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Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell... Background: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. Methods: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. Results: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm , 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p =0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. Conclusions: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, T , and CD20+ B-cells, supporting the notion that regs multiple cell types may potentiate tumor regression following PD-1 blockade. Keywords: PD-1, PD-L1, Merkel cell, Multispectral immunofluorescence, Pembrolizumab * Correspondence: jtaube1@jhmi.edu Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 2 of 11 Background immune cell subsets which may contribute to PD-1 Merkel cell carcinoma (MCC) is an aggressive uncommon biomarker relevance. cutaneous malignancy, for which two main etiologies have been described: Merkel cell polyomavirus (MCPyV) Methods infection, associated with approximately 80% of cases; and Case selection ultraviolet light exposure, which accounts for the This study was approved by the institutional review remaining 20% [1]. Patients with MCC often exhibit board of each participating institution. All participants oligoclonal lymphocyte-mediated and antibody-mediated provided written informed consent. Twenty-six patients immunity against MCC tumor antigens [2–4], with with stage IIIB or IV MCC were enrolled from January complex arrangements of immune cells, including B cells, 2015 until December 2015 and received at least one dose CD4+ and CD8+ T cells, macrophages and regulatory T of pembrolizumab on a phase 2, single-cohort, multicen- cells [5]. CD8+ tumor-infiltrating lymphocytes (TIL) [4, 6] ter clinical trial [11]. Objective responses were assessed and tumor cell PD-L1 expression [7] have been associated by the investigators according to RECIST, version 1.1 with improved patient survival, indicating that the [15]. A patient was considered to have an objective re- immune system is able to exert some control over this sponse to therapy if they demonstrated either a complete aggressive neoplasm. response (CR) or partial response (PR), per data analysis PD-1 is an inhibitory receptor expressed on various on 08/01/2016. A single representative pre-treatment immune cell subsets, including CD8+ and CD4+ T cells, formalin-fixed paraffin-embedded (FFPE) tumor speci- B cells, and natural killer cells [8]. The interaction be- men was chosen from each patient for additional studies. tween PD-1 and its ligands downregulates immune cell The minimum criteria for inclusion was a contiguous activation, proliferation, survival and cytokine produc- viable tumor deposit measuring >1 mm in size. tion [8, 9]. For this reason, therapeutic blockade of the A second cohort of 16 FFPE specimens acquired PD-1/PD-L1 checkpoint has been embraced as a strategy between 11/2002–04/2011 from 16 unique patients with to enhance antitumor immunity, with durable efficacy in stage IA- IV MCC was obtained from the Johns Hopkins some patients with multiple tumor types [10]. We Hospital (JHH) surgical pathology archives [7]. This recently reported that patients with advanced MCC cohort did not receive anti-PD-1 therapy. H&E slides receiving first-line treatment with pembrolizumab from each case were reviewed by a board-certified (anti-PD-1) experienced an objective response rate of dermatopathologist to confirm the diagnosis. A single 56% [11]. Also, patients treated with avelumab representative FFPE tumor block was chosen for add- (anti-PD-L1) showed a 66% and 32% response rate when itional studies. received in the first and second/third line settings, [12, 13] respectively. Many efforts to discover and validate Single IHC or IF stains biomarkers of response to anti-PD-(L)1 are currently Serial 4 μm FFPE pre-treatment tumor specimens from underway. The best-studied biomarker is tumor PD-L1 patients receiving anti-PD-1 were stained for CD8 (n = protein expression, measured by immunohistochemistry 23/26, mAb clone 144B, Dako, Carpinteria, CA), PD-1 (IHC) and graded by a pathologist as either “positive” or (n = 16/26, goat polyclonal Ab, R&D Systems, Minneap- “negative”. Across multiple solid tumor types, it has been olis, MN) or PD-L1 (n = 25/26, mAb clone 22C3, Merck shown that patients whose pre-treatment tumors are Research Laboratories, White House Station, NJ) by PD-L1+ demonstrate an enriched objective response rate IHC/IF, as previously described [11]. Archival specimens to anti-PD-(L)1, compared to their PD-L1- counterparts from patients not treated with anti-PD-1 were stained [14]. However, we found that the simple presence or for NKp46 using IHC. Appropriate positive and negative absence of tumor cell PD-L1 expression in MCC did not controls for each marker were run with every batch. correlate with anti-PD-1 response [11]. In the current study, we expanded our histopathologic analysis of the Pathologist interpretation of PD-L1 IHC MCC TME using next-generation digital pathology- PD-L1 expression on tumor cells (TC) and immune cells assisted quantitative methods, including topographic (IC) was scored by two pathologists blinded to patient quantitative density analyses and spatial proximity ana- outcomes (AS, JC). PD-L1 expression was assessed as lyses, to assess the density, distribution, and proximity of none (< 1%), 1%, 2–4%, 5–9%, 10–19%, and at increasing CD8+, PD-1+ and PD-L1+ cell populations. We found 10% intervals. PD-L1+ TC or IC had ≥1% positive cells. that the density of PD-1+ cells or PD-L1+ cells, and the number of PD-1+ cells in close proximity to PD-L1+ Digital image analysis for cell densities and proximity cells, each correlated with clinical response. While it is analysis using single IHC/IF stains assumed that PD-1 is mostly involved in regulation of Slides stained for PD-L1, PD-1 or CD8 were scanned CD8+ T-cell activity in the TME, we discerned multiple using Aperio ScanScope (Leica Biosystems Imaging, Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 3 of 11 Buffalo, IL). NKp46-stained slides were scanned using a Multiplex immunofluorescence (mIF) NanoZoomer XR (Hamamatsu Photonics, Hamamatsu Tumor specimens were available for additional study City, Japan). The resultant slide images were assessed with mIF from 6 patients who received anti-PD-1 [pro- using digital image analysis software (HALO V2.0, Indica gressive disease (PD), n =1; PR, n =4; CR, n = 1]. Speci- Labs, Corrales, NM). The tumor border was annotated mens were stained by mIF for CD8, PD-1, PD-L1, CD68, by a pathologist, and the area encompassed by this FoxP3 and neuron-specific enolase (NSE, tumor cells) as region was designated as intratumoral (IT). A 100 μm previously described [11]. In addition, 16 archival MCC distance beyond the tumor-stroma interface was desig- tumor specimens from patients who did not receive nated as the peritumoral (PT) region, Fig. 1. The num- anti-PD-1 therapy were stained by mIF for PD-1, CD8, ber of positive cells per mm displaying CD8, PD-1, CD4, CD20, Fox-P3, and NSE. PD-L1 or NKp46 was assessed as a continuous variable in the IT, PT or total (PT and IT) TME regions. PD-L1 Panel 1: (PD-L1, PD-1, NSE, CD68, CD8, DAPI) expression was quantified in the total fraction of tissue 4 μm-thick slides from FFPE tissue were heated at 57 °C surface area (total pixels positive/total pixels). Acellular overnight, and the residual paraffin was removed using and necrotic areas were excluded from analysis. xylene. After tissue rehydration using a series of graded al- To determine the proximity between cell membranes cohols to distilled water, antigen retrieval was performed displaying PD-L1 and either PD-1 or CD8, we used the using Tris-EDTA buffer and microwave treatment. Slides Serial Sections Alignment tool and Spatial Analysis were washed, and blocking was performed with 3% H2O2 Module in HALO. Specifically, serial sections that had blocking solution followed by Dako antibody diluent. The been stained for the markers of interest were registered, first primary antibody (“Position 1” in Table 1) was then i.e., Z-stacked, allowing for the assessment of two applied. Opal polymer HRP Ms. + Rb (Perkin Elmer, Hop- markers originally detected on two consecutive slides. kington, MA) was used as the secondary antibody. The The algorithm works by calculating the number of cells slides were washed, and the tyramide signal amplification within a given distance of another cell. First, the number (TSA)-dye (Opal 7 color kit, Perkin Elmer, Hopkington, of CD8+ or PD-1+ cells with their cell surfaces <20 μm MA) for Position 1 was applied. Slides were then micro- from of a PD-L1+ cell was determined across the total waved to strip the primary and secondary antibodies, TME (defined as the