Histone H3K27 dimethyl loss is highly specific for malignant peripheral nerve sheath tumor and distinguishes true PRC2 loss from isolated H3K27 trimethyl loss.

Malignant peripheral nerve sheath tumors contain loss of histone H3K27 trimethylation (H3K27me3) due to driver mutations affecting the polycomb repressive complex 2 (PRC2). Consequently, loss of H3K27me3 staining has served as a diagnostic marker for this tumor type. However, recent reports demonstrate H3K27me3 loss in numerous other tumors, including some in the differential diagnosis of malignant peripheral nerve sheath tumor. Since these tumors lose H3K27me3 through mechanisms distinct from PRC2 loss, we set out to determine whether loss of dimethylation of H3K27, which is also catalyzed by PRC2, might be a more specific marker of PRC2 loss and malignant peripheral nerve sheath tumor. Using mass spectrometry, we identify a near complete loss of H3K27me2 in malignant peripheral nerve sheath tumors and cell lines. Immunohistochemical analysis of 72 malignant peripheral nerve sheath tumors, seven K27M-mutant gliomas, 43 ependymomas, and 10 Merkel cell carcinomas demonstrates that while H3K27me3 loss is common across these tumor types, H3K27me2 loss is limited to malignant peripheral nerve sheath tumors and is highly concordant with H3K27me3 loss (33/34 cases). Thus, increased specificity does not come at the cost of greatly reduced sensitivity. To further compare H3K27me2 and H3K27me3 immunohistochemistry, we investigated 42 melanomas and 54 synovial sarcomas, histologic mimics of malignant peripheral nerve sheath tumor with varying degrees of H3K27me3 loss in prior reports. While global H3K27me3 loss was not seen in these tumors, weak and limited H3K27me3 staining was common. By contrast, H3K27me2 staining was more clearly retained in all cases, making it a superior binary classifier. This was confirmed by digital image analysis of stained slides. Our findings indicate that H3K27me2 loss is highly specific for PRC2 loss and that PRC2 loss is a rarer phenomenon than H3K27me3 loss. Consequently, H3K27me2 loss is a superior diagnostic marker for malignant peripheral nerve sheath tumor.

Evaluation of CAR-T cell cytotoxicity: Real-time impedance-based analysis.

This chapter describes the most common method for evaluating cytotoxicity of chimeric antigen receptor (CAR) T cells, the xCELLigence real-time cell analysis (RTCA) platform (Agilent Technologies, Inc., Santa Clara, CA). Though there are a variety of assays used to evaluate conventional and engineered T cell cytotoxicity, the benefit of the xCELLigence platform is the depth of real-time data collected. This chapter begins by providing information on the conceptual basis underlying the xCELLigence assay, followed by a detailed protocol for the application of this assay to evaluate your own CAR-T cells, as well as specific insight and helpful tips for assay design, usage, and data analysis. Application of the information and methods discussed within this chapter will provide a greater understanding for evaluating cytotoxicity of CAR-T cells using this in vitro model system.

GUCY2C as a biomarker to target precision therapies for patients with colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is one of the most-deadly malignancies worldwide. Current therapeutic regimens for CRC patients are relatively generic, based primarily on disease type and stage, with little variation. As the field of molecular oncology advances, so too must therapeutic management of CRC. Understanding molecular heterogeneity has led to a new-found promotion for precision therapy in CRC; underlining the diversity of molecularly targeted therapies based on individual tumor characteristics. AREAS COVERED: We review current approaches for the treatment of CRC and discuss the potential of precision therapy in advanced CRC. We highlight the utility of the intestinal protein guanylyl cyclase C (GUCY2C), as a multi-purpose biomarker and unique therapeutic target in CRC. Here, we summarize current GUCY2C-targeted approaches for treatment of CRC. EXPERT OPINION: The GUCY2C biomarker has multi-faceted utility in medicine. Developmental investment of GUCY2C as a diagnostic and therapeutic biomarker offers a variety of options taking the molecular characteristics of cancer into account. From GUCY2C-targeted therapies, namely cancer vaccines, CAR-T cells, and monoclonal antibodies, to GUCY2C agonists for chemoprevention in those who are at high risk for developing colorectal cancer, the utility of this protein provides many avenues for exploration with significance in the field of precision medicine.

