAX/ipilimumab treatment found a particularly strong association with prolonged revealed that PBDC and MDSC subsets were affected by survival for patients displaying a combination of high prostate GVAX/ipilimumab therapy, with on-treatment levels of on-treatment cDC1/cDC3/monocyte activation, increases in DC activation and mMDSC frequencies. Of low pretreatment mMDSC rates and high pretreatment note, a myeloid profile of low pretreatment frequencies + + frequencies of CD4 CTLA4 T cells (designated clustered of mMDSC and high treatment-induced cDC1, cDC3, group 2, median OS 46 months, see Figure 5A and 5B). and monocyte activation levels demonstrated predictive Nevertheless, patients with relatively high pretreatment value for OS on treatment. As this was only a small ex- + + frequencies of CD4 CTLA4 T cells showed significant ploratory study of 28 patients, no multivariate analyses survival benefit whether these were accompanied by con- were performed and the candidate biomarkers should be certed high levels of on-treatment cDC1/cDC3/monocyte validated prospectively in larger randomized trials. activation and low pretreatment mMDSC rates or not It has been reported that frequencies and activation (designated clustered groups 2 + 1, median OS 40 months; status of circulating DC are significantly lower in cancer see Figure 5A and 5C). patients compared with healthy individuals [12,15,27,28]. Percent survival % CD14 positive cells median Fl. Index CD40 Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 6 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 AB C cDC1 cDC2 cDC3 15 5 p=0.0271 p=0.0274 0 0 0 no IRAE no IRAE no IRAE IRAE IRAE IRAE DE pDC monocytes p=0.0117 no IRAE no IRAE IRAE IRAE Figure 3 PBDC activation in relation to immune-related adverse events (IRAE). Fold increase in activation of A) cDC1, B) cDC2, C) cDC3, D) pDC and E) monocytes was determined at week 4 after start of prostate GVAX/ipilimumab treatment by dividing the median Fluorescence Index (med. FI) of CD40 at week four (i.e. 2 vaccinations and 1 ipilimumab infusion) by the med. FI of CD40 at start of treatment and displayed for patients that experienced IRAE or no IRAE during therapy. Differences in fold increase in activation between groups of patients were analyzed with the two-sample Mann-Whitney U test. Differences were considered significant when p < 0.05, as indicated with the given p-value. In our study, similar results were observed, with signifi- vaccination (Van Mens et al, manuscript in preparation). cantly lower cDC1 frequencies and an inferior activation However, absolute PBDC subset numbers showed more state of cDC1, cDC2 and monocytes in mCRPC patients moderate decreases whereas they revealed a significant (see Additional file 3: Figure S3A and B). Interestingly, on-treatment increase in cDC3. The latter fits well with prostate GVAX/ipilimumab treatment resulted in a fur- the inflammatory nature of cDC3 or SLAN-DC, being ther reduction of circulating cDC and pDC subsets, and mobilized by cytokines like GM-CSF [33]. this reduction was paralleled by increases in their activa- MDSC represent a heterogeneous population of imma- tion status. The observed activation may be explained by ture myeloid cells, and have been recognized to play an the production of GM-CSF by the vaccine, as GM-CSF important role in suppression of (anti-tumor) immune has been described to activate DC in vivo [20,21]. Alter- responses. Elevated levels of either monocytoid or gran- natively, or additionally, ipilimumab may have altered ulocytic MDSC have been observed in a variety of hu- DC activation state through blocking CTLA-4/B7 inter- man cancers [34-38]. Moreover, their presence in actions at the Treg/DC interface or by direct binding of peripheral blood or at the tumor site has been linked CTLA-4 on DC [30-32]. Nevertheless, a lack of associ- with poor prognosis and may thus serve as a prognostic ation between ipilimumab dose and DC activation seems or predictive marker for clinical outcome [36,39-41]. In to support an overriding role for GVAX-derived GM- keeping with previous findings, mCRPC patients displayed CSF in this respect. The enhanced activation and simul- significantly higher levels of mMDSC compared with age- taneous reduction of cDC and pDC subset frequencies and sex-matched healthy controls (see Additional file 3: in blood is suggestive of their recruitment to effector Figure S3C). In fact, significantly prolonged OS was ob- sites (e.g. tumor and vaccination sites). This hypothesis served for patients displaying low pre-treatment levels of is further supported by our own observation of en- mMDSC, i.e. levels that were similar to those found in hanced recruitment of antigen-presenting cells to the healthy volunteers, suggesting that indeed mMDSC levels Prostate GVAX vaccination sites following repeated may serve as a predictive marker for clinical outcome. fold increase CD40 med FI fold increase CD40 med FI Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 7 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Our data demonstrated modest increases of mMDSC frequencies following multiple Prostate GVAX/ipilimu- - + - Monocytic MDSC (Lin CD14 HLA-DR ) mab doses in a subgroup of patients. As described, in- creases in the frequency of circulating MDSC can be related to progression [39,42-44]. No such relation was observed in our study, as no difference in mMDSC ex- R3 pansion was observed between PR, SD and PD patients (not shown). The observed mMDSC expansion might have been induced by the Prostate GVAX vaccine, since R2 GM-CSF-based vaccines have been shown to expand MDSC in mice and man [22,23]. Perhaps surprisingly, the HLA-DR CD14 post-vaccination increases of circulating mMDSC frequen- cies did not impact survival in our study, suggesting that 2.0 prostate GVAX/ipilimumab therapy may reduce the sup- pressive function of mMDSC. Indeed, reduction of MDSC 1.5 suppressive function has been described for CTLA-4 blockade therapy in an in vitro and in vivo murine ovarian 1.0 carcinoma model [24-26]. Moreover, as mMDSC have been shown to further develop into more mature macro- phage and DC-like cells [42], GM-CSF-driven differenti- 0.5 ation may have interfered with their suppressive ability. A major problem in CTLA-4 blockade therapy is the 0.0 development of potentially life-threatening IRAE like w0v1 w8v5 w16v9 w24v13 fu1 colitis, hepatitis, alveolitis and hypophysitis [45,46]. To date, attempts to reduce the development of these IRAE high pretreatment have been unsuccessful [47]. Therefore, it is important low pretreatment to identify biomarkers for patient selection or develop methods that enable the early detection of IRAE. Within N=13; med. surv. 52 mths the myeloid compartment, no pre-treatment marker with putative clinical value for IRAE risk prediction could be identified. Yet, our data showed that the mag- nitude of early (i.e. at week 4, after only one single ipili- N=11; med. mumab administration) CD40 up-regulation on cDC1, surv. 20 mths p = 0.0046 cDC3 and monocytes correlated with on-treatment Cut-off 0.3% IRAE development. Unfortunately, the clinical applic- 0 20 40 60 80 100 ability of this on-treatment change as a marker for IRAE months risk is questionable, as there is considerable overlap for Figure 4 High pretreatment frequency of mMDSC is associated CD40 activation levels on the indicated subsets between with reduced OS. mMDSC frequencies were determined before the IRAE positive and negative group (Figure 3). (week 0/visit 1 (w0v1)), during (w8v5, w16v9 and w24v13) and after Our profiling data are consistent with clinical benefit (follow-up (fu)) prostate GVAX/ipilimumab therapy by flow cytometry. − + −/lo A) A representative analysis of Lin CD14 HLA-DR mMDSC. B) and survival advantage for patients with high DC activa- Percentage of mMDSC over follow-up. C) Kaplan meier curve for tion and low levels of MDSC. These data echo our find- pre-treatment frequencies of mMDSC. Number of patients and ing of a predictive T cell profile of activated T cells and corresponding median survival for each group are given. Differences low pre-treatment Treg frequencies in the same group betweenpre-and on-orpost-treatmentwere analyzedwiththe of patients [17], and are in line with an ever growing repeated measures ANOVA with a post-hoc Dunnett’smultiple comparisons test. Differences were considered significant when number of studies stressing the importance of a pro- p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01). Statistical inflammatory, non-suppressive immune status for optimal significance of the survival distribution was analyzed by log-rank efficacy of immunotherapy in cancer patients. Interest- testing and indicated with the given