IT and PT regions combined) [16]. washed, and blocked again using blocking solution. The Then, the number of PD-L1+ cells with their cell sur- second primary antibody (“Position 2”) was applied, and faces < 20 um from a CD8+ cell or a PD-1+ cell was the process was repeated through amplification of the assessed. sixth primary antibody labeling. After the last step of Fig. 1 Tumor regions were annotated on high-resolution digital scans of slides stained by IHC/IF. a Peritumoral (PT, 100 um) and intratumoral (IT) regions were annotated. b Representative images for CD8 (brown), PD-1 (green) and PD-L1 (brown) staining from a non-responder (NR) and complete responder (CR) Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 4 of 11 Table 1 Primary antibody information for Multiplex IHC/IF panels Position Antibody Clone (host)/Company Dilution Incubation (min) TSA dye Panel 1 1 PD-L1 SP142 (rabbit)/Spring Bio 1:800 60 620 2 PD-1 EPR4877(2) (rabbit)/AbCam 1:1000 30 650 3 NSE BBS/NC/VI-H14(mouse)/Dako 1:1000 60 570 4 CD68 PGM-1(mouse)/Dako 1:500 30 540 5 CD8 4B11(mouse)/AbD 1:100 30 520 6 DAPI Perkin Elmer Opal 7-color kit 2 drops/ml 5 NA Panel 2 1 FoxP3 236A/E7(mouse)/abcam 1:100 30 570 2 NSE BBS/NC/VI-H14(mouse)/Dako 1:400 30 620 3 PD1 EPR4877(2)(rabbit)/abcam 1:500 120 650 4 CD4 EP204(rabbit)/Sigma 1:50 120 540 5 CD20 L26(mouse)/Leica 1:800 30 520 6 CD8 4B11(mouse)/AbD 1:100 30 690 7 DAPI Perkin Elmer Opal 7-color kit 2 drops/ml 5 NA antibody striping, DAPI was applied. After unbound After unbound DAPI was washed off, slides were cover- DAPI was washed off, slides were coverslipped using slipped using ProLong™ Diamond Antifade Mountant (Life VectaShield Antifade Mounting Medium (Vector Labs, Technologies, Waltham, MA). Burlingame,CA).Panel 1was performed using aman- ual method for staining. Slide scanning and analysis for multispectral IF/IHC Stained slides were scanned using the Vectra 3.0 Quantita- tive Pathology Imaging System (Perkin Elmer, Waltham, Panel 2: (PD-1, NSE, CD4, CD8, CD20, FoxP3, DAPI) MA). Ten high-power fields (HPF) along the tumor-stroma An automated protocol was used for Panel 2. Slides were interface enriched in immune cells (“hot-spots”)were heated at 60 °C for 30 min then Dewax (Leica Biosys- chosen for analysis in each specimen. InForm 2.3 Image tems, Buffalo Grove, IL) applied to remove any paraffin. Analysis software (Perkin Elmer) was used for spectral Antigen retrieval was performed using ER2 (Leica unmixing, cell segmentation, and identification and quanti- Biosystems, Buffalo Grove, IL) at 100 °C for 40 min fication of cellular subsets. The fraction of cells in each followed by a washing step. Non-specific staining was lineage was normalized by the number of tumor cells in blocked using Blocking/Ab Diluent (Perkin Elmer, each analyzed field. Hopkington, MA) for 5 min, then the first primary anti- body was applied, Table 1, followed by a washing step. ImmPRESS™ HRP Anti-Mouse IgG (Vector Laboratories, Tumor Merkel cell Polyomavirus (MCPyV) status Burlingame, CA) was applied for 15 min. The slides Tumor specimens were assessed for the presence of were washed, and the TSA-dye (Opal 7 color kit, Perkin MCPyV as previously described [7, 11]. Elmer, Hopkington, MA) for Position 1 was applied. Slides were then heated using ER1 (Leica Biosystems, Buffalo Grove, IL) at 95 °C for 20 min to strip the pri- Statistics mary and secondary antibodies, washed, and blocked Data are reported as the median and range in the text and again using Blocking/Ab Diluent. The second primary median ± IQR for figures. Two-sided Mann–Whitney antibody (Position 2) was applied, followed by Opal U-test was used to compare tumor-infiltrating immune polymer HRP Ms. + Rb (Perkin Elmer, Hopkington, cell densities between responders (R) vs. non-responders MA). The corresponding Opal was applied, and the anti- (NR); patients with PD-L1+ vs. PD-L1(−) tumors; and bodies stripped. The staining process was repeated for MCPyV+ vs. MCPyV- tumors. Chi-square test was used positions 3–6. After the last step of antibody striping, to compare the fraction of PD-L1+ tumors and the PD-L1 the slides were removed from the Bond Rx (Leica expression gradient (TC and IC) between R vs. NR, and Biosystems, Buffalo Grove, IL), and DAPI was applied. MCPyV+ vs. MCPyV- tumors. Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 5 of 11 Results interchangeable biomarkers of response to anti-PD-1 in Patient and specimen characteristics patients with MCC. Twenty-six MCC patients received anti-PD-1 therapy and had pretreatment tumor tissue available for study. PD-L1 density and total PD-L1 expression area correlate As of data analysis on 08/01/2016, 17 demonstrated an with response to anti-PD-1 objective response (CR = 5, PR = 12), 8 showed no PD-L1 status on tumor cells (TC) + immune cells (IC), or response (1 with stable disease and 7 with PD), and one TC or IC alone, was assigned by a pathologist as PD-L1+ patient demonstrated a transient PR that did not meet or PD-L1- using a 1% threshold. An association with clin- RECIST criteria. Fourteen tumors were primary lesions ical response was not observed, Additional file 1:Figure and twelve were metastases. The median interval S3A. When the percentage of all cells (TC + IC) in the between specimen acquisition and treatment initiation TME expressing PD-L1, as well as the percentage of TC was 5 months (range 7 days - 8 years), with 85% of the or IC, were studied at discrete intervals rather than specimens in the cohort being taken within 2 years of binomially, there was still no association with response, treatment initiation. PD-L1 IHC was performed on 25/ Additional file 1:FigureS3B. 26 specimens, CD8 IHC on 23/26, and PD-1 IF on 16/ We next evaluated the correlation between digitally- 26, depending on tissue availability. The median PD-1+ quantified PD-L1 expression (a continous variable) and and CD8+ cell densities in patients demonstrating CR response to anti-PD-1. Increased densities of PD-L1+ vs. PR were not significantly different, thus supporting cells (Fig. 2c) and an increased fraction of tissue surface the grouping of these patients as “Responders”. The area expressing PD-L1 (Fig. 2d) both correlated with im- density of PD-1+ and CD8+ cells also did not differ by proved response to PD-1 checkpoint inhibition (p = 0.02, whether the studied specimen was from a primary lesion p = 0.03, respectively). Similar associations held when or a metastasis. Six specimens had sufficient material for the IT and PT regions were examined separately, Add- IF multiplexing with a panel for NSE, PD-L1, PD-1, itional file 1: Figure S4. CD68, CD8, and FoxP3 expression. To further dissect potential associations between the To discern which cell types in the MCC TME express degree of CD8 cytotoxic T-cell infiltration and PD-1/ PD-1, a second cohort of 16 archival surgical pathology PD-L1 expression, the cohort was divided into quartiles specimens was studied. Six specimens were from of PD-L1+, CD8+ and PD-1+ total cell densities. All primary lesions and 10 from metastases. The archival patients in the highest quartile for each of these three specimens were previously characterized with regard to markers demonstrated a response, Additional file 1: MCPyV status, PT CD8+ density, and tumor cell PD-L1 Figure S5. None of the patients with the lowest PD-1+ expression, and these parameters were shown to signifi- cell density responded to therapy, while only one patient cantly associate with each other [7]. Among 26 patients among 5 in the lowest quartile of PD-L1 expression dem- who received anti-PD-1 therapy, similar findings were onstrated a response. In contrast, several patients in the observed, Additional file 1: Figure S1A. lowest quartile of CD8+ cell density showed a response. True comparison metrics of the sensitivity and specificity The density of PD-1+ cells, but not CD8+ cells or viral of these different markers will require studying larger status, correlates with response to anti-PD-1 cohorts, but these early findings suggest that PD-1+ cell We evaluated the correlation between the density and density in pretreatment tumor biopsies may be a better distribution of PD-1+ and CD8+ cells and response to predictor of response than PD-L1+ or CD8+ cell density. anti-PD-1. The total density of PD-1+ cells (PT + IT) was significantly higher in R vs. NR [median number of The density of PD-1+ lymphocytes adjacent to PD-L1+ positive cells/mm (range) 70.7(20.2–203.4) vs. 6.7(0–70), cells correlates with clinical response to anti-PD-1 therapy p = 0.03], Fig. 2a. In contrast, the total density of CD8+ We have previously reported examples of constitutive cells did not associate with response status [R 264/mm PD-L1 expression in the TME, i.e., not associated with (8.6–1712) vs. NR 216.6/mm (7.0–517.0), p = 0.17], Fig. an immune infiltrate [7, 17–20]. We posit