Adenocarcinoma of the Uterine Cervix Shows Impaired Recruitment Of

Published OnlineFirst March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3826

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Adenocarcinoma of the Uterine Cervix Shows Impaired Recruitment of cDC1 and CD8þ T Cells and Elevated b-Catenin Activation Compared with Squamous Cell Carcinoma Jossie Rotman1, A. Marijne Heeren2, Awa A. Gassama1, Sinead M. Lougheed2,Noelle€ Pocorni2, Anita G.M. Stam2, Maaike C.G. Bleeker4, Henry J.M.A.A Zijlmans3, Constantijne H. Mom1,2, Gemma G. Kenter1,3, Ekaterina S. Jordanova1,2,5, and Tanja D. de Gruijl2

ABSTRACT ◥ Purpose: Adenocarcinoma of the uterine cervix is the second analysis demonstrated higher expression of chemokines involved in most common type of cervical cancer after squamous cell carcinoma effector T-cell homing (CXCL9/10/11) in SCC primary tumors as (SCC). Although both subtypes are treated similarly, patients with compared with adenocarcinoma primary tumors, which was highly adenocarcinoma have a worse prognosis. In this study, immuno- correlated to a transcriptional signature for type I conventional logic features of the tumor microenvironment in these two subsets dendritic cells (cDC1). This was consistent with elevated fre- þ were pursued with potential therapeutic implications. quencies of CD141/BDCA3 cDC1 in primary tumor SCC sam- Experimental Design: The immune microenvironment of pri- ples relative to adenocarcinoma and correspondingly elevated mary tumors and nonmetastatic tumor-draining lymph nodes levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. (TDLN) was compared between patients with cervical adenocar- Hampered cDC1 recruitment in adenocarcinoma was in turn cinoma (n ¼ 16) and SCC (n ¼ 20) by polychromatic flow cytometry related to lower transcript levels of cDC1-recruiting chemokines and by transcriptional profiling of the primary tumors (n ¼ 299) and an elevated b-catenin activation score and was associated using publicly available data from The Cancer Genome Atlas with poor overall survival. (TCGA). Conclusions: Our data have identified an opportunity for the Results: Flow cytometric analyses revealed intact T-cell differ- investigation of potentially novel therapeutic interventions in entiation in TDLNs, but hampered effector T-cell trafficking to the adenocarcinoma of the cervix, that is, b-catenin inhibition and primary tumors in adenocarcinoma, as compared with SCC. TCGA cDC1 mobilization.

Introduction nocarcinoma accounts for approximately 20% of all cases, and is more often associated with HPV18 (2, 3). As a result of the implementation With an estimated 570,000 new cases in 2018 worldwide and over of population-based screening programs for the early detection of 311,000 deaths, cervical cancer is the fourth most common cancer in cervical neoplasia, the incidence of cervical cancer has decreased over women globally (1). The most prevalent histologic subtypes are the last 50 years and is expected to decrease further due to recently squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts introduced prophylactic HPV vaccination (4). However, the incidence for approximately 70% of all cases, and is mostly associated with an of cervical adenocarcinoma has shown a relative and absolute increase infection with human papillomavirus (HPV) type 16, whereas ade- compared with SCC over the past decades, especially in younger women (5–7). Several retrospective studies showed that patients with 1Cancer Center Amsterdam (CCA), Center for Gynecologic Oncology Amster- adenocarcinoma have a higher risk of developing metastases, resulting dam (CGOA), Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands. in a poorer prognosis (8–10). Cervical adenocarcinoma also differs 2Cancer Center Amsterdam (CCA), Department of Medical Oncology, Amster- from SCC in terms of prognostic factors, biological behavior, muta- dam UMC, Vrije Universiteit Amsterdam, the Netherlands. 3Center for Gyneco- tionalprofilesandsensitivitytochemo-and/orradiotherapy(2,11–15). logic Oncology Amsterdam (CGOA), The Netherlands Cancer Institute - Antoni However, patients with adenocarcinoma and SCC are still subjected to van Leeuwenhoek Hospital (NKI-AVL), Amsterdam, the Netherlands. 4Depart- the same conventional treatment modalities (ie, surgery and/or che- ment of Pathology, Amsterdam UMC, Vrije Universiteit Amsterdam, the Nether- lands. 5Department of Pathology, Leiden University Medical Center, Leiden, the moradiotherapy), often leading to infertility and reduced quality of Netherlands. life (16). Therefore, there is an urgent need to elucidate the differences between these histologic types, which could lead to more effective and Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). tumor-speci c treatment strategies. Cervical carcinogenesis is fueled by an active infection with high- E.S. Jordanova and T.D. de Gruijl contributed equally as co-senior authors of this article. risk types of HPV, and the immune system has been shown to be suppressed to prevent clearance of the tumor and maintain disease Corresponding Author: Tanja D. de Gruijl, Amsterdam UMC/Cancer Center fi þ progression (17, 18). Immunotherapy has proven its ef cacy in several Amsterdam, De Boelelaan 1117, Amsterdam 1081 HV, the Netherlands. Phone: 31 – þ tumor types (19 21), and the use of pembrolizumab in patients with 20 444 4063; Fax: 31 20 444 3620; E-mail: [email protected] þ advanced PD-L1 cervical cancer has recently been approved by the Clin Cancer Res 2020;XX:XX–XX FDA based on an overall response rate of 12.2% as reported by Chung doi: 10.1158/1078-0432.CCR-19-3826 and colleagues (22). Only five of 98 patients in this trial had cervical 2020 American Association for Cancer Research. adenocarcinoma, but interestingly, all were PD-L1 positive. In a

