Expression of SPINK1 (Serine protease inhibitor Kazal type I), also known as tumor associated trypsin inhibitor (TATI), has been demonstrated in a wide spectrum of benign, inflammatory, and neoplastic conditions. Based on our prior results of its expression in urothelial carcinoma, in this study we further characterized SPINK1 expression in a spectrum of urothelial lesions and investigated its potential diagnostic utility. A total of 396 samples comprising a spectrum of urothelial lesions including benign, premalignant, and malignant lesions were evaluated for SPINK1 expression by immunohistochemistry and amplification by fluorescence in situ hybridization (FISH). In a subset of lesions, immunohistochemistry for CK20, CD44, and p53 was also performed. SPINK1 expression was restricted to umbrella cells or lost in 93 % of normal urothelium. Overexpression of SPINK1 in reactive urothelial atypia, urothelial dysplasia, carcinoma in situ (CIS), and papillary urothelial carcinoma (invasive and non-invasive) was seen in 21 %, 36 %, 87 % and 54 % of cases, respectively. Increasing frequency of SPINK1 loss was observed with higher pathologic stage (48.5 % in pT1, 50 % in pT2, 62.5 % in pT3). When compared with other markers, SPINK1 positivity itself has a sensitivity of 90 % for detecting CIS, a 97 % sensitivity when combined with CK20, and a 98 % sensitivity when combined with p53. No amplification of SPINK1 was detected by FISH in any case. Our study illustrates the differential expression of SPINK1 in various urothelial lesions and shows that SPINK1 immunohistochemistry can be utilized as an ancillary tool with high sensitivity and specificity for diagnosing urothelial dysplasia and CIS in challenging cases.

Supplemental Figure 1: Flow chart for database Supplemental Figure 2: Representation of Tissue segmentation Supplemental Figure 3: Representation of Cell Segmentation Supplemental Figure 4: Representation of Phenotyping Supplemental Figure 5: Representation of Scoring Supplemental Figure 6: Supplementary Distance and Survival Data Supplementary ROC Curve and Univariable Cox results Supplemental Figure S7: ROC curve for 2y and 5y DSS

Demographics for DSS Cohort.

PDF file - 310KB, Figure S7: Comparison of copy number ratios at the 1q21.2 (MCL1) locus and 1q23.3; Figure S8: IGV screenshots of the 1q23.3 amplified region (Figure 3) for both cohorts; Figure S9: Kaplan-Meier curves for the second 1q23.3 GISTIC peak, located around FCGR3B; Figure S10: Kaplan-Meier curves for the PBX1 GISTIC peak at 1q23.3 (peak 3); Figure S11: Venn diagram of patients with amplification (log2 > 0.9) of the three GISTIC peaks in the (a) Spanish, (b) DFCI and (c) TCGA cohorts; Figure S12: Boxplots visualizing the correlation of mRNA expression and copy number in the Spanish cohort for all genes in the 1q23.3 GISTIC peak 1; Figure S13: Boxplots visualizing the correlation of mRNA expression and copy number in the DFCI cohort for all genes in the 1q23.3 GISTIC peak 1; Figure S14: Panel (a) shows the correlation of copy number and Immunohistochemistry (IHC) staining of the PVRL4 gene in the Spanish cohort; Table S3: Associations of GISTIC peaks with overall survival after start of chemotherapy, adjusted for ECOG performance status and visceral disease (Spanish cohort); Table S4: Copy numbers in all bladder cancer cohorts at all three 1q23.3 GISTIC peaks. Numbers of patients in the TCGA cohort correspond to patients with clinical information; Table S5: Associations of ECOG PS, visceral disease, 1q23.3 amplification (peak 2, FCGR3B gene) and OS; Table S6: Associations of ECOG PS, visceral disease, 1q23.3 amplification (peak 3, PBX1 gene) and OS; Table S7: For the genes in 1q23.3, this table lists the correlations of copy number and mRNA expression and the correlations of overall survival and mRNA expression.

