ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms.

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.

NTRK point mutations and their functional consequences.

The neurotrophic receptor tyrosine kinase (NTRK) family of genes, including NTRK1, NTRK2, and NTRK3, encodes membrane-bound receptors that normally regulate cell survival and differentiation upon binding of growth factors. Not surprisingly, mutations in these genes are known to contribute to the growth of a diverse number of cancers. With the recent FDA approval of two first-generation tyrosine-kinase inhibitors (TKIs) for adult and pediatric patients with solid tumors harboring NTRK gene fusions, much of the literature has focused on the biology behind these types of NTRK abnormalities; however, point mutations can also contribute to oncogenesis or resistance to TKI therapy, albeit at a lower frequency than fusions. This review focuses on NTRK gene mutations that are associated with resistance to directed therapies, mutations detected in the primary setting that confer increased oncogenic activity, and evidence that suggests that some of these variants may be treated using specific targeted therapies. Finally, this review focuses on the detection of point mutations, including the utility of cell-free DNA (cfDNA) for monitoring the acquisition of resistance mutations.

Interpretative differences of combined cytogenetic and molecular profiling highlights differences between MRC and ELN classifications of AML.

Acute myeloid leukemia (AML) is typically characterized clinically for prognostic purposes using both cytogenetic and molecular characteristics. However, both cytogenetic and molecular risk stratification schemas are varied and few reports have studied correlations between these schemas. We have performed a single institution retrospective review of cytogenetic and molecular classifications of AMLs seen at Penn Medicine between 2013 and 2018. One-hundred fourty-four cases were characterized according to European Leukemia Net (ELN) or Medical Research Council (MRC) criteria for cytogenetics and results compared to molecular profiling. When we analyzed the most common sequencing study results within the risk groupings, negative sequencing studies and FLT3 mutations were common in favorable AMLs, intermediate AMLs had mutations in FLT3, NPM1, DNMT3A and IDH2, while adverse AMLs had a high prevalence of TP53 mutations. We next grouped the genes on the panel by their proteins' functions and found mutations in signaling pathway genes to be common in favorable AMLs while tumor suppressors were commonly mutated in adverse AMLs. AMLs grouped by the type of chromosomal abnormality present showed that FLT3 mutations were common in AMLs with a trisomy while TP53 mutations were common in AMLs with a monosomy or a deletion. TP53 mutations are especially common in AMLs with a monosomal karyotype and often overlap with 17p loss. Interestingly, although all AMLs with TP53 mutations have a defect in the response to DNA damage, expression of P53 protein before and after irradiation is not consistently predicted by phenotype. Overall, these studies confirm the genetic complexity of AML which does not fall into simple patterns of cooperating mutations.

Targeted massively parallel sequencing of mature lymphoid neoplasms: assessment of empirical application and diagnostic utility in routine clinical practice.

Massively parallel sequencing (MPS) has become a viable diagnostic tool to interrogate genetic profiles of numerous tumors but has yet to be routinely adopted in the setting of lymphoma. Here, we report the empirical application of a targeted 40-gene panel developed for use in mature lymphoid neoplasms (MLNs) and report our experience on over 500 cases submitted for MPS during the first year of its clinical use. MPS was applied to both fresh and fixed specimens. The most frequent diagnoses were diffuse large B-cell lymphoma (116), chronic lymphocytic leukemia/small lymphocytic lymphoma (60), marginal zone lymphoma (52), and follicular lymphoma (43), followed by a spectrum of mature T-cell neoplasms (40). Of 534 cases submitted, 471 generated reportable results in MLNs, with disease-associated variants (DAVs) detected in 241 cases (51.2%). The most frequent DAVs affected TP53 (30%), CREBBP (14%), MYD88 (14%), TNFRSF14 (10%), TNFAIP3 (10%), B2M (7%), and NOTCH2 (7%). The bulk of our findings confirm what is reported in the scientific literature. While a substantial majority of mutations did not directly impact diagnosis, MPS results were utilized to either change, refine, or facilitate the final diagnosis in ~10.8% of cases with DAVs and 5.5% of cases overall. In addition, we identified preanalytic variables that significantly affect assay performance highlighting items for specimen triage. We demonstrate the technical viability and utility of the judicious use of a targeted MPS panel that may help to establish general guidelines for specimen selection and diagnostic application in MLNs in routine clinical practice.

Primary Pulmonary Myxoid Sarcoma and Myxoid Angiomatoid Fibrous Histiocytoma: A Unifying Continuum With Shared and Distinct Features.