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analyses between immune profiling data from all available SCC Translational Relevance primary tumor and metastasis-free lymph node (LN-) samples and Immune checkpoint blockade (ICB) has recently been approved data from all the available adenocarcinoma primary tumor and LN- for cancer of the uterine cervix, although response rates are very samples. Although they were not a priori matched, no obvious low. Patients with adenocarcinoma and squamous cell carcinoma differences in clinical characteristics were apparent between these two (SCC) undergo the same standard treatment, even though these small SCC and adenocarcinoma patient cohorts (see Table 1). The histologic subtypes differ substantially in terms of HPV and clinical characteristics per patient from both adenocarcinoma and SCC mutation status, immune infiltrate, response to therapy, and cohorts and the collected tissue samples per patient (primary tumor, patient outcome. In this study, we comprehensively studied immu- lymph node, or both) are shown in Supplementary Tables S1 and S2). nologic differences in tumors and draining lymph nodes between Of note, primary tumors and corresponding lymph nodes were the two histologic subtypes in an effort to uncover specific potential collected for only eight patients (SCC, n ¼ 5; adenocarcinoma, n ¼ treatment options. Our findings show that adenocarcinoma tumors 3). Thus, the performed analyses were not paired. are less T-cell inflamed than SCC tumors and that this is linked to b-catenin activation, a defective chemokine response, and low Fresh tissue collection and processing frequencies of conventional type I dendritic cells. Importantly, Cells were scraped from a halved lymph node with a surgical blade, The Cancer Genome Atlas analyses show an association between as described previously (25). After collection of samples for this study, high transcript levels of these factors and overall survival benefit. the patient material was used for routine pathologic diagnostics. In Our findings point to specific therapeutic intervention options for case of a primary tumor, the specimen was first cut into small cervical adenocarcinoma that may facilitate effective ICB. fragments. All samples were collected in dissociation medium, con- sisting of RPMI1640 without HEPES (Lonza), 0.1% DNAse I (Roche), 0.14% collagenase A (Roche), 5% FCS (Hyclone), and a penicillin– smaller cohort of patients (n ¼ 24), an overall response rate of streptomycin–glutamine (PSG, Gibco) solution. Next, the cell suspen- 17% was reported, but only one patient with adenocarcinoma was sion was transferred to a sterile dissociation flask followed by incu- included (23). In these studies, no subgroup analyses were performed bation in a water bath at 37C on a magnet stirrer. Incubation time for adenocarcinoma versus SCC. Furthermore, immune checkpoint differed depending on the sample type, ranging from 30 minutes for blockade (ICB) with anti-CTLA-4 showed disappointing results in lymph node samples to three times 45 minutes (by refreshing disso- patients with recurrent and metastatic cervical cancer, and the only ciation medium each time) for a primary tumor biopsy. After incu- partial responder had SCC, while stable disease was observed in bation, the cell suspension was filtered through a 100-mm cell strainer patients with both adenocarcinoma and SCC subtypes (24). Several (BD Falcon), in Iscove's modified Dulbecco medium (IMDM; Lonza) (combination-) immunotherapy trials are currently ongoing (18), but containing 10% FCS, gentamicin/amphotericin B (Gibco), and PSG. again, no distinction is being made for histologic subtypes. Consid- Erythrocytes were lysed and viable cells were counted. Cells were ering the rising incidence rates of cervical adenocarcinoma and its poor directly used or cryopreserved until further use. prognosis, it is of great interest to study the immunologic behavior of the histologic subtypes. Because distinct therapeutic strategies for Multicolor flow cytometry adenocarcinoma and SCC have not yet emerged, this knowledge can Per sample, 1.5 105–2 105 cells were stained with the following contribute to the development of different immunotherapeutic com- surface antibodies: BDCA-3 (1:50, Miltenyi Biotec), BDCA-2 (1:20, binatorial approaches to improve response and tumor control. Miltenyi Biotec; labeled with FITC) CD3 (1:25, BD Biosciences), CD14 In this study, comprehensive flow cytometry analyses, functional (1:20, BD Biosciences; both labeled with PerCP-Cy5.5); CD4 (1:200, assays, and publicly available mRNA sequencing data of The Cancer BD Biosciences), CD45 (1:200, BioLegend; both labeled with AF700); Genome Atlas (TCGA) were used to determine immune cell subset CD8 (1:75, BD Biosciences; labeled with V500); CD25 (1:75, BD frequencies, activation status, and chemokine release in adenocarci- Biosciences), CD11c (1:100, BD Biosciences; both labeled with APC); noma versus SCC. By mapping out the immune landscape of cervical CD45RA (1:100, BD Biosciences; labeled with APC-Cy7/H7); CD27 adenocarcinoma and SCC, we aimed to uncover leads for novel (1:100, BD Biosciences; labeled with PE-Cy7); CD127 (1:50, BD immunotherapeutic options tailored for each histologic subtype. Biosciences), Tim-3 (1:20, BioLegend), EpCAM (1:50, BD Biosciences; all labeled with BV421); Lag-3 (1:20, eBioscience; labeled with PE-Cy7); CD123 (1:30, BD Biosciences; labeled with BV650), PD-1 Methods (1:20, BD Biosciences; labeled with BV786), CD1a (1:50, BD Bios- Patients ciences; labeled with PE), diluted in BD Horizon Brilliant Stain Buffer Patients (n ¼ 36) were included at the Antoni van Leeuwenhoek (BD Biosciences). After membrane staining, T cells were stained with Hospital (AVL) and the Amsterdam UMC, location AMC [Center for intracellular antibodies against FoxP3 (1:40, eBioscience; labeled with Gynecologic Oncology Amsterdam (CGOA)]. Clinical samples for PE); Ki-67 (1:50, BD Biosciences; labeled with FITC); and CTLA-4 immunomonitoring were processed at the Amsterdam UMC, location (1:100, BD Biosciences; labeled with PE-CF594) using a FoxP3 staining VUmc - Cancer Center Amsterdam (CCA). All included patients were kit according to the manufacturer's instructions (eBioscience). þ diagnosed with cervical SCC or adenocarcinoma and were scheduled Frequencies of CD4 T-cell populations were based on expression of for a (radical) hysterectomy, and/or pelvic lymphadenectomy. The FoxP3 and CD45RA as previously proposed by Miyara and collea- þ study was approved by the local Institutional Review Board (no. gues (26). We recently published examples of CD4 subpopulation NL25610.058.08) and executed in accordance with the ethical guide- T-cell gating strategies within cervical cancer (25). The gating strategy þ þ lines of the 1975 Declaration of Helsinki. Patients gave written of CD1a migratory cDCs, CD14 antigen-presenting cells (APC), the informed consent prior to participation. The patients’ clinicopatho- type 1 conventional dendritic cells (cDC1), and plasmacytoid DCs logic characteristics are shown in Table 1. We recently published data (pDC) are shown in Supplementary Fig. S1. Specifications of the on the SCC cohort (25). In this study, we have undertaken comparative antibodies used are listed in Supplementary Table S3. Multicolor flow