p-value. ingly, our unsupervised clustering analysis of combined myeloid and lymphoid biomarkers indicates two, partially This is in keeping with a recent report indicating a similar overlapping, major populations with survival benefit: predictive value of pre-treatment mMDSC frequencies for 1) patients with combined low pre-treatment frequen- outcome of tumor peptide vaccination therapy in patients cies of mMDSC and high on-treatment cDC activation with renal cell cancer [39]. levels, and 2) patients with high pre-treatment levels of - + - % Lin CD14 HLA-DR Percent survival Lineage (CD3/16/19/56) Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 8 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 AB Group 2 Group 3 -2.0 0.0 +2.0 N=12; med. surv. 46 mths patient codes Group 1 Group 2 Group 3 25 p = 0.0042 N=6; med. surv. mMDSC pre 18.5 mths CD4 Teff/Tem increase CD4/CD25int/FoxP3 increase 0 20 40 60 80 100 Treg increase Time CD8 Teff/Tem/Tcm increase Group 1 + 2 cDC1 activation Group 3 cDC3 activation monocyte activation Tregs pre N=19; med. surv. 40 mths CD4 Teff/Tem pre CD4CTLA4 pre CD4PD-1 pre p = 0.0028 N=6; med. surv. 18.5 mths 0 20 40 60 80 100 Time Figure 5 High on-treatment cDC1/cDC3/monocyte activation and low pretreatment mMDSC frequencies predict clinical benefit after therapy. A) Unsupervised cluster analysis of the expression of the indicated treatment-induced and pre-treatment myeloid and lymphoid markers. To identify clusters of correlated markers, hierarchical cluster analysis using TIGR software was performed, and average linkage analysis was done by Pearson correlation analysis. Values of the treatment-induced and pre-treatment parameters are given relative to the cut-off value (determined by Cox regression model as described in materials and methods); below cut-off in green and above cut-off in red. Kaplan Meier curves for B) group 2 (with high on-treatment cDC1/cDC3/monocyte activation and low pretreatment mMDSC frequencies: highlighted by white boxes in the heat plot) versus group 3 (with generally low on-treatment cDC1/cDC3/monocyte activation and high mMDSC frequencies) and + + C) group 1 + 2 (with high pre-treatment frequencies of CD4 CTLA4 T cells: lower white box in heat plot) versus group 3. Statistical significance of the survival distribution was analyzed by log-rank testing and indicated with the given p-value. Number of patients and corresponding median survival for each group are given. + + CD4 CTLA4 T cells, see Figure 5A, white-lined boxes. and PC-3 (CG1940), which have been transduced with Whereas the latter biomarker profile may signal particular an adeno-associated viral vector to secrete GM-CSF. susceptibility to check point inhibition [17], the first may These cell lines were propagated, frozen, and irradiated be representative of a favorable immune state and as such to arrest further cell division [6,48]. The product was hold predictive value for immunotherapy in general. These stored and shipped in gaseous nitrogen phase, and admin- profiles warrant further assessment and validation of their istered within 60 minutes after thawing. All manufactur- utility for patient selection in other, preferably randomized, ing was conducted according to good manufacturing clinical trials of GM-CSF-based therapy and/or CTLA-4 practice. Ipilimumab (formerly MDX-010), a fully human blockade or other forms of cancer immunotherapy. IgG1κ monoclonal antibody directed against CTLA-4, was provided by Medarex/Bristol-Myers Squibb (Plainsboro, Conclusions NJ, USA). This study provides evidence that circulating myeloid sub- sets are affected by combined Prostate GVAX and ipilimu- mab therapy and that a myeloid lineage profile of low Study population and sampling of peripheral blood pretreatment mMDSC frequencies and high treatment- As described [11], 28 chemonaive patients with asymp- induced cDC activation levels may contribute to the identi- tomatic mCRPC received 13 bi-weekly vaccinations of the fication of patients with possible clinical benefit of Prostate Prostate GVAX vaccine and 6 four-weekly infusions of ipi- GVAX/ipilimumab treatment. limumab from the time of prime vaccination. In the first 12 patients, ipilimumab was administered at escalating Methods doses of 0.3, 1, 3 and 5 mg/kg (3 patients each). In the ex- Prostate GVAX and ipilimumab pansion phase, 16 additional patients were included at The Prostate GVAX vaccine is a cellular vaccine consist- 3 mg/kg ipilimumab. This study is registered with the ing of two prostate cancer cell lines, LNCaP (CG8711) Central Committee on Research involving Human Subjects Percent survival Percent survival Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 9 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 hi hi in the Netherlands, number P03.1786C, and ClinicalTrials. were defined as CD11c CD14 (highly positive; see gov, number NCT01510288. also Additional file 2: Figure S2). Monocytoid MDSC − + neg/lo Responses to treatment were defined as described [49]. (mMDSC) were defined as Lin CD14 HLA-DR In brief, PSA partial response (PR) was defined as >50% [28] (see also Figure 4A). For all the above mentioned PSA decline from baseline, which was confirmed by a populations a live gate was used based on FSC-SSC second PSA test 3 or more weeks later. PSA progressive properties of the lymphocyte and monocyte populations. disease (PD) was defined as >25% PSA increase and an Activation status of above mentioned cDC, pDC and absolute increase of 2 ng/ml or more from baseline, monocyte subsets was determined by calculating the me- whereas stable disease (SD) was defined as no PR and dian Fluorescence Index (med. FI) of CD40 expression by no PD on treatment. dividing the med. fluorescence (Med. fl) of the CD40 anti- For immune monitoring, blood samples were taken body by the med. fl of the isotype-control antibody. from thepatientsbeforestart of therapyand everyfour weeks thereafter until four weeks after the last treat- Statistical analyses ment (i.e. follow-up (fu)). Peripheral blood mononuclear Differences between immune parameters before treatment cells (PBMC) were isolated by density centrifugation (w0v1; w = week; v = visit; i.e. baseline levels) and during (NycomedAS, Oslo, Norway). PBMC were either directly and/or after treatment (w4v3, w8v5, w12v7, w16v9, used for PBDC or monocyte analysis or cryopreserved for w20v11, w24v13 and follow up [fu, i.e. 1 month after last later MDSC flow cytometric analysis. Prostate GVAX and 2 months after last ipilimumab ad- ministration]) were analyzed with the repeated measures Antibodies and 4-color flow cytometry ANOVA with a post-hoc Dunnett’s multiple comparisons PBDC and MDSC frequencies and activation status were test. To determine whether the identified immune param- assessed before, during and after treatment by flow cy- eters were indicative for response to treatment or useful tometry staining as described [17]. Cell surface antibody for survival prediction, optimal cut-off points were deter- staining of PBMC was performed in PBS/0.1% BSA/ mined by Cox regression analysis, according to which pa- 0.02% Sodium-Azide (hereafter referred to as FACS buf- tients were subsequently divided into two groups. OS for fer) for 30 minutes at 4°C. The following antibodies were the two groups was plotted using the Kaplan-Meier used: fluorescein isothiocyanate- (FITC), phycoerythrin- method and statistical significance of the survival distribu- (PE), peridinin chlorophyll protein-Cy5.5- (PerCP) or tion was analyzed by log-rank testing. To analyze whether allophycocyanin (APC)-labeled Abs directed against hu- prognosis impacted the value of the identified response/ man CD3, CD11c, CD14, CD16, CD19, CD33, CD56, survival parameters, the median Halabi Predicted Survival CD123 and HLA-DR (all BD Bioscience), CD40 (Beckman (HPS) was also determined for both groups ([29,53]; see Coulter, Marseille, France), Fab-M-FITC (Southern Bio- also Table 1). Differences in HPS between groups of pa- tec, Birmingham, AL) and blood DC antigens BDCA1, tients and in parameters between prostate cancer patients BDCA2, BDCA3 (all from Milteny Biotec, Bergisch- and age and sex-matched healthy volunteers were ana- Gladbach, Germany) and MDC8 (a kind gift from Dr. lyzed with the two-sample Mann-Whitney U or the E.P. Rieber, Dresden, Germany) and matching isotype Fisher’s exact test (both two-tailed). Above listed statis- control antibodies. Stained cells were analyzed on aFACS- tical analyses were performed either with Prism GraphPad calibur (BD Biosciences) using Cell Quest software. Events or SPSS software. Differences were considered significant collected were 120,000-150,000 per sample. when p < 0.05. To identify clusters of correlated markers, hierarchical cluster