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Synovial sarcoma is an aggressive translocation-associated soft tissue tumor driven by an SS18-SSX fusion oncoprotein. TRPS1 is a recently identified marker for breast carcinoma, but less is known about its expression in other tumor types. We encountered a case of synovial sarcoma showing strong and diffuse expression of TRPS1. To better characterize this observation, we examined the immunohistochemical expression of TRPS1 in 165 cases of synovial sarcoma, including 70 primary, 21 recurrent, and 74 metastatic tumors, using tissue microarrays. TRPS1 expression was observed in 86% of cases. Among the positive cases, TRPS1 labeled50% of tumor cells in 57% of cases, and staining intensity was strong or moderate in 68%. Metastatic tumors more frequently demonstrated strong and diffuse TRPS1 expression compared to primary tumors. To understand the mechanism of TRPS1 expression, we interrogated publicly available gene expression and ChIP-seq datasets and found that TRPS1 transcript levels are increased in synovial sarcoma compared to other soft tissue sarcomas. Data from ChIP-seq experiments showed enrichment of SS18-SSX protein at the TRPS1 locus and co-localization with RNA pol II. The TRPS1 locus is also enriched in several histone modifications associated with active transcription. In functional knockdown data, repression of SS18::SSX in synovial sarcoma cell lines is associated with reduced TRPS1 transcript levels, further supporting a model whereby TRPS1 expression is mediated, at least in part, by the SS18-SSX fusion oncoprotein. Knowledge of TRPS1 expression in synovial sarcoma may help avoid potential diagnostic pitfalls in the immunohistochemical workup of tumors.

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Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

Supplementary Figure 1. Immune profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 2. Myeloid cell profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 3. Increased contact between CD8 T cells and CD68 myeloid cells in non-responding patients to anti-CTLA-4 and anti-PD-1 therapy at pre-treatment CTLA-4 blockade, pre-treatment PD-1 blockade, and on-treatment PD-1 blockade time points. Supplementary Figure 4. Immune profiling of pre anti-PD-1, on-treatment anti-PD-1 and progression anti-PD-1 samples by immunohistochemistry. Supplementary Figure 5. Longitudinal increase in CD8, PD-1, and PD-L1 expression in responders to anti-PD-1 therapy. Supplementary Figure 6. Relative increase in CD8 T cell infiltrate at tumor center in responders to anti-PD-1 on treatment. Supplementary Figure 7. Significant increase in immune infiltrate between responders and non-responders to PD-1 blockade in absence of prior anti-CTLA-4 therapy. Supplementary Figure 8. Immune profiling of myeloid cells atpre-treatment and on-treatment PD-1 blockade time pointsby immunohistochemistry. Supplementary Figure 9. Heatmap of 54 NanoString samples. Supplementary Figure 10. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression CTLA-4 blockade samples. Supplementary Figure 11. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression PD-1 blockade samples. Supplementary Figure 12. Prior CTLA-4 blockade is not required for PD-1 early on-treatment profile. Supplementary Figure 13. Hierarchical clustering of gene expression across 54 samples confirms lack of batch effect.

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Supplementary Table S1a. Patient Cohort Clinical Summary. Supplementary Table S1b. Patient clinical characteristics. Supplementary Table S1c. Immune profiling by IHC sample log. Supplementary Table S1d. Nanostring 54 sample log. Supplementary Table S2. Summary of immune profiling by immunohistochemistry. Supplementary Table S3. Summary of immune profiling by 4 additional myeloid markers. Supplementary Table S4. Summary of immune profiling by IHC of additional CTLA-4 blockade-naïve samples. Supplementary Table S5. Nanostring Gene List. Supplementary Table S6a. Nanostring summary 54 samples. Supplementary Table S6b. Differentially upregulated and downregulated genes in pre-treatment anti-CTLA-4 samples. Supplementary Table S6c. Differentially upregulated and downregulated genes in on-treatment anti-CTLA-4 samples. Supplementary Table S6d. Differentially upregulated and downregulated genes in pre-treatment anti-PD-1 samples. Supplementary Table S6e. Differentially upregulated and downregulated genes in on-treatment anti-PD-1 samples. Supplementary Table S7. Differentially upregulated and downregulated genes from pre- to on-treatment anti-CTLA-4. Supplementary Table S8. Differentially upregulated and downregulated genes from pre- to on-treatment anti-PD-1. Supplementary Table S9a. Nanostring gene list - chip used for comparison of CTLA-4 blockade-experienced vs -naive anti-PD1 samples (28 samples). Supplementary Table S9b. NanoString additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S9c. NanoString normalized data of additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S10. Commonly differentially regulated genes between pre- to on-treatment CTLA-4 blockade and PD-1 blockade. Supplementary Table S11a. Fold changes of significant change by anti-PD-1 therapy for paired samples. Supplementary Table S11b. Frequency of significant change by anti-PD1 therapy for paired samples.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Uveal melanoma (UM) is a rare and lethal malignancy with very limited therapeutic options characterized by mutually exclusive activating mutations in GNAQ and GNA11, resulting in overactive proliferative signaling of Gαq/11 heterotrimeric G proteins. Additionally, tumors typically present with mutually exclusive mutations in one of three proteins with little functional overlap: BAP1, SF3B1, or EIF1AX. Metastatic progression of late-stage uveal melanoma is highly associated with chromosome 3 copy-loss, a unique chromosomal event (monosomy 3 or M3). However, the underlying biology of this distinct copy-loss event, and whether this event directly contributes towards the metastatic phenotype observed in late-stage disease, has largely been unexplored partially due to a lack of available models in the field. Here, using CRISPR-based centromere targeting we artificially created multiple isogenic clones exhibiting monosomy 3 (M3) from a well characterized disomy 3 (D3) UM cell model. Remarkably, the selective loss of one copy of chromosome 3 was sufficient to recapitulate clinical hallmarks of metastatic UM, including pigmentation loss, alterations in cell morphology and expression of late-stage disease markers. In addition, we also show how chromosome 3 copy-loss increases invasive potential and site-specific liver metastasis upon orthotopic implantation. Furthermore, successful development of this isogenic system has allowed for a comprehensive understanding of the molecular signaling, transcriptomic changes and chromatin-level modifications directly regulated by chromosome 3 copy-loss. Specifically, the monosomy 3 event emerged as the major contributor towards the molecular variance across D3 vs. M3 UM contexts and the primary driving force underpinning the large-scale epigenomic rewiring observed in M3 isogenic clones via transposase-accessible chromatin with sequencing (ATACseq). These observations together supported a scenario where the epigenomic rewiring associated to a selective genomic alteration, in this case the loss of one copy of chromosome 3, directs the acquisition of a new and aggressive phenotype. Current studies are focused on defining how these epigenomic alterations contribute towards the metastatic phenotype and identifying context-specific vulnerabilities associated with chromosome 3 copy-loss to develop novel therapeutics strategies to treat late-stage metastatic disease. Citation Format: Johnathon L. Rose, Sanjana Srinivasan, Meng He, Rosalba Minelli, Chiu-Yi Liu, Jason Gay, Joseph R. Daniele, Michael Peoples, Melinda Soeung, Vandhana Ramamoorthy, Anastasia Lopez, Charles Deckard, Brooke Meyers, Edoardo Del Poggetto, Daniel Zamler, Justin Huang, Luigi Perelli, Khalida Wani, Christopher Bristow, Ningping Feng, Christopher P. Vellano, Joe Marszalek, Alexander J. Lazar, Andrew Futreal, Giulio F. Draetta, Scott E. Woodman, Timothy Heffernan, Giannicola Genovese, Alessandro Carugo, Virginia Giuliani. Advanced preclinical modeling reveals unique monosomy 3 associated biology in late-stage uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 94.