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Table 1. Clinical and histopathologic characteristics of the study population.

Clinical characteristics TDLN ACa (n ¼ 11) TDLN SCC (n ¼ 13) P PT AC (n ¼ 11) PT SCC (n ¼ 10) P

Age, mean SD 47.2 6.5 43.8 9.4 0.34 43.45 8.9 41.9 12.1 0.48 FIGO stageb 0.26 0.54 IB1 9 (82) 6 (46) 7 (64) 7 (70) IB2 1 (9) 3 (23) 3 (27) 2 (20) IIA1 1 (9) 0 1 (9) 0 IIA2 0 1 (8) 0 1 (10) IIB 0 2 (15) 0 0 IIIB 0 1 (8) 0 0 Vaginal involvementc 0.15 >0.99 Yes 1 (9) 0 0 1 (10) No 10 (91) 10 (77) 10 (100) 9 (90) Unknown 0 3 (23) 0 0 Parametrium invasionc 0.13 >0.99 Yes 1 (9) 5 (38) 1 (9) 0 No 10 (91) 7 (54) 10 (91) 10 (100) Unknown 0 1 (8) 0 0 HPV type -- 16 4 (37) 7 (54) 1 (9) 5 (50) 18 1 (9) 0 2 (18) 0 31 0 1 (8) 0 0 33 0 0 0 1 (10) 59 0 0 0 1 (10) Unknown 1 (9) 4 (30) 1 (9) 2 (20) P16þd 3 (27) 1 (8) 4 (37) 1 (10) Negative 1 (9) 0 0 0 hrHPVþe 1 (9) 0 3 (27) 0

Note: Values in the table expressed as n (%). Abbreviations: AC, adenocarcinoma; FIGO, International Federation of Gynecology and Obstetrics; HPV, human papillomavirus; PT, primary tumor; SCC, squamous cell carcinoma. aTwo lymph nodes were collected from the same patient (sentinel and nonsentinel). bFIGO-staging was based on clinical characteristics. cVaginal and parametrium invasion were based on pathology reports and in case this information was missing, based on clinical findings. dIn case no HPV typing was performed, results of the p16 staining are shown. ehrHPV-positive, but exact type unknown. cytometry was performed using the LSR Fortessa X-20 flow cytometer Chemokine release with TLR7/8 ligands and CBA read-out (BD Biosciences). Data were analyzed using the Kaluza flow cytometry Per well, 1 105 disaggregated cells from primary tumors (n ¼ 8 for analysis software version 1.3 (Beckman Coulter). SCC, n ¼ 4 for adenocarcinoma) were plated into a U-bottom 96-well plate in 200-mL IMDM medium with 10% FCS and PSG. Cells mRNAseq TCGA patient cohort were cultured without stimulation, or with the toll-like receptor Differential gene expression was examined using a publically avail- 7/8 (TLR7/8) ligand resiquimod (R848) (10 mg/mL, InvivoGen). After able dataset. The R2 genomics analysis and visualization platform an incubation of 24 hours at 37C, supernatant was collected and (https://r2.amc.nl) has the TCGA dataset on gene expression in stored at -20C until further analysis. A Cytometric Bead Array (CBA) cervical cancer available (27). For our analyses, RSEM-normalized was performed with the human chemokine flex kit (BD Biosciences) mRNA expression data from patients with adenocarcinoma and SCC to measure CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC), were used (n ¼ 299). For the generation of gene expression signatures, CXCL8 (IL8), CCL2 (MCP-1), and CCL5 (RANTES) production in fi normalized expression values were log2 transformed and the mean the collected supernatants. For speci cations of the used beads, see value of the various signature defining transcripts was calculated. The Supplementary Table S3. Per sample, 15-mL supernatant was used and following gene signatures were used, based on previously published the samples were analyzed on the BD LSR Fortessa flow cytometer. The þ data: CD8 effector T-cell score (CD8A, CD8B, IFNG, PRF1, GZMB), quantity of each chemokine was calculated using FCAP array software CXCL-score (CXCL9, CXCL10, CXCL11; ref. 28), cDC1-score (BATF3, version 3.0 (BD Biosciences), reported in pg/mL, and values below the XCR1, CLEC9A, CLNK; refs. 29, 30), cDC1 chemoattractant score detection limit were set to zero. (CCL5, XCL1, XCL2; ref. 29), and a b-catenin signaling activation score (ALCAM, AQP4, AQP5, BMP4, CDH16, CHGA, CXCL8, EPCAM, Statistical analysis FOXA2, HHIP, IGF2BP1, LGR5, POU3F2, SHH, SIX1, SOX11, TH, To compare clinicopathologic characteristics of patients in the TMSB15A, TSPAN8, WIF1; ref. 31). Recurrence-free and overall adenocarcinoma versus SCC cohort, a Fisher exact test (2 2 tables) survival analyses were performed using Kaplan–Meier Plotter or a x2 test was used to assess significant differences. Normal distri- (kmplot.com), which was previously used for studies involving bution was assessed with the D’Agostino–Pearson omnibus test or several tumor types (32–35).Thresholds for follow-up were set at Shapiro–Wilk test. An unpaired Student t test (in case of normally 60 months. distributed data) or the Mann–Whitney test were applied to assess