that this pat- 2b. We further subdivided the TME into PT and IT tern may explain why a proportion of patients with regions and analyzed the PD-1+ and CD8+ cell densities PD-L1+ tumors do not respond to anti-PD-1/PD-L1, in each area separately, and similar results were [14, 21] as it is adaptive PD-L1 expression that indicates observed, Additional file 1: Figure S2. We also found an endogenous antitumor immunity [22]. One way to that while CD8+ cell densities, especially peritumoral, denote adaptive (as opposed to constitutive) PD-L1 associated with the presence of MCPyV, PD-1+ cell expression is the close proximity of PD-L1+ cells in the densities and response to therapy did not, Additional file TME to TILs [17]. As such, we calculated the density of 1: Figure S1b and Nghiem, et al. 2016. These findings PD-1+ or CD8+ TILs proximate to a PD-L1+ cell, indicate that CD8+ and PD-1+ cell densities are not Fig. 3a, as well as the density of PD-L1+ cells proximate Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 6 of 11 Fig. 2 PD-1+ and PD-L1+ cell densities correlate with clinical response to anti-PD-1. a Responders (R) had significantly higher median densities (±IQR) of PD-1+ when compared to non-responders (NR) b CD8+ cell densities were not significantly different between the two groups. c, d R had higher median densities (±IQR) of PD-L1+ cells as well as PD-L1+ tissue area when compared to NR. *p < 0.05. Assessments were made on the total TME (IT+PT). Results for each individual region are presented in Additional file 1: Figures S2 and Figure S4 to a PD-1+ or CD8 + cell. The density of PD-1+ cells response to anti-PD-1 therapy in patients with melan- adjacent to a PD-L1+ cell was significantly higher in R oma [23]. 2 2 vs. NR [69.9/mm (10.5–141.8) vs. 5.15/mm (0–32.4), p = 0.03], Fig. 3b. In contrast, the density of CD8+ cells in Multiplex analysis demonstrates that PD-1 is expressed close proximity to a PD-L1+ cell was not correlated with on multiple cell types in MCC clinical response to anti-PD-1 therapy [R 326.9/mm (67.3– We performed multiplex IHC/IF on six pre-treatment 748.8) vs. NR 152/mm (1–593.7), p = 0.46]. When the specimens from patients receiving anti-PD-1 therapy to transposed metric of PD-L1+ cell density proximate to a further characterize the MCC TME. We observed that PD-1+ or CD8 + cell was assessed for the relationship to while a substantial population of cells expressed both response, similar results were observed, Additional file 1: CD8 and PD-1, there were subpopulations that Figure S6. We next controlled for the density of PD-1+ and expressed one or the other marker, Fig. 4a. PD-1 can be PD-L1+ cells in each sample, and Responders still exhibited expressed not only by CD8+ but also by CD4+, CD20+, a significantly higher density of PD-1+ cells in proximity to T and NK cells. As such, a second multiplex panel reg PD-L1+ cells than Non-Responders, indicating that the was designed to assess the relative proportion of PD-1 proximity measurement reflects more than simply another expressed by these different immune cell subsets in a representation of PD-1+ and PD-L1+ cell density. cohort of archival MCC specimens, Fig. 4b.ANK Preliminary results have also been reported noting marker was not included in the multiplex panel due to the association between PD-1/PD-L1 ‘interaction’ and the very low density of NK cells in the MCC specimens Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 7 of 11 Fig. 3 The density of PD-1+ cells adjacent to a PD-L1+ cell correlates with clinical response to anti-PD-1. a Representative composite image depicting proximity analysis between PD-1+ and PD-L1+ cells performed using the HALO software Spatial Analysis module, Supplemental Methods. The distance between each PD-1+ cell and the nearest PD-L1+ cell (green circles) was calculated, and only those at a distance ≤20 μm (black lines) are quantified (red circles). b Responders (R) had significantly higher median densities (±IQR) of PD-1+, but not CD8+, cells interacting with PD-L1+ cells compared to non-responders (NR). *p < 0.05. The density of PD-L1+ cells within 20 μm of a PD-1+ or CD8+ cell was also calculated, and a similar association with response was observed, Additional file 1: Figure S6 shown on single IHC staining using anti-NKp46 (median nor the juxtaposition of CD8+ cells to PD-L1+ cells density 1 cell/mm ). correlate with response in this setting. The median density of CD8 + PD-1+ cells was 46.4/ Galon and colleagues demonstrated the prognostic mm (range 0.1–199.4), and the median density of utility of quantitative density assessments of lymphocyte CD4 + PD-1+ cells (T and T ) was similar [50.5/ subsets in specific geographic tumor regions for patients reg eff mm (0.0–278.0)]. On average, FoxP3+ cells represented with colorectal carcinoma. In their seminal studies, they approximately 22% of the CD4 + PD-1+ population. identified CD3+, CD8+ and CD45RO+ cells in the IT Scattered CD20 + PD-1+ cells were also found [median and PT regions with IHC and showed that the “Immu- 5.3/mm (0–30.2)]. One exceptional case demonstrated noscore” tiered scoring system based on cell density broad, constitutive PD-1 expression on tumor cells, Fig. measurements in these areas had the power to not only 4c. PD-1 expression on multiple immune cell types was augment, but sometimes surpass the predictive value of observed and was independent of the degree of inflam- TNM staging [24, 25]. Newly available multiplexed mation or viral status, Fig. 4d. imaging platforms have facilitated even more finely resolved spatial metrics, allowing for the enumeration of Discussion relationships between individual cells. For example, MCC appears to be highly responsive to anti-PD-1 ther- studies in pancreatic carcinoma and head and neck apy, regardless of viral status. Although anti-PD-1 respon- squamous cell carcinoma (HNSCC) have shown the siveness in some other cancer types has been correlated association between improved prognosis and proximity with PD-L1 expression (“positive” or “negative”)inpre- of specific cell types, e.g., CD8+ cells adjacent to cancer treatment tumor specimens; this has not been shown for cells, and between the number of CD8+ cells next to a MCC [11]. In the current study, we used sophisticated PD-L1+ or T cell, respectively [26, 16]. Similar reg digital image analysis for cell density along with carto- approaches were used to map the PD-L1+ microenviron- graphic assessments and found that higher-resolution mental niche for Reed-Sternberg cells in Hodgkin digitally-assisted quantitative measurements of the PD-1/ lymphoma [27]. PD-L1 axis do, in fact, associate with response to therapy. In addition to assisting with prognostication, immune Significant factors include PD-1+ cell density, PD-L1+ cell cell density measurements in the IT and PT regions have density, total surface area within the tumor mass display- been studied as predictive biomarkers for response to ing PD-L1, and the expression of PD-1 in close proximity anti-PD-1 [22, 28, 29]. The emphasis in most of the stud- to a PD-L1+ cell. Importantly, we also showed a divergent ies to date has been on CD8, rather than PD-1 expression. result for CD8+ cells, whereby neither CD8+ cell densities Our findings suggest that the precise quantification of Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 8 of 11 Fig. 4 Multiplex immunofluorescence studies demonstrate that PD-1 is expressed on multiple cell types in MCC, including CD8+ cells, CD4+ cells, T (CD4 + FoxP3+), CD20+ B-cells, and even sometimes on tumor cells. a Representative photomicrograph of multiplex panel (CD8, yellow; reg CD68, magenta; FoxP3, red; NSE (tumor), orange; PD-1, cyan; PD-L1, green and DAPI) from a responder in the cohort of patients treated with anti- PD-1. Higher magnification photomicrograph shows that while there is a significant proportion of PD-1+/CD8+ cells (arrow), there are also PD-1+ cells that are CD8- (arrowhead), and CD8+ cells that are PD-1- (asterisk). Left and right panels: 200× and 400× original magnification, respectively. b A second multiplex panel (PD-1, cyan; CD8, yellow; CD4, magenta; FoxP3, red; CD20, white; NSE (tumor), orange, and DAPI) was applied to archival MCC specimens to further characterize cell types expressing PD-1. Left panel: Representative photomicrograph showing host-tumor interface, 200× original magnification. Upper-right panel (1): Cell types expressing PD-1 include CD4 + FoxP3+ cells (arrow) and PD-1 + FoxP3- (arrowhead) cells, 400× original magnification. Lower-right panel (2): CD20+ B-cells (arrowhead) were also noted to express PD-1. (only CD20 and PD-1 channels are shown in the inset, 400× original magnfication). c In one case, low-level, constitutive PD-1 expression on nearly every tumor cell was observed (arrowhead). High levels of PD-1 expression were also seen on TIL (arrow). d PD-1+ cell densities across n = 16 tumor specimens show that PD-1 expression on multiple cell types is observed across different levels of inflammation. The virus status of each specimen is displayed below each specimen number PD-1+ cell densities could be of value to predict the disrupting the PD-1/PD-L1 interface. By adding a distance response to anti-PD-1 therapy. Because PD-1 is the direct assessment between these two molecules, we provide a target of anti-PD-1 drugs, it stands to reason that the more explicit marker of the PD-1/PD-L1 interaction. This amount of PD-1 in the TME may be a key component of effectively ‘corrects’ for the potential expression of one next generation biomarker panels. More specifically, immunoactive partner too far away from a likely anti-PD-1 agents are thought to exert their action by receptor-ligand pairing or in the absence of the other, for Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 9 of 11 example, in the case of oncogene-driven or “constitutive” cohorts. Due to the limited FFPE material, we were not tumor expression. able to apply our multiplex panels on specimens from To our knowledge, this is the first study reporting an patients treated with anti-PD-1. Thus we were not able association between PD-1+ cells densities and proximity to assess relative impact of PD-1 expression on CD8+ to a PD-L1+ cell and reponse to anti-PD-1 treatment. and CD4+ cells as well as PD-L1 expression on macro- One previous study assessed PD-1/PD-L1 distance and phages and tumor cells as they relate to anti-PD-1 association with response to anti-PD-1 in patients with response. Lastly, it will also likely be of value to study melanoma but reported a co-expression score (number on-treatment specimens, which have the potential to of microscopic fields/random disks where both PD-1 further inform mechanisms of response and resistance and PD-L1 were expressed) [22]. Such an approach does to anti-PD-1 in this tumor type. not provide an actual distance between PD-1+ and PD-L1+ cells, and in fact, could erroneously count cells Conclusions that are dual positive for PD-1 and PD-L1. In that study, The complexity of the TME has surpassed the digital the CD8 T-cells also represented the primary cellular reads of single-stain IHC as positive vs. negative. Value source of PD-1 expression. is gained by quantitating the density of cells expressing The differential association between PD-1+ and CD8+ PD-L1 or PD-1. The addition of spatial metrics, such as TIL densities with response to anti-PD-1 in MCC the density of PD-1+ cells within a given distance of a prompted us to explore other cell types in the MCC PD-L1+ cell, adds a new feature to predictive bio- TME expressing PD-1. We found that in addition to markers. By incorporating both partners of the CD8+ cells and a singular case of constitutive tumor cell receptor-ligand pair, this parameter more accurately re- expression, PD-1 was frequently expressed on CD4+ flects the fundamental mechanism underlying PD-1/ effector cells, T , and occasional CD20+ B-cells. In regs PD-L1 blockade. Lastly, evolving multiplexing technolo- fact, approximately half of the PD-1+ TILs were CD4+ gies facilitate studies of marker co-expression. We were (T or T ), which is consistent with studies of archival eff reg able to use these techniques to identify cell types beyond HNSCC, ovarian cancer, and Hodgkin lymphoma FFPE CD8 in the MCC TME expressing PD-1. While future specimens studied by IHC/IF; [27, 30–32] and melan- studies are needed to characterize the relative contribu- oma, renal cell carcinoma, and MCC specimens studied tions of each cell type participating in the anti-tumor by flow cytometry [33–35]. In vitro studies show that response, our study supports the evolving concept that PD-L1 engagement of PD-1 receptors on CD4+ cells lymphocytic populations beyond CD8+ cytotoxic T-cells causes T-cell dysfunction. CD4+ helping capacities (e.g., may promote tumor regression following anti-PD-1 IFN-γ and TNF-α production which promote CD8+ administration. T-cell effector programs) can be restored following administration of anti-PD-1 [36, 37]. Patients with Additional file advanced melanoma treated with pembrolizumab showed increased Ki-67 expression not only on CD8+ Additional file 1 Figure S1. CD8+ cell densities correlate with the presence of McPyV, but PD-1 densities do not correlate with viral status. cells, but also CD4+ cell populations, lending in vivo Figure S2. PD-1+ cell densities in both the peritumoral and intratumoral support to these in vitro findings [38]. Intriguing studies regions correlate with anti-PD-1 response, but CD8+ cell densities in suggest that antigen-specific CD4+ cells may assume these regions do not. Figure S3. Pathologist scores for PD-L1 expression levels did not associate with response to anti-PD-1 in patients with MCC. cytotoxic anti-tumor capabilities following immune Figure S4. Computer-assisted quantitation of PD-L1 in the PT and IT checkpoint blockade [39, 40]. This mechanism may be regions of tumor can help distinguish anti-PD-1 responders (R) from non- particularly relevant in patients with MCC and Hodgkin responders (NR). Figure S5. CD8+, PD-1+, and PD-L1+ TME cell densities by quartile from MCC patients receiving anti-PD1. Figure S6. The density lymphoma, both of which demonstrate high response of PD-L1+ cells adjacent to a PD-1+ cell correlates with clinical response rates to PD-1/PD-L1 checkpoint blockade despite to anti-PD-1. (PDF 611 kb) reduced MHC class I expression [41, 42]. The functional role of PD-1 on B-cells and T is not as well studied, regs Acknowledgements but recent results suggest that anti-PD-1 antibodies may The authors would like to thank Clifford Hoyt from Perkin Elmer and Darren also exert anti-tumor functions by arresting suppressive Locke from Bristol Myers Squibb for technical assistance, as well as Jessica Esandrio for administrative assistance. B-cells and T-cells, both of which express high levels of PD-1 [43, 44]. Funding MCC is an extremely rare cancer, affecting fewer than This work was supported by the Melanoma Research Alliance (JMT, SLT); 3000 patients each year in the US, and as such, the pri- Bristol-Myers Squibb (JMT, SLT); Sidney Kimmel Cancer Center Core Grant P30 CA006973 (JMT); National Cancer Institute R01 CA142779 (JMT, SLT); NIH mary limitation of this study is the number of specimens T32 CA193145 (TRC, AS, JES); NCI K24 CA139052 (PN); NIH Grant UM1 and amount of material per specimen available for study. CA154967 (MAC); NIH/NCI Cancer Center Support Grant P30 CA015704; the Our findings will need to be confirmed in larger MCC Bloomberg-Kimmel Institute for Cancer Immunotherapy; the Cancer Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 10 of 11 Immunotherapy Trials Network, and Stand Up To Cancer–Cancer Research In- 6. Paulson KG, Iyer JG, Simonson WT, Blom A, Thibodeau RM, Schmidt M, et al. stitute Cancer Immunology Translational Cancer Research Grant SU2C- CD8+ lymphocyte intratumoral infiltration as a stage-independent predictor AACR-DT1012. Stand Up To Cancer is a program of the Entertainment Industry of Merkel cell carcinoma survival: a population-based study. Am J Clin Foundation administered by the American Association for Cancer Research. Pathol. 2014;142:452–8. 7. Lipson EJ, Vincent JG, Loyo M, Kagohara LT, Luber BS, Wang H, et al. PD-L1 expression in the Merkel cell carcinoma microenvironment: association with Availability of data and materials inflammation, Merkel cell polyomavirus and overall survival. Cancer The datasets used and/or analyzed during the current study available from Immunol Res. 2013;1:54–63. the corresponding author on reasonable request. 8. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677–704. 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Evaluation of archived pathology specimens described in this manuscript is Avelumab in patients with chemotherapy-refractory metastatic Merkel cell covered under a study approved by the Johns Hopkins University carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Institutional Review Board which does not require individual patient consent. Oncol. 2016;17:1374–85. 13. D’Angelo SP, Russell J, Lebbé C, Chmielowski B, Gambichler T, Grob J-J, et al. Consent for publication Efficacy and safety of first-line Avelumab treatment in patients with stage IV Not applicable. metastatic Merkel cell carcinoma: a preplanned interim analysis of a clinical trial. JAMA Oncol. 2018. Competing interests 14. Giraldo NA, Taube JM. PD-L1 and Other Immunological Diagnosis Tools. EJL: consultant for Bristol-Myers Squibb, EMD Serono, Merck, Novartis; Oncoimmunology [Internet]. Springer, Cham;2018[cited2017Dec 19]. research funding from Bristol-Myers Squibb. p. 371–85. 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Copyright © 2018 by The Author(s).