Uveal Melanoma (UM) is a rare tumor characterized by mutually-exclusive activating mutations in GNAQ or GNA11, followed by a mutation event in BAP1, SF3B1 or EIF1AX. Notably, a large subset of patients present with a unique chromosome 3 copy-loss event, which is highly associated with metastatic progression in late-stage disease. To date, the biology underlying the causes and consequences of this unique copy-loss event (Monosomy 3 or M3), and its role in promoting metastases remains largely unexplored. This can be attributed to both the lack of preclinical models and in-depth characterization of paired primary and metastatic tumors in patients. To address this, we derived a preclinical isogenic model of chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Isogenic Disomy (D3) and Monosomy (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired, in vivo, primary and metastatic settings. This modeling has enabled us to employ spatial transcriptomics in a PDX setting to characterize gene expression signatures across unique D3 and M3 UM clones. Investigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage). We have also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, where we are able to overcome the lack of same-species microenvironment controls. Our methodology allows for deep characterization of sub-clonal heterogeneity in both D3 and M3 settings and informs on the unique biology underlying invasiveness and outgrowth of M3 tumors. Finally, we complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors. More broadly, comparing these D3 vs. M3 signatures provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival. Citation Format: Sanjana Srinivasan, Johnathon Rose, Joseph R. Daniele, Michael Peoples, Meng He, Rosalba Minelli, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Khalida Wani, Luigi Perelli, Dmitriy Loza, Ningping Feng, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, P. Andrew Futreal, Scott E. Woodman, Alexander J. Lazar, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Deploying spatial transcriptomics to inform intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 92.

Introduction: ATM loss of function (LOF) in cancer may serve as a predictive biomarker of response for antitumor therapies. However, clinical studies of targeted treatments in ATM-aberrant patients have yielded mixed results; thus, identifying the optimal strategy for selecting patients with ATM LOF tumors remains a critical area of unmet need. Methods: In order to investigate the heterogeneity observed in ATM aberrant tumors, we conducted a pan-cancer genomic and proteomic profiling of ATM LOF, and also performed retrospective review of clinical outcomes for a subset of patients with ATM LOF treated with platinum or ATR inhibition. Results: We identified 10,609 ATM variants in 8,587 patients with cancer. The prevalence of deleterious (Tier 1) versus variants of unknown significance (VUS)/benign (Tier 2) variants differed by cancer tissue type, as did selective pressure for loss of heterozygosity (LOH). Analysis of ATM protein staining in 471 patient tumors showed tumors with null, inactivating variants were significantly (p Conclusions: Our data highlight that there is heterogeneity in ATM LOF in patients due to multiple variables, including notable tissue-specific differences in the type of variants seen and the relationship between ATM variant status and ATM protein. This genomic heterogeneity and tissue-specificity has implications for predictive biomarker development and clinical trial designs. Citation Format: Patrick G. Pilie, Daniel Mcgrail, Wei-Lien Wang, Natalie Ngoi, Keith Kyewalabye, Khalida Wani, Hung Le, Erick Campbell, Vijaykumar Holla, Kenna R. Shaw, Funda Meric-Bernstam, Alexander J. Lazar, Virginia Giuliani, Timothy Heffernan, Timothy A. Yap. Ataxia-telangiectasia mutated loss of function displays variant and tissue specific differences across tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3432.

BACKGROUND AND AIMS Complete loss of histone H3 lysine 27 trimethylation (H3K27me3) has recently emerged as a biomarker for malignant peripheral nerve sheath tumours (MPNST). Loss of H3K27me3 staining has also been reported in post-radiation MPNST; however, it has not been evaluated in a large series of radiation-associated sarcomas of different histological subtypes. The aim of this study was to assess H3K27me3 labelling by immunohistochemistry in radiation-associated sarcomas and to determine the prevalence of H3K27me3 loss in these tumours. METHODS AND RESULTS Radiation-associated sarcomas (n = 119) from two tertiary care referral centres were evaluated for loss of H3K27me3, defined as complete loss of staining within tumour cells in the presence of a positive internal control. Twenty-three cases (19%) showed H3K27me3 loss, including nine of 10 (90%) MPNST, seven of 77 (9%) undifferentiated spindle cell/pleomorphic sarcomas, five of 25 (20%) angiosarcomas, one of five (20%) leiomyosarcomas and one of two (50%) osteosarcomas. CONCLUSIONS Complete H3K27me3 loss was present in 19% of radiation-associated sarcomas in our series. Our findings demonstrate that loss of H3K27me3 is not specific for radiation-associated MPNST and may also occur in other histological subtypes of RAS, including radiation-associated undifferentiated spindle cell/pleomorphic sarcoma, angiosarcoma, leiomyosarcoma and osteosarcoma.