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Figure 1. T-cell frequencies and subsets in primary tumor (PT) samples of patients with cervical adenocarcinoma (AC) and SCC. A, Lower percentages of CD45þ leukocytes (left), lower CD45þ:EpCAM ratio (middle) and percentages of tumor-inﬁltrating CD3þ lymphocytes in adenocarcinoma primary tumor as compared with SCC primary tumor. B, No signiﬁcant differences in CD4þ, CD8þ, double negative (DN; CD4CD8) T-cell frequencies, and double positive (DP; CD4þCD8þ) T-cell frequencies in adenocarcinoma primary tumor compared with SCC primary tumor. C, Higher frequencies of CD4þ non-Tregs in adenocarcinoma primary tumor, and more CD4þ total Tregs in SCC primary tumors (left). Lower frequencies of na€ve (nCD4þ, FoxP3CD45RAþ)and FCD4þ (FoxP3CD45RA)inSCC primary tumor, same frequencies of FþCD4þ (FoxP3intCD45RA)conventional CD4þ T cells, and resting regulatory T cells (rCD4þTregs, FoxP3intCD45RAþ), but higher percentages of activated Tregs (aCD4þTregs, FoxP3hiCD45RA)in SCC primary tumor (right). Error bars, SEM (, P ≤ 0.05; , P ≤ 0.01).

statistically significant differences between groups. Correlations between SCC and adenocarcinoma primary tumors. However, within þ were determined by the Pearson r test. All statistical analyses were the CD4 T-cell population, the distribution between regulatory T performed using GraphPad Prism 8.0 (GraphPad Software Inc.). cells (Treg) and non-Tregs was skewed in favor of Tregs in SCC Differences were considered statistically significant when P < 0.05. primary tumor (Fig. 1C, left), with relatively more na€ve T cells þ þ (nCD4 ; FoxP3 CD45RA ), albeit at very low frequencies, and þ memory T cells (F CD4 ; FoxP3 CD45RA ) present in the adeno- Results carcinoma primary tumor (Fig. 1C, right). Specifically, the frequencies Lower level of T-cell infiltration in adenocarcinoma tumors of activated, that is, functionally suppressive (26), Tregs (aTregs; þ First, we compared the immune infiltration rates in the primary FoxP3 CD45RA ) were significantly higher in SCC primary tumor tumor of patients with cervical SCC with those of patients with (Fig. 1C, right). Taken together, these data indicate that compared adenocarcinoma. On the basis of expression of the marker CD45 with adenocarcinoma, the TME of SCC is more T-cell infiltrated, but within the total cell population, significantly higher percentages of also more immune suppressed, as judged by Treg activation. þ þ CD45 immune cells were found in SCC (Fig. 1A, left). Next, CD45 infiltration was related to the number of tumor cells, based on the Immune checkpoint expression and differentiation state of þ expression of the tumor marker EpCAM (Fig. 1A, middle). Indeed, CD8 T cells in primary tumors and TDLNs of patients with þ higher ratios of infiltrating immune cells to EpCAM tumor cells were adenocarcinoma þ found in SCC. Also, CD3 T cells were observed at higher frequencies We next analyzed immune checkpoint expression on effector within the SCC tumor microenvironment (TME; Fig. 1A, right). T cells in primary tumor and TDLN samples. Overall, higher þ þ Subsequently, the frequencies of CD4 T cells, CD8 T cells, expression levels of the various immune checkpoints were observed þ double-negative (DN; CD4 CD8 ), and double-positive cells (DP; on CD8 T cells in primary tumors of patients with SCC as þ þ CD4 CD8 ) were determined (Fig. 1B). No differences were observed compared with adenocarcinoma (Fig. 2A). Specifically, the

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Figure 2. Effector–memory differentiation of CD8þ T cells within TDLN and primary tumor (PT) of patients with adenocarcinoma (AC) versus SCC. A, In adenocarcinoma primary tumor, significantly more cells had intermediate PD-1 expression as compared with SCC primary tumor, where more cells had high expression of PD-1. (Co) expression of PD-1 and TIM-3 was significantly higher in SCC primary tumor. B, Immune checkpoint expression in TDLN of adenocarcinoma versus SCC patients. Significantly higher (co) expression of PD-1, LAG-3 and TIM-3 was observed in SCC TDLN. Within the tumor microenvironment, frequencies of CD8þ central memory (Tcm, CD27þCD45RA), effector memory (Tem, CD27CD45RA), effector memory RA (Temra, CD27CD45RAþ) CD8þ T cells and na€ve CD8þ T cells (Tn, CD27þCD45RAþ; C). No significant differences in percentages of CD8þ T-cell subsets were observed. D, Only in TDLN differences were observed in percentages of Tcm, Tem, and Tn for adenocarcinoma versus SCC, with more effector memory differentiation of CD8þ T cells within the adenocarcinoma TDLN compared with SCC. Error bars, SEM (, P ≤ 0.05; , P ≤ 0.01; , P ≤ 0.001).

