Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone.

CONTEXT.­: An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. OBJECTIVE.­: To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). DESIGN.­: Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. RESULTS.­: LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. CONCLUSIONS.­: Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

Development of a LAG-3 immunohistochemistry assay for melanoma.

AIMS: A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumour tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardised, well-characterised assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision and reproducibility of this assay. METHODS: The specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a ≥1% cut-off to determine LAG-3 positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. RESULTS: The LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator and inter-reagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). CONCLUSIONS: We have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC.

Coexisting Alterations of MHC Class I Antigen Presentation and IFNγ Signaling Mediate Acquired Resistance of Melanoma to Post-PD-1 Immunotherapy.

Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti-programmed death 1 (anti-PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti-PD-1 plus anti-lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti-PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.

HYPERsol: High-Quality Data from Archival FFPE Tissue for Clinical Proteomics.

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing an invaluable resource for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present high-yield protein extraction and recovery by direct solubilization (HYPERsol), an optimized workflow using ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). When applied to archival samples, HYPERsol resulted in high-quality data from FFPE specimens in storage for up to 17 years, and may enable the discovery of new immunohistochemical markers.

Sclerosing Epithelioid Fibrosarcoma of the Bone With Rare EWSR1-CREB3L3 Translocation Driving Upregulation of the PI3K/mTOR Signaling Pathway.

Sclerosing epithelioid fibrosarcoma (SEF) is an uncommon neoplasm that rarely presents in bone. It is characterized by epithelioid cells arranged in nests and single-file cords within a sclerotic stromal background which may mimic neoplastic bone. SEF harbors an EWSR1 translocation, which may complicate its distinction from Ewing sarcoma in cases with histomorphologic overlap. We present a diagnostically challenging case of SEF in the mandible of a 16-year-old girl. Our experience highlights the lack of specificity of traditional morphology and EWSR1 break-apart fluorescent in situ hybridization. Open-ended RNA-based fusion gene testing coupled with MUC4 immunohistochemistry aided the eventual diagnosis in this case. Herein, we report the third case of SEF with EWSR1-CREB3L3 translocation and show that this fusion leads to aberrant upregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway in heterologous cell models.

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.

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.

Proteomic approaches for cancer epigenetics research.

Introduction: Epigenetic dysregulation drives or supports numerous human cancers. The chromatin landscape in cancer cells is often marked by abnormal histone post-translational modification (PTM) patterns and by aberrant assembly and recruitment of protein complexes to specific genomic loci. Mass spectrometry-based proteomic analyses can support the discovery and characterization of both phenomena. Areas covered: We broadly divide this literature into two parts: 'modification-centric' analyses that link histone PTMs to cancer biology; and 'complex-centric' analyses that examine protein-protein interactions that occur de novo as a result of oncogenic mutations. We also discuss proteomic studies of oncohistones. We highlight relevant examples, discuss limitations, and speculate about forthcoming innovations regarding each application. Expert commentary: 'Modification-centric' analyses have been used to further understanding of cancer's histone code and to identify associated therapeutic vulnerabilities. 'Complex-centric' analyses have likewise revealed insights into mechanisms of oncogenesis and suggested potential therapeutic targets, particularly in MLL-associated leukemia. Proteomic experiments have also supported some of the pioneering studies of oncohistone-mediated tumorigenesis. Additional applications of proteomics that may benefit cancer epigenetics research include middle-down and top-down histone PTM analysis, chromatin reader profiling, and genomic locus-specific protein identification. In the coming years, proteomic approaches will remain powerful ways to interrogate the biology of cancer.

MDM2 RNA In Situ Hybridization for the Diagnosis of Atypical Lipomatous Tumor: A Study Evaluating DNA, RNA, and Protein Expression.