Palbociclib has been evaluated in early phase trials for well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) patients, with reported median progression-free survival (PFS) of 18 weeks. Here, we report on real-world use and surgical outcomes associated with palbociclib treatment. We retrospectively reviewed 61 consecutive patients with retroperitoneal WDLPS (n = 14) or DDLPS (n = 47) treated with palbociclib monotherapy between 1 March 2016 and 28 February 2021 at The University of Texas MD Anderson Cancer Center. At palbociclib initiation, median age was 64 (interquartile range [IQR] 56-72). In WDLPS and DDLPS cohorts, the median number of prior systemic treatments was 0 (IQR 0-0) and 2 (IQR 0-4), respectively. Median number of prior surgeries was 2 (WDLPS IQR 1-2.75) and 2 (DDLPS IQR 1-3). Median PFS was 9.2 (WDLPS IQR 3.9-21.9) and 2.6 months (DDLPS IQR 2.0-6.1), with median time on treatment of 7.4 months (WDLPS IQR 3.5-14.2) and 2.7 months (DDLPS IQR 2.0-5.7). Twelve patients ultimately underwent surgical resection. Resections were macroscopically complete (R0/R1) in half (n = 6/12), among whom only one patient experienced relapse after resection (median follow-up 7.5 months). All patients who underwent macroscopically incomplete resections progressed after surgery with median time to progression of 3.3 months (IQR 2.3-4.4). Surgery after palbociclib treatment was not associated with improved overall survival. Efficacy of palbociclib monotherapy for patients with advanced WDLPS and DDLPS is disappointing. While palbociclib may have been used to delay surgery, there was no clear benefit from treatment and few patients achieved prolonged tumor control.

Another benefit of dietary fiber The gut microbiome can modulate the immune system and influence the therapeutic response of cancer patients, yet the mechanisms underlying the effects of microbiota are presently unclear. Spencer et al . add to our understanding of how dietary habits affect microbiota and clinical outcomes to immunotherapy. In an observational study, the researchers found that melanoma patients reporting high fiber (prebiotic) consumption had a better response to checkpoint inhibitor immunotherapy compared with those patients reporting a low-fiber diet. The most marked benefit was observed for those patients reporting a combination of high fiber consumption and no use of over-the-counter probiotic supplements. These findings provide early insights as to how diet-related factors may influence the immune response. —PNK

Simple Summary We conducted a network analysis of microRNA–mRNA associations in melanoma tissue and cell lines to identify the microRNAs central to melanoma biology and their associated gene expression profiles. Further, we evaluated expression of these microRNAs in melanoma patient biopsies and found that increased expression of miR-100-5p and miR-125b-5p were associated with improved outcomes with anti-PD-1 immunotherapy. Further investigation of these microRNAs as biomarkers and potential targets to improve immunotherapy response in melanoma is warranted. Abstract Metastatic melanoma is a deadly malignancy with poor outcomes historically. Immuno-oncology (IO) agents, targeting immune checkpoint molecules such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1), have revolutionized melanoma treatment and outcomes, achieving significant response rates and remarkable long-term survival. Despite these vast improvements, roughly half of melanoma patients do not achieve long-term clinical benefit from IO therapies and there is an urgent need to understand and mitigate mechanisms of resistance. MicroRNAs are key post-transcriptional regulators of gene expression that regulate many aspects of cancer biology, including immune evasion. We used network analysis to define two core microRNA–mRNA networks in melanoma tissues and cell lines corresponding to ‘MITF-low’ and ‘Keratin’ transcriptomic subsets of melanoma. We then evaluated expression of these core microRNAs in pre-PD-1-inhibitor-treated melanoma patients and observed that higher expression of miR-100-5p and miR-125b-5p were associated with significantly improved overall survival. These findings suggest that miR-100-5p and 125b-5p are potential markers of response to PD-1 inhibitors, and further evaluation of these microRNA–mRNA interactions may yield further insight into melanoma resistance to PD-1 inhibitors.

Introduction: Melanoma-associated antigen A4 (MAGE-A4) is a cancer testis antigen expressed in multiple solid tumors. Afami-cel is an autologous, specific peptide enhanced affinity receptor T-cell therapy targeting MAGE-A4 in HLA-A*02+ patients, which has shown promising efficacy in heavily pretreated metastatic synovial sarcoma (SS) and myxoid liposarcoma (MLPS) with cellular features (myxoid/round cell liposarcoma, MRCLS). Assessing MAGE-A4 expression across a wider range of adult and pediatric sarcoma subtypes could identify new therapeutic indications for afami-cel. Methods: Formalin-fixed, paraffin-embedded tissue microarrays from resections of primary and metastatic sarcomas were collected from 1981 to 2015 at The University of Texas MD Anderson Cancer Center. Included sarcomas were: SS (n=70), osteosarcomas (OS; n=107), Ewing sarcomas (ES; n=125), angiosarcoma (n=101), non-uterine leiomyosarcoma (n=186), malignant peripheral nerve sheath tumor (MPNST; n=95), pleomorphic liposarcoma (n=42), MLPS (n=78), uterine leiomyosarcoma (ULMS; n=216), and undifferentiated pleomorphic sarcoma (n=82). Tumors from patients ≤21 years were considered pediatric sarcomas. MAGE-A4 expression intensity (0 to 3+) was assessed by a research-only, non-clinical-trial use, immunohistochemistry assay developed at MD Anderson using a monoclonal antibody (Origene, Rockville, MD). The staining was reported as H-score (range 0–300; [(% positive cells intensity 1) x 1] + [(% positive cells intensity 2+) x 2] + [(% positive cells intensity 3+) x 3]) and percentage of MAGE-A4 expression ≥1+ or 2+. Samples with 30% of tumor cells showing ≥2+ MAGE-A4 expression were considered positive. Results: We analyzed 1102 samples from 702 sarcoma patients. The mean (range) H-score was 26.2 (0–101.9), and 532 (59%) samples were from female patients. Patients’ ages ranged from 5 to 90 years; 124 (11.3%) samples were pediatric. Most common SS was monophasic histology (72%), and most MLPS lacked a cellular component (65%). SS had the highest percentage of MAGE-A4 positivity (47.1%, mean H-score: 101.9), followed by MLPS (16.7%, mean H-score: 36.6), pleomorphic liposarcoma (11.9%), angiosarcoma (9.9%), ULMS (7.4%), MPNST (6.3%), OS (6.5%), LMS (2.7%), and UPS (2.4%). All Ewing sarcoma samples were negative. In pediatric patients, MAGE-A4 positivity was seen only in SS, OS, and angiosarcoma (MAGE-A4 positive rate: 33.3% [5/15], 7.9% [5/62], 25% [1/4], respectively). In MLPS, MRCLS showed significantly more MAGE-A4 positive cases than uniformly myxoid tumors (30% vs 11.8%, Tukey's test, a=0.05). In ULMS, recurrent tumors had significantly more MAGE-A4 positive cases than metastatic tumors (12.1% vs 2.4%, Tukey's test, a=0.05). Conclusions: MAGE-A4 was heterogeneously expressed across 10 sarcoma subtypes. The rates of MAGE-A4 positivity were the highest in SS and MLPS and were lower in other sarcoma subtypes. MAGE-A4 testing in sarcoma subtypes other than SS and MLPS warrants evaluating afami-cel in these ultrarare subtypes of soft tissue sarcomas where treatment options are very limited. Citation Format: Swethajit Biswas, Caddie Laberiano, Khalida Wani, Davis Ingram, Wei Lu, Rossana Lazcano, Wei-Lien Wang, Yao Chen, Dennis Williams, Cheryl McAlpine, Alexander J. Lazar, Luisa M. Solis Soto. Identifying new therapeutic indications for afamitresgene autoleucel (“afami-cel” [formerly ADP-A2M4]) in adult and pediatric sarcomas using MAGE-A4 immunohistochemistry [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A023.