þ combination of more frequent CD8 T cells with high PD-1 effector T cells. We tested this hypothesis by analyzing publicly þ expression levels and higher rates of CD8 T cells (co-) expressing available cervical cancer mRNAseq data of the TCGA database. We TIM-3, LAG-3, and/or PD-1, pointed to a generally more generated a CXCL9/10/11 score and compared the expression for þ “exhausted” state of CD8 T cells in SCC. Also in the TDLNs of adenocarcinoma versus SCC. As hypothesized, significantly higher þ þ þ patients with SCC, more CD8 T cells were TIM-3 ,PD-1 TIM- expression of these chemokines was observed in SCC (Fig. 3A,left). þ þ 3 ,andLAG-3 (Fig. 2B). Similarly, higher immune checkpoint We further assessed whether this chemokine score was correlated þ þ þ expression levels were found on conventional CD4 Th cells in with the presence of CD8 effector T cells, by creating a CD8 T- SCC, both in tumors and TDLNs (Supplementary Fig. S2). cell effector score comprising the following genes: CD8A, CD8B, þ While the distribution between CD8 T-cell differentiation states IFNG, PRF1,andGZMB. Indeed, a strong correlation was observed þ within the primary tumors of adenocarcinoma versus SCC was the betweenthechemokinescoreandtheeffectorCD8 T-cell score, þ same (based on relative percentages within the whole CD8 T-cell with adenocarcinoma tumors generally scoring low on both population, see Fig. 2C), surprisingly, in the TDLNs of patients with (Fig. 3A, right), thus supporting our hypothesis of hampered þ adenocarcinoma, we observed more CD8 memory T-cell differenti- trafficking of effector T cells due to low-level chemokine release. þ þ ation. Here, increased rates of central memory CD8 T cells (Tcm; Recent studies have identified BDCA3/CD141 cDC1 as an essen- þ þ CD27 CD45RA ) and effector memory CD8 T cells (Tem; tial source of T-cell attracting chemokines in the TME (28, 36). We þ CD27 CD45RA ) were found and lower frequencies of na€ve CD8 therefore assessed the presence of cDC1 in the primary tumor TME þ þ T cells (Tn; CD27 CD45RA ) when compared with SCC TDLNs of the two histologic subtypes. Indeed, a higher cDC1 transcrip- (Fig. 2D). tional signature (which included CLEC9, XCR1, CLNK,andBATF3) was observed in SCC primary tumor (Fig. 3B;left).Furthermore, þ Lower CD8 effector T-cell rates and chemokine release levels this signature showed a positive correlation with CXCL9/10/11 in adenocarcinoma are related to cDC1 content transcript levels (Fig. 3B; right). To validate the observation of þ The combination of apparently intact CD8 T-cell differentia- lower expression levels of T-cell–attracting chemokines in adeno- tion in TDLN, but low levels of T-cell recruitment to the primary carcinoma, we performed overnight cultures with single-cell sus- tumors of patients with adenocarcinoma led us to hypothesize that pensions of adenocarcinoma and SCC primary tumors, with and the patients’ primary tumors might be impaired in the production without R848, a ligand for TLR8, which is known to be expressed by of effector T-cell–attracting chemokines, such as CXCL9, CXCL10, cDC1 (37). Subsequently, release of the chemokines CXCL8, CCL2, and CXCL11, which bind to the CXCR3 chemokine receptor on and-5,andCXCL9,-10,and-11wasassessedbyCBA(Fig. 3C).

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Figure 3. Chemokine transcript expression and protein release in adenocarcinoma (AC) versus SCC primary tumor (PT) is related to cDC1 content. A, Mean of CXCL9/10/11 was used to compare the transcriptional signature of the chemokines in adenocarcinoma versus SCC primary tumor (left). The CXCL9/10/11 signature score was plotted against the CD8þ effector T-cell score (CD8A, CD8B, IFNG, PRF1, GZMB, right) showing a signiﬁcant correlation (right). B, TCGA analyses of mRNA expression data shows that the cDC1 score (CLEC9A, XCR, CLNK, BATF3) is higher within the SCC primary tumor samples compared with adenocarcinoma primary tumor (left). (Continued on the following page.)

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Figure 4. Factors responsible for attraction and maintenance of cDC1 in TME. Higher transcript levels of the cDC1 chemoattractant signature score (composed of XCL1, XCL2, and CCL5) and CCL4 and lower transcript levels of the b-catenin response signature (activation) score (ALCAM, AQP4, AQP5, BMP4, CDH16, CHGA, CXCL8, EPCAM, FOXA2, HHIP, IGF2BP1, LGR5, POU3F2, SHH, SIX1, SOX11, TH, TMSB15A, TSPAN8, WIF1) in SCC samples compared with adenocarcinoma (AC). Significant positive correlations were found for the cDC1 chemoattractant score and CCL4 versus the cDC1 score. A significant negative correlation was found for the b-catenin signature score versus the cDC1 score. No significant difference for FLT3L and only a weak correlation with the cDC1 score was found between adenocarcinoma and SCC. r, Pearson correlation coefficient (, P ≤ 0.001; , P ≤ 0.0001).