The distinction of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from its benign counterpart, lipoma, may represent a challenge. MDM2 DNA amplification is used as the gold standard as MDM2 immunohistochemistry lacks specificity and sensitivity. Herein, we investigate the diagnostic utility of MDM2 RNA in situ hybridization (RNA-ISH) and compare the test with MDM2 immunohistochemistry and MDM2 DNA fluorescence in situ hybridization (FISH) in benign and malignant lipomatous neoplasms. We evaluated 109 neoplasms including 27 lipomas, 25 spindle cell lipomas, 32 ALTs/WDLs, and 25 dedifferentiated liposarcomas (DDL). The validation cohort included 14 lipoma-like neoplasms that lacked unequivocal features of ALT/WDL and in which MDM2 immunohistochemistry was either equivocal, negative or falsely positive. Immunohistochemistry, automated RNA-ISH and DNA-FISH for MDM2 were performed. Tumors with diffuse nuclear staining or >50 dots per cell on RNA-ISH were considered positive. All lipomas and lipoma variants were negative for RNA-ISH while all ALTs/WDLs and DDLs were positive. Eighty percent (24/30) and 92% (22/24) of ALTs/WDLs and DDLs were positive for MDM2 immunohistochemistry. Lipomas and its variants were negative for MDM2 amplification; 92% and 100% of ALTs/WDLs and DDLs showed MDM2 DNA amplification. The mean percentage of ALT/WDL tumor cells showing MDM2 RNA-ISH positivity was 73% compared with 24% on MDM2 immunohistochemistry. RNA-ISH correctly classified all 10 ALTs/WDLs and all 4 lipomas in the validation cohort. The performance of MDM2 RNA-ISH and MDM2 DNA-FISH are equivalent. MDM2 RNA-ISH can be of diagnostic value in histologically challenging lipomatous neoplasms. The automated MDM2 RNA-ISH assay should allow for more widespread use of MDM2 testing and for a more sensitive and specific diagnosis of ALT/WDL.

Clear Cell Chondrosarcoma With Chondroblastoma-Like Features: A Case for Team Diagnosis.

Clear cell chondrosarcoma (CCCS) is a rare variant of conventional chondrosarcoma with low-grade malignant features that may be confused radiographically and histologically with chondroblastoma. We report a case of a 50-year-old female who presented with 6 months of left hip pain. Initial radiographs demonstrated an osteolytic lesion with adjacent area of sclerosis in the proximal left femur. Magnetic resonance imaging demonstrated a marrow-infiltrative lesion with periosteal reaction and thickened enhancing periosteum. Biopsy of the sclerotic area demonstrated chondroblastoma-like findings, whereas biopsy of the lytic area showed features suggestive of CCCS. The patient eventually underwent en bloc resection and reconstruction with a proximal femoral megaprosthesis. The final diagnosis was CCCS. We present this unusual case with review of the radiographic and histologic features of CCCS with attention to its ability to mimic chondroblastomas. This case highlights the importance of sampling radiographically heterogeneous areas within a bone lesion to facilitate accurate diagnosis and appropriate management.

Imaging and histological appearance of pleomorphic hyalinizing angiectatic tumors: A case series and literature review.

Pleomorphic hyalinizing angiectatic tumors (PHATs) are rare mesenchymal soft tissue tumors of uncertain lineage and intermediate malignancy. The present study assesses two cases of PHAT and discusses the histological and immunophenotypical features, as well as the imaging appearance of these tumors on ultrasound, computed tomography (CT), magnetic resonance imaging and positron emission tomography/CT scans. The current study also reviews the literature and discusses the clinical management of these tumors. Wide local excision with tumor free margins is the current recommended treatment for PHAT. Surgical excision may be combined with low-dose radiation to reduce the risk of local recurrence. Patients should be followed up with serial imaging, as PHAT lesions tend to recur locally.

Actionable mutations in canine hemangiosarcoma.

BACKGROUND: Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. METHODS: Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. RESULTS AND CONCLUSIONS: Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Epigenetic Alterations in Bone and Soft Tissue Tumors.

Human malignancies are driven by heritable alterations that lead to unchecked cellular proliferation, invasive growth and distant spread. Heritable changes can arise from changes in DNA sequence, or, alternatively, through altered gene expression rooted in epigenetic mechanisms. In recent years, high-throughput sequencing of tumor genomes has revealed a central role for mutations in epigenetic regulatory complexes in oncogenic processes. Through interactions with or direct modifications of chromatin, these proteins help control the accessibility of genes, and thus the transcriptional profile of a cell. Dysfunction in these proteins can lead to activation of oncogenic pathways or silencing of tumor suppressors. Although epigenetic regulators are altered across a broad spectrum of human malignancies, they play a particularly central role in tumors of mesenchymal and neuroectodermal origin. This review will focus on recent advances in the understanding of the molecular pathogenesis of a subset of tumors in which alterations in the polycomb family of chromatin modifying complexes, the SWI/SNF family of nucleosome remodelers, and histones play a central role in disease pathogenesis. Although this review will focus predominantly on the molecular mechanisms underlying these tumors, each section will also highlight areas in which an understanding of the molecular pathogenesis of these diseases has led to the adoption of novel immunohistochemical and molecular markers.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

Denosumab-treated Giant Cell Tumor of Bone Exhibits Morphologic Overlap With Malignant Giant Cell Tumor of Bone.