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Medicine & Public Health; Oncology; Immunology
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Abstract

Background: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. Methods: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. Results: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm , 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p =0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. Conclusions: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, T , and CD20+ B-cells, supporting the notion that regs multiple cell types may potentiate tumor regression following PD-1 blockade. Keywords: PD-1, PD-L1, Merkel cell, Multispectral immunofluorescence, Pembrolizumab * Correspondence: jtaube1@jhmi.edu Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 2 of 11 Background immune cell subsets which may contribute to PD-1 Merkel cell carcinoma (MCC) is an aggressive uncommon biomarker relevance. cutaneous malignancy, for which two main etiologies have been described: Merkel cell polyomavirus (MCPyV) Methods infection, associated with approximately 80% of cases; and Case selection ultraviolet light exposure, which accounts for the This study was approved by the institutional review remaining 20% [1]. Patients with MCC often exhibit board of each participating institution. All participants oligoclonal lymphocyte-mediated and antibody-mediated provided written informed consent. Twenty-six patients immunity against MCC tumor antigens [2–4], with with stage IIIB or IV MCC were enrolled from January complex arrangements of immune cells, including B cells, 2015 until December 2015 and received at least one dose CD4+ and CD8+ T cells, macrophages and regulatory T of pembrolizumab on a phase 2, single-cohort, multicen- cells [5]. CD8+ tumor-infiltrating lymphocytes (TIL) [4, 6] ter clinical trial [11]. Objective responses were assessed and tumor cell PD-L1 expression [7] have been associated by the investigators according to RECIST, version 1.1 with improved patient survival, indicating that the [15]. A patient was considered to have an objective re- immune system is able to exert some control over this sponse to therapy if they demonstrated either a complete aggressive neoplasm. response (CR) or partial response (PR), per data analysis PD-1 is an inhibitory receptor expressed on various on 08/01/2016. A single representative pre-treatment immune cell subsets, including CD8+ and CD4+ T cells, formalin-fixed paraffin-embedded (FFPE) tumor speci- B cells, and natural killer cells [8]. The interaction be- men was chosen from each patient for additional studies. tween PD-1 and its ligands downregulates immune cell The minimum criteria for inclusion was a contiguous activation, proliferation, survival and cytokine produc- viable tumor deposit measuring >1 mm in size. tion [8, 9]. For this reason, therapeutic blockade of the A second cohort of 16 FFPE specimens acquired PD-1/PD-L1 checkpoint has been embraced as a strategy between 11/2002–04/2011 from 16 unique patients with to enhance antitumor immunity, with durable efficacy in stage IA- IV MCC was obtained from the Johns Hopkins some patients with multiple tumor types [10]. We Hospital (JHH) surgical pathology archives [7]. This recently reported that patients with advanced MCC cohort did not receive anti-PD-1 therapy. H&E slides receiving first-line treatment with pembrolizumab from each case were reviewed by a board-certified (anti-PD-1) experienced an objective response rate of dermatopathologist to confirm the diagnosis. A single 56% [11]. Also, patients treated with avelumab representative FFPE tumor block was chosen for add- (anti-PD-L1) showed a 66% and 32% response rate when itional studies. received in the first and second/third line settings, [12, 13] respectively. Many efforts to discover and validate Single IHC or IF stains biomarkers of response to anti-PD-(L)1 are currently Serial 4 μm FFPE pre-treatment tumor specimens from underway. The best-studied biomarker is tumor PD-L1 patients receiving anti-PD-1 were stained for CD8 (n = protein expression, measured by immunohistochemistry 23/26, mAb clone 144B, Dako, Carpinteria, CA), PD-1 (IHC) and graded by a pathologist as either “positive” or (n = 16/26, goat polyclonal Ab, R&D Systems, Minneap- “negative”. Across multiple solid tumor types, it has been olis, MN) or PD-L1 (n = 25/26, mAb clone 22C3, Merck shown that patients whose pre-treatment tumors are Research Laboratories, White House Station, NJ) by PD-L1+ demonstrate an enriched objective response rate IHC/IF, as previously described [11]. Archival specimens to anti-PD-(L)1, compared to their PD-L1- counterparts from patients not treated with anti-PD-1 were stained [14]. However, we found that the simple presence or for NKp46 using IHC. Appropriate positive and negative absence of tumor cell PD-L1 expression in MCC did not controls for each marker were run with every batch. correlate with anti-PD-1 response [11]. In the current study, we expanded our histopathologic analysis of the Pathologist interpretation of PD-L1 IHC MCC TME using next-generation digital pathology- PD-L1 expression on tumor cells (TC) and immune cells assisted quantitative methods, including topographic (IC) was scored by two pathologists blinded to patient quantitative density analyses and spatial proximity ana- outcomes (AS, JC). PD-L1 expression was assessed as lyses, to assess the density, distribution, and proximity of none (< 1%), 1%, 2–4%, 5–9%, 10–19%, and at increasing CD8+, PD-1+ and PD-L1+ cell populations. We found 10% intervals. PD-L1+ TC or IC had ≥1% positive cells. that the density of PD-1+ cells or PD-L1+ cells, and the number of PD-1+ cells in close proximity to PD-L1+ Digital image analysis for cell densities and proximity cells, each correlated with clinical response. While it is analysis using single IHC/IF stains assumed that PD-1 is mostly involved in regulation of Slides stained for PD-L1, PD-1 or CD8 were scanned CD8+ T-cell activity in the TME, we discerned multiple using Aperio ScanScope (Leica Biosystems Imaging, Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 3 of 11 Buffalo, IL). NKp46-stained slides were scanned using a Multiplex immunofluorescence (mIF) NanoZoomer XR (Hamamatsu Photonics, Hamamatsu Tumor specimens were available for additional study City, Japan). The resultant slide images were assessed with mIF from 6 patients who received anti-PD-1 [pro- using digital image analysis software (HALO V2.0, Indica gressive disease (PD), n =1; PR, n =4; CR, n = 1]. Speci- Labs, Corrales, NM). The tumor border was annotated mens were stained by mIF for CD8, PD-1, PD-L1, CD68, by a pathologist, and the area encompassed by this FoxP3 and neuron-specific enolase (NSE, tumor cells) as region was designated as intratumoral (IT). A 100 μm previously described [11]. In addition, 16 archival MCC distance beyond the tumor-stroma interface was desig- tumor specimens from patients who did not receive nated as the peritumoral (PT) region, Fig. 1. The num- anti-PD-1 therapy were stained by mIF for PD-1, CD8, ber of positive cells per mm displaying CD8, PD-1, CD4, CD20, Fox-P3, and NSE. PD-L1 or NKp46 was assessed as a continuous variable in the IT, PT or total (PT and IT) TME regions. PD-L1 Panel 1: (PD-L1, PD-1, NSE, CD68, CD8, DAPI) expression was quantified in the total fraction of tissue 4 μm-thick slides from FFPE tissue were heated at 57 °C surface area (total pixels positive/total pixels). Acellular overnight, and the residual paraffin was removed using and necrotic areas were excluded from analysis. xylene. After tissue rehydration using a series of graded al- To determine the proximity between cell membranes cohols to distilled water, antigen retrieval was performed displaying PD-L1 and either PD-1 or CD8, we used the using Tris-EDTA buffer and microwave treatment. Slides Serial Sections Alignment tool and Spatial Analysis were washed, and blocking was performed with 3% H2O2 Module in HALO. Specifically, serial sections that had blocking solution followed by Dako antibody diluent. The been stained for the markers of interest were registered, first primary antibody (“Position 1” in Table 1) was then i.e., Z-stacked, allowing for the assessment of two applied. Opal polymer HRP Ms. + Rb (Perkin Elmer, Hop- markers originally detected on two consecutive slides. kington, MA) was used as the secondary antibody. The The algorithm works by calculating the number of cells slides were washed, and the tyramide signal amplification within a given distance of another cell. First, the number (TSA)-dye (Opal 7 color kit, Perkin Elmer, Hopkington, of CD8+ or PD-1+ cells with their cell surfaces <20 μm MA) for Position 1 was applied. Slides were then micro- from of a PD-L1+ cell was determined across the total waved to strip the primary and secondary antibodies, TME (defined