Both in steady state and upon R848-mediated activation, generally CXCL9, Fig. 3E). A strong positive correlation between the cDC1 gene þ higher levels of these chemokines were observed in SCC, except for signatureandtheCD8 effector T-cell score lent further support for a þ CXCL11, which remained below detection levels. Upon stimulation, role of the cDC1 in attracting CD8 effector T cells to the TME in cervical significant increases in the release of CXCL8, CCL5, and CXCL10 were cancer (Fig. 3F). This correlation further showed the adenocarcinoma þ found, but only in SCC, thus confirming an intrinsically hampered TME to be relatively low on both cDC1 and CD8 effector T cells. ability for effector chemokine release in adenocarcinoma. To assess whether this could be related to cDC1 content, the frequencies of b-Catenin activation and a lack of cDC1-attracting chemokines different APC subsets in primary tumor samples from patients with as underlying cause for low cDC1 content in adenocarcinoma adenocarcinoma versus SCC were determined by flow cytometry. Next, factors involved in the attraction of the cDC1 to the TME were Consistent with the TCGA analysis, the flow cytometry analysis assessed for SCC and adenocarcinoma based on TCGA gene expres- þ þ þ þ revealed that BDCA3 cDC1 (CD45 CD11c CD14 CD1a BDCA3 ) sion scores, and all were correlated to the cDC1 score (Fig. 4, top). were significantly more frequent in SCC (both as a ratio per Expression of CCL5, XCL1, and XCL2 (ie, the cDC1 chemoattractant þ þ þ EpCAM as well as a percentage of all CD45 CD11c cells), whereas score; ref. 29) was significantly higher in the SCC samples when þ þ þ þ no differences for CD1a DCs (CD45 CD11c CD14 CD1a ), compared with adenocarcinoma. No differences were found for the þ þ þ þ CD14 APCs (CD45 CD11c CD14 ), or plasmacytoid DCs (pDC, expression of fms-like tyrosine kinase 3 ligand (FLT3L), a growth þ þ þ CD45 BDCA2 CD123 ) were observed between SCC and adeno- factor for cDC1, between SCC and adenocarcinoma. Transcript levels carcinoma (Fig. 3D). In keeping with the notion that cDC1 might be a of CCL4, a chemokine recently shown in melanoma to attract cDC1 to major source of effector T-cell-attracting chemokines in the TME, low the TME and to be produced in the absence of active Wnt/b-catenin CXCL9- or -10 producing tumors (ie, lower than mean) had indeed signaling (38), were also increased in SCC. Interestingly, and consis- lower frequencies of cDC1, whereas high chemokine-producing tent with observations made by Spranger and colleagues, transcripts tumors had correspondingly higher cDC1 frequencies (P < 0.01 for of known response genes of the Wnt/b-catenin pathway as

(Continued.) A significant correlation (right) exists for the CXCL9/10/11 score versus cDC1 score. C, The production of the chemokines CXCL8, CXCL9, CXCL10, CCL5, CCL2, and CXCL11 in single cells suspensions of adenocarcinoma (n ¼ 4) and SCC (n ¼ 8) primary tumor in medium and R848 condition was assessed by using a CBA assay. Wilcoxon matched-pairs signed-rank test was used to compare differences in chemokine production upon R848 stimulation. Significantly higher chemokine production of CXCL8, CXCL10, and CCL5 was observed in SCC PT samples upon R848 stimulation compared to no stimulation. D, Flow cytometry analyses reveal a lower ratio per EpCAMþ cell of BDCA3þCD11cþCD45þ DCs, double-negative (DN) for CD14 and CD1a (DN BDCA3þ) and lower percentages of DN BDCA3þ as a percentage of all CD11cþCD45þ cells, in adenocarcinoma compared with SCC primary tumor. No differences in CD14þ and CD1aþ myeloid cells and pDCs were observed for adenocarcinoma versus SCC primary tumor. E, Percentages of BDCA3þ DN cells versus chemokine production of CXCL9 (left) and CXCL10 (right), based on mean release in unstimulated condition. F, A significant correlation for mRNA expression data of the cDC1 versus CD8þ T-cell signatures. Error bars, SEM. r, Pearson correlation coefficient (, P ≤ 0.01; , P ≤ 0.0001).