Background: We evaluated the activity of neoadjuvant ICB in localized resectable DDLPS (n=17) and undifferentiated pleomorphic sarcomas (UPS; n=10). DDLPS and UPS patients were randomized to neoadjuvant nivolumab or ipilimumab+nivolumab, with UPS patients receiving concurrent radiotherapy. We assessed genomic markers of early relapse (progression before surgery or relapse within 52 weeks following surgery) using longitudinally acquired tumor samples. Methods: RNA sequencing (RNAseq) and whole genome sequencing (WGS) were performed on longitudinally acquired samples (baseline biopsies and surgical specimens). Differential gene expression between any two groups of patients (i.e., non-early relapse [non-relapsers] vs early relapse [relapsers]) were selected (fold change>1.5 and p value Citation Format: Elise F. Nassif, Chia-Chin Wu, Kadir Akdemir, Russell G. Witt, Raymond Traweek, Brandon Cope, Prapassorn Thirasastr, Taylor Tate, Grace Mathew, Shadarra Crosby, Randy Chu, Mohammad Mohammad, Kenna Shaw, Ingram Davis, Khalida Wani, Alexander J. Lazar, Wei-Lien Wang, Sheila Duncan, Ashleigh B. Guadagnolo, Andrew J. Bishop, Valerae Lewis, Justin E. Bird, Keila E. Torres, Kelly K. Hunt, Barry W. Feig, Christopher P. Scally, Ravin Ratan, Shreyaskumar Patel, Robert S. Benjamin, Robert Satcher, Kevin McBride, Wolf H. Fridman, Ignacio Wistuba, Andrew Futreal, Jennifer A. Wargo, Neeta Somaiah, Christina L. Roland, Emily Z. Keung. Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR002.

International audience; Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1β in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Durable remission rates with immune checkpoint inhibitor (ICI) monotherapy remain low. Combination ICI therapies could overcome immune resistance but are associated with higher rates of immune-related adverse events (irAEs). To elucidate the underlying irAE immunobiology, we profiled human intestinal tissue, enterocolitis (irEC), and tumor tissue from ICI-treated patients using NanoString PanCancer Immune Panel (NanoPCIP) and multiplex immunohistochemistry (mIHC); hypotheses generated from these translational results for potential irAE treatments were tested in parallel preclinical mouse model studies. In 23 patients identified with both normal intestinal and irEC tissue, we found genes encoding interleukin-6 (IL-6), Th17 differentiating cytokine, and neutrophil and monocyte chemotactic molecules were the highest significantly upregulated in irEC compared to patient-matched normal intestine. Using NanoPCIP cell scoring (23 patients) and mIHC (27 patients), Th17 cells were significantly higher in irEC than Th1 cells. T-cell subsets were similar between anti-CTLA-4 based regimen versus anti-PD-1monotherapy induced irEC, except Th17 memory cells were significantly higher CTLA-4i-induced irEC. We compared the degree of differential gene expression in irEC (normal intestine versus irEC) to the differential expression in tumors (baseline versus on-treatment tumors). The genes highest upregulated in irEC (including Th17 differentiating cytokines IL-6 and IL-1B) were not upregulated in responding tumors from patients receiving ICI. NanoPCIP Th17 cell score was significantly more upregulated than Th1 cells and neutrophils more upregulated than CD8 T cells in the irEC analysis, but not in the responding tumor analysis where IL-12 and CD8 cell score was significantly upregualted . Based on these discoveries, we used C57BL/6 and Balb/c mouse strains to address whether the antitumor effect of ICIs can be enhanced by IL-6 blockade while also improving irAEs. We found that IL-6 blockade + ICI was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and MDSCs compared to ICI alone. Furthermore, in the experimental autoimmune encephalomyelitis (EAE) mouse model, the pathogenesis of which is well documented to be Th17 mediated (Robinson et al., 2014), we demonstrated that combined IL-6 blockade and ICI enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICI alone. Based on the totality of the basic science, translational, and clinical data we hypothesize that IL-6 blockade combined with ICIs could de-couple autoimmunity from antitumor immunity. We are currently conducting a prospective phase 2 trials of combination ipilimumab (3mg/kg) + Nivolumab (1mg/kg) + Tocilizumab in patients with metastatic melanoma, EGFR mutant NSCLC, and urothelial carcinoma ( NCT04940299). Citation Format: Daniel H. Johnson, Yared Hailemichael, Salah-Eddine Bentebibel, Noha Abdel-Wahab, Sungnam Cho, Wai C. Foo, Khalida Wani, Stephanie S. Watowich, Suhendan Ekmekcioglu, Adi Diab. Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4177.