as the IT and PT regions combined) [16]. washed, and blocked again using blocking solution. The Then, the number of PD-L1+ cells with their cell sur- second primary antibody (“Position 2”) was applied, and faces < 20 um from a CD8+ cell or a PD-1+ cell was the process was repeated through amplification of the assessed. sixth primary antibody labeling. After the last step of Fig. 1 Tumor regions were annotated on high-resolution digital scans of slides stained by IHC/IF. a Peritumoral (PT, 100 um) and intratumoral (IT) regions were annotated. b Representative images for CD8 (brown), PD-1 (green) and PD-L1 (brown) staining from a non-responder (NR) and complete responder (CR) Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 4 of 11 Table 1 Primary antibody information for Multiplex IHC/IF panels Position Antibody Clone (host)/Company Dilution Incubation (min) TSA dye Panel 1 1 PD-L1 SP142 (rabbit)/Spring Bio 1:800 60 620 2 PD-1 EPR4877(2) (rabbit)/AbCam 1:1000 30 650 3 NSE BBS/NC/VI-H14(mouse)/Dako 1:1000 60 570 4 CD68 PGM-1(mouse)/Dako 1:500 30 540 5 CD8 4B11(mouse)/AbD 1:100 30 520 6 DAPI Perkin Elmer Opal 7-color kit 2 drops/ml 5 NA Panel 2 1 FoxP3 236A/E7(mouse)/abcam 1:100 30 570 2 NSE BBS/NC/VI-H14(mouse)/Dako 1:400 30 620 3 PD1 EPR4877(2)(rabbit)/abcam 1:500 120 650 4 CD4 EP204(rabbit)/Sigma 1:50 120 540 5 CD20 L26(mouse)/Leica 1:800 30 520 6 CD8 4B11(mouse)/AbD 1:100 30 690 7 DAPI Perkin Elmer Opal 7-color kit 2 drops/ml 5 NA antibody striping, DAPI was applied. After unbound After unbound DAPI was washed off, slides were cover- DAPI was washed off, slides were coverslipped using slipped using ProLong™ Diamond Antifade Mountant (Life VectaShield Antifade Mounting Medium (Vector Labs, Technologies, Waltham, MA). Burlingame,CA).Panel 1was performed using aman- ual method for staining. Slide scanning and analysis for multispectral IF/IHC Stained slides were scanned using the Vectra 3.0 Quantita- tive Pathology Imaging System (Perkin Elmer, Waltham, Panel 2: (PD-1, NSE, CD4, CD8, CD20, FoxP3, DAPI) MA). Ten high-power fields (HPF) along the tumor-stroma An automated protocol was used for Panel 2. Slides were interface enriched in immune cells (“hot-spots”)were heated at 60 °C for 30 min then Dewax (Leica Biosys- chosen for analysis in each specimen. InForm 2.3 Image tems, Buffalo Grove, IL) applied to remove any paraffin. Analysis software (Perkin Elmer) was used for spectral Antigen retrieval was performed using ER2 (Leica unmixing, cell segmentation, and identification and quanti- Biosystems, Buffalo Grove, IL) at 100 °C for 40 min fication of cellular subsets. The fraction of cells in each followed by a washing step. Non-specific staining was lineage was normalized by the number of tumor cells in blocked using Blocking/Ab Diluent (Perkin Elmer, each analyzed field. Hopkington, MA) for 5 min, then the first primary anti- body was applied, Table 1, followed by a washing step. ImmPRESS™ HRP Anti-Mouse IgG (Vector Laboratories, Tumor Merkel cell Polyomavirus (MCPyV) status Burlingame, CA) was applied for 15 min. The slides Tumor specimens were assessed for the presence of were washed, and the TSA-dye (Opal 7 color kit, Perkin MCPyV as previously described [7, 11]. Elmer, Hopkington, MA) for Position 1 was applied. Slides were then heated using ER1 (Leica Biosystems, Buffalo Grove, IL) at 95 °C for 20 min to strip the pri- Statistics mary and secondary antibodies, washed, and blocked Data are reported as the median and range in the text and again using Blocking/Ab Diluent. The second primary median ± IQR for figures. Two-sided Mann–Whitney antibody (Position 2) was applied, followed by Opal U-test was used to compare tumor-infiltrating immune polymer HRP Ms. + Rb (Perkin Elmer, Hopkington, cell densities between responders (R) vs. non-responders MA). The corresponding Opal was applied, and the anti- (NR); patients with PD-L1+ vs. PD-L1(−) tumors; and bodies stripped. The staining process was repeated for MCPyV+ vs. MCPyV- tumors. Chi-square test was used positions 3–6. After the last step of antibody striping, to compare the fraction of PD-L1+ tumors and the PD-L1 the slides were removed from the Bond Rx (Leica expression gradient (TC and IC) between R vs. NR, and Biosystems, Buffalo Grove, IL), and DAPI was applied. MCPyV+ vs. MCPyV- tumors. Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 5 of 11 Results interchangeable biomarkers of response to anti-PD-1 in Patient and specimen characteristics patients with MCC. Twenty-six MCC patients received anti-PD-1 therapy and had pretreatment tumor tissue available for study. PD-L1 density and total PD-L1 expression area correlate As of data analysis on 08/01/2016, 17 demonstrated an with response to anti-PD-1 objective response (CR = 5, PR = 12), 8 showed no PD-L1 status on tumor cells (TC) + immune cells (IC), or response (1 with stable disease and 7 with PD), and one TC or IC alone, was assigned by a pathologist as PD-L1+ patient demonstrated a transient PR that did not meet or PD-L1- using a 1% threshold. An association with clin- RECIST criteria. Fourteen tumors were primary lesions ical response was not observed, Additional file 1:Figure and twelve were metastases. The median interval S3A. When the percentage of all cells (TC + IC) in the between specimen acquisition and treatment initiation TME expressing PD-L1, as well as the percentage of TC was 5 months (range 7 days - 8 years), with 85% of the or IC, were studied at discrete intervals rather than specimens in the cohort being taken within 2 years of binomially, there was still no association with response, treatment initiation. PD-L1 IHC was performed on 25/ Additional file 1:FigureS3B. 26 specimens, CD8 IHC on 23/26, and PD-1 IF on 16/ We next evaluated the correlation between digitally- 26, depending on tissue availability. The median PD-1+ quantified PD-L1 expression (a continous variable) and and CD8+ cell densities in patients demonstrating CR response to anti-PD-1. Increased densities of PD-L1+ vs. PR were not significantly different, thus supporting cells (Fig. 2c) and an increased fraction of tissue surface the grouping of these patients as “Responders”. The area expressing PD-L1 (Fig. 2d) both correlated with im- density of PD-1+ and CD8+ cells also did not differ by proved response to PD-1 checkpoint inhibition (p = 0.02, whether the studied specimen was from a primary lesion p = 0.03, respectively). Similar associations held when or a metastasis. Six specimens had sufficient material for the IT and PT regions were examined separately, Add- IF multiplexing with a panel for NSE, PD-L1, PD-1, itional file 1: Figure S4. CD68, CD8, and FoxP3 expression. To further dissect potential associations between the To discern which cell types in the MCC TME express degree of CD8 cytotoxic T-cell infiltration and PD-1/ PD-1, a second cohort of 16 archival surgical pathology PD-L1 expression, the cohort was divided into quartiles specimens was studied. Six specimens were from of PD-L1+, CD8+ and PD-1+ total cell densities. All primary lesions and 10 from metastases. The archival patients in the highest quartile for each of these three specimens were previously characterized with regard to markers demonstrated a response, Additional file 1: MCPyV status, PT CD8+ density, and tumor cell PD-L1 Figure S5. None of the patients with the lowest PD-1+ expression, and these parameters were shown to signifi- cell density responded to therapy, while only one patient cantly associate with each other [7]. Among 26 patients among 5 in the lowest quartile of PD-L1 expression dem- who received anti-PD-1 therapy, similar findings were onstrated a response. In contrast, several patients in the observed, Additional file 1: Figure S1A. lowest quartile of CD8+ cell density showed a response. True comparison metrics of the sensitivity and specificity The density of PD-1+ cells, but not CD8+ cells or viral of these different markers will require studying larger status, correlates with response to anti-PD-1 cohorts, but these early findings suggest that PD-1+ cell We evaluated the correlation between the density and density in pretreatment tumor biopsies may be a better distribution of PD-1+ and CD8+ cells and response to predictor of response than PD-L1+ or CD8+ cell density. anti-PD-1. The total density of PD-1+ cells (PT + IT) was significantly higher in R vs. NR [median number of The density of PD-1+ lymphocytes adjacent to PD-L1+ positive cells/mm (range) 70.7(20.2–203.4) vs. 6.7(0–70), cells correlates with clinical response to anti-PD-1 therapy p = 0.03], Fig. 2a. In contrast, the total density of CD8+ We have previously reported examples of constitutive cells did not associate with response status [R 264/mm PD-L1 expression in the TME, i.e., not associated with (8.6–1712) vs. NR 216.6/mm (7.0–517.0), p = 0.17], Fig. an immune infiltrate [7, 17–20]. We posit that this pat- 2b. We further