analysis using TIGR software was per- PBDC and MDSC subset and activation definitions formed and complete linkage analysis was done by Pearson PBDC frequencies were determined on the basis of ex- correlation analysis. For this purpose, the values of the pression of BDCA or MDC8 markers: two major mye- treatment-induced and pre-treatment parameters were loid or conventional DC (cDC) subsets as recognized taken for each patient, and divided by the cut-off value, after by the Nomenclature Committee of the International which the resulting ratios were log-transformed (base 2). Union of Immunological Societies were identified as Threepatients wereexcludedfromthisanalysissince <70% hi − − + CD11c CD19 CD14 BDCA-1/CD1c DC (designated of the analyzed biomarkers wereavailablefor thesepatients + − + cDC1) and as CD11c CD14 BDCA-3 DC (designated due to withdrawal from the study or sampling failure. cDC2) [50,51]; in addition we assessed frequencies of a third myeloid subset, designated cDC3, and defined as Additional files + lo + CD11c CD14 MDC8 DC (also known as 6-sulfo Lac- NAc non-classical monocytes or SLAN-DC [33,52]) Additional file 1: Figure S1. IRAE in relation to treatment response and survival. A) Distribution of IRAE within the different treatment response as well as plasmacytoid DC (pDC), defined as groups (i.e. partial response (PR), Stable Disease (SD) and Progressive − − hi + CD11c CD14 CD123 BDCA-2 [28]. Classic monocytes Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 10 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Acknowledgements Disease (PD)) is given. Black bars: patients experiencing IRAE and white/ This research was financially supported by awards and grants from the open bars: patients without IRAE during treatment. B) Kaplan Meier Prostate Cancer Foundation (PCF Competitive Research Award to T.D.G.), curve for the patients that experienced IRAE. Number of patients and StichtingVUmc-CCA, and the Dutch Cancer Society (KFW; VU 2006-3697). corresponding median survival for each group are given. Differences in distribution of IRAE between treatment response groups were analyzed Author details with a two-tailed Fisher’s exact test. Statistical significance of the survival Department of Medical Oncology, VU University Medical Center, Cancer distribution was analyzed by log-rank testing. Differences were considered Center Amsterdam, Amsterdam, The Netherlands. Department of Pathology, significant when p < 0.05. VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The 3 4 Additional file 2: Figure S2. Peripheral Blood DC (PBDC) gating Netherlands. Cell Genesys Inc, South San Francisco, CA, USA. Medarex, strategy. A) First, live cells were gated based on FSC-SSC properties of Bloomsbury, NJ/Bristol-Myers Squibb Company, Wallingford, CT, USA. the lymphocyte and monocyte populations (not shown), after which the cDC1, cDC2, cDC3 and pDC populations were identified through BDCA1 Received: 9 April 2014 Accepted: 6 August 2014 (and CD19 , not shown), BDCA3, MDC8 and BDCA2 expression. By backgating on CD11c and CD14 as indicated, the identity of the different DC subsets was confirmed. Activation status of abovementioned cDC, pDC and monocyte subsets was determined by calculating the median References Fluorescence Index (med. FI) of CD40 expression by dividing the med. 1. Drake CG: Prostate cancer as a model for tumour immunotherapy. Nat fluorescence (Med. fl) of the CD40 antibody by the med. fl of the Rev Immunol 2010, 10:580–593. isotype-control antibody. B) Isotype control and CD40 histograms are 2. Tannock IF, De WR, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, depicted for the cDC1 (left) and cDC3 (right) subsets at week (w) 0 visit Theodore C, James ND, Turesson I, Rosenthal MA, Eisenberger MA: (v) 1 (i.e. baseline) and w4v3 for a representative patient. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004, 351:1502–1512. Additional file 3: Figure S3. PBDC frequencies and activation status 3. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern and MDSC frequencies in mCRPC patients and healthy individuals. CH, Ferrari AC, Dreicer R, Sims RB, Xu Y, Frohlich MW, Schellhammer PF: Frequencies of cDC, pDC, monocytes and gMDSC and mMDSC subsets, Sipuleucel-T immunotherapy for castration-resistant prostate cancer. and activation status of cDC, pDC and monocytes was determined in N Engl J Med 2010, 363:411–422. mCRPC patients before prostate GVAX/ipilimumab therapy and age- and 4. Kantoff PW, Schuetz TJ, Blumenstein BA, Glode LM, Bilhartz DL, Wyand M, sex-matched healthy donors (HD). Percentage A) and activation status B) Manson K, Panicali DL, Laus R, Schlom J, Dahut WL, Arlen PM, Gulley JL, of cDC1, cDC2, cDC3, pDC and monocytes and percentage of C) mMDSC Godfrey WR: Overall survival analysis of a phase II randomized controlled are shown. Differences in percentage or activation between mCRPC and trial of a Poxviral-based PSA-targeted immunotherapy in metastatic HD were analyzed with the two-sample Mann-Whitney U test. Differences castration-resistant prostate cancer. J Clin Oncol 2010, 28:1099–1105. were considered significant when p < 0.05, as indicated with the given 5. Yang JC, Hughes M, Kammula U, Royal R, Sherry RM, Topalian SL, Suri KB, p-value. Levy C, Allen T, Mavroukakis S, Lowy I, White DE, Rosenberg SA: Ipilimumab Additional file 4: Figure S4. Differential leukocyte analysis in mCRPC (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer patients before and during prostate GVAX/ipilimumab therapy. Absolute associated with enteritis and hypophysitis. J Immunother 2007, 30:825–830. lymphocytes (white squares) and monocyte (black squares) counts were 6. Small EJ, Sacks N, Nemunaitis J, Urba WJ, Dula E, Centeno AS, Nelson WG, determined before (week 0/visit 1 (w0v1)), during (w4v3, w8v5, w16v9) Ando D, Howard C, Borellini F, Nguyen M, Hege K, Simons JW: Granulocyte and after (follow-up (fu)) prostate GVAX/ipilimumab therapy. Mean macrophage colony-stimulating factor–secreting allogeneic cellular absolute counts ± SEM is given in 10 6 per ml of blood for lymphocytes immunotherapy for hormone-refractory prostate cancer. Clin Cancer Res (white squares), monocytes (black squares) and the sum of lymphocytes 2007, 13:3883–3891. and monocytes (i.e. PBMC, grey squares). Differences between pre- and 7. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, on- or post-treatment were analyzed with the repeated measures ANOVA Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den with a post-hoc Dunnett’s multiple comparisons test. Differences were Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, considered significant when p < 0.05, as indicated with an asterisk Ottensmeier CH, Lebbe C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, (* p < 0.05, ** p < 0.01). Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010, 363:711–723. Abbreviations 8. Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, Davis T, cDC: Conventional dendritic cell; CTLA-4: CTL antigen-4; CRCP: Castration Henry-Spires R, Macrae S, Willman A, Padera R, Jaklitsch MT, Shankar S, resistant prostate cancer; fu: Follow-up; HPS: Halabi predicted survival; Chen TC, Korman A, Allison JP, Dranoff G: Biologic activity of cytotoxic T IRAE: Immune related adverse events; MDSC: Myeloid-derived suppressor cell; lymphocyte-associated antigen 4 antibody blockade in previously OS: Overall survival; pDC: Plasmacytoid dendritic cell; PD: Progressive disease; vaccinated metastatic melanoma and ovarian carcinoma patients. PR: Partial response; SD: Stable disease. Proc Natl Acad Sci U S A 2003, 100:4712–4717. 9. Fong L, Kwek SS, O’Brien S, Kavanagh B, McNeel DG, Weinberg V, Lin AM, Rosenberg J, Ryan CJ, Rini BI, Small EJ: Potentiating endogenous antitumor Competing interests immunity to prostate cancer through combination immunotherapy with IL owns stock and/or stock options from Bristol-Myers Squibb. AJMvdE and CTLA4 blockade and GM-CSF. Cancer Res 2009, 69:609–615. WRG have served as consultants and received honoraria from Bristol-Myers 10. Hurwitz AA, Yu TF, Leach DR, Allison JP: CTLA-4 blockade synergizes with Squibb. TDG and WRG received an educational grant from Cell Genesys Inc. tumor-derived granulocyte-macrophage colony-stimulating factor for All other authors declare that they have no competing interests. treatment of an experimental mammary carcinoma. Proc Natl Acad Sci USA 1998, 95:10067–10071. 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Halabi S, Lin CY, Kelly WK, Fizazi KS, Moul JW, Kaplan EB, Morris MJ, Small EJ: Updated prognostic model for predicting overall survival in first-line chemotherapy for patients with metastatic castration-resistant prostate cancer. J Clin Oncol 2014, 32:671–677. doi:10.1186/s40425-014-0031-3 Cite this article as: Santegoets et al.: Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab. Journal for ImmunoTherapy of Cancer 2014 2:31. 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Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab