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described by Luke and colleagues (31) were found to be increased in immune infiltration in SCC may have actually triggered the suppres- adenocarcinoma samples. Strong significant correlations with the sive features that we observed in this histologic subtype, that is, cDC1 score were observed for the cDC1 chemoattractant score, increased Treg rates and higher immune checkpoint expression levels þ CCL4, and the b-catenin response gene score (see Fig. 4, bottom), on CD8 T cells. So they may have facilitated immune escape from an thus confirming b-catenin activation and a lack of CCL4-mediated ongoing antitumor response in the TME. Indeed, previous IHC-based cDC1 recruitment to likely be at the root of hampered T-cell studies showed that Tregs were more frequently present in SCC than in recruitment to the primary tumor in adenocarcinoma. adenocarcinoma, which in SCC correlated with a poor survival (42). In contrast, the presence of Tregs correlated positively with survival in þ Expression of gene signatures related to effector CD8 T cell adenocarcinoma, presumably because it was also indicative of a higher and cDC1 recruitment correlate with improved patient survival effector T-cell infiltration rate (43). By the same token, the upregula- Finally, we determined whether the transcriptional signatures that tion of HLA-DR and HLA-E on tumor cells that we previously þ we identified to be connected to cDC1 and/or effector CD8 T-cell reported in cervical adenocarcinoma, may have been induced by IFNg, recruitment, were related to overall survival (OS) and recurrence-free derived from any infiltrating T cells present. Consequently, these þ survival (RFS). Indeed, higher expression of CD8 effector T-cell and parameters may have served as surrogate markers for an ongoing effector T-cell-attracting chemokine (CXCL9, -10, -11) signature effector response, which could explain their association with improved genes was significantly associated with improved OS and RFS, respec- survival despite their obvious roles in immune suppression (44, 45). tively (Fig. 5; Supplementary Fig. S3). Also, a high cDC1 signature SCC were found to express other inhibitory receptors at higher levels as score was significantly associated with a better OS (Fig. 5). Finally, well, such as the C-type lectin MGL and PD-L1 on tumor cells and expression of genes involved in the attraction to or maintenance of tumor-associated macrophages, consistent with the more urgent need cDC1 within the TME (as determined by the cDC1 chemoattractant for immune escape in order for the tumor to grow and spread. score, b-catenin activation score, and FLT3L transcript levels) were Importantly, in lung cancer and esophageal cancer, differences in the also significantly associated with OS and/or RFS (Fig. 5; Supplemen- immune landscape were also reported among the histologic subtypes, tary Fig. 3). These data show that the prognosis of patients with cervical with evidence for different immune escape mechanisms and hence a þ cancer is related to processes involved in cDC1 and CD8 effector need for different immune interventions (15, 46, 47). T-cell recruitment to the TME, underlining the importance of these An obvious explanation for the lower T-cell content of adenocar- immune-cell subsets in orchestrating effective antitumor immunity. cinoma tumors might have been a lower intrinsic immunogenicity of Of note, adenocarcinoma numbers with available follow-up data were adenocarcinoma. However, this notion was refuted by the higher þ too low to perform separate survival analyses based on histological frequencies of CD8 effector memory and central memory T cells subtype. Nevertheless, the obvious difference in these signatures found in the tumor-free TDLNs of patients adenocarcinoma compared between adenocarcinoma and SCC may offer an underlying cause with SCC TDLNs, a clear sign of T-cell priming and differentiation. for the difference in prognosis associated with these two histological Rather, we interpreted this as being indicative of impaired trafficking subtypes as well as provide new leads for the effective treatment of of differentiated T cells to the tumor. Expression of the effector T-cell– patients with adenocarcinoma. attracting chemokines CXCL9, CXCL10, and CXCL11 has been associated with increased numbers of T cells in the TME and favorable outcome in patients with melanoma or colorectal cancer (28, 48–51). Discussion Similarly, we found that in cervical cancer chemokine gene transcripts þ In this study, we explored immunological differences between showed a positive correlation with a CD8 effector T-cell signature for cervical adenocarcinoma and SCC that would provide novel thera- both SCC and adenocarcinoma, with higher levels significantly asso- peutic targets for the treatment of adenocarcinoma. There is a clear ciating with improved OS. Noteworthy, patients with melanoma with unmet need in this regard, because patients with adenocarcinoma high pretreatment CXCL9 levels had an improved response to treat- currently receive the same treatment regimen as patients with SCC, ment with anti-PD-L1 (52), underlining the importance of the presence despite their clearly worse response to therapy. In freshly collected of these chemokines for an effective antitumor immune response. In primary tumor and TDLN tissues, we observed that the immune keeping with the lower T-cell content of adenocarcinoma tumors, they microenvironment in adenocarcinoma of the cervix is less immune also expressed significantly lower levels of CXCL9/-10/-11 transcripts. þ infiltrated compared with SCC in terms of frequencies of CD45 cells One of the crucial CXCL9- and CXCL10-producing immune-cell þ and CD3 T cells. Also, more evidence of immune suppression was subsets in the TME are cDC1 (28). The presence of cDC1has been observed in SCC with elevated levels of immune checkpoints (such as associated with patient survival and response to ICB across different PD-1 and TIM-3) and increased rates of Tregs. cDC1, that play an cancer types (53–55). Indeed, using the cervical cancer TCGA important role in the orchestration of an effective immune response by mRNAseq dataset, we found a strong positive correlation between both priming and attracting effector T cells, were found at lower CXCL9/-10/-11 transcripts and the gene expression signature for frequencies in adenocarcinoma. Our findings indicate that different cDC1. Furthermore, we observed higher numbers of cDC1 within immunotherapy strategies for patients with these different histological SCC tumors with accordingly higher CXCL9 and CXCL10 produc- subtypes of cervical cancer should be explored. tion levels. Altogether, these data point to the low frequencies of This is the first study to undertake an in-depth analysis of the cDC1as responsible for lower CXCL9 and CXCL10 expression levels differences in infiltrating T-cell content between the two main histo- and correspondingly lower T-cell content in adenocarcinoma. This logic subtypes of cervical cancer. In adenocarcinoma, lower frequen- also fits with the observation that increased CXCL9 and CXCL10 cies of T cells were found. This is of importance, because the presence production was fueled by 24-hour R848 stimulation of SCC but not þ of CD8 T cells in the TME has been shown to be prognostically of adenocarcinoma single-cell suspensions. favorable in multiple cancer types including cervical cancer and, As described by Spranger and colleagues (38), activation of the importantly, it has also been recognized as a possible predictive oncogenic WNT/b-catenin signaling pathway might prevent the pro- biomarker for response to ICB (39–41). Interestingly, this increased duction of CCL4 by tumor cells, and through that effect defective cDC1

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Figure 5. TCGA derived mRNAseq gene signatures and their correlation with overall survival. Prognostic value of the CD8þ effector T-cell score, CXCL9/10/11 score, cDC1 score, cDC1 chemoattractant score, and b-catenin activation score comparing top and bottom quartiles for overall survival within the cervical cancer TCGA dataset, obtained via kmplot.com. recruitment to the tumor. In this study, gene expression analyses showed higher in adenocarcinoma. In keeping with this observation, Noordhuis that there was a positive correlation between CCL4 and the cDC1 and colleagues previously reported on higher levels of membrane signature and indeed a negative correlation between the b-catenin expressed (ie, inactive) b-catenin in SCC primary tumor (56). Vice activation score and the cDC1 transcript levels. In seeming contradiction, versa, we found that CCL4 transcript levels were significantly lower in Luke and colleagues (31) recently reported a relatively weak association adenocarcinoma as compared with SCC. This clearly links active b-cate- þ between b-catenin activation and immune exclusion in cervical cancer, nin signaling in adenocarcinoma with defective cDC1 and CD8 effector but in that study no distinction was made between histologic subtypes. In T-cell recruitment and points the way to specific therapeutic intervention our study, we found that the b-catenin response score was significantly options for adenocarcinoma of the uterine cervix.