Demographics for Unanalyzable Cohort

Supplementary Table 1 PDF file - 98K, p53 somatic point mutations in PTEN-null CRPC

PDF file - 423KB, Figure S1: Broad copy numbers at the p- and q-arms in the Spanish cohort; Figure S2: Broad copy numbers at the p- and q-arms in the DFCI cohort. (see Figure S1 for an explanation of this plot); Figure S3: These GISTIC output files show the correlation between gene number and frequency of armlevel events in the (a) Spanish cohort and the (b) DFCI cohort; Figure S4: Recurrent copy number gains in the (a) Spanish and (b) DFCI cohort. Figure S5: Recurrent deletions in the (a) Spanish and (b) DFCI cohorts (see Figure S4 for an explanation of this plot); Table S1: Significant (q-value < 0.25) GISTIC broad aberrations and their association with overall survival after start of chemotherapy (Spanish cohort); Table S2: Significant (q-value < 0.25) GISTIC broad aberrations and their association with overall survival after recurrence (DFCI cohort).

Supplementary ROC Curve and Univariable Cox results

PDF file 196K, The Data Supplement contains Supplementary Tables 1-3, which provide additional detail about the clinical trial as well as Supplementary Methods and References

XLS file, 43K, List of studies reviewed for but excluded from the meta-analysis.

PDF file - 626K, One supplementary table detailing the relationship between aCGH abnormalities and the clinicopathological characteristics of the study cohort. Three supplementary figures illustrating i) the design of the FISH probes ii) key biological processes, gene signatures and pathways enriched among the top differentially expressed genes and iii)Ingenuity Pathway Analysis for potential Pax6 targets.

Supplementary Pearson Correlation Matrix

PDF file, 130K, Selection process for the study inclusion in the meta-analysis.

PDF file, 143K, Characteristics for all men and by ERG-overexpression status among 90 men diagnosed with prostate cancer during transurethral resection of the prostate (TURP)a, PHS and HPFS cohorts 1982-2011.

PDF file - 38KB, Chromosomal Instability. Analysis testing for potential confounding of pathologic stage and tissue type on chromosomal instability. Figure S6: Fraction of genome altered (FGA, 1) in both cohorts.

PDF file - 452KB, Independent validation; Validation of 1q23.3 survival association in independent cohorts; Figure S15: Expression of genes on 1q23.3 in an independent cohort 3; S16: Expression of genes on 1q23.3 as in Supplemental Figure S15, but in the Spanish (a-b) and DFCI (c-d) cohorts. Panels (a) and (c) are for samples with 1q23.3 gain or amplification (log2 copy number ratio > 0.15), (b) and (d) for patients without amplification; S17: Panel (a): No association of overall survival after surgery and 1q23.3 amplification in an inde- pendent cohort from DFCI. (b) No significant association of 1q23.3 amplification and OS after recurrence, possibly due to small patient numbers; Table S8: Overall survival (OS) Hazard Ratios (HRs) of genes in the three peaks, available on the Affymetrix HGU133 Plus 2.0 genechip and displaying patterns of gene expression different from background noise as identified by the Sleipnir library 1. The LMX1A gene was flat, i.e., showed minimum expression intensity in all patients. Table S9: Overall survival (OS) Hazard Ratios (HRs) of genes in 1q21.2 as in Supplementary Table S8.

Supplementary Figures PDF file - 5047K, supplementary figures for opposing effects of androgen deprivation and targeted therapy on prostate cancer prevention