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Copyright © 2014 by Santegoets et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology
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10.1186/s40425-014-0031-3
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26196012
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Abstract

Background: Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome. Methods: Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit. Results: Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) + + + was observed, which in the case of BDCA1/CD1c cDC1 and MDC8 /6-sulfoLacNAc inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related + − adverse events. High pre-treatment levels of CD14 HLA-DR monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction + + with our previously identified lymphoid biomarker of high pretreatment CD4 CTLA4 T cell frequencies. Conclusions: Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment. Keywords: Ipilimumab, Prostate GVAX, Biomarker, Patient selection, Survival prediction * Correspondence: td.degruijl@vumc.nl Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article © 2014 Santegoets et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 2 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Background To study whether PBDC and MDSC subsets are af- Prostate cancer is the third leading cause of cancer- fected by prostate GVAX/ipilimumab therapy, extensive related death in men worldwide [1]. Curative treatment myeloid subset monitoring was performed. Myeloid sub- options are only available for localized disease. In pa- sets were analyzed prospectively and followed during tients that develop metastatic castration-resistant prostate treatment, after which cut-off points for response to cancer (mCRPC) the median survival is 16-21 months treatment and/or survival were determined in retrospect. [2-4]. Recent advances have led to novel immunotherapy Our data demonstrate that PBDC are activated by pros- options with proven clinical efficacy in patients with tate GVAX/ipilimumab therapy and that a specific mye- metastatic CRPC (mCRPC), such as PROSTVAC and loid lineage marker profile (i.e. high post-treatment cDC Sipuleucel-T [1,3,4]. In addition, the CTLA-4 blocking activation and low pre-treatment frequencies of mono- antibody ipilimumab has shown clinical activity in a cytic MDSC) proved predictive for clinical benefit after variety of cancer types, including prostate cancer [5-7]. Prostate GVAX and/or ipilimumab immunotherapy. CTLA-4 blockade enhanced antitumor efficacy when combined with other immunomodulating agents, in- Results cluding Granulocyte Macrophage-Colony Stimulating Clinical results Factor (GM-CSF) and GM-CSF-secreting cancer vac- mCRPC patients (n = 28) received 13 bi-weekly injec- cines, e.g. GVAX immunotherapy [8-10]. In line with tions of the prostate GVAX vaccine and 6 four-weekly this, we recently reported the combined immunotherapy infusions of ipilimumab. As described previously [11], of a GM-CSF-engineered allogeneic tumor cell-based vac- five patients experienced serum-PSA-based PR with PSA cine (Prostate GVAX) and ipilimumab in patients with declines of more than 50% and 12 demonstrated disease mCRPC to be safe and clinically active [11]; clinical results stabilization (SD); PR/SD was significantly correlated included partial responses (PR) and a relatively long sur- with prolonged overall survival (med. survival of 41 ver- vival as compared to survival rates observed in control sus 21 months; p = 0.0034). Nine patients, all of which arms of recent Phase III trials [2-4]. received 3 or 5 mg/kg ipilimumab and five of which ex- Recent clinical findings have indicated that the induc- perienced a PR, developed immune-related adverse tion of an effective antitumor immune response relies events (IRAE) [11]. Interestingly, although IRAE were on the proper differentiation, maturation and functional- more frequent in patients that benefited from treatment ity of myeloid antigen-presenting cells (APC), and that (i.e. PR and SD; p = 0.0015), the development of IRAE the accumulation of myeloid-derived suppressor cells was not associated with survival ([11]; Additional file 1: (MDSC) and functionally impaired (immature) dendritic Figure S1). cells (DC) in tumor, blood or lymph nodes of cancer pa- tients is a poor prognostic factor for survival [12-16]. PBDC and monocyte frequency and activation in relation Therefore, cancer immunotherapeutic approaches aiming to survival and IRAE at the normalization of myeloid differentiation are of inter- To assess the effects of prostate GVAX/ipilimumab treat- est for clinical application in support of immunotherapy. ment on circulating myeloid DC subsets, frequency and We have reported the effects of prostate GVAX/ activation status of circulating conventional DC (cDC) ipilimumab immunotherapy on circulating T cell sub- subsets cDC1, cDC2, cDC3 and plasmacytoid DC (pDC) sets [17]. However, the net effect of combined prostate were determined before, during and after treatment. cDC1 hi − − + GVAX/ipilimumab immunotherapy on DC and MDSC were identified as CD11c CD19 CD14 BDCA-1/CD1c ; + − + hi subsets in peripheral blood is currently unknown. It cDC2 as CD11c CD14 BDCA-3 ; cDC3 as CD11c C- lo + − − hi has been described that under proper maturational D14 MDC8 and pDC as CD11c CD14 CD123 BDCA- conditions, peripheral blood DC (PBDC) have the po- 2 (see also Additional file 2: Figure S2 for gating tential to develop into functional DC with the capacity strategies). Similar to previous observations in cancer to induce antitumor T-cell responses [18,19]. In par- patients by us and by others [12,15,27,28], frequencies ticular GM-CSF has been implicated in the recruit- and activation status of circulating DC and monocytes ment and activation of DC in vivo [20,21]. Importantly were generally lower in CRPC patients as compared however, GM-CSF has also been linked to the systemic in- with healthy individuals (see Additional file 3: Figure S3A duction/expansion of MDSC in mice and man [22,23]. and 3B). On-treatment activation (shown in Figure 1A by Furthermore, CTLA-4 antibody blockade has been shown CD40 expression levels) was observed for all DC subsets to reduce MDSC suppressive potency in vitro,and in vivo (as previously reported by us for cDC1 [11]). Interestingly, in a murine ovarian carcinoma model, and this effect was this activation was paralleled by decreases in cDC1, cDC2, achieved both indirectly through inhibiting T cell-MDSC and pDC frequencies (Figure 1B). These decreases were interaction [24,25] and directly through binding to CTLA- observed as early as four weeks after start of treatment 4 expressed on MDSC [26]. and were maintained during treatment (Figure 1B). Of Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 3 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 25 25 1.25 AB ** ** 1.00 20 20 ** ** cDC3 0.75 * * 0.50 15 15 * * cDC1 0.5 0.4 ** 10 10 ** ** ** ** 0.3 ** pDC 5 5 cDC1 cDC1 0.2 ** * ** * ** * BDCA1/CD1c BDCA1/CD1c 0.1 0.10 cDC2 50 50 0.05 ** * * * * 0.00 40 40 w0 w4 w8 w12 w16 w20 w24 fu 30 30 0.025 *** 20 20 0.020 cDC3 10 10 cDC2 cDC2 0.015 BDCA3/CD141 BDCA3/CD141 0 0 cDC1 0.010 20 20 * * 0.005 pDC 15 15 0.0015 * * 0.0010 cDC2 10 10 0.0005 0.0000 w0 w4 w8 w16 fu 5 5 cDC3 cDC3 dim dim CD14 CD14 /MDC8 /MDC8 cDC1 and cDC3 activation cDC1 and cDC3 activation 0 0 100 100 Activation Activation of both of both 20 20 Activation Activation of of one one or or none none * * 75 75 15 15 N=15; med N=15; med. . surv surv.. 52 52 mths mths ** ** 10 10 50 50 5 5 P<0.0001 P<0.0001 pDC pDC 25 25 BDCA2/CD123 BDCA2/CD123 N=13; med N=13; med. . surv surv.. w0 w4 w8 w12 w16 w20 w24 fu 16mths 16mths 0 0 0 0 20 20 40 40 60 60 80 80 100 100 months months Figure 1 Prostate GVAX/ipilimumab therapy-induced activation of peripheral blood (PB) DC subsets is associated with prolonged survival. PBDC activation and frequency was determined before (week 0/visit 1 (w0v1)), during (w4v3, w8v5, w12v7, w16v9, w20v11, w24v13 and after (follow-up (fu)) prostate GVAX/ipilimumab therapy by flow cytometry. A) Activation state over treatment –by Median Fluorescence Index hi − lo + + − + + lo + − − hi + (MFI)ofCD40ofCD11c CD19 CD14 BDCA1 cDC1, CD11c CD14 BDCA-3 cDC2, CD11c CD14 MDC8 cDC3 and CD11c CD14 CD123 BDCA-2 pDC. Grey bars denote the mean ± SEM range at baseline. B) DC subset frequencies (as percentages of PBMC) and C) absolute numbers per ml blood, over treatment, cDC1: solid black squares, cDC2: open black squares, cDC3: solid grey squares, pDC: open grey squares. Means ± SEM of 28 patients are shown. D) Kaplan Meier curve for on-treatment increases in cDC1 and cDC3 activation. Number of patients and corresponding median survival for each group is given. Differences between pre- and on- or post-treatment were analyzed with the repeated measures ANOVA with a post-hoc Dunnett’s multiple comparisons test. Differences were considered significant when p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01) within the respective squares. Statistical significance of the survival distribution was analyzed by log-rank testing. note, decreases in absolute cDC1, CDC2, and pDC num- reached significance (Figure 1C). These differences be- bers per volume blood were much less pronounced and tween DC frequencies and absolute numbers may in large on-treatment increased absolute cDC3 numbers even part be explained by a sustained increase in absolute CD40 MedFI absolute count (in 1x10e6 per ml of blood) % cells (as % PBMC) Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 4 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 lymphocyte numbers over the course of treatment (see activation were significantly higher in patients that eventu- Additional file 4: Figure S4). Increased PBDC activation ally developed IRAE, suggesting that these treatment- was generally maintained during treatment (see Figure 1A) induced increases might serve as an indicator for risk of and increases of >70% of CD40 med. FI on the cDC1 IRAE. and cDC3 subsets (see for representative histograms Additional file 2: Figure S2) were associated with signifi- MDSC frequencies in relation to survival cantly prolonged overall survival (OS; median survival To assess the effects of prostate GVAX/ipilimumab 38.5 vs. 15.5 months, p = 0.0004 and median survival 40 treatment on circulating MDSC, the frequency of mono- ־ + ־/lo vs. 19 months, p = 0.0031, respectively (Table 1). Survival cytoid MDSC (mMDSC; Lin CD14 HLA-DR ,see benefit was even more pronounced for patients who dis- Figure 4A) was determined. Significantly higher levels played treatment-induced activation of both cDC1 and of circulating mMDSC were detected in CRPC patients cDC3 subsets (median survival 52 vs. 16 months, p < compared with age- and sex-matched healthy individ- 0.0001; Figure 1C). No relationship with survival was uals. Significant post-treatment increases in mMDSC found for either pDC or cDC2. frequencies were observed (Figure 4B), yet these in- When patients were divided by treatment response, creases were only modest and did not correlate with re- decreases in the frequency of monocytes were found to sponse to treatment (not shown) or survival (Table 1). be selectively associated with PR (Figure 2A), which, like In contrast, patients who displayed high pre-treatment observed for PBDC subsets, was paralleled by increased levels of mMDSC had a significantly shorter OS than pa- activation (Figure 2B). Of note, although these were tients who did not (Figure 4C, median survival 20 vs. 52 clear trends, they did not reach statistical significance. months, HR = 4.26, 95% CI = 1.37 - 13.25, p = 0.0046). Similarly to the cDC1/BDCA1 and cDC3/SLAN-DC subsets, treatment-induced increases of CD40 med. FI A myeloid marker profile of high DC activation and low on CD14 monocytes was associated with prolonged OS frequencies of MDSC is predictive for survival (median survival 57 vs. 21 months, p = 0.0749; Figure 2C To assess whether clinical prognosis impacted the puta- and Table 1). tive predictive value for treatment outcome of any of the To determine whether changes in DC and monocyte identified myeloid parameters, the median Halabi Pre- activation status could possibly serve as early marker for dicted Survival (HPS) was determined for the patient the ipilimumab-associated development of IRAE, we groups above and below the designated cut-offs [29]. No compared the rise in DC or monocyte activation status at significant differences were observed, indicating that bet- week four of treatment (i.e. after two GVAX administra- ter prognosis before treatment was not the determining tions and only one ipilimumab infusion) between patients factor for any of these parameters (Table 1). with or without IRAE. As shown in Figure 3, treatment- We previously identified a lymphoid biomarker profile induced increases in cDC1, cDC3, and monocyte with predictive value for OS benefit, which was dominated Table 1 Characteristics and survival distribution of treatment-induced changes in PBDC activation and in mMDSC frequencies † § ׀׀ Immune parameter Mean treatment- Cut-off Median survival between p-value Hazard ratio Mean Halabi p-value induced increase groups‡ (# of patients (95% CI HR) Predicted * ¶ (range) in each group) Survival CD40 on cDC1 313% (-67 to +1194) 70% 38.5 vs. 15.5(22 vs. 6) 0.0004 0.052 (0.010-0.267) 18.7 vs. 16.6 0.614 CD40 on cDC2 170% (-52 to +612) 150% 36.5 vs. 25.5 (10 vs. 18) 0.397 0.688 (0.299-1.582) 16.8 vs. 19.1 0.338 CD40 on cDC3 160% (-16 to +765) 70% 40.0 vs. 19.0 (19 vs. 9) 0.0031 0.179 (0.057-0.560) 18.6 vs. 17.7 0.684 CD40 on pDC 290% (-19 to +1814) 450% 26.0 vs. 31.5 (4 vs. 26) 0.739 1.226 (0.373-4.032) 14.2 vs. 19.0 0.168 CD40 on monocytes 243% (-28 to +1001) 160% 57.0 vs. 21.0 (9 vs. 19) 0.0749 0.469 (0.204-1.079) 20.1 vs. 17.4 0.192 + − Lin-CD14 HLA-DR mMDSC 87% (-71 to +543) 60% 35.0 vs. 36.0 (8 vs. 11) 0.270 0.559 (0.198-1.572) 16.7 vs. 21.4 0.139 Mean and range of treatment-induced increases are given in percentages relative to pre-treatment values. Cut-off points for survival prediction were determined using the Cox regression model and the relative increments are given as percentage of pre-treatment values. Median Survival for both groups was calculated using the Kaplan-Meier method and given in months. Statistical significance of the survival distribution was analyzed by log-rank testing and considered significant when p < 0.05 (in bold). Mean Halabi Predicted Survival (HPS) ± standard error (in months) was determined for patients with biomarker increments above or under designated cut-offs. NB: Halabi scores were determined based on Halabi et al. J. Clin. Oncol. 2003 [29], but Halabi scores based on Halabi et al. J. Clin. Oncol. 2014 [53] similarly showed prognosis before treatment (based on HPS) not to be the determining factor for any of these myeloid markers in terms of prediction of median survival upon treatment (not shown). ׀׀ Differences in HPS between groups were determined by Mann-Whitney U test and were considered significant when p < 0.05 (in bold). Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 5 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 PR SD PD w0 w4 w8 w12 w16 w20 w24 fu1 w0 w4 w8 w12 w16 w20 w24 fu1 v1 v3 v5 v7 v9 v11 v13 v1 v3 v5 v7 v9 v11 v13 160% increase < 160% increase N=9; med.surv. 57 mths N=17; med.surv. 21 p = 0.0749 mths Cut-off 160% 0 20 40 60 80 100 months Figure 2 Increased monocyte activation following Prostate GVAX/ipilimumab therapy is associated with prolonged survival. Frequencies and activation status of circulating CD14 monocytes were determined before (w0), during (w4, w8, w12, w16, w20, w24) and after (fu) Prostate GVAX/ ipilimumab treatment by flowcytometric analysis. A) Mean percentage (of PBMC) ± SEM and B) mean activation ± SEM of CD14+ monocytes is shown before, during and after Prostate GVAX/ipilimumab treatment for 28 patients, divided by clinical PSA response: partial PSA response (PR; black squares), disease stabilization or (SD; white squares) or disease progression (PD; grey squares). Activation is given as med. Fluorescence Index (med. FI) and calculated by dividing the med. fluorescence (med. fl) of CD40 antibody by the med. fl of the isotype control antibody. C) Kaplan Meier curve for treatment-induced increases in activation of monocytes. Number of patients and corresponding median survival for each group are given. Differences between pre- and on- or post-treatment were analyzed with the repeated measures ANOVA with a post-hoc Dunnett’s multiple comparisons test. Differences were considered significant when p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01). + + by high CD4 CTLA4 T cell frequencies prior to therapy Discussion [17]. When combining all predictive lymphoid [17] and PBDC and MDSC profiling of patients with CRPC re- myeloid markers in an unsupervised cluster analysis, we ceiving combined Prostate GVAX/ipilimumab treatment found a particularly strong association with prolonged revealed that PBDC and MDSC subsets were affected by survival for patients displaying a combination of high prostate GVAX/ipilimumab therapy, with on-treatment levels of on-treatment cDC1/cDC3/monocyte