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Response rates to ICB in patients with cervical cancer remain low. recruitment that offers novel and specific treatment options for By enhancing cDC1 abundance and function, we might engender a adenocarcinoma of the uterine cervix. An important future direc- more T-cell–inflamed TME in adenocarcinoma tumors (ie, “cold” to tion for biomarker research would be to study b-catenin protein “hot” conversion) and possibly improve outcome of ICB with anti-PD- expression in relation to T-cell markers by IHC, as well as to study (L)1. This might be achieved by targeting the Wnt/b-catenin pathway, the possible role of b-catenin as a predictive biomarker upon anti- eg, by Wnt-inhibitors or by (oncolytic) viruses encoding constitutively PD-1 therapy in patients with cervical cancer. Besides, it would be active GSK3b (57), intratumoral injection of NK cells (29), or by the interesting to explore whether similar observations would hold true application of DC mobilizing and activating compounds like FLT3-L, for the same histologic subtype across different anatomically deter- CpG, or GM-CSF; refs. 58, 59). Importantly, as well-differentiated mined cancer types. þ CD8 T cells seem to be abundant in the adenocarcinoma TDLNs, targeting these TDLNs by intratumoral therapies, to leverage these T Disclosure of Potential Conflicts of Interest cells, would be an interesting option. No potential conflicts of interest were disclosed. As there is a wide range in b-catenin response scores among T-cell fl nonin amed adenocarcinoma tumors, other oncogenic signaling Authors’ Contributions pathways might as well be involved in the apparent immune exclusion Conception and design: J. Rotman, A.M. Heeren, E.S. Jordanova, T.D. de Gruijl and could be considered for therapeutic targeting. For instance, the Development of methodology: J. Rotman, A.M. Heeren, A.G.M. Stam, JAK-2/Stat3 and PI3K signaling pathways have also been described to E.S. Jordanova, T.D. de Gruijl affect T-cell migration and exclusion (60, 61). However, PI3K muta- Acquisition of data (provided animals, acquired and managed patients, provided tions have been described to occur more often in SCC of the cervix than facilities, etc.): J. Rotman, A.M. Heeren, A.A. Gassama, N. Pocorni, A.G.M. Stam, in adenocarcinoma (15). M.C.G. Bleeker, H.J.M.A.A. Zijlmans, C.H. Mom Analysis and interpretation of data (eg, statistical analysis, biostatistics, Otherchemokinesresponsiblefor the attraction of cDC1 into computational analysis): J. Rotman, A.M. Heeren, A.A. Gassama, A.G.M. Stam, tumors are CCL5, XCL1 and XCL2 which are produced by NK cells E.S. Jordanova, T.D. de Gruijl and by the tumor itself (36). In this study, the transcript levels of Writing, review, and/or revision of the manuscript: J. Rotman, M.C.G. Bleeker, CCL5, XCL1 and XCL2, were associated with the cDC1-specific H.J.M.A.A. Zijlmans, C.H. Mom, G.G. Kenter, E.S. Jordanova, T.D. de Gruijl gene signature and found to be significantly lower in adenocarci- Administrative, technical, or material support (ie, reporting or organizing data, noma. Bottcher€ and colleagues, described that in COX-deficient constructing databases): J. Rotman, A.M. Heeren, S.M. Lougheed Study supervision: G.G. Kenter, E.S. Jordanova, T.D. de Gruijl tumors, PGE2-mediated DC suppression was lifted and cDC1 were attracted to the tumor by the chemokines XCL1 and CCL5 (36). Indeed, in adenocarcinoma, higher COX-2 expression has been Acknowledgments observed when compared with SCC (62). Therefore, COX-2 inhi- The authors wish to thank Dr. Sanne Samuels for patient inclusion, all the patients who participated in this study and the staff of the CGOA, and pathology departments bition should also be considered in the treatment of cervical of AMC and AvL (both in Amsterdam, the Netherlands) for providing lymph node adenocarcinoma and could be combined with other agents acting and primary tumor samples. This work was supported by research grants from the to recruit cDC1 and/or effector T cells to the TME. Dutch Cancer Society (KWF VU 2013-6015), Stichting VUmc-CCA (CCA2015-306) As for SCC tumors, direct activation of the tumor-associated cDC1 and the Louise Vehmeijer Foundation. Dr. Ekaterina S. Jordanova was directly by (intratumorally delivered) TLR3 and/or (as applied ex vivo in this supported by these grants. study) TLR7/8 ligands seems sufficient to trigger CXCL9 and CXCL10 secretion. Combining this effector T-cell recruitment strategy The costs of publication of this article were defrayed in part by the payment of page with anti-PD-L1 with anti-TIM-3 or Treg-depleting agents such as charges. This article must therefore be hereby marked advertisement in accordance anti-CTLA-4, seems an attractive immunotherapeutic approach for with 18 U.S.C. Section 1734 solely to indicate this fact. the treatment of SCC. In conclusion, we have uncovered a link between oncogenic Received November 23, 2019; revised January 14, 2020; accepted March 23, 2020; þ b-catenin signaling and impaired cDC1 and CD8 effector T-cell published first March 27, 2020.

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Adenocarcinoma of the Uterine Cervix Shows Impaired Recruitment of cDC1 and CD8 + T Cells and Elevated β -Catenin Activation Compared with Squamous Cell Carcinoma

Jossie Rotman, A. Marijne Heeren, Awa A. Gassama, et al.

Clin Cancer Res Published OnlineFirst March 27, 2020.

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