Primary pulmonary myxoid sarcoma (PPMS) is a recently reported, exceedingly rare low-grade lung neoplasm characterized by reticular/lace-like growth of spindle to epithelioid cells embedded in an abundant myxoid matrix. Morphologically, it overlaps with a myxoid variant of angiomatoid fibrous histiocytoma (AFH) of the soft tissue. Genetically, they were both reported to harbor EWSR1-CREB1 fusion, while EWSR1-ATF1 has only been reported in AFH thus far. We report a case of primary pulmonary low-grade myxoid spindle cell tumor with morphologic and immunohistochemical features of PPMS but with an EWSR1-ATF1 fusion gene. In addition, we also encountered a case of endobronchial AFH with EWSR1-CREB1 translocation but also focal morphologic features of PPMS. These findings provide new evidence supporting the concept that PPMS and a myxoid variant of AFH represent a continuum with overlapping histologic, immunohistochemical, and genetic features.

Epigenomic profiling of neuroblastoma cell lines.

Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma.

CAMKV Is a Candidate Immunotherapeutic Target in MYCN Amplified Neuroblastoma.

We developed a computational pipeline designed to use RNA sequencing (n = 136) and gene expression profiling (n = 250) data from neuroblastoma tumors to identify cell surface proteins predicted to be highly expressed in MYCN amplified neuroblastomas and with little or no expression in normal human tissues. We then performed ChIP-seq in the MYCN amplified cell lines KELLY, NB-1643, and NGP to identify gene promoters that are occupied by MYCN protein to define the intersection with the differentially-expressed gene list. We initially identified 116 putative immunotherapy targets with predicted transmembrane domains, with the most significant differentially-expressed of these being the calmodulin kinase-like vesicle-associated gene (CAMKV, p = 2 × 10-6). CAMKV encodes a protein that binds calmodulin in the presence of calcium, but lacks the kinase activity of other calmodulin kinase family members. We confirmed that CAMKV is selectively expressed in 7/7 MYCN amplified neuroblastoma cell lines and showed that the transcription of CAMKV is directly controlled by MYCN. From membrane fractionation and immunohistochemistry, we verified that CAMKV is membranous in MYCN amplified neuroblastoma cell lines and patient-derived xenografts. Finally, immunohistochemistry showed that CAMKV is not expressed on normal tissues outside of the central nervous system. Together, these data demonstrate that CAMKV is a differentially-expressed cell surface protein that is transcriptionally regulated by MYCN, making it a candidate for targeting with antibodies or antibody-drug conjugates that do not cross the blood brain barrier.

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms.

CONTEXT.­: Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. OBJECTIVE.­: To validate an anchored multiplex polymerase chain reaction-based panel for the detection of fusion transcripts in a university hospital-based clinical molecular diagnostics laboratory. DESIGN.­: We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. RESULTS.­: Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. CONCLUSIONS.­: This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

Validation of a next-generation sequencing oncology panel optimized for low input DNA.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes.

CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05-1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

The epigenetic modifier ubiquitin-specific protease 22 (USP22) regulates embryonic stem cell differentiation via transcriptional repression of sex-determining region Y-box 2 (SOX2).

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs, and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs.

Inhibition of the single downstream target BAG1 activates the latent apoptotic potential of MYC.

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

Enzymatic assays for assessing histone deubiquitylation activity.

While the post-translational modification of histones by the addition of ubiquitin was discovered decades ago, it has only recently been appreciated that the dynamic regulation of histone ubiquitylation patterns is an important mechanism for controlling a variety of biological processes. The processes include transcription, the recognition and repair of genomic damage and DNA replication, among others. Enzymes that catalyze the addition of ubiquitin to histones, such as the polycomb family, have been well-studied. In contrast, the enzymes that remove ubiquitin from histones are less well understood. The assay strategies described here provide a platform for the thorough in vitro and in vivo analysis of histone deubiquitylation. In some cases, these poorly characterized enzymes are likely to provide new opportunities for therapeutic targeting and a detailed understanding of their biochemical and biological activities is a prerequisite to these clinical advances.

Chemotherapy-resistant side-population of colon cancer cells has a higher sensitivity to TRAIL than the non-SP, a higher expression of c-Myc and TRAIL-receptor DR4.

Cancer stem cells are resistant to chemotherapy and provide an important target for drug development. We found that, surprisingly, the dye-effluxing side population (SP) within SW480 human colon tumor cells, a population defined to possess stem cell characteristics, expresses a 10-fold higher level of pro-apoptotic TRAIL receptor DR4 as compared to non-SP cells. The TRAIL receptors are activated by the anti-tumor host immune system through the TRAIL ligand. SW480 SP-cells express similar levels of another TRAIL receptor (DR5), as non-SP cells. SP-cells from multiple tumorigenic human cell lines, which are most often resistant to chemotherapeutic agents such as etoposide, cisplatin and 5-FU, are more sensitive to TRAIL than non-SP cells. SP-cells express higher levels of c-Myc than non-SP cells which may explain their sensitivity to TRAIL. We have found c-Myc activates DR4 transcription through E-box DNA-response elements located in the DR4 promoter, thereby increasing the expression of cell-surface pro-apoptotic death receptors in TRAIL-resistant cell lines. TRAIL sensitivity of SP-cells may represent a safeguard against malignancy, and therefore, offers a therapeutic window and opportunity.