activation, increases in DC activation and mMDSC frequencies. Of low pretreatment mMDSC rates and high pretreatment note, a myeloid profile of low pretreatment frequencies + + frequencies of CD4 CTLA4 T cells (designated clustered of mMDSC and high treatment-induced cDC1, cDC3, group 2, median OS 46 months, see Figure 5A and 5B). and monocyte activation levels demonstrated predictive Nevertheless, patients with relatively high pretreatment value for OS on treatment. As this was only a small ex- + + frequencies of CD4 CTLA4 T cells showed significant ploratory study of 28 patients, no multivariate analyses survival benefit whether these were accompanied by con- were performed and the candidate biomarkers should be certed high levels of on-treatment cDC1/cDC3/monocyte validated prospectively in larger randomized trials. activation and low pretreatment mMDSC rates or not It has been reported that frequencies and activation (designated clustered groups 2 + 1, median OS 40 months; status of circulating DC are significantly lower in cancer see Figure 5A and 5C). patients compared with healthy individuals [12,15,27,28]. Percent survival % CD14 positive cells median Fl. Index CD40 Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 6 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 AB C cDC1 cDC2 cDC3 15 5 p=0.0271 p=0.0274 0 0 0 no IRAE no IRAE no IRAE IRAE IRAE IRAE DE pDC monocytes p=0.0117 no IRAE no IRAE IRAE IRAE Figure 3 PBDC activation in relation to immune-related adverse events (IRAE). Fold increase in activation of A) cDC1, B) cDC2, C) cDC3, D) pDC and E) monocytes was determined at week 4 after start of prostate GVAX/ipilimumab treatment by dividing the median Fluorescence Index (med. FI) of CD40 at week four (i.e. 2 vaccinations and 1 ipilimumab infusion) by the med. FI of CD40 at start of treatment and displayed for patients that experienced IRAE or no IRAE during therapy. Differences in fold increase in activation between groups of patients were analyzed with the two-sample Mann-Whitney U test. Differences were considered significant when p < 0.05, as indicated with the given p-value. In our study, similar results were observed, with signifi- vaccination (Van Mens et al, manuscript in preparation). cantly lower cDC1 frequencies and an inferior activation However, absolute PBDC subset numbers showed more state of cDC1, cDC2 and monocytes in mCRPC patients moderate decreases whereas they revealed a significant (see Additional file 3: Figure S3A and B). Interestingly, on-treatment increase in cDC3. The latter fits well with prostate GVAX/ipilimumab treatment resulted in a fur- the inflammatory nature of cDC3 or SLAN-DC, being ther reduction of circulating cDC and pDC subsets, and mobilized by cytokines like GM-CSF [33]. this reduction was paralleled by increases in their activa- MDSC represent a heterogeneous population of imma- tion status. The observed activation may be explained by ture myeloid cells, and have been recognized to play an the production of GM-CSF by the vaccine, as GM-CSF important role in suppression of (anti-tumor) immune has been described to activate DC in vivo [20,21]. Alter- responses. Elevated levels of either monocytoid or gran- natively, or additionally, ipilimumab may have altered ulocytic MDSC have been observed in a variety of hu- DC activation state through blocking CTLA-4/B7 inter- man cancers [34-38]. Moreover, their presence in actions at the Treg/DC interface or by direct binding of peripheral blood or at the tumor site has been linked CTLA-4 on DC [30-32]. Nevertheless, a lack of associ- with poor prognosis and may thus serve as a prognostic ation between ipilimumab dose and DC activation seems or predictive marker for clinical outcome [36,39-41]. In to support an overriding role for GVAX-derived GM- keeping with previous findings, mCRPC patients displayed CSF in this respect. The enhanced activation and simul- significantly higher levels of mMDSC compared with age- taneous reduction of cDC and pDC subset frequencies and sex-matched healthy controls (see Additional file 3: in blood is suggestive of their recruitment to effector Figure S3C). In fact, significantly prolonged OS was ob- sites (e.g. tumor and vaccination sites). This hypothesis served for patients displaying low pre-treatment levels of is further supported by our own observation of en- mMDSC, i.e. levels that were similar to those found in hanced recruitment of antigen-presenting cells to the healthy volunteers, suggesting that indeed mMDSC levels Prostate GVAX vaccination sites following repeated may serve as a predictive marker for clinical outcome. fold increase CD40 med FI fold increase CD40 med FI Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 7 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Our data demonstrated modest increases of mMDSC frequencies following multiple Prostate GVAX/ipilimu- - + - Monocytic MDSC (Lin CD14 HLA-DR ) mab doses in a subgroup of patients. As described, in- creases in the frequency of circulating MDSC can be related to progression [39,42-44]. No such relation was observed in our study, as no difference in mMDSC ex- R3 pansion was observed between PR, SD and PD patients (not shown). The observed mMDSC expansion might have been induced by the Prostate GVAX vaccine, since R2 GM-CSF-based vaccines have been shown to expand MDSC in mice and man [22,23]. Perhaps surprisingly, the HLA-DR CD14 post-vaccination increases of circulating mMDSC frequen- cies did not impact survival in our study, suggesting that 2.0 prostate GVAX/ipilimumab therapy may reduce the sup- pressive function of mMDSC. Indeed, reduction of MDSC 1.5 suppressive function has been described for CTLA-4 blockade therapy in an in vitro and in vivo murine ovarian 1.0 carcinoma model [24-26]. Moreover, as mMDSC have been shown to further develop into more mature macro- phage and DC-like cells [42], GM-CSF-driven differenti- 0.5 ation may have interfered with their suppressive ability. A major problem in CTLA-4 blockade therapy is the 0.0 development of potentially life-threatening IRAE like w0v1 w8v5 w16v9 w24v13 fu1 colitis, hepatitis, alveolitis and hypophysitis [45,46]. To date, attempts to reduce the development of these IRAE high pretreatment have been unsuccessful [47]. Therefore, it is important low pretreatment to identify biomarkers for patient selection or develop methods that enable the early detection of IRAE. Within N=13; med. surv. 52 mths the myeloid compartment, no pre-treatment marker with putative clinical value for IRAE risk prediction could be identified. Yet, our data showed that the mag- nitude of early (i.e. at week 4, after only one single ipili- N=11; med. mumab administration) CD40 up-regulation on cDC1, surv. 20 mths p = 0.0046 cDC3 and monocytes correlated with on-treatment Cut-off 0.3% IRAE development. Unfortunately, the clinical applic- 0 20 40 60 80 100 ability of this on-treatment change as a marker for IRAE months risk is questionable, as there is considerable overlap for Figure 4 High pretreatment frequency of mMDSC is associated CD40 activation levels on the indicated subsets between with reduced OS. mMDSC frequencies were determined before the IRAE positive and negative group (Figure 3). (week 0/visit 1 (w0v1)), during (w8v5, w16v9 and w24v13) and after Our profiling data are consistent with clinical benefit (follow-up (fu)) prostate GVAX/ipilimumab therapy by flow cytometry. − + −/lo A) A representative analysis of Lin CD14 HLA-DR mMDSC. B) and survival advantage for patients with high DC activa- Percentage of mMDSC over follow-up. C) Kaplan meier curve for tion and low levels of MDSC. These data echo our find- pre-treatment frequencies of mMDSC. Number of patients and ing of a predictive T cell profile of activated T cells and corresponding median survival for each group are given. Differences low pre-treatment Treg frequencies in the same group betweenpre-and on-orpost-treatmentwere analyzedwiththe of patients [17], and are in line with an ever growing repeated measures ANOVA with a post-hoc Dunnett’smultiple comparisons test. Differences were considered significant when number of studies stressing the importance of a pro- p < 0.05, as indicated with asterisks (* p < 0.05, ** p < 0.01). Statistical inflammatory, non-suppressive immune status for optimal significance of the survival distribution was analyzed by log-rank efficacy of immunotherapy in cancer patients. Interest- testing and indicated with the given p-value. ingly, our unsupervised clustering analysis of combined myeloid and lymphoid biomarkers indicates two, partially This is in keeping with a recent report indicating a similar overlapping, major populations with survival benefit: predictive value of pre-treatment