The use of immune-checkpoint inhibitors (CPI) has become an important modality in the treatment of metastatic melanoma (MM). However, most patients (pts) do not experience durable responses and new treatment options are needed to improve clinical outcomes. Our pre-clinical studies have demonstrated that intratumoral CD40 activation synergizes with anti-PD-1 based therapy and induces systemic and distant anti-tumor effects. In this ongoing phase I/II study, we assessed intratumoral sotigalimab (APX005M), a CD40 agonist antibody, in combination with systemic pembrolizumab in CPI treatment naïve, unresectable stage III or IV MM. A total of 40 participants will be enrolled. As of December 15, 2021, 30 pts were enrolled. Pts received sotigalimab every 3 weeks for a total of 4 doses. The dose escalation portion of the trial has been completed, with 14 pts enrolled in 5 dosing cohorts of sotigalimab at 0.1, 0.5, 1, 3 and 10 mg. The primary objectives include safety and tolerability, determination of the recommended phase 2 dose (RP2D), and assessment of the overall response rate (ORR) by RECIST v1.1. Biomarker analyses of blood and tumor samples were performed to measure immune activation using immunophenotyping including imaging mass cytometry, TCR sequencing, and a cross-cohort comparison of gene expression data (sotigalimab plus pembrolizumab versus anti-PD1 monotherapy). The combination therapy has been well-tolerated, and there were no study discontinuations or death due to treatment-related adverse events (TRAEs). Most common TRAEs were injection-site reactions; six pts experienced grade-3 immune-related adverse events. Efficacy analysis of 30 pts with post-baseline disease evaluations demonstrated an ORR of 50% (5 CR and 10 PR) in distant lesions and a disease control rate of 67%. The ORR at the RP2D of 10 mg is 55% (12/22). Responses were observed in PD-L1 negative pts and those with elevated LDH. Comprehensive transcriptome and immune cell profiling of peripheral blood mononuclear cells and tumor biopsies obtained from local lesions at baseline and 24 hours post sotigalimab injection demonstrate that sotigalimab effectively engaged CD40 pathway. In comparison to anti-PD1 monotherapy, the combination therapy significantly increased expression of genes associated with antigen presentation and effector T cells in local lesions accompanied by an increase in T cell activation genes at distant lesions. Additionally, T cell repertoire analysis demonstrated a significant increase in T cell clonality with expansion of new clones shared between local and distant tumors. Importantly, these immunologic changes were correlated with clinical response. Collectively, this combination therapy is well tolerated and has a notable clinical response rate, accompanied by broad innate and adaptive immune activation at both local and distant lesions. Citation Format: Salah-Eddine Bentebibel, Daniel Johnson, Rodabe Amariae, Daniel McGrail, Srisuda Lecagoonporn, Cara Haymaker, Dzifa Duose, Khalida Wani, Houssein Safa, Isabella Claudia Glitza, Sapna Pradyuman Patel, Michael K. Wong, Hussein Tawbi, Jared Burks, Xiaodong Yang, Patrick Hwu, Cassian Yee, Michael A. Davies, Ravi Murthy, Chantale Bernatchez, Suhendan Ekmekcioglu, Adi Diab, Gregory Lizée. Intratumoral CD40 agonist sotigalimab with pembrolizumab induces broad innate and adaptive immune activation in local and distant tumors in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT039.

Immune checkpoint blockade (ICB) therapy frequently induces immune-related adverse events. To elucidate the underlying immunobiology, we performed a deep immune analysis of intestinal, colitis, and tumor tissue from ICB-treated patients with parallel studies in preclinical models. Expression of interleukin-6 (IL-6), neutrophil, and chemotactic markers was higher in colitis than in normal intestinal tissue; T helper 17 (Th17) cells were more prevalent in immune-related enterocolitis (irEC) than T helper 1 (Th1). Anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) induced stronger Th17 memory in colitis than anti-program death 1 (anti-PD-1). In murine models, IL-6 blockade associated with improved tumor control and a higher density of CD4

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (��-CTLA-4 or ��-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten���/���). Short-term treatment with ��-PD-1 and ��-CTLA-4 monotherapies were relatively ineffective, while treatment with ��-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined ��-OX40/��-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/��-CTLA-4/��-PD-1 or DT/��-OX40/��-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but a high rate of immune-related adverse events (irAE). Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 advanced melanoma patients treated with CICB, with a high rate of any ≥Grade 3 irAEs (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1b in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Treatment with molecularly-targeted therapy has revolutionized cancer care, including BRAF/MEK-targeted melanoma therapy. However responses are heterogenous and frequently not long-lasting. Novel strategies to target resistance are needed. We studied a cohort of patients with resectable metastatic melanoma treated with neoadjuvant BRAF/MEK-targeted therapy (n=52) and noted a strong sexual dimorphism in response to treatment, with female patients demonstrating significantly higher rates of a major pathologic response (MPR) (p=0.0001). RNA sequencing of tumors demonstrated enrichment of androgen-related genes in those failing to achieve MPR. Pre-clinical studies validated these findings, with significantly increased tumor growth in male vs female mice treated with BRAF/MEK inhibitors (BRAF/MEKi) (p=0.0005). Androgen receptor (AR) expression was upregulated in tumors of BRAF/MEKi-treated mice, and modulation of AR signaling via AR-blockade or castration was associated with significantly slower tumor growth (p=0.0001 and p=0.00004, respectively). Together, these results have important implications in the context of treatment with BRAF/MEKi-targeted therapy.