Epigenomic Reordering Induced by Polycomb Loss Drives Oncogenesis but Leads to Therapeutic Vulnerabilities in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive sarcoma with recurrent loss-of-function alterations in polycomb-repressive complex 2 (PRC2), a histone-modifying complex involved in transcriptional silencing. To understand the role of PRC2 loss in pathogenesis and identify therapeutic targets, we conducted parallel global epigenomic and proteomic analysis of archival formalin-fixed, paraffin-embedded (FFPE) human MPNST with and without PRC2 loss (MPNSTLOSS vs. MPNSTRET). Loss of PRC2 resulted in increased histone posttranslational modifications (PTM) associated with active transcription, most notably H3K27Ac and H3K36me2, whereas repressive H3K27 di- and trimethylation (H3K27me2/3) marks were globally lost without a compensatory gain in other repressive PTMs. Instead, DNA methylation globally increased in MPNSTLOSS. Epigenomic changes were associated with upregulation of proteins in growth pathways and reduction in IFN signaling and antigen presentation, suggesting a role for epigenomic changes in tumor progression and immune evasion, respectively. These changes also resulted in therapeutic vulnerabilities. Knockdown of NSD2, the methyltransferase responsible for H3K36me2, restored MHC expression and induced interferon pathway expression in a manner similar to PRC2 restoration. MPNSTLOSS were also highly sensitive to DNA methyltransferase and histone deacetylase (HDAC) inhibitors. Overall, these data suggest that global loss of PRC2-mediated repression renders MPNST differentially dependent on DNA methylation to maintain transcriptional integrity and makes them susceptible to therapeutics that promote aberrant transcription initiation. SIGNIFICANCE: Global profiling of histone PTMs and protein expression in archival human MPNST illustrates how PRC2 loss promotes oncogenesis but renders tumors vulnerable to pharmacologic modulation of transcription.See related commentary by Natarajan and Venneti, p. 3172.

Promotion of growth factor signaling as a critical function of ß-catenin during HCC progression.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. ß-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that ß-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, ß-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of ß-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease.

Papillary Thyroid Carcinoma Emerging from Hashimoto Thyroiditis Demonstrates Increased PD-L1 Expression, Which Persists with Metastasis.

There is evidence that programmed death-ligand 1 (PD-L1) is expressed by thyroid follicular epithelium in thyroiditis, but the role of PD-L1 in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to determine whether (1) the presence of background chronic lymphocytic thyroiditis (CLT) or Hashimoto thyroiditis (HT) influenced the expression of PD-L1 in benign follicular epithelium or in PTC and (2) if PD-L1 expression in PTC persisted with lymph node metastasis. We performed immunohistochemistry (IHC) for PD-L1 on formalin-fixed paraffin-embedded tissues. We first studied five cases of unremarkable thyroid, five cases of CLT, and five cases of HT without carcinoma. We subsequently performed PD-L1 IHC on ten cases of PTC arising in normal thyroid, ten cases of PTC arising in CLT, and ten cases of PTC arising in HT. Whenever available, we evaluated corresponding synchronous lymph node metastases from all cases for PD-L1 expression. PD-L1 expression was increased (10-90%) in all five cases of HT, only minimal expressed (1-5%) in two of five cases of CLT, and not expressed in five cases of unremarkable thyroid. PTC arising in normal thyroid or CLT nearly uniformly lacked PD-L1 expression. In contrast, PTC arising in HT demonstrated significant PD-L1 expression, which persisted in corresponding lymph node metastases. Background non-neoplastic follicular epithelium in the HT cases also demonstrated PD-L1 expression. Thyroid follicular epithelium in HT demonstrates increased PD-L1 expression, and PTC arising in a background of HT shows increased PD-L1 expression, which is retained with metastasis.