mMDSC frequencies for 1) patients with combined low pre-treatment frequen- outcome of tumor peptide vaccination therapy in patients cies of mMDSC and high on-treatment cDC activation with renal cell cancer [39]. levels, and 2) patients with high pre-treatment levels of - + - % Lin CD14 HLA-DR Percent survival Lineage (CD3/16/19/56) Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 8 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 AB Group 2 Group 3 -2.0 0.0 +2.0 N=12; med. surv. 46 mths patient codes Group 1 Group 2 Group 3 25 p = 0.0042 N=6; med. surv. mMDSC pre 18.5 mths CD4 Teff/Tem increase CD4/CD25int/FoxP3 increase 0 20 40 60 80 100 Treg increase Time CD8 Teff/Tem/Tcm increase Group 1 + 2 cDC1 activation Group 3 cDC3 activation monocyte activation Tregs pre N=19; med. surv. 40 mths CD4 Teff/Tem pre CD4CTLA4 pre CD4PD-1 pre p = 0.0028 N=6; med. surv. 18.5 mths 0 20 40 60 80 100 Time Figure 5 High on-treatment cDC1/cDC3/monocyte activation and low pretreatment mMDSC frequencies predict clinical benefit after therapy. A) Unsupervised cluster analysis of the expression of the indicated treatment-induced and pre-treatment myeloid and lymphoid markers. To identify clusters of correlated markers, hierarchical cluster analysis using TIGR software was performed, and average linkage analysis was done by Pearson correlation analysis. Values of the treatment-induced and pre-treatment parameters are given relative to the cut-off value (determined by Cox regression model as described in materials and methods); below cut-off in green and above cut-off in red. Kaplan Meier curves for B) group 2 (with high on-treatment cDC1/cDC3/monocyte activation and low pretreatment mMDSC frequencies: highlighted by white boxes in the heat plot) versus group 3 (with generally low on-treatment cDC1/cDC3/monocyte activation and high mMDSC frequencies) and + + C) group 1 + 2 (with high pre-treatment frequencies of CD4 CTLA4 T cells: lower white box in heat plot) versus group 3. Statistical significance of the survival distribution was analyzed by log-rank testing and indicated with the given p-value. Number of patients and corresponding median survival for each group are given. + + CD4 CTLA4 T cells, see Figure 5A, white-lined boxes. and PC-3 (CG1940), which have been transduced with Whereas the latter biomarker profile may signal particular an adeno-associated viral vector to secrete GM-CSF. susceptibility to check point inhibition [17], the first may These cell lines were propagated, frozen, and irradiated be representative of a favorable immune state and as such to arrest further cell division [6,48]. The product was hold predictive value for immunotherapy in general. These stored and shipped in gaseous nitrogen phase, and admin- profiles warrant further assessment and validation of their istered within 60 minutes after thawing. All manufactur- utility for patient selection in other, preferably randomized, ing was conducted according to good manufacturing clinical trials of GM-CSF-based therapy and/or CTLA-4 practice. Ipilimumab (formerly MDX-010), a fully human blockade or other forms of cancer immunotherapy. IgG1κ monoclonal antibody directed against CTLA-4, was provided by Medarex/Bristol-Myers Squibb (Plainsboro, Conclusions NJ, USA). This study provides evidence that circulating myeloid sub- sets are affected by combined Prostate GVAX and ipilimu- mab therapy and that a myeloid lineage profile of low Study population and sampling of peripheral blood pretreatment mMDSC frequencies and high treatment- As described [11], 28 chemonaive patients with asymp- induced cDC activation levels may contribute to the identi- tomatic mCRPC received 13 bi-weekly vaccinations of the fication of patients with possible clinical benefit of Prostate Prostate GVAX vaccine and 6 four-weekly infusions of ipi- GVAX/ipilimumab treatment. limumab from the time of prime vaccination. In the first 12 patients, ipilimumab was administered at escalating Methods doses of 0.3, 1, 3 and 5 mg/kg (3 patients each). In the ex- Prostate GVAX and ipilimumab pansion phase, 16 additional patients were included at The Prostate GVAX vaccine is a cellular vaccine consist- 3 mg/kg ipilimumab. This study is registered with the ing of two prostate cancer cell lines, LNCaP (CG8711) Central Committee on Research involving Human Subjects Percent survival Percent survival Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 9 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 hi hi in the Netherlands, number P03.1786C, and ClinicalTrials. were defined as CD11c CD14 (highly positive; see gov, number NCT01510288. also Additional file 2: Figure S2). Monocytoid MDSC − + neg/lo Responses to treatment were defined as described [49]. (mMDSC) were defined as Lin CD14 HLA-DR In brief, PSA partial response (PR) was defined as >50% [28] (see also Figure 4A). For all the above mentioned PSA decline from baseline, which was confirmed by a populations a live gate was used based on FSC-SSC second PSA test 3 or more weeks later. PSA progressive properties of the lymphocyte and monocyte populations. disease (PD) was defined as >25% PSA increase and an Activation status of above mentioned cDC, pDC and absolute increase of 2 ng/ml or more from baseline, monocyte subsets was determined by calculating the me- whereas stable disease (SD) was defined as no PR and dian Fluorescence Index (med. FI) of CD40 expression by no PD on treatment. dividing the med. fluorescence (Med. fl) of the CD40 anti- For immune monitoring, blood samples were taken body by the med. fl of the isotype-control antibody. from thepatientsbeforestart of therapyand everyfour weeks thereafter until four weeks after the last treat- Statistical analyses ment (i.e. follow-up (fu)). Peripheral blood mononuclear Differences between immune parameters before treatment cells (PBMC) were isolated by density centrifugation (w0v1; w = week; v = visit; i.e. baseline levels) and during (NycomedAS, Oslo, Norway). PBMC were either directly and/or after treatment (w4v3, w8v5, w12v7, w16v9, used for PBDC or monocyte analysis or cryopreserved for w20v11, w24v13 and follow up [fu, i.e. 1 month after last later MDSC flow cytometric analysis. Prostate GVAX and 2 months after last ipilimumab ad- ministration]) were analyzed with the repeated measures Antibodies and 4-color flow cytometry ANOVA with a post-hoc Dunnett’s multiple comparisons PBDC and MDSC frequencies and activation status were test. To determine whether the identified immune param- assessed before, during and after treatment by flow cy- eters were indicative for response to treatment or useful tometry staining as described [17]. Cell surface antibody for survival prediction, optimal cut-off points were deter- staining of PBMC was performed in PBS/0.1% BSA/ mined by Cox regression analysis, according to which pa- 0.02% Sodium-Azide (hereafter referred to as FACS buf- tients were subsequently divided into two groups. OS for fer) for 30 minutes at 4°C. The following antibodies were the two groups was plotted using the Kaplan-Meier used: fluorescein isothiocyanate- (FITC), phycoerythrin- method and statistical significance of the survival distribu- (PE), peridinin chlorophyll protein-Cy5.5- (PerCP) or tion was analyzed by log-rank testing. To analyze whether allophycocyanin (APC)-labeled Abs directed against hu- prognosis impacted the value of the identified response/ man CD3, CD11c, CD14, CD16, CD19, CD33, CD56, survival parameters, the median Halabi Predicted Survival CD123 and HLA-DR (all BD Bioscience), CD40 (Beckman (HPS) was also determined for both groups ([29,53]; see Coulter, Marseille, France), Fab-M-FITC (Southern Bio- also Table 1). Differences in HPS between groups of pa- tec, Birmingham, AL) and blood DC antigens BDCA1, tients and in parameters between prostate cancer patients BDCA2, BDCA3 (all from Milteny Biotec, Bergisch- and age and sex-matched healthy volunteers were ana- Gladbach, Germany) and MDC8 (a kind gift from Dr. lyzed with the two-sample Mann-Whitney U or the E.P. Rieber, Dresden, Germany) and matching isotype Fisher’s exact test (both two-tailed). Above listed statis- control antibodies. Stained cells were analyzed on aFACS- tical analyses were performed either with Prism GraphPad calibur (BD Biosciences) using Cell Quest software. Events or SPSS software. Differences were considered significant collected were 120,000-150,000 per sample. when p < 0.05. To identify clusters of correlated markers, hierarchical cluster analysis using TIGR software was per- PBDC and MDSC subset and activation definitions formed and complete linkage analysis was done by Pearson PBDC frequencies were determined on the basis of ex- correlation analysis. For this purpose, the values of the pression of BDCA