Giant cell tumor (GCT) of bone is a locally aggressive benign neoplasm characterized by an abundance of osteoclastic giant cells that are induced by the neoplastic mononuclear cells; the latter express high levels of receptor activator of nuclear factor κ-B ligand (RANKL). Denosumab, a RANKL inhibitor, which is clinically used to treat GCT, leads to a marked alteration in the histologic appearance of the tumor with giant cell depletion and new bone deposition, leading to substantial histologic overlap with other primary tumors of bone. Most significantly, denosumab-treated GCT (tGCT) with abundant bone deposition may mimic de novo osteosarcoma, or GCT that has undergone malignant transformation. To histologically characterize tGCT, we identified 9 cases of GCT biopsied or resected after denosumab treatment. tGCT cases included 16 specimens from 9 patients including 6 female and 3 male individuals aged 16 to 47 (median 32) years. Duration of treatment varied from 2 to 55 months. We compared these tumors with malignant neoplasms arising in GCTs (n=9). The histology of tGCT was variable but appeared to relate to the length of therapy. All tGCTs showed marked giant cell depletion. Early lesions were highly cellular, and the combination of cellularity, atypia, and haphazard bone deposition caused the lesion to resemble high-grade osteosarcoma. Unlike de novo high-grade osteosarcoma or malignancies arising in GCT, however, tGCT showed less severe atypia, reduced mitotic activity, and lack of infiltrative growth pattern. Tumor in patients on prolonged therapy showed decreased cellularity and abundant new bone, deposited as broad, rounded cords or long, curvilinear arrays. The latter morphology was reminiscent of low-grade central osteosarcoma, but, unlike low-grade central osteosarcoma, tGCT was negative for MDM2 and again lacked an infiltrative growth pattern. Overall, tGCT may have a wide range of morphologic appearances. Because the treated tumors bear little resemblance to their pretreatment counterparts, careful attention to the history of denosumab administration is crucial to avoid a misdiagnosis with an important impact on therapy. Unlike malignant GCTs, tGCTs lack significant nuclear atypia, mitotic activity, and infiltration of preexisting bone, but instead show a unique pattern of intralesional bone deposition.

Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare, aggressive malignant neoplasm characterized by small nests and linear arrays of epithelioid cells embedded in a dense collagenous matrix. Very few primary SEFs of bone have been reported. Recognition is critical, as the dense extracellular collagenous matrix can be interpreted as osteoid, leading to misdiagnosis as-osteosarcoma. MUC4 and SATB2 are 2 recently characterized immunohistochemical markers for SEF and osteosarcoma, respectively. In reports to date, osteosarcomas are positive for SATB2 and negative for MUC4, whereas soft tissue SEFs have shown the opposite immunohistochemical profile (SATB2-/MUC4+). The purpose of this study was to characterize the clinicopathologic and immunohistochemical features of 8 primary SEFs of bone. The patients presented at a wide range of ages (25 to 73 y; median 52 y). Tumors mostly involved long bones of the extremities, with 3 cases involving the femur, 2 involving the ulna, and 1 involving the humerus. Other sites of involvement included the second rib (1) and the C6 vertebra (1). Follow-up information was available for 7 patients, 3 of whom developed metastases within 2 years of diagnosis. The other 4 patients were free of local recurrence or metastases at 1, 5, 12, and >84 months of follow-up, respectively. Radiographically, the tumors were predominantly lytic and poorly marginated. Histologically, 6 tumors showed pure SEF morphology, and 2 showed hybrid SEF/low-grade fibromyxoid sarcoma morphology. Focal dystrophic mineralization was seen in 1 case but was limited to areas of necrosis. None of the tumors showed the lace-like pattern of mineralization typical of osteosarcoma. The majority (6/8) of the tumors strongly expressed MUC4. SATB2 was negative in all but 1 case, which showed variable weak to moderate staining in ∼50% of nuclei. In general, the combination of morphology, MUC4 expression, and the absence of SATB2 expression was highly useful in arriving at the correct diagnosis.