subdivided the TME into PT and IT tern may explain why a proportion of patients with regions and analyzed the PD-1+ and CD8+ cell densities PD-L1+ tumors do not respond to anti-PD-1/PD-L1, in each area separately, and similar results were [14, 21] as it is adaptive PD-L1 expression that indicates observed, Additional file 1: Figure S2. We also found an endogenous antitumor immunity [22]. One way to that while CD8+ cell densities, especially peritumoral, denote adaptive (as opposed to constitutive) PD-L1 associated with the presence of MCPyV, PD-1+ cell expression is the close proximity of PD-L1+ cells in the densities and response to therapy did not, Additional file TME to TILs [17]. As such, we calculated the density of 1: Figure S1b and Nghiem, et al. 2016. These findings PD-1+ or CD8+ TILs proximate to a PD-L1+ cell, indicate that CD8+ and PD-1+ cell densities are not Fig. 3a, as well as the density of PD-L1+ cells proximate Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 6 of 11 Fig. 2 PD-1+ and PD-L1+ cell densities correlate with clinical response to anti-PD-1. a Responders (R) had significantly higher median densities (±IQR) of PD-1+ when compared to non-responders (NR) b CD8+ cell densities were not significantly different between the two groups. c, d R had higher median densities (±IQR) of PD-L1+ cells as well as PD-L1+ tissue area when compared to NR. *p < 0.05. Assessments were made on the total TME (IT+PT). Results for each individual region are presented in Additional file 1: Figures S2 and Figure S4 to a PD-1+ or CD8 + cell. The density of PD-1+ cells response to anti-PD-1 therapy in patients with melan- adjacent to a PD-L1+ cell was significantly higher in R oma [23]. 2 2 vs. NR [69.9/mm (10.5–141.8) vs. 5.15/mm (0–32.4), p = 0.03], Fig. 3b. In contrast, the density of CD8+ cells in Multiplex analysis demonstrates that PD-1 is expressed close proximity to a PD-L1+ cell was not correlated with on multiple cell types in MCC clinical response to anti-PD-1 therapy [R 326.9/mm (67.3– We performed multiplex IHC/IF on six pre-treatment 748.8) vs. NR 152/mm (1–593.7), p = 0.46]. When the specimens from patients receiving anti-PD-1 therapy to transposed metric of PD-L1+ cell density proximate to a further characterize the MCC TME. We observed that PD-1+ or CD8 + cell was assessed for the relationship to while a substantial population of cells expressed both response, similar results were observed, Additional file 1: CD8 and PD-1, there were subpopulations that Figure S6. We next controlled for the density of PD-1+ and expressed one or the other marker, Fig. 4a. PD-1 can be PD-L1+ cells in each sample, and Responders still exhibited expressed not only by CD8+ but also by CD4+, CD20+, a significantly higher density of PD-1+ cells in proximity to T and NK cells. As such, a second multiplex panel reg PD-L1+ cells than Non-Responders, indicating that the was designed to assess the relative proportion of PD-1 proximity measurement reflects more than simply another expressed by these different immune cell subsets in a representation of PD-1+ and PD-L1+ cell density. cohort of archival MCC specimens, Fig. 4b.ANK Preliminary results have also been reported noting marker was not included in the multiplex panel due to the association between PD-1/PD-L1 ‘interaction’ and the very low density of NK cells in the MCC specimens Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 7 of 11 Fig. 3 The density of PD-1+ cells adjacent to a PD-L1+ cell correlates with clinical response to anti-PD-1. a Representative composite image depicting proximity analysis between PD-1+ and PD-L1+ cells performed using the HALO software Spatial Analysis module, Supplemental Methods. The distance between each PD-1+ cell and the nearest PD-L1+ cell (green circles) was calculated, and only those at a distance ≤20 μm (black lines) are quantified (red circles). b Responders (R) had significantly higher median densities (±IQR) of PD-1+, but not CD8+, cells interacting with PD-L1+ cells compared to non-responders (NR). *p < 0.05. The density of PD-L1+ cells within 20 μm of a PD-1+ or CD8+ cell was also calculated, and a similar association with response was observed, Additional file 1: Figure S6 shown on single IHC staining using anti-NKp46 (median nor the juxtaposition of CD8+ cells to PD-L1+ cells density 1 cell/mm ). correlate with response in this setting. The median density of CD8 + PD-1+ cells was 46.4/ Galon and colleagues demonstrated the prognostic mm (range 0.1–199.4), and the median density of utility of quantitative density assessments of lymphocyte CD4 + PD-1+ cells (T and T ) was similar [50.5/ subsets in specific geographic tumor regions for patients reg eff mm (0.0–278.0)]. On average, FoxP3+ cells represented with colorectal carcinoma. In their seminal studies, they approximately 22% of the CD4 + PD-1+ population. identified CD3+, CD8+ and CD45RO+ cells in the IT Scattered CD20 + PD-1+ cells were also found [median and PT regions with IHC and showed that the “Immu- 5.3/mm (0–30.2)]. One exceptional case demonstrated noscore” tiered scoring system based on cell density broad, constitutive PD-1 expression on tumor cells, Fig. measurements in these areas had the power to not only 4c. PD-1 expression on multiple immune cell types was augment, but sometimes surpass the predictive value of observed and was independent of the degree of inflam- TNM staging [24, 25]. Newly available multiplexed mation or viral status, Fig. 4d. imaging platforms have facilitated even more finely resolved spatial metrics, allowing for the enumeration of Discussion relationships between individual cells. For example, MCC appears to be highly responsive to anti-PD-1 ther- studies in pancreatic carcinoma and head and neck apy, regardless of viral status. Although anti-PD-1 respon- squamous cell carcinoma (HNSCC) have shown the siveness in some other cancer types has been correlated association between improved prognosis and proximity with PD-L1 expression (“positive” or “negative”)inpre- of specific cell types, e.g., CD8+ cells adjacent to cancer treatment tumor specimens; this has not been shown for cells, and between the number of CD8+ cells next to a MCC [11]. In the current study, we used sophisticated PD-L1+ or T cell, respectively [26, 16]. Similar reg digital image analysis for cell density along with carto- approaches were used to map the PD-L1+ microenviron- graphic assessments and found that higher-resolution mental niche for Reed-Sternberg cells in Hodgkin digitally-assisted quantitative measurements of the PD-1/ lymphoma [27]. PD-L1 axis do, in fact, associate with response to therapy. In addition to assisting with prognostication, immune Significant factors include PD-1+ cell density, PD-L1+ cell cell density measurements in the IT and PT regions have density, total surface area within the tumor mass display- been studied as predictive biomarkers for response to ing PD-L1, and the expression of PD-1 in close proximity anti-PD-1 [22, 28, 29]. The emphasis in most of the stud- to a PD-L1+ cell. Importantly, we also showed a divergent ies to date has been on CD8, rather than PD-1 expression. result for CD8+ cells, whereby neither CD8+ cell densities Our findings suggest that the precise quantification of Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 8 of 11 Fig. 4 Multiplex immunofluorescence studies demonstrate that PD-1 is expressed on multiple cell types in MCC, including CD8+ cells, CD4+ cells, T (CD4 + FoxP3+), CD20+ B-cells, and even sometimes on tumor cells. a Representative photomicrograph of multiplex panel (CD8, yellow; reg CD68, magenta; FoxP3, red; NSE (tumor), orange; PD-1, cyan; PD-L1, green and DAPI) from a responder in the cohort of patients treated with anti- PD-1. Higher magnification photomicrograph shows that while there is a significant proportion of PD-1+/CD8+ cells (arrow), there are also PD-1+ cells that are CD8- (arrowhead), and CD8+ cells that are PD-1- (asterisk). Left and right panels: 200× and 400× original magnification, respectively. b A second multiplex panel (PD-1, cyan; CD8, yellow; CD4, magenta; FoxP3, red; CD20, white; NSE (tumor), orange, and DAPI) was applied to archival MCC specimens to further characterize cell types expressing PD-1. Left panel: Representative photomicrograph showing host-tumor interface, 200× original magnification. Upper-right panel (1): Cell types expressing PD-1 include CD4 + FoxP3+ cells (arrow) and PD-1 + FoxP3- (arrowhead) cells, 400× original magnification. Lower-right panel (2): CD20+ B-cells (arrowhead) were also noted to express PD-1. (only CD20 and PD-1 channels are shown in the inset, 400× original magnfication). c In one case, low-level, constitutive PD-1 expression on nearly every tumor cell was observed (arrowhead). High levels of PD-1 expression were also seen on TIL (arrow). d PD-1+ cell densities across n = 16 tumor specimens show that