or MDC8 markers: two major mye- treatment-induced and pre-treatment parameters were loid or conventional DC (cDC) subsets as recognized taken for each patient, and divided by the cut-off value, after by the Nomenclature Committee of the International which the resulting ratios were log-transformed (base 2). Union of Immunological Societies were identified as Threepatients wereexcludedfromthisanalysissince <70% hi − − + CD11c CD19 CD14 BDCA-1/CD1c DC (designated of the analyzed biomarkers wereavailablefor thesepatients + − + cDC1) and as CD11c CD14 BDCA-3 DC (designated due to withdrawal from the study or sampling failure. cDC2) [50,51]; in addition we assessed frequencies of a third myeloid subset, designated cDC3, and defined as Additional files + lo + CD11c CD14 MDC8 DC (also known as 6-sulfo Lac- NAc non-classical monocytes or SLAN-DC [33,52]) Additional file 1: Figure S1. IRAE in relation to treatment response and survival. A) Distribution of IRAE within the different treatment response as well as plasmacytoid DC (pDC), defined as groups (i.e. partial response (PR), Stable Disease (SD) and Progressive − − hi + CD11c CD14 CD123 BDCA-2 [28]. Classic monocytes Santegoets et al. Journal for ImmunoTherapy of Cancer 2014, 2:31 Page 10 of 12 http://www.immunotherapyofcancer.org/content/2/1/31 Acknowledgements Disease (PD)) is given. Black bars: patients experiencing IRAE and white/ This research was financially supported by awards and grants from the open bars: patients without IRAE during treatment. B) Kaplan Meier Prostate Cancer Foundation (PCF Competitive Research Award to T.D.G.), curve for the patients that experienced IRAE. Number of patients and StichtingVUmc-CCA, and the Dutch Cancer Society (KFW; VU 2006-3697). corresponding median survival for each group are given. Differences in distribution of IRAE between treatment response groups were analyzed Author details with a two-tailed Fisher’s exact test. Statistical significance of the survival Department of Medical Oncology, VU University Medical Center, Cancer distribution was analyzed by log-rank testing. Differences were considered Center Amsterdam, Amsterdam, The Netherlands. Department of Pathology, significant when p < 0.05. VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The 3 4 Additional file 2: Figure S2. Peripheral Blood DC (PBDC) gating Netherlands. Cell Genesys Inc, South San Francisco, CA, USA. Medarex, strategy. A) First, live cells were gated based on FSC-SSC properties of Bloomsbury, NJ/Bristol-Myers Squibb Company, Wallingford, CT, USA. the lymphocyte and monocyte populations (not shown), after which the cDC1, cDC2, cDC3 and pDC populations were identified through BDCA1 Received: 9 April 2014 Accepted: 6 August 2014 (and CD19 , not shown), BDCA3, MDC8 and BDCA2 expression. By backgating on CD11c and CD14 as indicated, the identity of the different DC subsets was confirmed. Activation status of abovementioned cDC, pDC and monocyte subsets was determined by calculating the median References Fluorescence Index (med. FI) of CD40 expression by dividing the med. 1. Drake CG: Prostate cancer as a model for tumour immunotherapy. Nat fluorescence (Med. fl) of the CD40 antibody by the med. fl of the Rev Immunol 2010, 10:580–593. isotype-control antibody. B) Isotype control and CD40 histograms are 2. Tannock IF, De WR, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, depicted for the cDC1 (left) and cDC3 (right) subsets at week (w) 0 visit Theodore C, James ND, Turesson I, Rosenthal MA, Eisenberger MA: (v) 1 (i.e. baseline) and w4v3 for a representative patient. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004, 351:1502–1512. Additional file 3: Figure S3. PBDC frequencies and activation status 3. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern and MDSC frequencies in mCRPC patients and healthy individuals. CH, Ferrari AC, Dreicer R, Sims RB, Xu Y, Frohlich MW, Schellhammer PF: Frequencies of cDC, pDC, monocytes and gMDSC and mMDSC subsets, Sipuleucel-T immunotherapy for castration-resistant prostate cancer. and activation status of cDC, pDC and monocytes was determined in N Engl J Med 2010, 363:411–422. mCRPC patients before prostate GVAX/ipilimumab therapy and age- and 4. Kantoff PW, Schuetz TJ, Blumenstein BA, Glode LM, Bilhartz DL, Wyand M, sex-matched healthy donors (HD). Percentage A) and activation status B) Manson K, Panicali DL, Laus R, Schlom J, Dahut WL, Arlen PM, Gulley JL, of cDC1, cDC2, cDC3, pDC and monocytes and percentage of C) mMDSC Godfrey WR: Overall survival analysis of a phase II randomized controlled are shown. Differences in percentage or activation between mCRPC and trial of a Poxviral-based PSA-targeted immunotherapy in metastatic HD were analyzed with the two-sample Mann-Whitney U test. Differences castration-resistant prostate cancer. J Clin Oncol 2010, 28:1099–1105. were considered significant when p < 0.05, as indicated with the given 5. Yang JC, Hughes M, Kammula U, Royal R, Sherry RM, Topalian SL, Suri KB, p-value. Levy C, Allen T, Mavroukakis S, Lowy I, White DE, Rosenberg SA: Ipilimumab Additional file 4: Figure S4. Differential leukocyte analysis in mCRPC (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer patients before and during prostate GVAX/ipilimumab therapy. Absolute associated with enteritis and hypophysitis. J Immunother 2007, 30:825–830. lymphocytes (white squares) and monocyte (black squares) counts were 6. Small EJ, Sacks N, Nemunaitis J, Urba WJ, Dula E, Centeno AS, Nelson WG, determined before (week 0/visit 1 (w0v1)), during (w4v3, w8v5, w16v9) Ando D, Howard C, Borellini F, Nguyen M, Hege K, Simons JW: Granulocyte and after (follow-up (fu)) prostate GVAX/ipilimumab therapy. Mean macrophage colony-stimulating factor–secreting allogeneic cellular absolute counts ± SEM is given in 10 6 per ml of blood for lymphocytes immunotherapy for hormone-refractory prostate cancer. Clin Cancer Res (white squares), monocytes (black squares) and the sum of lymphocytes 2007, 13:3883–3891. and monocytes (i.e. PBMC, grey squares). Differences between pre- and 7. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, on- or post-treatment were analyzed with the repeated measures ANOVA Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den with a post-hoc Dunnett’s multiple comparisons test. Differences were Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, considered significant when p < 0.05, as indicated with an asterisk Ottensmeier CH, Lebbe C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, (* p < 0.05, ** p < 0.01). Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010, 363:711–723. Abbreviations 8. Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, Davis T, cDC: Conventional dendritic cell; CTLA-4: CTL antigen-4; CRCP: Castration Henry-Spires R, Macrae S, Willman A, Padera R, Jaklitsch MT, Shankar S, resistant prostate cancer; fu: Follow-up; HPS: Halabi predicted survival; Chen TC, Korman A, Allison JP, Dranoff G: Biologic activity of cytotoxic T IRAE: Immune related adverse events; MDSC: Myeloid-derived suppressor cell; lymphocyte-associated antigen 4 antibody blockade in previously OS: Overall survival; pDC: Plasmacytoid dendritic cell; PD: Progressive disease; vaccinated metastatic melanoma and ovarian carcinoma patients. PR: Partial response; SD: Stable disease. Proc Natl Acad Sci U S A 2003, 100:4712–4717. 9. Fong L, Kwek SS, O’Brien S, Kavanagh B, McNeel DG, Weinberg V, Lin AM, Rosenberg J, Ryan CJ, Rini BI, Small EJ: Potentiating endogenous antitumor Competing interests immunity to prostate cancer through combination immunotherapy with IL owns stock and/or stock options from Bristol-Myers Squibb. AJMvdE and CTLA4 blockade and GM-CSF. Cancer Res 2009, 69:609–615. WRG have served as consultants and received honoraria from Bristol-Myers 10. Hurwitz AA, Yu TF, Leach DR, Allison JP: CTLA-4 blockade synergizes with Squibb. TDG and WRG received an educational grant from Cell Genesys Inc. tumor-derived granulocyte-macrophage colony-stimulating factor for All other authors declare that they have no competing interests. treatment of an experimental mammary carcinoma. Proc Natl Acad Sci USA 1998, 95:10067–10071. 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Halabi S, Lin CY, Kelly WK, Fizazi KS, Moul JW, Kaplan EB, Morris MJ, Small EJ: Updated prognostic model for predicting overall survival in first-line chemotherapy for patients with metastatic castration-resistant prostate cancer. J Clin Oncol 2014, 32:671–677. doi:10.1186/s40425-014-0031-3 Cite this article as: Santegoets et al.: Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab. Journal for ImmunoTherapy of Cancer 2014 2:31. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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