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Abstract Leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4) is expressed on myeloid cells and inhibits myeloid cell activation through binding Major Histocompatibility class I (MHC-I) molecules. These include Human Leukocyte Antigen-G (HLA-G), which is highly expressed in the tumor microenvironment (TME) of many patients and is known to drive immunosuppressive signaling. JTX-8064 is a highly selective IgG4 monoclonal antibody that binds to LILRB2 and blocks interactions with MHC-I. Cancer types have been identified with the potential for higher likelihood of deriving clinical benefit by JTX-8064 through the analysis of single cell RNAseq and bulk tumor RNAseq from early stage and advanced metastatic tumors, and an evaluation of the TME by immunohistochemistry (IHC). LILRB2 expression was analyzed using over 400,000 single cell transcriptomes from 143 patients across 7 cancer types. We observed that LILRB2 mRNA is highly expressed on tumor-associated macrophages (TAMs). Using these data, we generated an RNA signature of genes highly correlated with LILRB2 on TAMs. This TAM signature was used to score tumors in The Cancer Genome Atlas (TCGA) and other datasets for the infiltration of target cells across many cancer types. Cancer types were ranked using the TAM signature as well as other metrics such as Interferon gamma (IFNg) signatures that represent the levels of cytotoxic immune infiltrate. We also evaluated these signatures in relation to response to immune checkpoint blockade. In a dataset of almost 300 patients treated with a PD-L1 inhibitor, we identified an association between pre-treatment LILRB2 expression and an IFNg signature with patient's response to treatment. Non-responders (SD, PD) compared to responders (CR, PR) had significantly higher intra-tumoral LILRB2 levels relative to the IFNg signature (p <0.01) suggesting LILRB2 may be involved in primary resistance to checkpoint blockade. The data presented provides evidence for the development of JTX-8064 in combination with PD-1(L1) inhibitors. Inhibition of LILRB2 by JTX-8064 on myeloid cells directly increases cell activation, antigen presentation, and secretion of pro-inflammatory cytokines. In Mixed Lymphocyte Reactions (MLRs), JTX-8064 induces indirect T cell activation, proliferation and IFNg secretion by inhibiting LILRB2 on monocyte-derived myeloid cells. The activities of JTX-8064 in cell culture and high LILRB2 expression in non-responding patients with high levels of IFNg signature expression provides a rationale that JTX-8064 may be able to overcome anti-PD-1 primary resistance mechanisms. JTX-8064 is currently in Phase 1 clinical development as a monotherapy and combination with anti-PD-1 inhibitors in settings with both anti-PD-1(L1) naïve and anti-PD-1(L1) experienced patients in specific cancer types. Citation Format: Lara McGrath, Amy Mueller, Tanzila Rahman, Jeffrey Smith, Mark Yore, Edward Stack, Kristin O'Malley, Andrew Dunn, Kristen Legendre, Reva Shenwai, Margaret Willer, Allison Naumovski, Johan Baeck, Ben Umiker. Tumor associated macrophages and resistance to immune checkpoint blockade: consideration of cancer indications for the clinical development of JTX-8064, an anti-LILRB2/ILT4 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1727.

TPS2672 Background: Leukocyte Immunoglobulin-like receptor B2 [LILRB2; immunoglobulin-like transcript 4 (ILT4)] is an immunoinhibitory protein expressed on the surface of myeloid cells and is a therapeutic target of interest in immuno-oncology. Published data showed that antagonism of LILRB2 resulted in the repolarization of human macrophages from an M2 (suppressive) to M1 (pro-inflammatory) phenotype, and enhancement of anti-tumor immunity in a mouse model (Chen 2018). JTX-8064 is a novel humanized IgG4 monoclonal antagonist antibody that selectively binds LILRB2 and prevents it from binding its ligands, classical and non-classical MHC I molecules. By blocking the ability of LILRB2 to bind HLA-A/B and/or HLA-G, a marker of immunotolerance on cancer cells, JTX-8064 has been shown to enhance pro-inflammatory cytokine production in macrophages (Cohen 2019). Additionally, blocking HLA-A/B-LILRB2 binding with JTX-8064 may augment antigen presentation and has been shown to lead to enhanced T cell activation and IFNg production (McGrath 2021). Using an ex vivo tumor explant model, we observed an IFNg-associated pharmacodynamic response in tumor tissue treated with JTX-8064 and a PD-1 inhibitor (PD-1i) that was not observed with PD-1i alone. Biomarkers were identified that predicted this JTX-8064 driven response (Hashambhoy-Ramsay 2020). It is hypothesized that JTX-8064 is a novel macrophage immune checkpoint inhibitor that may overcome mechanisms of resistance to PD-1i in tumors not responsive to JTX-8064 or PD-1i alone. Methods: The primary objectives of this open-label, phase 1, first-in-human, multicenter trial are to determine the safety and tolerability, and the recommended phase 2 dose (RP2D) of JTX-8064 as a monotherapy and in combination with a PD-1i, JTX-4014 (a Jounce investigational agent) or pembrolizumab, in patients with advanced solid tumors (NCT04669899). The INNATE study will consist of 4 stages: 1) JTX-8064 monotherapy dose escalation, 2) JTX-8064 dose escalation in combination with a PD-1i, 3) JTX-8064 monotherapy in indication-specific expansion cohorts and 4) JTX-8064 in combination with a PD-1i in indication-specific expansion cohorts. Stages 1 and 2 will employ an innovative interval i3 + 3 design with Bayesian decision framework to guide dose escalation. Safety, pharmacokinetic and receptor occupancy data will be considered during dose escalation. INNATE will assess pharmacodynamic and potential predictive biomarkers of response, and the expansion cohorts will explore multiple patient populations, including PD-(L)1i sensitive and PD-(L)1i-resistant (primary or acquired) patients to address current unmet medical needs. Enrolment in INNATE began in January 2021. Clinical trial information: NCT04669899.