PD-1 expression on multiple cell types is observed across different levels of inflammation. The virus status of each specimen is displayed below each specimen number PD-1+ cell densities could be of value to predict the disrupting the PD-1/PD-L1 interface. By adding a distance response to anti-PD-1 therapy. Because PD-1 is the direct assessment between these two molecules, we provide a target of anti-PD-1 drugs, it stands to reason that the more explicit marker of the PD-1/PD-L1 interaction. This amount of PD-1 in the TME may be a key component of effectively ‘corrects’ for the potential expression of one next generation biomarker panels. More specifically, immunoactive partner too far away from a likely anti-PD-1 agents are thought to exert their action by receptor-ligand pairing or in the absence of the other, for Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 9 of 11 example, in the case of oncogene-driven or “constitutive” cohorts. Due to the limited FFPE material, we were not tumor expression. able to apply our multiplex panels on specimens from To our knowledge, this is the first study reporting an patients treated with anti-PD-1. Thus we were not able association between PD-1+ cells densities and proximity to assess relative impact of PD-1 expression on CD8+ to a PD-L1+ cell and reponse to anti-PD-1 treatment. and CD4+ cells as well as PD-L1 expression on macro- One previous study assessed PD-1/PD-L1 distance and phages and tumor cells as they relate to anti-PD-1 association with response to anti-PD-1 in patients with response. Lastly, it will also likely be of value to study melanoma but reported a co-expression score (number on-treatment specimens, which have the potential to of microscopic fields/random disks where both PD-1 further inform mechanisms of response and resistance and PD-L1 were expressed) [22]. Such an approach does to anti-PD-1 in this tumor type. not provide an actual distance between PD-1+ and PD-L1+ cells, and in fact, could erroneously count cells Conclusions that are dual positive for PD-1 and PD-L1. In that study, The complexity of the TME has surpassed the digital the CD8 T-cells also represented the primary cellular reads of single-stain IHC as positive vs. negative. Value source of PD-1 expression. is gained by quantitating the density of cells expressing The differential association between PD-1+ and CD8+ PD-L1 or PD-1. The addition of spatial metrics, such as TIL densities with response to anti-PD-1 in MCC the density of PD-1+ cells within a given distance of a prompted us to explore other cell types in the MCC PD-L1+ cell, adds a new feature to predictive bio- TME expressing PD-1. We found that in addition to markers. By incorporating both partners of the CD8+ cells and a singular case of constitutive tumor cell receptor-ligand pair, this parameter more accurately re- expression, PD-1 was frequently expressed on CD4+ flects the fundamental mechanism underlying PD-1/ effector cells, T , and occasional CD20+ B-cells. In regs PD-L1 blockade. Lastly, evolving multiplexing technolo- fact, approximately half of the PD-1+ TILs were CD4+ gies facilitate studies of marker co-expression. We were (T or T ), which is consistent with studies of archival eff reg able to use these techniques to identify cell types beyond HNSCC, ovarian cancer, and Hodgkin lymphoma FFPE CD8 in the MCC TME expressing PD-1. While future specimens studied by IHC/IF; [27, 30–32] and melan- studies are needed to characterize the relative contribu- oma, renal cell carcinoma, and MCC specimens studied tions of each cell type participating in the anti-tumor by flow cytometry [33–35]. In vitro studies show that response, our study supports the evolving concept that PD-L1 engagement of PD-1 receptors on CD4+ cells lymphocytic populations beyond CD8+ cytotoxic T-cells causes T-cell dysfunction. CD4+ helping capacities (e.g., may promote tumor regression following anti-PD-1 IFN-γ and TNF-α production which promote CD8+ administration. T-cell effector programs) can be restored following administration of anti-PD-1 [36, 37]. Patients with Additional file advanced melanoma treated with pembrolizumab showed increased Ki-67 expression not only on CD8+ Additional file 1 Figure S1. CD8+ cell densities correlate with the presence of McPyV, but PD-1 densities do not correlate with viral status. cells, but also CD4+ cell populations, lending in vivo Figure S2. PD-1+ cell densities in both the peritumoral and intratumoral support to these in vitro findings [38]. Intriguing studies regions correlate with anti-PD-1 response, but CD8+ cell densities in suggest that antigen-specific CD4+ cells may assume these regions do not. Figure S3. Pathologist scores for PD-L1 expression levels did not associate with response to anti-PD-1 in patients with MCC. cytotoxic anti-tumor capabilities following immune Figure S4. Computer-assisted quantitation of PD-L1 in the PT and IT checkpoint blockade [39, 40]. This mechanism may be regions of tumor can help distinguish anti-PD-1 responders (R) from non- particularly relevant in patients with MCC and Hodgkin responders (NR). Figure S5. CD8+, PD-1+, and PD-L1+ TME cell densities by quartile from MCC patients receiving anti-PD1. Figure S6. The density lymphoma, both of which demonstrate high response of PD-L1+ cells adjacent to a PD-1+ cell correlates with clinical response rates to PD-1/PD-L1 checkpoint blockade despite to anti-PD-1. (PDF 611 kb) reduced MHC class I expression [41, 42]. The functional role of PD-1 on B-cells and T is not as well studied, regs Acknowledgements but recent results suggest that anti-PD-1 antibodies may The authors would like to thank Clifford Hoyt from Perkin Elmer and Darren also exert anti-tumor functions by arresting suppressive Locke from Bristol Myers Squibb for technical assistance, as well as Jessica Esandrio for administrative assistance. B-cells and T-cells, both of which express high levels of PD-1 [43, 44]. Funding MCC is an extremely rare cancer, affecting fewer than This work was supported by the Melanoma Research Alliance (JMT, SLT); 3000 patients each year in the US, and as such, the pri- Bristol-Myers Squibb (JMT, SLT); Sidney Kimmel Cancer Center Core Grant P30 CA006973 (JMT); National Cancer Institute R01 CA142779 (JMT, SLT); NIH mary limitation of this study is the number of specimens T32 CA193145 (TRC, AS, JES); NCI K24 CA139052 (PN); NIH Grant UM1 and amount of material per specimen available for study. CA154967 (MAC); NIH/NCI Cancer Center Support Grant P30 CA015704; the Our findings will need to be confirmed in larger MCC Bloomberg-Kimmel Institute for Cancer Immunotherapy; the Cancer Giraldo et al. Journal for ImmunoTherapy of Cancer (2018) 6:99 Page 10 of 11 Immunotherapy Trials Network, and Stand Up To Cancer–Cancer Research In- 6. Paulson KG, Iyer JG, Simonson WT, Blom A, Thibodeau RM, Schmidt M, et al. stitute Cancer Immunology Translational Cancer Research Grant SU2C- CD8+ lymphocyte intratumoral infiltration as a stage-independent predictor AACR-DT1012. Stand Up To Cancer is a program of the Entertainment Industry of Merkel cell carcinoma survival: a population-based study. Am J Clin Foundation administered by the American Association for Cancer Research. Pathol. 2014;142:452–8. 7. Lipson EJ, Vincent JG, Loyo M, Kagohara LT, Luber BS, Wang H, et al. PD-L1 expression in the Merkel cell carcinoma microenvironment: association with Availability of data and materials inflammation, Merkel cell polyomavirus and overall survival. Cancer The datasets used and/or analyzed during the current study available from Immunol Res. 2013;1:54–63. the corresponding author on reasonable request. 8. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677–704. 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Evaluation of archived pathology specimens described in this manuscript is Avelumab in patients with chemotherapy-refractory metastatic Merkel cell covered under a study approved by the Johns Hopkins University carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Institutional Review Board which does not require individual patient consent. Oncol. 2016;17:1374–85. 13. D’Angelo SP, Russell J, Lebbé C, Chmielowski B, Gambichler T, Grob J-J, et al. Consent for publication Efficacy and safety of first-line Avelumab treatment in patients with stage IV Not applicable. metastatic Merkel cell carcinoma: a preplanned interim analysis of a clinical trial. JAMA Oncol. 2018. Competing interests 14. Giraldo NA, Taube JM. PD-L1 and Other Immunological Diagnosis Tools. EJL: consultant for Bristol-Myers Squibb, EMD Serono, Merck, Novartis; Oncoimmunology [Internet]. Springer, Cham;2018[cited2017Dec 19]. research funding from Bristol-Myers Squibb. p. 371–85. 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Journal for ImmunoTherapy of CancerSpringer Journals

Published: Oct 1, 2018

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