BackgroundTo date, no systemic therapy, including immunotherapy, exists to improve clinical outcomes in metastatic uveal melanoma (UM) patients. To understand the role of immune infiltrates in the genesis, metastasis, and response to treatment for UM, we systematically characterized immune profiles of UM primary and metastatic tumors, as well as samples from UM patients treated with immunotherapies.MethodsRelevant immune markers (CD3, CD8, FoxP3, CD68, PD-1, and PD-L1) were analyzed by immunohistochemistry on 27 primary and 31 metastatic tumors from 47 patients with UM. Immune gene expression profiling was conducted by NanoString analysis on pre-treatment and post-treatment tumors from patients (n=6) receiving immune checkpoint blockade or 4-1BB and OX40 dual costimulation. The immune signature of UM tumors responding to immunotherapy was further characterized by Ingenuity Pathways Analysis and validated in The Cancer Genome Atlas data set.ResultsBoth primary and metastatic UM tumors showed detectable infiltrating lymphocytes. Compared with primary tumors, treatment-naïve metastatic UM showed significantly higher levels of CD3+, CD8+, FoxP3+ T cells, and CD68+ macrophages. Notably, levels of PD-1+ infiltrates and PD-L1+ tumor cells were low to absent in primary and metastatic UM tumors. No metastatic organ-specific differences were seen in immune infiltrates. Our NanoString analysis revealed significant differences in a set of immune markers between responders and non-responders. A group of genes relevant to the interferon-γ signature was differentially up-expressed in the pre-treatment tumors of responders. Among these genes, suppressor of cytokine signaling 1 was identified as a marker potentially contributing to the response to immunotherapy. A panel of genes that encoded pro-inflammatory cytokines and molecules were expressed significantly higher in pre-treatment tumors of non-responders compared with responders.ConclusionOur study provides critical insight into immune profiles of UM primary and metastatic tumors, which suggests a baseline tumor immune signature predictive of response and resistance to immunotherapy in UM.

Elevated serum lactate dehydrogenase (sLDH) is associated with poor clinical outcomes in patients with stage IV metastatic melanoma (MM). It is currently unknown if sLDH elevation correlates with distinct molecular, metabolic, or immune features of melanoma metastases. The identification of such features may identify rational therapeutic strategies for patients with elevated sLDH. Thus, we obtained sLDH levels for melanoma patients with metastases who had undergone molecular and/or immune profiling. Our analysis of multi‐omics data from independent cohorts of melanoma metastases showed that elevated sLDH was not significantly associated with differences in immune cell infiltrate, point mutations, DNA copy number variations, promoter methylation, RNA expression, or protein expression in melanoma metastases. The only significant association observed for elevated sLDH was with the number of metastatic sites of disease. Our data support that sLDH correlates with disease burden, but not specific molecular or immunological phenotypes, in metastatic melanoma., The association between elevated serum lactate dehydrogenase (sLDH) and poor prognosis in metastatic melanoma patients is well‐established but poorly understood. Our multi‐omics analysis of multiple cohorts of patients with melanoma metastases identified no significant intratumoral molecular, immunological, or metabolic associations with sLDH, supporting that sLDH serves at least partially as a surrogate of tumor burden but not of adverse tumor features.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response., Immunotherapies now dominate the treatment landscape for melanoma, but why they only work in a subset of patients remains unclear. Here, the authors perform an immunogenomic analysis on 67 intratumor sub-regions of a PD-1 inhibitor resistant melanoma, and 2 additional metastases from a single patient, mapping the spatial relationships between genomic and immune heterogeneity at high resolution.

// Adriana Olar 1 , Lindsey D. Goodman 2 , Khalida M. Wani 3 , Nicholas S. Boehling 4 , Devi S. Sharma 5 , Reema R. Mody 5 , Joy Gumin 6 , Elizabeth B. Claus 7, 8 , Frederick F. Lang 6 , Timothy F. Cloughesy 5 , Albert Lai 5 , Kenneth D. Aldape 9 , Franco DeMonte 6 and Erik P. Sulman 3, 10 1 Medical University of South Carolina & Hollings Cancer Center, Departments of Pathology and Laboratory Medicine & Neurosurgery, Charleston, SC, USA 2 Neurosciences Graduate Group, Perlman School of Medicine, University of Pennsylvania, Department of Biology, Philadelphia, PA, USA 3 The University of Texas MD Anderson Cancer Center, Department of Translational Molecular Pathology, Houston, TX, USA 4 St. Charles Cancer Center, Department of Radiation Oncology, Bend, OR, USA 5 The University of California at Los Angeles, Department of Neurology, David Geffen School of Medicine, Los Angeles, CA, USA 6 The University of Texas MD Anderson Cancer Center, Department of Neurosurgery, Houston, TX, USA 7 Brigham and Women’s Hospital, Harvard Medical School, Department of Neurosurgery, Boston, MA, USA 8 School of Public Health, Yale University, Department of Biostatistics, New Haven, CT, USA 9 MacFeeters-Hamilton Brain Tumour Centre, Princess Margaret Cancer Centre, Toronto, Ontario, Canada 10 The University of Texas MD Anderson Cancer Center, Departments of Radiation Oncology and Genomic Medicine, Houston, TX, USA Correspondence to: Adriana Olar, email: adriana_olar@yahoo.com ; olar@musc.edu Erik P. Sulman, email: epsulman@mdanderson.org Keywords: meningioma; gene expression; affymetrix; recurrence risk; predictor algorithm Received: August 02, 2017 Accepted: February 01, 2018 Epub: February 15, 2018 Published: March 23, 2018 ABSTRACT BACKGROUND : Meningioma is the most common primary brain tumor and has a variable risk of local recurrence. While World Health Organization (WHO) grade generally correlates with recurrence, there is substantial within-grade variation of recurrence risk. Current risk stratification does not accurately predict which patients are likely to benefit from adjuvant radiation therapy (RT). We hypothesized that tumors at risk for recurrence have unique gene expression profiles (GEP) that could better select patients for adjuvant RT. METHODS :We developed a recurrence predictor by machine learning modeling using a training/validation approach. RESULTS : Three publicly available AffymetrixU133 gene expression datasets (GSE9438, GSE16581, GSE43290) combining 127 primary, non-treated meningiomas of all grades served as the training set. Unsupervised variable selection was used to identify an 18-gene GEP model (18-GEP) that separated recurrences. This model was validated on 62 primary, non-treated cases with similar grade and clinical variable distribution as the training set. When applied to the validation set, 18-GEP separated recurrences with a misclassification error rate of 0.25 (log-rank p=0.0003). 18-GEP was predictive for tumor recurrence [p=0.0008, HR=4.61, 95%CI=1.89-11.23)] and was predictive after adjustment for WHO grade, mitotic index, sex, tumor location, and Simpson grade [p=0.0311, HR=9.28, 95%CI=(1.22-70.29)]. The expression signature included genes encoding proteins involved in normal embryonic development, cell proliferation, tumor growth and invasion (FGF9, SEMA3C, EDNRA), angiogenesis (angiopoietin-2), cell cycle regulation (CDKN1A), membrane signaling (tetraspanin-7, caveolin-2), WNT-pathway inhibitors (DKK3), complement system (C1QA) and neurotransmitter regulation (SLC1A3, Secretogranin-II). CONCLUSIONS : 18-GEP accurately stratifies patients with meningioma by recurrence risk having the potential to guide the use of adjuvant RT.

Summary Background Dual BRAF and MEK inhibition produces a response in a large number of patients with stage IV BRAF-mutant melanoma. The existing standard of care for patients with clinical stage III melanoma is upfront surgery and consideration for adjuvant therapy, which is insufficient to cure most patients. Neoadjuvant targeted therapy with BRAF and MEK inhibitors (such as dabrafenib and trametinib) might provide clinical benefit in this high-risk p opulation. Methods We undertook this single-centre, open-label, randomised phase 2 trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Eligible participants were adult patients (aged ≥18 years) with histologically or cytologically confirmed surgically resectable clinical stage III or oligometastatic stage IV BRAFV600E or BRAFV600K (ie, Val600Glu or Val600Lys)-mutated melanoma. Eligible patients had to have an Eastern Cooperative Oncology Group performance status of 0 or 1, a life expectancy of more than 3 years, and no previous exposure to BRAF or MEK inhibitors. Exclusion criteria included metastases to bone, brain, or other sites where complete surgical excision was in doubt. We randomly assigned patients (1:2) to either upfront surgery and consideration for adjuvant therapy (standard of care group) or neoadjuvant plus adjuvant dabrafenib and trametinib (8 weeks of neoadjuvant oral dabrafenib 150 mg twice per day and oral trametinib 2 mg per day followed by surgery, then up to 44 weeks of adjuvant dabrafenib plus trametinib starting 1 week after surgery for a total of 52 weeks of treatment). Randomisation was not masked and was implemented by the clinical trial conduct website maintained by the trial centre. Patients were stratified by disease stage. The primary endpoint was investigator-assessed event-free survival (ie, patients who were alive without disease progression) at 12 months in the intent-to-treat population. This trial is registered at ClinicalTrials.gov, number NCT02231775. Findings Between Oct 23, 2014, and April 13, 2016, we randomly assigned seven patients to standard of care, and 14 to neoadjuvant plus adjuvant dabrafenib and trametinib. The trial was stopped early after a prespecified interim safety analysis that occurred after a quarter of the participants had been accrued revealed significantly longer event-free survival with neoadjuvant plus adjuvant dabrafenib and trametinib than with standard of care. After a median follow-up of 18·6 months (IQR 14·6–23·1), significantly more patients receiving neoadjuvant plus adjuvant dabrafenib and trametinib were alive without disease progression than those receiving standard of care (ten [71%] of 14 patients vs none of seven in the standard of care group; median event-free survival was 19·7 months [16·2–not estimable] vs 2·9 months [95% CI 1·7–not estimable]; hazard ratio 0·016, 95% CI 0·00012–0·14, p Interpretation Neoadjuvant plus adjuvant dabrafenib and trametinib significantly improved event-free survival versus standard of care in patients with high-risk, surgically resectable, clinical stage III–IV melanoma. Although the trial finished early, limiting generalisability of the results, the findings provide proof-of-concept and support the rationale for further investigation of neoadjuvant approaches in this disease. This trial is currently continuing accrual as a single-arm study of neoadjuvant plus adjuvant dabrafenib and trametinib. Funding Novartis Pharmaceuticals Corporation.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

SUMMARY The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients., Graphical abstract, In brief Terranova et al. provide a comprehensive epigenome resource for melanoma encompassing 284 chromatin maps. They find key regulatory roles for bivalent and broad domains in expression of pro-metastatic genes and identify EZH2 plus MEK inhibition as a therapeutic strategy for NRAS mutant melanomas.

AIMS Multiple genetic alterations, including alternative lengthening of telomeres (ALT) and NOTCH mutations, have been described in angiosarcoma. Loss of α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death domain-associated protein 6 (DAXX) expression is frequently associated with the ALT phenotype. Additionally, inhibition of NOTCH signalling induces the development of malignant vascular tumours in mice, indicating a tumour suppressive role of the NOTCH pathway in the pathogenesis of angiosarcoma. The aim of this study was to evaluate the immunohistochemical expression of ATRX, DAXX and NOTCH receptors (NOTCH1 and NOTCH2) in a large cohort of angiosarcomas, and study their clinicopathological and prognostic significance. METHODS AND RESULTS One hundred and forty cases of angiosarcoma were stained for ATRX, DAXX, NOTCH1 and NOTCH2. ATRX loss (

Meningioma is the most common primary brain tumor and carries a substantial risk of local recurrence. Methylation profiles of meningioma and their clinical implications are not well understood. We hypothesized that aggressive meningiomas have unique DNA methylation patterns that could be used to better stratify patient management. Samples (n = 140) were profiled using the Illumina HumanMethylation450BeadChip. Unsupervised modeling on a training set (n = 89) identified 2 molecular methylation subgroups of meningioma (MM) with significantly different recurrence-free survival (RFS) times between the groups: a prognostically unfavorable subgroup (MM-UNFAV) and a prognostically favorable subgroup (MM-FAV). This finding was validated in the remaining 51 samples and led to a baseline meningioma methylation classifier (bMMC) defined by 283 CpG loci (283-bMMC). To further optimize a recurrence predictor, probes subsumed within the baseline classifier were subject to additional modeling using a similar training/validation approach, leading to a 64-CpG loci meningioma methylation predictor (64-MMP). After adjustment for relevant clinical variables [WHO grade, mitotic index, Simpson grade, sex, location, and copy number aberrations (CNAs)] multivariable analyses for RFS showed that the baseline methylation classifier was not significant (p = 0.0793). The methylation predictor, however, was significantly associated with tumor recurrence (p

Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte–associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. Significance: These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827–37. ©2016 AACR. See related commentary by Teng et al., p. 818. This article is highlighted in the In This Issue feature, p. 803

Background: Undifferentiated pleomorphic sarcoma (UPS) are genetically complex sarcomas often showing a relatively higher immune infiltration among sarcomas, and a demonstrated benefit for immunotherapy in a subset of patients. This study characterizes the immune microenvironment of UPS including tumor infiltrating lymphocytes (TILs), as well as immune checkpoint (IC) and related biomarkers. Methods: We used FFPE surgical resected UPS from 90 patients (43 primary, 28 recurrence and 19 metastasis) placed in a tissue microarray and stained with immunohistochemistry for the following biomarkers: IC (stimulatory: OX40 & ICOS; inhibitory: PD-L1, LAG3, IDO1, & PD1), TILs (CD3 & CD8), monocytes-macrophages (CD163), Adenosine Pathway (CD73 & CD39), and pSTAT3. We scored TIL and IC biomarkers as positive cell densities, and PD-L1, pSTAT3, CD73 and CD39 as percentage of expression in malignant cells. Clinicopathological data and clinical outcome were available for all patients. Spearman correlation was used to assess the correlation between two biomarkers. Cox proportional hazard regression models were used to identify the prognostic biomarkers for overall survival (OS), recurrence-free survival (RFS), and metastasis-free survival (MFS). Results: In our cohort, PD-L1, pSTAT3, CD73 and CD39 positive expression (cut-off ≥1%) was 19, 73 78 and 68% respectively. These biomarkers were positively correlated with immune infiltrates: CD3 (p=0.013, 0.009, 0.009, Conclusions: In this study, we characterized the immune cell infiltrates and the expression of PD-L1, pSTAT3, CD73 and CD39 in UPS. These biomarkers differentially correlated with clinicopathological characteristics in primary, recurrent and metastatic tumors. Lower TILs and low CD73/PD-L1 expression were associated with inferior survival outcomes. Citation Format: Carmelia M. Barreto, Luisa M. Solis-Soto, Ruth Salazar, Swati Gite, Edwin R. Parra, Barbara Mino, Davis Ingram, Khalida M. Wani, Cheuk H. Leung, Heather Lin, Ignacio I. Wistuba, Alexander Lazar, Wei-Lien Wang. Characterization of immune cell biomarkers in undifferentiated pleomorphic sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3857.

3629 Background: ATM is frequently mutated in cancer, and defects may serve as a putative predictive biomarker. However, the functional impact of most ATM variants is not well known. In this study, we examined the relationship between ATM variants and ATM protein expression to better discern ATM functional defects in patients (pts) with advanced cancer. Methods: We retrospectively identified pts seen at MD Anderson Cancer Center who had ATM variants detected on CLIA-certified next generation sequencing (NGS) assays. ATM immunohistochemistry (IHC) was performed on available tumors. We then prospectively assessed ATM IHC on tumors from pts who were referred for DNA damage repair inhibitor (DDRi) trials. Functional classification of the variants was performed via published in silico tools and/or precision oncology decision support (PODS). An IHC cut-off of 100% loss in tumor cell nuclei defined ATM loss of protein (LOP). Results: Of 1394 ATM-mutant tumors identified retrospectively, ATM alterations were classified as 16% (N = 216) inactivating, 12% (N = 163) potentially inactivating, 71% (N = 993) variant of unknown significance (VUS), and 2% (N = 22) benign. Coding variants were seen across the ATM exonic structure/splice sites, and 20 individual variants were shared in > 10 pts. 263/297 available retrospective tumor samples had interpretable IHC results; 27% (N = 72) had ATM LOP. LOP was most prevalent in tumors with inactivating ATM variants (39/100, 39%); but, importantly, LOP was seen in 20% (N = 33/162) of potentially inactivating/VUS, thus better clarifying their functional impact. In the prospective cohort of 217 pt tumors, 17% (N = 37) had ATM LOP. 29% (N = 62/217) of this cohort also had ATM variants. ATM LOP was seen in 48% of tumors with inactivating variants (N = 14/29), 25% of tumors with potentially/VUS(N = 9/36), and 9% (N = 14/156) of tumors without ATM variants identified. ATM LOP was detected most commonly in colorectal (24%; N = 8/34), cholangiocarcinoma (20%; N = 6/30), prostate (16%; N = 16/104) and pancreatic (9%; N = 1/11) cancers among this cohort of pts referred for DDRi trials. Conclusions: ATM coding variants occurred across the gene, with certain variants shared across tumor types. The functional impact of most ATM variants was VUS, and ATM LOP can help clarify function in up to 25% of these VUS. Also, ATM LOP can be seen even in tumors without ATM variants identified, suggesting epigenetic or post-translational loss. Future prospective studies assessing predictive capability of paired DNA and protein-level profiling of ATM are warranted.

Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

AIMS: TERT promoter mutations have been reported in 22% of solitary fibrous tumors (SFT) and have been associated with poor outcomes. We performed testing for TERT hotspot mutations in a large series of solitary fibrous tumors in order to confirm this finding and explore clinicopathologic correlates of mutation status. METHODS AND RESULTS: PCR for TERT hotspot mutations C250T and C228T was performed on DNA extracted from 216 SFT, and mutation status correlated with clinicopathological factors including predicted risk for metastasis using a previously published model. Testing was successful in 189 tumors from 172 patients, and mutations were present in 29%. Presence of TERT promoter mutation was associated with larger primary tumor size, necrosis and older patient age. TERT promoter mutations were most common in high risk tumors (9/20, 45%), and were present in 11/26 (42%) moderate risk tumors, and 14/67 (21%) low risk tumors (p=0.004). Overall, TERT mutations were associated with shorter time to first metastasis (p=0.04), but had no impact on overall survival. TERT promoter mutation status was found not to provide additional prognostic information in low and high risk SFT, but did identify a group of patients with intermediate risk SFT who had an increased risk of metastasis. CONCLUSIONS: TERT promoter mutations were more frequent in SFT with higher risk of metastasis, but TERT promoter mutation status was not a reliable predictor of clinical outcome by itself. However, mutations in the TERT promoter may be useful in further stratifying patients with intermediate risk tumors. This article is protected by copyright. All rights reserved.